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Received: 10 April 2020 / Revised: 8 June 2020 / Accepted: 19 June 2020 / Published: 22 June 2020

Rice-based intensive cropping systems require high input levels making them less profitable and vulnerable to the reduced availability of labor and water in Asia. With continuous conventional puddled rice transplanting, the situation is exacerbated by damaged soil structure, declining underground water and decreasing land and water productivity. To minimize these negative effects a range of new crop establishment practices have been developed (zero tillage, dry direct seeding, wet direct seeding, water seeding, strip planting, bed planting, non-puddled transplanting of rice, mechanical transplanting of rice crop and combinations thereof) with varying effects on soil health, crop productivity, resource saving and global warming mitigation potential. Some of these allow Conservation Agriculture (CA) to be practiced in the rice-based mono-, double- and triple cropping systems. Innovations in machinery especially for smallholder farms have supported the adoption of the new establishment techniques. Non-puddling establishment of rice together with increased crop residue retention increased soil organic carbon by 79% and total N (TN) in soil by 62% relative to conventional puddling practice. Rice establishment methods (direct seeding of rice, system of rice intensification and non-puddled transplanting of rice) improve soil health by improving the physical (reduced bulk density, increased porosity, available water content), chemical (increased phosphorus, potassium and sulphur in their available forms) and biological properties (microbiome structure, microbial biomass C and N) of the soil. Even in the first year of its practice, the non-puddled transplanting method of rice establishment and CA practices for other crops increase the productivity of the rice-based cropping systems. Estimates suggest global warming potential (GWP) (the overall net effect) can be reduced by a quarter by replacing conventional puddling of rice by direct-seeded rice in the Indo-Gangetic Plains for the rice-based cropping system. Moreover, non-puddled transplanting of rice saves 35% of the net life cycle greenhouse gases (GHGs) compared with the conventional practice by a combination of decreasing greenhouse gases emissions from soil and increasing soil organic carbon (SOC). Though the system of rice intensification decreases net GHG emission, the practice releases 1.5 times greater N ₂O due to the increased soil aeration. There is no single rice establishment technology that is superior to others in all circumstances, rather a range of effective technologies that can be applied to different agro-climates, demography and farm typologies. View Full-Text

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Alam, M.K.; Bell, R.W.; Hasanuzzaman, M.; Salahin, N.; Rashid, M.H.; Akter, N.; Akhter, S.; Islam, M.S.; Islam, S.; Naznin, S.; Anik, M.F.A.; Apu, M.M.R.B.; Saif, H.B.; Alam, M.J.; Khatun, M.F. Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields. Agronomy 2020, 10, 888. https://doi.org/10.3390/agronomy10060888

AMA Style

Alam MK, Bell RW, Hasanuzzaman M, Salahin N, Rashid MH, Akter N, Akhter S, Islam MS, Islam S, Naznin S, Anik MFA, Apu MMRB, Saif HB, Alam MJ, Khatun MF. Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields. Agronomy. 2020; 10(6):888. https://doi.org/10.3390/agronomy10060888

Chicago/Turabian Style

Alam, Md. K., Richard W. Bell, Mirza Hasanuzzaman, N. Salahin, M.H. Rashid, Nadia Akter, S. Akhter, Mahammad S. Islam, S. Islam, S. Naznin, M.F.A. Anik, Md. M.R.B. Apu, Hasib B. Saif, M.J. Alam, and Mst. F. Khatun 2020. "Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields" Agronomy 10, no. 6: 888. https://doi.org/10.3390/agronomy10060888

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# data[].attributes.attachment_link.title
Rice (Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields

# data[].attributes.attachment_link.description
Rice-based intensive cropping systems require high input levels making them less profitable and vulnerable to the reduced availability of labor and water in Asia. With continuous conventional puddled rice transplanting, the situation is exacerbated by damaged soil structure, declining underground water and decreasing land and water productivity.

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Abstract

Introduction

Lodging, which is the permanent displacement of the stem of a free-standing crop plant from the vertical ( Pinthus, 1973; Berry et al., 2004), has attracted the attention of scientists particularly on rice for over 80 years. Over 70 years ago ( Ramaiah and Mudaliar, 1934), it was shown that the more lodging-resistant types of rice had stems with a thicker band of sclerenchyma at the periphery of the stem than lodging-susceptible strains. More recently, Chaturvedi et al. (1995) showed that lodging-tolerant submerged lowland rice varieties had more vascular bundles, both peripheral and in the inner section of the outer layers, than susceptible varieties.

These studies did not show any causal link, via mechanical strength, between anatomy and lodging susceptibility. By contrast, Ooawa et al. (1993) carried out studies on differences in physical and chemical characteristics of culms associated with lodging resistance in paddy rice. They discovered that differences in the degree of lodging and the lodging index were largely dependent on variation in the breaking strength of the basal internode. The chemical differences were related to the physical characteristics, pointing to the fact that the latter derives from the former.

The resultant effects of these studies have always been the selection of shorter varieties with more rigid straws and culms. As in wheat ( Berry et al., 2004) these efforts have allowed breeders to increase the yield without increasing lodging susceptibility, despite the greater weight of the panicle. However, lodging remains a problem in cereals and, more recently, attention has started to be focused on the mechanics of roots and root systems vis-à-vis lodging. According to Crook and Ennos (1994), one of the reasons for the incomplete success of plant breeding programmes in reducing the occurrence of lodging may be that the mechanical basis of lodging has not been fully explored; breeders have tended to rely on a more empirical approach to selection. Analysing the basis of lodging further, they stated that the susceptibility of a variety to lodging will depend on three factors: (i) the size and dynamics of the forces to which it is subjected ( Pinthus, 1973); (ii) the bending strength of the stem and its resistance to lodging ( Neenan and Spencer-Smith, 1975); and (iii) the anchorage strength of the root system.

Other scientists worldwide have implicated the root system and its failure as being responsible for lodging in rice. Terashima et al. (1994, 1995) worked on the eco-physiological characteristics related to lodging tolerance in direct-sown rice and found out that rice varieties resistant to lodging developed more roots in deeper soil layers than lodging-susceptible varieties, and that the contribution of unit root weight to lodging tolerance was higher in deeper than in shallower soil layers. Thus their work confirmed that, in rice, a greater ability to form roots with a higher bulk density in subsoil was one of the most important characteristics for root lodging tolerance.

The implication of roots, root systems, and soil factors in lodging phenomena became clearer with the studies of Ennos (1991) and Crook and Ennos (1993, 1994) on the mechanics of lodging in winter wheat, studies put into context by Ennos's review of the mechanics of root anchorage ( Ennos, 2000). The two studies by Crook and Ennos (1993, 1994) looked into the mechanics of lodging and related them to the stem and root characteristics associated with lodging in winter wheat. From these studies, it was clear that lodging resistance was not related to the strength and stiffness of the stems, which were usually adequate to withstand the force to which they were subjected, but rather was dependent on a cone of rigid coronal roots which emerge from around the stem base. A theoretical model of anchorage in the studies suggested that lodging resistance should be dependent on the diameter of the root-soil cone, coronal root bending strength, and soil shear strength. The earlier study by Ennos (1991) on spring wheat also found that the root system was weaker than the shoot system in wet soil, notwithstanding the differences in the root morphology between the two types of wheat.

The root system in rice is different from that in wheat. Izumi et al. (1996) showed the branching pattern in rice roots to be largely herringbone type. Though, this does not particularly indicate the specific anchorage system in rice, the time-course changes in the growth of the roots vis-à-vis the branching system may influence the resistance or otherwise to root lodging in rice. A clearer picture of the influence of the root system of rice and its nature was presented by Ogata and Matsue (1996), who found that the thickness of the crown roots at different periods after sowing is influential in determining lodging resistance in direct-sown rice. It is, therefore, imperative to obtain a better understanding of the mechanics of root anchorage and lodging resistance in rice.

Lately, lodging has been responsible for limiting the yield and the potential for increases in yields as well as losses in eventual and harvestable yields in cereals globally ( Berry et al., 2004). This has been implicated in wheat ( Laude and Pauli, 1956; Weibel and Pendleton, 1964; Crook and Ennos, 1994, 1995), oats ( Pendleton, 1954), barley ( Day, 1957), grain sorghum ( Larson and Maranville, 1977), and rice ( Basak et al., 1962; Setter et al., 1997). In rice, yield losses can be as much as 1% for every 2% of the crop lodged ( Setter et al., 1997) and grain quality is also affected. The prevalence of lodging in rice is very variable, as it depends on a wide range of factors, and in bad years yield losses in Japan can reach up to 50% ( Setter et al., 1997). Unfortunately, the most sophisticated research has been concentrated on barley, oats, and especially wheat ( Berry et al., 2004).

The research described in this paper was aimed at making the first step in understanding how to prevent root lodging in rice by investigating its structural development and stability using the techniques pioneered for wheat by Crook and Ennos (1993, 1994).

Materials and methods

Planting

The Nerica 1 variety of upland rice Oryza sativa L. which was supplied by the West African Rice Development Association at IITA, Ibadan, Nigeria, was used in the two trials carried out. The seeds were sown in a plastic tray lined with a thin layer of John Innes compost no. 3, and were well watered. Plants were grown in a greenhouse which was heated to an average daytime temperature of 26 °C, falling to 12 °C at night. Supplementary lighting was supplied to give a 16 h day and maximum irradiance was essentially that of full sunlight. The seedlings, which emerged 3 d after sowing, were watered daily and observed for good growth until about 2 weeks when they were transplanted into polystyrene trays measuring 78 cm×38 cm×21 cm and filled with John Innes compost no. 3. The compost was mixed with horticultural grit of 3-6 mm size at a 3:1 ratio to aid drainage. This soil-based compost should mimic a relatively weak natural soil. Each tray contained 10 rice plants transplanted at a spacing of 20 cm×12.5 cm. There were 18 such trays in the first trial and 15 in the second. Each tray, which had perforations underneath to allow drainage, was watered on a daily basis, while nutrients (Phostrogen) were applied at weekly intervals starting from 12 weeks after transplanting.

Two trials were carried out: an exploratory first trial, in which the techniques developed for previous studies of wheat ( Crook and Ennos, 1993, 1994) were customized for rice and the mechanics of lodging investigated; and a second trial in which the development was studied more regularly and methodically, as in the study of Crook et al. (1994) of the structural development of wheat.

Stem and root morphology and mechanical properties

Shoot morphology:

At the 4th week after planting (WAT), five plants, randomly selected from the whole population were carefully uprooted, ensuring that all the soil particles were removed without damaging the roots. The plants' roots were immediately immersed in water and plants were taken to the laboratory for morpho-physical studies. Parameters taken per plant were plant height, centre of gravity, stem cross-sectional area at 5 cm height, number of leaves, fresh shoot weight, number of adventitious roots, and number of tillers. These data were also measured at 11 subsequent fortnightly harvests, spanning six key growth stages: tillering (4 WAT), stem elongation (10 WAT), booting (18 WAT), anthesis (20 WAT), grain filling, (22 WAT), and maturity (26 WAT). Details of the methods used are given below

On arrival in the laboratory, about 0.5 h after uprooting, the number of tillers and leaves were recorded, and the coronal root system was cut away from the stem, both the root systems and the bases of the stems being kept immersed in water to maintain turgor until just before measurements were made. Each shoot system was separated into its individual tillers and the plant height, fresh weight, and centre of gravity were recorded. Plant height was determined by measuring the distance from the base to the tip of the last leaf using a ruler. The centre of gravity was determined by placing separate tillers across an outstretched index finger and moving the tiller along the finger until the balance point was reached-the height of the centre of gravity being the distance from the base of the stem to the balance point ( Crook and Ennos, 1994). Plant fresh weight was determined using a top loading balance (Denver Instrument no. 60665). Stem diameter was measured 5 cm from the base in two (right-angled) directions: the long axis and short axis of an approximate ellipse. The cross-sectional area of the stem base was then calculated 5 cm from the base using the formula

where A is the cross-sectional area, and b and c are the diameters of the stem along the long and short axis, respectively.

Mechanical properties of the stems:

Under the applied force of wind, bending failure of the stem in most cereals is most likely to occur towards the base of the stem ( Ennos, 2000; Berry et al., 2004). For this reason, mechanical tests were limited to the lower region of the stems. The main tillers of five plants at each harvest were prepared for mechanical testing. Basal sections, 10 cm long, were cut from the tillers so that the bending strength and rigidity could be measured using a universal testing machine (Instron model 4301, fitted with a 100 N load cell).

Each section was then subjected to a three-point bending test. Stem sections were placed on two metal supports set 60 mm apart, while a blunt rubber probe of diameter 20 mm, attached to the crosshead of the Instron, was moved down at a speed of 50 mm min −1, touching the stem midway between the supports and bending it. A force/displacement graph was simultaneously recorded on an interfacing computer and used to calculate the mechanical properties of the stem section. The bending strength, S, of a uniform beam is given by

where F max is the minimum force the stem will withstand before it fails and L is the distance between the supports. The bending rigidity, EI, of a uniform beam is given by the formula

where dF/ dY is the initial slope of the force/deflection curve. These equations are appropriate because each length of the stem exhibited very little taper. Since the stems were solid, local buckling by means of ovalization did not occur.

Morphology and mechanical properties of the roots:

The coronal root system which had been removed was washed carefully to clean away the soil and then prepared for mechanical testing; the thick coronals were cut away from the plant base and the length of rigid root, with a diameter of ≥0.5 mm, was measured. Roots with rigid basal regions ≥25 mm long ( Crook and Ennos, 1993, 1994) were then subjected to three-point bending tests on the Instron to determine their bending strength. In this case, the supports were set 20 mm apart, the probe diameter was 4 mm and the crosshead speed of the Instron was 20 mm min −1. Over this distance the roots were not noticeably curved. Roots were out of the water >5 min before being tested.

For each plant, the number and bending strength of rigid roots ≥25 mm long were recorded and the sum of bending strength of all the roots was calculated.

Anchorage mechanics and anchorage strength:

In the first trial, the anchorage mechanics of rice were examined visually in plants from 12 to 26 WAT using the trenching method developed by Coutts (1983) for trees, and used in wheat by Crook and Ennos (1993). The movements of the roots and soil during the overturning of five plants were observed when plants were pulled over parallel to a vertical trench cut alongside the stem, so revealing a 'cross-section' of the root system.

In the second trial starting from the eighth week after transplanting (8 WAT) until maturity, eight plants were randomly selected every fortnight for anchorage tests. Soil cores, each with a diameter of 103 mm, were used to remove the rice plants from the polystyrene trays while ensuring that disturbance to the root system and the surrounding ball of earth was minimal ( Crook and Ennos, 1993). The plants were taken to the laboratory where they were watered overnight to bring the soil medium to field potential. This gave simulated soil conditions typical of waterlogged conditions after rain, when anchorage failure is most likely to occur; it produced soil strengths measured using a shear-vane of 14.9 ±4.6 kPa in the soil cores. The plants were then subjected to anchorage tests using the universal testing machine, Instron, fitted with a lodging meter which enabled it to deliver a rotational force centred around the base of the stem at soil level ( Ennos, 1991; Crook and Ennos, 1993). The apparatus simultaneously pushed the plant over and measured the restoring anchorage moment supplied by the root system, applying a force at a height of 17 cm from the stem base and pushing it over at a rotational velocity of 50° min −1. The interfacing computer calculated two measurements of anchorage: (i) the stiffness, given as the initial slope of the moment/angular deflection curve; and (ii) the strength, given as the restoring moment provided by the plant at a deflection from the vertical of 30°. This deflection was chosen to make the results comparable with those for wheat ( Crook and Ennos, 1993).

Results

Shoot morphology

The plants displayed consistent increases in plant height, centre of gravity, and fresh shoot weight right up to maturity ( Fig. 1), although height growth did tend to flatten off after around 12-15 WAT. The centre of gravity remained at around 30% of plant height. There were similar steady increases in the numbers of leaves and tillers, and in the total cross-sectional area of the tillers ( Fig. 2).

Mechanical properties of the stems

The bending strength and flexural rigidity of individual stems also increased steadily with time in the rice plant ( Fig. 3).

Morphology and mechanical properties of the roots

Figure 4 shows the number of adventitious roots produced per plant, as well as the bending strength of both the individual roots and the sum of root strengths of each plant over time. The number of the roots produced per plant increased over time before levelling off at about 18 WAT. By contrast, the individual root strength levelled off after only 8 WAT, with a slight, but not significant, decline at maturity. Consequently the total root bending strength also rose to a maximum at 18 WAT, and fell slightly but not significantly thereafter, being influenced more by the number of roots than by individual root strength.

Anchorage tests

Observation of the cross-sections of the root systems of rice plants showed that plants showed very similar anchorage failure to winter wheat ( Crook and Ennos, 1993); rotation occurred around a point 3-5 cm below ground and on the windward side of the plant (opposite the direction of push-over force). Consequently, as the plant was pushed over, the soil on the leeward side and underneath the plant was compressed and sheared and the stem bent a little towards the leeward side. Anchorage failure occurred before the stems failed. After the test, plants did not return to their original upright position, but leaned at an angle of 10-30° to vertical, pointing to the fact that anchorage failure had occurred.

Figure 5 shows the anchorage stiffness and anchorage strength of plants over time. Resistance to the anchorage failure increased with time until around 14 WAT ( Fig. 5b), after which it levelled off. The trend for anchorage stiffness ( Fig. 5a) was similar except that there was an unexplained peak at 14 WAT. The large standard deviation suggests that this was due to a single rogue point.

Self-weight moments and factors of safety

The results of the morphological and mechanical tests on the plants were combined to calculate two further sets of parameters: the self-weight moments of individual tillers and whole plants; and self-weight safety factors of the plant against shoot and root lodging which were first devised by Crook and Ennos (1994).

Push over forces caused by wind on a plant will be transmitted to the ground and may eventually cause the stem to bend permanently. Once a plant is leaning, the weight of the aerial parts of the stem will help to push the plant over further. Given the mass distribution of the stem and the angle of its inclination, an estimate of its 'self-weight' moment, M P, of a single tiller can be calculated according to the following equation ( Crook and Ennos, 1994)

where m is the mass and h is the height of centre of gravity of each tiller, g is the acceleration due to gravity, and θ is the angle of inclination of each stem from the vertical. The self-weight moment of individual tillers was calculated at an inclination of 30° from the vertical for the rice plant ( Fig. 6a). The self-weight moment for the entire plant was also calculated by summing the values for each tiller ( Fig. 6b). Both parameters increased with age of the plant throughout the study period.

For a plant to remain structurally intact, both the stem and the anchorage system must be strong enough to withstand the overturning moments generated by the wind and by the weight of the plant. Estimates of safety factors against the self-weight component of the turning moment were calculated by combining measurements on stem and anchorage strength with the calculated self-weight moments ( Crook and Ennos, 1994).

Self-weight safety factors against stem failure

The ability of each stem to avoid breaking depends upon the basal bending strength. The safety factor against stem breakage ( SF S), is expressed by the following equation

where S S is the bending strength of the base of the stem and M T is the self-weight moment generated by a single tiller at an angle, θ°, from the vertical.

Values of the safety factor for the main tillers were calculated for a stem inclination from the vertical of 30° ( Fig. 7a). The self-weight safety factors against stem breakage in rice increased from around 30 to 150 as the plant advanced in age before levelling off at around 21 WAT.

Self-weight safety factors against anchorage failure

Self-weight safety factors against anchorage failure, SF A, were also calculated according to the following equation

where S A is the anchorage moment at θ° from the vertical and M P is the self-weight moment generated per plant at θ° from the vertical. Figure 7b shows the safety factor against anchorage failure at an inclination of 30° from the vertical during plant development. The safety factors against anchorage failure, unlike those obtained for the safety factors against stem failure, decreased after about 12 WAT. The safety factors for the anchorage failure were also much lower than for stem failure, falling from 14 to around 5. Note that there are no error bars for the safety factors against anchorage failure, because anchorage strength and plant morphological data had been collected from different plants, but are likely to be around 50% of the mean values.

Discussion

The results of this study show that, unsurprisingly, the Nerica 1 variety of rice has broadly similar patterns of development and broadly similar mechanics to the two winter wheat varieties studied by Crook et al. (1994), but that there were, in some cases, large quantitative differences between the two cereals that should affect the likelihood of stem and root lodging. These results also suggest that the techniques developed for the wheat study are appropriate, at least to start with, to investigate the effect of differences in genotype and management on the lodging resistance of rice.

Although the pattern of development of the shoot system of the rice was similar to that of wheat ( Crook et al., 1994), there were also some striking differences which should affect the likelihood of stem lodging. Despite being of similar height and mass, the tillers were around 15 times stronger than those of wheat. This difference is largely due to the greater cross-sectional area and hence strength of the elliptic stems in rice compared with the narrow circular stems of wheat. Consequently, the self-weight safety factor of the tillers against stem failure was high, rising from around 30 at 8 WAT to around 150 at maturity. By contrast, in wheat, the factor of safety fell from around 15 to around 5 ( Crook et al., 1994). This suggests that this upland rice variety would be far less likely to suffer from stem lodging than winter wheat, especially as rice grows in tropical regions that have much lower winds.

The anchorage system of rice was also broadly similar to that in wheat ( Crook et al., 1994). As in wheat, plants were anchored by a mass of rigid adventitious roots that emerged from the base of the tillers to form an inverted crown shape. Anchorage failure in both plants occurs in the same way; plants rotate about a point on the windward side of the crown, and the crown and the soil it surrounds compresses the soil beneath it. However, once again there were quantitative differences in both strength and development. The roots of the rice plants were actually only around one-third as strong in bending as those of wheat. However, the number of roots was around 10-15 times that seen in wheat. As a consequence, by harvest rice had an anchorage two to three times as strong as that of wheat. Because of the extra number and weight of the tillers, however, the factor of safety of rice against root lodging was similar to that of wheat and showed the same pattern over time; in both cases, factors of safety fell from around 15 to 3 or 4. This suggests that the rice plants, like wheat, are more likely to be susceptible to root lodging than stem lodging.

Of course, it must be remembered that this is only a preliminary and small-scale study, and there are many caveats that must be put on any conclusions drawn here. First, it is not possible to extrapolate from this study directly to all rice varieties since there are huge differences between deepwater, paddy, and upland rice.

Secondly, the rice plants were grown in Manchester, UK, and therefore by necessity were grown in unnatural greenhouse conditions, which must have greatly affected their morphology and mechanics. Apart from the relatively low light and temperatures, which slowed development, the soil was by no means natural. The most important difference, however, was probably the lack of wind in the greenhouse. Plants grown in windier conditions show thigmomorphogenetic responses, which generally make them shorter and stronger ( Ennos, 1997). Crook and Ennos (1996) have shown, for instance, that supporting the stems of wheat plants against stem sway reduces stem strength by around 20% and anchorage strength by about one-third. Further research should be carried out on plants grown in field conditions.

Thirdly, the analysis of safety factors used here only takes into account the self-weight of the stems, and ignores the usually larger aerodynamic forces on plants and the dynamic forces caused by stem sway. Although Crook and Ennos (1994) showed that there was reasonable correlation between the simple self-weight safety factors and lodging resistance in wheat, subsequent research has developed much more sophisticated analyses of lodging based on the dynamics and aerodynamics of plants ( Baker et al., 1998; Farquar and Meyer-Phillips, 2001; Spatz and Zebrowski, 2001; Berry et al., 2004; Py et al., 2005). Future quantitative work analysing the effect of genotype and management on rice lodging should also use these sorts of models.

Nevertheless, from this research, two important points seem to have emerged. The factor of safety of rice against stem lodging is far greater than that in wheat, so rice plants should be very unlikely to stem lodge. By contrast, rice shows similarly low factors of safety against root lodging as wheat, so like wheat it is likely to be relatively prone to root lodging. To avoid the occurrence of lodging, therefore, efforts should be channelled towards increasing the anchorage strength rather than stem strength. These can be approached in two ways: (i) rice plants with stronger and stiffer roots can be bred, without reducing the number of roots per plant; and (ii) the soil medium could be altered to make it stronger.

The findings of this research also point to areas in which further research could improve our understanding of lodging in rice and help suggest how it can be prevented. Further studies could be carried out to compare rice varieties with contrasting lodging susceptibilities, and to investigate the effects of different soil types on the rice anchorage system and, in effect, on its lodging susceptibility. There is also a need for field trials that will help in further understanding how plants lodge, the environmental factors that affect plant stability, and how to prevent lodging in the future.

We would like to thank Dr O Osiname of the West African Rice Development Association for the supply of the Nerica 1 rice variety seeds. We are also grateful to Thurston W Heaton and his staff at the Botanical Unit of Owens Park, University of Manchester, Manchester, UK, for assisting in looking after the plants in the greenhouse. Dr Oladokun was funded by a Royal Society international short visit grant.

References

# data[].attributes.attachment_link.title
Structural development and stability of rice Oryza sativa L. var. Nerica 1

# data[].attributes.attachment_link.description
Lodging, which is the permanent displacement of the stem of a free-standing crop plant from the vertical ( Pinthus, 1973; Berry et al., 2004), has attracted the attention of scientists particularly on rice for over 80 years.

# data[].attributes.attachment_link.readable_text
Weedy rice is highly variable and can be a serious weed at direct seeded rice (IRRI).

Latin name

Oryza sativa L.

Family

Poaceae

Common name

Red rice, weedy rice

Synonyms

None

Geographical distribution

Asia: Japan and Korea.

South and Southeast Asia: Bangladesh, India, Indonesia, Malaysia, Pakistan, Philippines, and Vietnam.

Rest of the world: widespread in North and South America.

Awns can be absent, short (a), or long (b) (IRRI).

Morphology

A variable, erect, stout or slender annual rice with varying height and form.

Stem: tufted, erect, hollow and slender or stout, smooth and hairless, 80−120 cm tall.

Leaf: blade flat with parallel veins, 15−30 cm long; ligule and auricle present; ligule usually 10−20 mm long.

Inflorescence: an erect or nodding loose panicle, spikelet about 7 mm long, flat, one-flowered, with or without awns of varying length.

Biology and ecology

Weedy rice is highly variable and thought to originate from outcrosses to wild species (O. nivara, O. rufipogon) or as a result of outcrossing within domestic rice. It is distinguished by the key weedy traits of ready panicle shattering and variable secondary dormancy. It is highly adapted to a wide range of environmental conditions, dry land to deepwater culture. It commonly possesses red pericarp, which requires additional milling, thereby reducing the quality of milled rice.

Agricultural importance

Weedy rice is a major weed in direct-seeded rice. It reduces rice yields and is an alternate host of rice insects, diseases, and nematodes. Often spread by contaminated rice seeds.

Management

Cultural control: the use of crop seed free from wild rice, crop rotation, soil puddling, hand weeding of initial infestations, and water management are possible components of an integrated approach to control weedy rice. Established infestations will need to be addressed through the use of stale seedbeds or water seeding or transplanting.

Chemical control: the application of glyphosate before land preparation or seeding reported to be effective.

Selected references

Holm L, Doll J, Holm E, Pancho J, Herberger J. 1997. World weeds: natural histories and distribution. John Wiley & Sons, Inc., USA. 1,129 p.

Holm LG, Plucknett DL, Pancho JV, Herberger JP. 1977. The world's worst weeds: distribution and biology. Honolulu, Hawaii (USA): University of Hawaii Press. 609 p.

Hsu C. 1978. Gramineae (Poaceae). Flora of Taiwan. 5:373-783.

Moody K. 1989. Weeds reported in rice in South and Southeast Asia. Manila (Philippines): International Rice Reseach Institute. 442 p.

Merrill ED. 1976. A flora of Manila. Manila (Philippines): Department of the Interior, Bureau of Science. 491 p.

Reissig WH, Heinrichs EA, Litsinger JA, Moody K, Fiedler L, Mew TW, Barrion AT. 1986. Illustrated guide to integrated pest management in rice in tropical Asia. Manila (Philippines): International Rice Research Institute. 411 p.

Vergara BS. 1992. A farmer's primer on growing rice. Manila (Philippines): International Rice Research Institute. 219 p.

W3TROPICOS at http://mobot.mobot.org/Pick/Search/pick.html.

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Weedy rice is highly variable and thought to originate from outcrosses to wild species (O. nivara, O. rufipogon) or as a result of outcrossing within domestic rice. It is distinguished by the key weedy traits of ready panicle shattering and variable secondary dormancy.

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Rice plants have been grown under water culture to study the growth yield and uptake of nutrients with graded doses of 0, 5, 10, 20, 40, 60, 100, 150 and 200 p.p.m. nitrogen.

Maximum grain yield was obtained at 40 p.p.m. and at higher levels of nitrogen, the growth was considerably retarded due to nitrogen excess in the plant, thereby depressing the grain yield.

Four distinct stages of nitrogen utilisation have been observed: (i) the deficiency stage from 0-10 p.p.m. where grain yield and nitrogen content in grain both increase with increase in nitrogen supply, (ii) the normal stage from 20-40 p.p.m. where the grain yield increases with increase in nitrogen level but increase in nitrogen content of grain is not as sharp. The optimum nitrogen level lies in the range of 30-40 p.p.m., (iii) the luxury stage from 40-60 p.p.m. when the nitrogen content increases with increasing supply of nitrogen whereas there is practically no difference in the yield of grain or straw, (iv) the excess stage from 60-200 p.p.m. where the growth of crop is considerably retarded and yield decreases with increase in nitrogen supply, though nitrogen content of both grain and straw continues to rise.

Results obtained give an idea regarding optimum levels of nitrogen. 30-40 p.p.m. (corresponding to roughly 30-40 kg./ha. under field conditions) is considered optimum for indica varieties. Higher nitrogen levels are shown to result in malnutrition giving low yield of grain.

The authors are indebted to Dr. K. Ramiah, Rice Expert of F.A.O., Bangkok, for suggestion and improvement in the text of the paper.

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Studies on the nutrition of rice plant (Oryza sativa L.) - Proceedings: Plant Sciences

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Rice plants have been grown under water culture to study the growth yield and uptake of nutrients with graded doses of 0, 5, 10, 20, 40, 60, 100, 150 and 200 p.p.m. nitrogen.Maximum grain yield was obtained at 40 p.p.m.

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17.1.1 Seminal Identification of the GROWTH-REGULATING FACTORs

Oryza sativa GROWTH-REGULATING FACTOR1 ( OsGRF1) is the founding member of the GROWTH-REGULATING FACTOR family of TFs. It was identified as one of the genes induced in deep-water rice ( O. sativa L.) internodes in response to gibberellins (GA; Van Der Knaap et al., 2000). This plant hormone promotes the growth of rice stems after flooding by activating cell proliferation in the intercalary meristem (Kende et al., 1998), a region at the base of internodes where new cells are produced. OsGRF1 was detected in regions of the rice plant that contain meristematic tissues and was induced just before cell proliferation genes in response to GA, suggesting that this gene stimulates growth and mitosis, hence prompting this gene family's name.

Sequence analysis of OsGRF1 homologs highlighted two highly conserved protein domains: the QLQ (Gln, Leu, Gln), involved in protein-protein interactions, and the WRC (Trp, Arg, Cys), containing a putative nuclear localization signal and a DNA-binding domain (Figure 17.1A and C). It was also shown that Arabidopsis thaliana GRFs ( AtGRFs) have transcriptional transactivation activity in yeast, further confirming their role as TFs (Kim and Kende, 2004).

Characterization of the GRF gene family in Arabidopsis revealed nine members (Figure 17.1A) that contain the same characteristic regions of OsGRF1: the QLQ and WRC domains (Kim et al., 2003). AtGRF genes were detected in actively growing and developing tissues, such as vegetative shoot apices, inflorescences, and roots tips. Triple insertional null mutants of AtGRF1, AtGRF2, and AtGRF3 had smaller leaves (Figure 17.1B) confirming that GRFs promote organ growth. Further analysis of grf mutants indicated that their smaller leaves contain fewer cells, which implicated a link between GRFs and the control of cell proliferation (Horiguchi et al., 2005; Kim and Kende, 2004; Kim et al., 2003).

The GRF family of TFs has not only been found in all land plant genomes, including representatives of angiosperms and gymnosperms, but also in the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. So far, no GRF has been identified in the genomes of chlorophytes or charophytes, or in species from other kingdoms, indicating that this family of TFs is specific of embryophytes, the more complex multicellular members of the Plantae kingdom.

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Introduction

Oryza sativa L. seeds of the Poaceae family, commonly known as Asian rice or simply rice, could be considered as one of the most important grains since it is consumed by the half of the world population. According to a United States Department of Agriculture (USDA) report, 453.4 million tonnes of rice was consumed worldwide in 2010/2011.

In Asia in particular rice comprises a basic component of the daily diet, and it occupies one of the top positions of commodities with the highest worldwide production according to FAOSTAT (the Statistics Division of the Food and Agriculture Organization of the United Nations). As expected, Asia is by far the main producer and China the most representative country, producing 200 million tonnes of rice and paddy in 2010 (Fig. 32.1).

The oil deriving from rice, namely rice bran oil (RBO), is one of the most commonly used cooking oils in Asia, and the major by-product of the rice milling industry. RBO represents a rich source of high added-value phytochemicals of nutritional, pharmaceutical, and cosmetic interest, such as tocopherols and tocotrienols (tocols or vitamin E), lecithin, carotenoids, and γ-oryzanol, and has therefore attracted considerable interest in recent years. γ-Oryzanol is of particular significance because it is abundant in RBO compared to other vegetable oils.

γ-Oryzanol was first isolated in 1954 by Kaneko and Tsuchiya from the unsaponifiable fraction of rice bran as a crystalline substance; it was considered to be a single compound and was named oryzanol in reference to the botanical species of rice, Oryza sativa. Three years later, Shimizu et al., using a different extraction method, discovered that γ-oryzanol was not a single entity and suggested that it was composed of three ferulic acid esters of triterpene alcohols, which were designated oryzanol A, B, and C. Subsequently the same scientific team identified oryzanol A as cycloartenyl ferulate, and in 1958 oryzanol C was identified as 24-methylene cycloartanyl ferulate. Finally, oryzanol B was found to be a mixture of oryzanol A and C. Since then, various studies have been carried out investigating the composition of γ-oryzanol, allowing the identification of more than 20 ferulic acid esters of triterpene alcohols and sterols by different methods.

Given that RBO is an important by-product in the rice industry, much attention has been paid to optimization of its production and further treatment processes. In the standard industrial procedure of RBO production a refining process takes place through a deacidification treatment with alkali, giving rise to soapstock. This residue is quite valuable, since the highest percentage of γ-oryzanol contained in the crude oil is lost in the soapstock (83%-95%, according to Krishna et al.). Therefore soapstoack is the richest source of γ-oryzanol and probably the most important by-product of rice processing.

γ-Oryzanol generally exhibits a wide spectrum of health-beneficial effects, including anticarginogenic, anti-inflammatory, antihyperlipidemic, antidiabetic, and neuroprotective, which are mainly attributed to its antioxidant capacity. This pleiotropic biological profile of γ-oryzanol, together with its high availability in industrial by-products, has contributed significantly to the observed increasing interest from academia and industry, especially in recent years.

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5.3.6.3 Monocotyledonous Plants

Rice ( Oryza sativa ), maize ( Zea mays), and sorghum ( S. bicolor) all belong to the order Poales and family Poaceae. Therefore, the common pattern for the Poaceae family has been more highly analyzed than other groups of plants. The bZIP, WRKY, NAC, AP2/EREBP, and ABI3/VP1 TF genes are slightly more divergent in monocots than other seed plants. The Ring finger, homeodomain protein, SRF-TF, and AS2 genes are less abundant in monocots than other seed plants. Among these three monocots, rice has more Myb and SRF-TF (MADS) genes, maize has more bZIP, bHLH, WRKY, AP2/EREBP, and AUX_IAA genes, and sorghum has more GRAS and Ring finger genes.

There are many small differences among seed plants, but their TF gene patterns are similar (Figure 5.3F). Notably, monocots and dicots are highly similar in their TF gene patterns. Therefore, physiological differences between the two groups may be associated with a few TF genes.

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Rice

Rice ( Oryza sativa ) cultivation probably originated in Asia, and rice is still a staple food. In the USA, more than 90% of the rice crop is produced in four states: Arkansas, Louisiana, Texas, and California. The rice grain consists of a hull and an edible berry (caryopsis). The outer fibrous layers cover the starchy endosperm and the germ. The endosperm contains mainly starch granules and protein bodies, with little matrix protein in evidence.

To isolate rice starch, broken rice is treated with dilute (0.3-0.5%) sodium hydroxide for about 12 h to soften the grain and aid protein removal. It is wet hammer-milled, the cell walls are removed by screening, and the protein is extracted. The rice starch slurry is purified by further centrifugal washing and then dried.

Rice starch is little used, because of its high cost of production relative to other commercial starches but may be used as a custard or pudding thickener in foods, as a dusting powder in cosmetics, and as a stiffening agent in the cold-starching of fabrics. Rice starch is used in Europe in baby foods, and in the laundry and paper industries.

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Rice ( Oryza sativa L.) is cultivated in all tropical and temperate regions of the world. Considering that almost 90% of the total rice production takes place in Asia, it is not surprising to learn that the majority of viral diseases of rice occurs in this continent. Among the plant viruses that affect rice production, the double-stranded RNA viruses of the family Reoviridae (rice dwarf, rice black streaked dwarf, rice gall dwarf, and rice ragged stunt viruses), and the single-stranded RNA viruses of the genus Tenuivirus (rice stripe, rice grassy stunt, and rice hoja blanca viruses) are some of the most important pathogens. Only the latter virus occurs outside Asia, in Latin America. Rice yellow mottle virus, transmitted by beetles, is the most important viral pathogen of rice in Africa. Rice stripe necrosis virus, transmitted by a soil-borne fungus, is the second and only other viral disease of rice in Africa and Latin America. However, one of the most important viral diseases of rice in Asia is 'tungro', caused by two different viruses: rice tungro bacilliform virus, and its assistor virus, rice tungro spherical virus. Other important viral diseases of rice include rice yellow stunt and rice necrosis mosaic.

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1 Introduction

Rice ( Oryza sativa ), a crop that provides staple food for more than half of the world's population, has served as an excellent model plant for functional genomic studies. This is due to its richness of genetic resources, genomic synteny with other cereals, amenable transformability, and a rich history of extensive research efforts. Rice has also provided a model for establishing technological platforms and testing strategies for genetic improvements to itself and to other crops. For the past decades, we have witnessed tremendous progress in gene discovery and genomic analysis of agronomically important traits and biological processes in rice, largely thanks to the availability of genetic resources and tools. The complex genetic makeup of rice, as with other crop species, allows continued progress in increasing yields, fending off biotic and abiotic stresses and improving nutritional quality-the ultimate goals in breeding elite rice. This genetic diversity allows novel and improved genetic traits fueled by new discoveries and synthetic biology. Biotechnologies, especially those related to altering the genetic makeup of rice varieties, have played and will continue to play an important role not only in the basic understanding of rice biology but also in the creation and introgression of novel genetic elements for improved agronomic traits. Knowledge obtained from rice as a model system can also be extrapolated to other cereal crops, in particular, and to all other crop plants, in general.

Recent advances in genome-editing technology allow precise, targeted genomic changes, including whole-gene insertion or deletion, stacking or pyramiding of genes, and precise modification of genetic elements that allow scientists to introduce, change, or optimize genetic traits of interest. Several classes of edit-enabling technologies have been developed in the past decades, including zinc finger nucleases, TAL effector nucleases (TALENs) and most recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas). The diverse genetic toolboxes, especially TALEN and CRISPR, represent the state-of-the-art genome-editing technologies and hold immense promise for defining the genetic and molecular basis for valuable traits, as well as for the introgression of valuable genes/traits into cultivated varieties in a timely and economically sound manner. The aims of this review chapter are, therefore, to provide a clear understanding of the molecular mechanisms involved in TALEN and CRISPR/Cas9 gene editing and to highlight the research endeavors in the plant science community for their utilization in rice improvement.

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1 Overall Health Benefits of Rice Bran Dietary Supplement

Rice ( Oryza sativa L.) is grown in over 100 countries and served as the staple food for around 3.5 billion people all over the world. While the milled and polished rice grain widely counts for human consumption, the outer covering layer, rice bran, is usually considered as a type of livestock feed, mainly because of traditional perceptions and the rapid development of hydrolytic rancidity in the bran during storage. Therefore, rice bran is known as an underutilized by-product of rice milling. To overcome the storage issue, heat stabilization is applied to inactivate rancidity-inducing lipoxygenases and lipases in rice bran; the increased shelf life and retained bioactivity have expanded the usage of rice bran for human health and nutrition. Rice bran is a natural mixture of a large variety of bioactive compounds. Rice bran metabolite profiling revealed a suite of 65 biochemical molecules with health properties, including amino acids, vitamins and cofactors, and secondary metabolites. The multifaceted health benefits of rice bran have been illuminated using whole rice bran and extracts such as rice bran oil, polysaccharides, proteins/peptides, and minerals. As a result, there is an increasing interest in dietary rice bran supplementation in animals and humans for the medicinal and nutritional properties.

In mice, consumption of rice bran induced an increase in fecal and serum immunoglobulin A (IgA), as well as increased antigen-presenting cells in mesenteric lymph nodes and lamina propria. Another study showed that rice bran glycoprotein was able to help cyclophosphamide-treated mice recover from immunosuppression by promoting the proliferation of splenic lymphocytes. These reports indicate the immunomodulatory effects of rice bran in enhancing mucosal and systemic immunity. In gnotobiotic (Gn) pigs, rice bran intake exhibited high protective efficacy against diarrhea induced by human rotavirus (HRV) and norovirus, the two most prevalent viral pathogens causing gastroenteritis. In human clinical trials, dietary rice bran ameliorated type 2 diabetes by lowering lipid and glycemic levels, and arabinoxylan rice bran has been evaluated with promising therapeutic effects on irritable bowel syndrome (IBS) and chronic hepatitis C virus infection. In addition, emerging scientific evidence indicates that dietary rice bran represents chemopreventive potential against several types of cancer such as liver, lung, breast, and colon cancer. Overall, rice bran is being recognized as an economical, natural, novel, and safe dietary supplement with broad beneficial effects on animal and human health, including treating gastrointestinal diseases.

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3.3.1 Rice

Rice ( Oryza sativa ) is not only a major cereal but also a model monocotyledonous plant. The transformation of rice is relatively easy (Hiei et al., 1994) and the DNA sequence of its rather small genome has been completely determined (International Rice Genome Project, 2005). In rice, all seven CSP orthologs are present in the genome (Banba et al., 2008; Chen et al., 2007, 2008, 2009; Gutjahr et al., 2008), and that is why rice performs AM symbiosis. AM fungi preferentially colonize large lateral roots that contain cortical cells (Gutjahr et al., 2009a). The initiation of the growth of large lateral roots is induced upon colonization by AM fungi, although the architecture of rice roots is somewhat different from that of dicots (Gutjahr et al., 2009a).

Studies on the presymbiotic phase of rice AM symbiosis have been basically lacking, except that it has been found that rice produces strigolactones like all other plants (Umehara et al., 2008). On the other hand, the endosymbiotic phase has been investigated well because of the ample genome information and cDNA array experiments. Paszkowski et al. (2002) identified OsPT11 as the major AM-inducible phosphate transporter gene among 13 genes for Pht1 phosphate transporters. Later, OsPT13 was also reported to be AM-inducible (Güimil et al., 2005). Slightly different expression patterns of the two genes were observed (Glassop et al., 2007). Although rice AM-inducible ammonium transporters have not yet been identified, we expect this in the very near future. Research on microRNAs of rice has been conducted (Sunkar et al., 2005, 2008), but their functions are unclear except for that of the rice ortholog of miR399 (Bari et al., 2006). Recently, OsSPX1 was reported to act via a negative feedback loop to optimize rice growth under phosphate-limited conditions (Wang et al., 2009a), like AtSPX3 in Arabidopsis (Duan et al., 2008).

Compared to the roots of other plants such as legumes, rice roots exhibit very low autofluorescence. Making use of this characteristic, we succeeded in real-time observation of pariarbuscular membranes in rice roots as follows (Kobae and Hata, 2010). Since arbuscules are temporal organs and develop mainly in the inner cortical cells of roots, they are optically and physically inaccessible. Therefore, if this difficulty is overcome because fluorescent marker protein-mediated in planta imaging becomes feasible, it would be an ideal method for investigating these dynamic organs. We prepared transgenic rice ( O. sativa) plants that express a fusion, that is, rice AM-inducible phosphate transporter OsPT11-GFP, and grew them with AM fungi. The OsPT11-GFP fusion resides exclusively on the periarbuscular membranes closely surrounding the fine branches of mature arbuscules, this area being called the "arbuscule branch domain" (Pumplin and Harrison, 2009). We also established a simple method for visualizing OsPT11-GFP fluorescence in the rhizosphere (Fig. 1.4). The T1 seeds of the transgenic rice were grown in 35 mm petri dishes with 27 mm coverslip windows at the bottom. As the glass bottom was covered with AM fungi inoculant, roots that extended and reached the bottom were effectively infected by AM fungi just above the coverslip window. Consequently, real-time OsPT11-GFP fluorescence can be successfully performed around 10 days after seed germination using an inverted fluorescence microscope. In cortical cells, arbuscules seem to be functional for only a couple of days, confirming morphometric studies performed long ago (Alexander et al., 1989; Smith and Read, 2008). Then, time-lapse imaging enabled pinpointing of the beginning of arbuscule degeneration by capturing of images for a period of 220 min (Fig. 1.5A). The images revealed a series of early‐stage degeneration events, from the early symptom of arbuscules to the disappearance of OsPT11-GFP. The initial morphological change of collapsing arbuscules comprised rapid shrinkage of arbuscule branches (Fig. 1.5A). It seems likely that the arbuscule branches suddenly became plastic or fragile, and then gradually but rapidly shrank, and, as a result, the periarbuscular membranes might have become stacked and the OsPT11-GFP signals might have become densely accumulated (Fig. 1.5A). The collapsed state of arbuscules was visible for approximately 2.5 h, and then the GFP fluorescence became undetectable. Therefore, it is probable that the constituents of periarbuscular membrane proteins were promptly digested or realigned during this short time period. Notably, the arbuscule shrinkage preceded the degradation of OsPT11-GFP. It seems likely that withdrawal or autolysis of the fungus induces the degradation event within the plant cells. The collapse of arbuscules occurred in the subsequent several days. Another example, showing that differentially fluorescent cells were present within a single colonization unit, is shown in Fig. 1.5B. It is notable that some arbuscules remained for over 24 h, but others formed and collapsed within 24 h. The images indicate that the OsPT11-GFP expression and the protein turnover in the periarbuscular membrane comprise a cell-autonomous process. In conclusion, the real-time imaging of OsPT11-GFP revealed one of the dynamic aspects of AM symbiosis, namely a period of nutrient exchange. This simple method can be applicable not only for observation of AM symbiosis but also for revealing dynamic aspects of any root proteins of interest, if fluorescent protein fusions are available.

Since AM fungi are basically aerobic microorganisms, they are generally believed to colonize rice plants only under aerobic conditions, that is, not on continuous submersion (Vallino et al., 2009). On the contrary, Secilia and Bagyaraj (1992, 1994) inoculated young rice seedlings with AM fungi in well-drained nursery boxes, transplanted the colonized seedlings to larger pots filled with well-puddled soil and then grew the rice plants for 145 days until harvest under flooded conditions with 5 cm of standing water. They reported that the rice roots were colonized, although the percent of AM colonization of roots was not very high, and that colonization by AM fungi increases the phosphorus absorption by plant bodies and the grain yields. Later, Solaiman and Hirata (1997, 1998) performed similar experiments and confirmed the above results. Moreover, they pointed out that AM colonization increased the absorption of not only phosphorus but also nitrogen from paddy soil by rice plants. We tried to infect rice roots with an AM fungus under water. Surprisingly, infection and colonization occurred even under flooded conditions (Fig. 1.6A and B). As the aerenchyma is formed through programmed death of cortical cells and AM fungi only colonize cortical cells (Gutjahr et al., 2009a), arbuscules were formed in the remaining cortical cells along with the aerenchyma (Fig. 1.6B). The root aerenchyma is connected to those in culms and leaves, enabling efficient air passage from the shoots to the roots. This unique characteristic of rice roots for overcoming the anaerobic and reduced conditions in paddy soil may make the microaerophillic region around the roots a comfortable area for AM fungi. To our knowledge, however, investigations as to how commonly AM fungi live in paddy fields have not yet been performed.

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2.08.5.2 Tricycles from ent-CPP and syn-CPP

In cell cultures of Oryza sativa (rice) that have been exposed to either UV light or chitin, ent-CPP ( 33) is cyclized to ent-sandaracopimaradiene ( 77), presumably by si-face attack of C-17 at C-13 via (( 75), Scheme 13). In the same system, syn-CPP ( 36) is cyclized to 9β-pimara-7,15-diene ( 78), presumably via ( 76). Neither ent-sandaracopimaradiene ( 77) nor 9β-pimara-7,15-diene ( 78) are formed in nonelicited cell cultures. The elicitation of the enzymes for the formation of ( 77) and ( 78) is of interest vis-à-vis the phytoalexins, such as the oryzalexins (e.g., 79) and momililactones (e.g., 81) which are formed in rice plants that have been exposed to UV light or fungal infection. However, no biosynthetic studies on the formation of ( 79) from ( 77) or ( 81) from ( 78) have been reported. ent-Sandaracopimaradiene ( 77) is also formed from GGPP in soluble enzyme preparations from seedlings of castor bean.

The ent-abieta-7,13-diene (( 80) Scheme 13), reported to occur in Helichrysum chionosphaerum, may be formed from ent-CPP ( 33) via ( 77) by a sequence similar to the formation of abieta-7,13-diene (( 59), Scheme 11).

There are no reports of the cyclization of ent- syn-CPP ( 39) to tricyclic diterpenes. The absolute stereochemistry, assigned to the ent-9β-pimara-7,15-dienes, reported to occur in Calceolaria species, has not been established.

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(i) Alpha Amylases from Cereals

Ten isoforms of this enzyme were isolated from rice ( Oryza sativa L.) cells. They differ subtly from one another in their optimum pH. Rice alpha amylase converted only 50% of starch during 3 h at 35 °C. Alpha amylase isolated from cholam ( Sorghum vulgare) contained a small proportion of beta amylase, which could be deactivated by heating the enzyme blend for 10-15 min at 62-63 °C. Cholam diastase is less active than barley diastase, and it leaves intact some dextrins produced from potato starch.

Some plants can secrete thermostable enzymes, as shown with a waxy, self-liquefying starch from hull-less barley. This enzyme performed better at 60 °C, producing predominantly maltose and maltohexaose. Optimal operational conditions for this enzyme were different from those for alpha amylase isolated from amygdalin. Barley is a good source of alpha amylase. The germinated substrate is better than the nongerminated one, and the enzyme isolated from it permits more-complete hydrolysis of starch. Alpha amylase is present in barley before its conversion into malt, but beta amylase also appears on malting. Both enzyme preparations generate no more than an 80% yield of maltose, regardless of the applied enzyme concentration, Independently of the cereal starch hydrolyzed, barley alpha amylase produces maltose and maltohexaose as the main products.

Four enzymes, namely alpha and beta amylases, α-glucosidase, and debranching enzyme were isolated from the barley seeds. Incubation of potato starch with malt alpha amylase gave 3% of glucose and 13% of maltose after 1 h of treatment, and after 48 h of processing there resulted 7.8% of glucose and 48.4% of maltose. Glucose was formed from the limit dextrins, but ptyalin could not release glucose from these dextrins.

A mathematical model, the so-called subsite model, based on kinetics predicts the result of hydrolysis. It was evaluated on thirty distillery barley malts checked for reaction progress and the limiting degree of starch hydrolysis. These malts showed some differences in behavior. All provided a limiting degree of hydrolysis in the range of 51.7-56.2%. In contrast to this report, 95.5% of hydrolysis was reported when the concentration of malt amylase was increased. Enzymatic digestion at 50 °C proceeds better than at lower temperature, and the optimal temperature range was settled as being between 50 and 55 °C, although for the hydrolysis of cooked starch, the optimum temperature for ptyalin and for barley amylase was established as 70 °C. With potato and malt starches, malt amylase performs best at 71 °C, providing a 10% yield of maltodextrins. The optimum pH for purified malt alpha amylase ranged between 4.4 and 5.4.

Amylase isolated from wheat flour is specific and differs from amylase isolated from other plants.

Alpha amylase isolated from the cereal materials is usually complex. Three alpha amylases were isolated from ragi ( Eleusine coracana). They hydrolyzed germinated ragi starches, but the yield did not exceed 70%. Two alpha amylases were isolated from seeds of winter triticale. Their hydrolyzing ability was tested on triticale starch granules. After 22 h of treatment, both amylases produced practically the same amount of reducing sugars, but the rates of hydrolysis were different, as shown in Fig. 5.

Three forms of alpha amylase isolated from maize have been distinguished. They differed from one another in their isoelectric point (pI). They all showed substantially different action patterns, particularly when hydrolysis of gelatinized and granular starch was compared.

Amylases isolated from various wheat cultivars exhibited some differences in their activity and susceptibility towards various substrates. Alpha amylase isoenzymes of germinating winter wheat were also distinguished from one another in their pI values. Those of pI 6.0-6.5, constituting 84% of total isolated isoenzymes, adsorbed readily on undamaged wheat starch granules and digested them. The remainder of these isolated enzymes did not adsorb on these granules.

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Oryza sativa GROWTH-REGULATING FACTOR1 ( OsGRF1) is the founding member of the GROWTH-REGULATING FACTOR family of TFs. It was identified as one of the genes induced in deep-water rice ( O. sativa L.) internodes in response to gibberellins (GA; Van Der Knaap et al., 2000).

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Abstract

A number of genes that contribute to the domestication traits of cultivated rice have been identified. These include Sh4, Rc, PROG1 and LABA1, which are associated with non-shattering rachis, white pericarp, erect growth and barbless awns, respectively. The mutations giving rise to the "domestication alleles" of these genes are either invariable in cultivated rice, or have variability that is strictly associated with the phenotypic trait. This observation forms the basis to those current rice domestication models that envisage a single origin for the domesticated phenotype. Such models assume that the domestication alleles are absent or rare in wild rice, emerged under cultivation and spread across all rice groups by introgressive hybridization. We examined whole-genome sequencing datasets for wild and cultivated rice to test the former two assumptions. We found that the rc and laba1 alleles occur in wild rice with broad geographical distribution, and reach frequencies as high as 13 and 15%, respectively. These results are in agreement with previous observations of the prog1 and sh4 domestication alleles in wild populations. We also show that the diversity of the genomic regions surrounding the rc, laba1, prog1 and sh4 alleles in wild accessions is greater than that in cultivated rice, suggesting that these alleles emerged prior to domestication. Our findings indicate that the possibility that independent rice groups obtained identical domestication alleles directly from the wild population needs to be considered.

Cultivated rice, Domestication genes, Origins of cultivation, Keywords: Oryza sativa, Whole-genome sequence data, Wild rice

Introduction

Cultivated Asian rice ( Oryza sativa L.) can be divided into several groups based on different culinary properties, ecology and genetics- O. sativa subsp. indica Kato; subsp. japonica Kato (with tropical and temperate subgroups); aromatic rice with specific flavours popular in India and Pakistan (basmati) and Iran (sadri), usually treated as a subgroup of subsp. japonica; and aus rice consisting of early-maturing, draught-tolerant ecotypes previously considered to be a subgroup of subsp. indica (Garris et al. 2005; Zhao et al. 2011; Civáň et al. 2015). Current ideas regarding the domestication of O. sativa are strongly driven by analyses of so called "domestication genes", i.e. genes contributing to the domestication phenotype of cultivated rice. The domestication phenotype is a set of characteristics differentiating cultivated rice from its wild progenitor O. rufipogon Griff., resulting from selective pressures imposed by humans during domestication. The most prominent of these changes are growth habit, reduction of seed dormancy, overall yield, quality of rachis, hulls and awns, adaptations to diverse habitats, and the culinary properties of the grain. Although most of these traits are quantitative in nature and hence controlled by a network of genes interacting with the environment, several major genes determining alternative modes of phenotypic variability in cultivated rice have been identified. Well-characterised examples of such major genes are Sh4 (Li et al. 2006), Rc (Sweeney et al. 2006), PROG1 (Tan et al. 2008) and LABA1 (Hua et al. 2015), whose recessive alleles are associated with non-shattering rachis, white pericarp, erect growth and barbless awns, respectively. Functional polymorphisms associated with the phenotypic changes have been identified as either microdeletions disrupting the open reading frame of the coding sequence ( rc, laba1), a mutation causing an amino acid change in the protein ( sh4), or a set of candidate mutations in the coding sequence and promoter region ( prog1). These mutations are either invariable in cultivated rice ( sh4, prog1), or their variability is strictly associated with the phenotypic trait ( rc, laba1). For example, all cultivated rice with a white pericarp (with exceptions in aus) has a 14 bp deletion in the Rc coding sequence, while all cultivated rice with a red pericarp has the reading frame uninterrupted by the deletion (Sweeney et al. 2007). Since the red grain colour is ubiquitous in O. rufipogon, Sweeney et al. ( 2007) did not search for the 14 bp deletion in wild populations and assumed that the rc allele emerged under cultivation. Based on this assumption, they compared the Rc haplotypes found in red japonica and red indica cultivars and concluded that the rc variant emerged in subsp. japonica and was transferred to subsp. indica and partially also to aus by introgressive hybridization (Sweeney et al. 2007). Similarly, Oikawa et al. ( 2015) assumed that the allelic variant of the Kala4 gene responsible for black pigmentation in some rice cultivars emerged during or after domestication, and did not examine genotypes in wild populations (where black pigmentation has not been found).

Since it has been shown that gene variants beneficial for cultivation can be selected from the standing variation of the progenitor (Weber et al. 2007, 2008; Studer et al. 2011) and that the genotype-phenotype association in wild populations may be obscured by interacting genes (Zhu et al. 2012), the assumption that any domestication allele emerged under cultivation is rather risky. Examination of allelic variability in wild populations has been conducted for other rice genes, although with various sample sizes. Hua et al. ( 2015) examined 43 O. rufipogon accessions and did not find a single case with the laba1 deletion. However, Tan et al. ( 2008) sequenced the PROG1 coding sequence in 30 accessions of wild rice with prostrate growth and found 14 cases where the wild haplotypes are identical or similar to the uniform allele of cultivated rice. This led the authors to conclude that other gene(s) may be involved in control of prostrate growth in wild rice. A similar conclusion has been reached regarding the Sh4 gene since multiple other loci affecting the degree of seed shattering have been discovered (Konishi et al. 2006; Subudhi et al. 2014; Yao et al. 2015) and it has been shown that the "non-shattering" sh4 allele is relatively common in wild populations, being present in 44 of 166 wild accessions tested by Zhu et al. ( 2012).

Knowing if the recessive variants of the Sh4, PROG1, Rc and LABA1 genes naturally occur in wild populations is crucial for interpretation of the rice domestication process. Absence of these alleles in wild populations would indicate that the mutations indeed emerged during domestication. Allelic uniformity in cultivated rice would then imply either a single origin of O. sativa, or spread of the domestication alleles among different groups of O. sativa by introgressive hybridization, as has been suggested (Sweeney et al. 2007; Fujino et al. 2010; Hua et al. 2015; Oikawa et al. 2015; Si et al. 2016). However, if the association between the mutations and the phenotype is not strict in O. rufipogon, and the "domestication alleles" are found in wild populations-as is the case with sh4 and prog1-then it is more difficult to make conclusions about the origin of those alleles in different cultivated groups. For example, subsp. indica could have received a domestication allele from subsp. japonica by introgressive hybridization (or vice versa), or both groups could have outsourced the allele from their wild progenitors in separate domestication events. Despite the fact that the prog1 and sh4 alleles are found in O. rufipogon and the occurrence of other domestication alleles in wild rice has not been thoroughly checked, models proposing a single origin of cultivated rice followed by spread of the domestication alleles by introgressive hybridization have become widely accepted (Kovach et al. 2007; Sang and Ge 2007; Izawa 2008; Huang et al. 2012; Gross and Zhao 2014). Here we examine the occurrence of the sh4, prog1, rc and laba1 alleles in O. rufipogon by searching published whole-genome sequencing datasets, and based on our findings suggest that the possibility that independent rice groups obtained identical domestication alleles directly from the wild population needs to be considered.

Methods

We downloaded raw sequence data for 1543 wild and cultivated rice accessions (Huang et al. 2012) from the Sequence Read Archive (ERP001143, ERP000729, ERP000106) and converted each file into FASTQ format using the fastq-dump command in sratoolkit 2.3.5. The full FASTQ dataset consisted of 9.174 billion Illumina reads totalling 2.129 TB and was processed on a Linux platform. Low-quality regions were removed by Trimmomatic-0.33 (Bolger et al. 2014).

These data sets have low sequencing depth (mean 1× and 2× for O. sativa and O. rufipogon, respectively; Huang et al. 2012) which does not allow reconstruction of entire genes or ungapped haplotypes. Nonetheless, known single nucleotide polymorphisms (SNPs) or indel variants can be scored in a subset of the samples where sequencing reads are available for the particular locus. Given the sample size (460 accessions of wild rice; 519 subsp. indica; 482 subsp. japonica; 30 aus; 5 aromatic; 47 other/unassigned), estimates of allelic frequencies can be obtained for subsp. indica, subsp. japonica and wild rice, despite the prevalence of missing data.

To determine indel variants of the Rc gene, we searched each of the 1543 trimmed FASTQ datasets using the grep command with 30 bp words (perfect match) spanning the 14 bp indel site in the Rc gene (5′-AAAGGCGCAAGTGGATGCCATCCAAGGTGA-3′ and reverse complement, matching the variant with deletion; 5′-CAAGTGGAACGCGAAAAGTCGGTGCCATCC-3′ and reverse complement, matching the wild type). Identified matches were further verified by aligning reads to reference sequences of the Rc gene with and without the deletion. We also searched for reads perfectly matching a 30 bp sequence spanning the 1 bp indel site in the LABA1 coding sequence (5′-AGCCATGGCTCTACTCAGTCTCGGTTCAGG-3′ and reverse complement, matching the variant with deletion; 5′-AGCCATGGCTCTACTCCAGTCTCGGTTCAG-3′ and reverse complement, matching the wild type), and verified these matches against the reference sequences for the LABA1 gene.

To obtain diversity estimates for the rc, laba1, sh4 and prog1 haplotypes, we searched for variability in 10 kb windows surrounding the causative mutations utilising the SNP matrix published by Huang et al. ( 2012) (downloaded from the Rice Haplotype Map Project database). The diversity was calculated as the average number of polymorphic sites in a 10 kb window surrounding the causative mutation, weighted per examined accession and the proportion of non-missing data. A maximum-parsimony tree of the Sh4 locus was constructed from the data published by Zhu et al. ( 2012) using dnapars program in the PHYLIP package with default parameters (Felsenstein 2005). Only the parsimony-informative sites were used and >1 bp gaps were recoded as single events. A majority consensus tree was constructed from 5171 equally parsimonious trees.

Results and discussion

We found one or more reads exactly matching the causative indel position in the Rc gene for 255 accessions of wild rice (Table ; Table S1). In 33 cases (12.9%), the rc variant with the deletion was detected. Although we do not posses detailed phenotypic information about the wild accessions, we assume that all of them have pigmented pericarp. We therefore show that the rc allele does exist in wild rice in moderate frequency and is not necessarily associated with white pericarp, similar to the situation previously reported for the sh4 and prog1 alleles. Consequently, the conclusion of Sweeney et al. ( 2007) that the rc allele originated in subsp. japonica and later spread to subsp. indica by introgressive hybridization is questioned, and the possibility that all cultivated rice groups obtained the rc allele directly from their wild progenitors has to be considered.

Table 1

In the case of the LABA1 gene, we found perfect matches for the indel position in 215 wild accessions. In 33 of those (~15%), the variant with the deletion was detected. In contrast, Hua et al. ( 2015) did not find a single case of the laba1 allele in their smaller sample of 43 O. rufipogon accessions.

Plots of the geographical locations of the wild accessions carrying the rc and laba1 alleles show that both alleles have a relatively broad distribution (Fig. ). This is consistent with the results of Liu et al. ( 2015), who found no correlation between genetic groups and geographic regions in wild rice, and ascribed the absence of a phylogeographic pattern to repeated extinctions and re-colonizations of wild populations during Quaternary glacial-interglacial cycles. If this explanation is correct, it may imply that both mutations emerged prior to the last glaciation.

It could be argued that the observation of the "domestication" alleles in wild populations does not necessarily reject the hypothesis of their origin under cultivation. Each of the recessive alleles could have emerged during the domestication process and escaped into wild populations by gene flow. This possibility needs to be evaluated critically. Gene flow from O. sativa to its wild relatives has been well documented due to concerns of transgene escape from genetically modified rice (Song et al. 2003; Chen et al. 2004; Wang et al. 2006; Shivrain et al. 2007). The recessive alleles of the Sh4, PROG1, Rc and LABA1 genes can be transferred by gene flow just like any other genomic segment. However, these alleles are either neutral (if no phenotypic change is manifested) or disadvantageous in wild populations. In the absence of positive selection, it is difficult to conceive that gene flow and retention of the alleles in the wild would occur to the extent resulting in the frequencies reported for sh4 (~26%; Zhu et al. 2012), rc and laba1 (~13 and 15%, respectively; this study). Moreover, high fixation index (F ST) values indicate that the reproductive barrier between cultivated rice and O. rufipogon is relatively strong. For example, Huang et al. ( 2012) calculated that the F ST between subsp. japonica and its assumed progenitor population is 0.36, which means that japonica rice and its wild progenitor-although sympatric-share less genetic variability than East-Asians do with people from sub-Saharan Africa (F ST = 0.19; Nelis et al. 2009).

The above considerations suggest that the presence of domestication alleles in wild rice is not wholly explained by gene flow from O. sativa, but empirical data are needed to confirm this point. One way of addressing the question is by exploration of associated diversity. If the domestication alleles found in O. rufipogon are derived from cultivated rice, then their nucleotide diversity in wild populations would not be expected to exceed their diversity in O. sativa. On the other hand, if the emergence of the causative mutations pre-dates domestication, then the domestication alleles found in O. rufipogon would have higher diversity compared to O. sativa. Our estimates of nucleotide diversity indicate that the latter interpretation is correct. The 10 kb regions surrounding the causative deletion contain ~4× more polymorphisms in rc-type and laba1-type O. rufipogon than they do in O. sativa (Table ). For the sh4 allele, it can be directly demonstrated that the causative G → T substitution changing asparagine for lysine in the protein product emerged in wild rice prior to domestication. This is established by a gene tree constructed from the polymorphism data in the Sh4 exon, partial intron and ~1.5 kb flanking region published by Zhu et al. ( 2012). The phylogenetic tree shows that most haplotypes with the G → T substitution form a clade where the sequences found in O. rufipogon occupy both the basal and sister positions in respect to the haplotypes found in O. sativa (Fig. ). Furthermore, in the SNP matrix by Huang et al. ( 2012) we found a polymorphic site approximately 1 kb downstream of the Sh4 coding sequence (chromosome 4; IRGSP4 position 34,628,688). All 99 subsp. indica accessions with data have A at this position, while 46 out of 67 non-missing japonica data points are C (68.7%). Both variants are found in O. rufipogon (Table S1), indicating that the sh4 haplotypes of O. sativa do not originate from a single wild genotype.

For the prog1 allele, the causative mutation has not been unambiguously identified from the set of candidate polymorphisms (Tan et al. 2008), and we therefore could not sort the wild population into prog1/PROG1 classes. Nonetheless, we identified variability that indicates independent genealogical histories in different groups of O. sativa. A SNP located 230 bp upstream from the PROG1 start codon (chromosome 7; IRGSP4 position 2,872,361) is uniform in subsp. indica (all 127 accessions with data have C; Table S1) but variable in subsp. japonica (58.9% C and 41.1% A; 129 accessions with data). A short distance further upstream-780 bp away from the PROG1 start codon (chromosome 7; IRGSP4 position 2,872,911)-another polymorphic position was found, this time monomorphic in subsp. japonica (all 128 accessions with data have C) and polymorphic in subsp. indica (77.6% C and 22.4% T; 143 accessions with data). Both of these positions are variable in O. rufipogon. Thus, we observe two haplotypes in subsp. indica (TC- prog1 and CC- prog1) and two haplotypes in subsp. japonica (CC- prog1 and CA- prog1). Subsp. indica could not have obtained the TC- prog1 haplotype from subsp. japonica since it does not occur there, and similarly, subsp. japonica could not have obtained the CA- prog1 haplotype from subsp. indica. However, both groups could have obtained both haplotypes from wild rice, which is the most parsimonious explanation.

The diversity associated with prog1 and sh4 therefore indicates different genealogical histories for these alleles in subsp. indica and subsp. japonica, but unfortunately, the whole-genome data sets do not allow conclusions to be drawn about the aus group. Interestingly, Sweeney et al. ( 2007) reported a second mutation disrupting the open reading frame of the Rc gene and leading to white pericarp in some aus varieties. Similarly, Hua et al. ( 2015) found several aus varieties with the LABA1 (wild type) allele but barbless awns. These observations indicate that alternative mutations underlie the domesticated phenotype of some aus cultivars, in agreement with our previous suggestion that this group has a unique domestication history (Civáň et al. 2015).

In conclusion, we show that the rc, laba1, prog1 and sh4 alleles are moderately frequent in O. rufipogon populations, where they display higher associated diversity than that present in O. sativa. This evidence suggests that the causative mutations determining white pericarp, barbless awns, erect growth and non-shattering ear in O. sativa each emerged in wild rice prior to domestication. Since these mutations are not associated with the domesticated phenotype in wild rice, a group of interacting genes is probably responsible for the phenotypic trait in each case. The implication is that, for each of these traits, selection in cultivated rice acts on a network of alleles rather than at an individual locus. A single gene can still determine the alternative phenotypes, but only in the appropriate allelic background.

Our results broaden the possible scope for models describing the events giving rise to the different groups of cultivated rice. The uniformity of the domestication alleles in cultivated rice has previously constrained those models, with focus largely on schemes in which the domestication phenotype originated in one type of rice and was subsequently transferred to other groups by introgressive hybridization (e.g. Sweeney et al. 2007; He et al. 2011; Huang et al. 2012; Yang et al. 2012; Hua et al. 2015; Oikawa et al. 2015). The underlying assumption on which these models are based, that the different groups of rice could not have acquired domestication alleles from standing variation in the wild population, is clearly incorrect. Conversely, models that propose independent domestications giving rise to subsp. indica, subsp. japonica and/or aus (e.g. Civáň et al. 2015) are not invalidated by the uniformity of the domestication alleles in these different groups.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Funding

This work was supported by European Research Council grant 339941 awarded to TAB.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.

Availability of data and materials

In this article, data produced by third parties were used. The original data repositories are referenced in the text and our data extractions are summarised in the supporting file.

Footnotes
Electronic supplementary material
The online version of this article (doi:10.1007/s10722-017-0518-0) contains supplementary material, which is available to authorized users.

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Origin of rice (Oryza sativa L.) domestication genes

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A number of genes that contribute to the domestication traits of cultivated rice have been identified. These include Sh4, Rc, PROG1 and LABA1, which are associated with non-shattering rachis, white pericarp, erect growth and barbless awns, respectively.

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From a wild Asian grass to a refined crop that is the staple diet of half the world's population, the domestication of Oryza sativa spans centuries, but the grain's ancestry is hotly contested.

Asian civilization was built on rice - on Oryza sativa, to be exact. The crop, which today is the primary food source for half of the world's population, transformed nomadic hunter-gatherers into stay-at-home farmers, spawned the first urban centres and built empires and dynasties. "Probably more so than any crop, it drove societies and economies to become densely populated, potentially more urbanized, and it also transformed landscapes," says Dorian Fuller, an archaeobotanist at University College London.

Despite - or possibly because of - rice's primacy, the history of the grain remains controversial, with little agreement on where, when and how many times humans tamed O. sativa in Asia to create the world's most important crop. (The only other domesticated rice species, Oryza glaberrima, has its roots in Africa. See 'The second story'.) "Almost every part of Asia had been pinpointed as the area where rice originated," says Michael Purugganan, an evolutionary geneticist at New York University who studies rice domestication. Unravelling the history of rice in Asia would illuminate a turning point in human civilization and give scientists fresh insight that could help improve the crop for the future. Thanks to advances in genetics and to new archaeological finds, that history is becoming clearer - and it is a lot more complicated and convoluted than anyone thought.

Oryza rufipogon, the Asian wild grass that is most closely related to O. sativa, is a sinewy, weedy plant. Its red grains are edible, but some modern rice growers consider it a pest. As humans started intentionally planting rice around 8,000 to 9,000 years ago, they sought out plants with the most desirable traits. Over time, the cultivated grass became stouter and straighter, producing heftier grains in greater quantities and that clung to the plant instead of tumbling to the soil - or 'shattering' - to facilitate spread. Claiming the title of the birthplace of rice would be a matter of great pride for a nation. This, along with the wide geographical range of O. rufipogon, has led to competing claims for domestication - including the Ganges valley in northern India, several locations in China, southeast Asia, the southern slope of the Himalayas and numerous other places. "Because rice is embedded within cultural identities within different nations in Asia, every-body wants to have had rice first," says Fuller.

For the past few decades, much of the focus on rice domestication has centred on China and India. In the 1960s and 1970s an influential Taiwanese agriculture scientist, the late Te-Tzu Chang, collected and cultivated wild rice from across Asia and proposed a band of domestication that stretches from northern India to southern China. As Fuller explains, this work encouraged Indian archaeologists to continue maintaining that rice came out of India, and the Chinese to claim that it came out of South China.

Perhaps both camps are correct. Han dynasty records dating back more than 2,000 years distinguish between two varieties of rice, Keng and Hsien, now known as japonica (short-grained) and indica (long-grained) respectively. Research1 comparing dozens of wild and domestic strains has suggested that japonica and indica are more closely related to distinct wild varieties than they are to each other, pointing to two separate domestications: japonica in China and indica in India.

Indica and japonica clearly come from different genetic stock, says Tao Sang, a plant geneticist at the Institute of Botany of the Chinese Academy of Sciences in Beijing. But a closer look at those genes shows some surprising - and telling - overlap. In 2006, Sang's team identified a variation in a gene in domesticated rice known as sh4 that prevents rice grains from shattering2. Japonica and indica share identical non-shatter mutations in sh4, suggesting that the two varieties had a shared history. Later studies suggested that the mutation arose in an ancestor of japonica rice first then found its way to indica. Since then, other teams have identified domestication genes that follow the same pattern: they appeared first in japonica before showing up in indica. One example is a mutation in a gene called Rc that lightened the seed coating, or pericarp3.

These discoveries sent rice scientists scrambling to explain how indica and japonica could be genetically distinct across most of their genomes yet share mutations responsible for the key traits that made rice easier to cultivate. Although the specifics are still hotly contested, most researchers have concluded that human-directed hybridization played an important part in the very early history of domesticated rice.

One leading theory comes from a 2011 study4 by Puruggunan and his colleagues that modelled the relationship of dozens of wild and domestic rice varieties. The study suggested that the domestication of japonica, most probably in China, was the watershed event in the history of Asian rice. From there it seems that japonica spread to India, where farmers intentionally hybridized it with their local rice to produce indica. The exact nature of that local rice is still a mystery. It could have been an early domesticated variety, or it could have been wild.

Whether the creation of indica from japonica counts as a separate domestication event "is somewhat a semantic issue", says Jeffrey Ross-Ibarra, an evolutionary geneticist at the University of California, Davis. The take-home message remains: the earliest fields of japonica - again, most probably in China - provided the key materials for the rice revolution in Asia.

Exactly where in China japonica was domesticated is another lively debate. The lower stretches of the Yangtze River valley in eastern China had long laid claim to early domestication, based on the discovery of roughly 8,000-year-old grains in archaeological sites there. And so a team led by Bin Han, a geneticist at the Shanghai Institute for Biological Sciences of the Chinese Academy of Sciences, raised eyebrows in 2012 when they proposed the Pearl River valley in deep southern China as the birthplace. Han's team had compared the genomes of more than 1,000 domesticated strains of rice and another 446 wild varieties from across Asia. They found that both indica and japonica were most closely related to wild varieties growing in the Pearl River valley. There is no archaeological evidence supporting early rice cultivation in the region, which is one reason why the claim remains controversial. But Han says that this could be because of the comparative poverty of its ancient inhabitants, who did not keep large stores of rice, unlike the richer Yangtze valley dwellers.

Archaeological work remains focused on the Yangtze. Since 2004, Fuller has excavated at a site in the lower Yangtze River valley that has been touted as an early centre of rice domestication. "I had always been taught, and all the textbooks will tell you, that there's early rice domestication here in 5000 BC," he says. But the history, he says, turns out to be a bit more nuanced. "My impression of visiting this site is that it's full of storage pits that are full of acorns. Nobody mentions the acorns."

In Fuller's view, the early inhabitants of this and other Yangtze valley sites were hunter-gatherers who dabbled in rice cultivation but mostly ate acorns, water chestnuts and fish. In a 2009 paper5 that analysed thousands of microscopic rice remains from the site, Fuller's team documented a slight increase in the presence of domestic-looking rice, beginning around 5000 BC. But these rice grains kept changing shape - a sign of a crop not yet fully domesticated. Not until the shape stabilized thousands of years later did acorns disappear from Chinese archaeological sites. Rice domestication, as Fuller sees it, was a slow, haphazard enterprise.

In the hope of untangling rice domestication further, researchers are turning to the same ancient-DNA technologies that have revolutionized the understanding of human evolution. If they can recover DNA from ancient rice remains, researchers may be able to determine when and where the key domestication genes emerged. Most rice from archaeological sites is charred, and past efforts to glean DNA from grains have failed. But modern sequencing machines that can decode very short strands of DNA - likely to be the only ones left in rice that is thousands of years old - mean it may be possible to get DNA and even whole genomes from ancient rice. "The techniques are here, and the revolution is going to happen," says Fuller, who has begun a project with a team at the University of Warwick, UK, to sequence ancient rice DNA from China, India and Thailand.

Humans continue to shape the rice genome, but some researchers worry that intensive breeding for high yield and pest resistance has narrowed the genetic diversity of cultivated rice, leaving crops more susceptible to disease and less adaptable to the effects of climate change. Sang and other scientists studying rice domestication argue that contemporary rice breeders should take advantage of the handiwork of the humans who transformed wild grasses into the world's most successful crop. Ancient rice varieties no longer under widespread cultivation could provide attributes to help high-yielding strains to feed a world of nine billion people, Sang says. "There's great potential in those ancient cultivars."

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Domestication: The birth of rice - Nature

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From a wild Asian grass to a refined crop that is the staple diet of half the world's population, the domestication of Oryza sativa spans centuries, but the grain's ancestry is hotly contested. Asian civilization was built on rice - on Oryza sativa, to be exact.

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In this study, we employed a DNA-microarray-based transcriptomic approach to investigate the gene regulation in response to M. oryzae MSP1 protein. Rice leaves were treated with PBS (control) and MSP1 recombinant protein and harvested at 6 hpt and 24 hpt. A total of 4386 DEGs were detected in response to MSP1 treatment. Further bioinformatics analyses, including functional data mining and pathway participation, were employed to identify specific key transcriptional changes in response to MSP1 treatment.

MSP1 downregulates photosynthesis and plant shifts to non-assimilatory metabolism

During pathogen attack, recognition of pathogens or elicitors leads to a massive reprogramming of plant cells to activate and deploy defense responses ( Bolton, 2009; Rojas et al., 2014). Assignment of recourses for immune response and biosynthesis of protective compounds leads to an increased demand for energy and carbon skeleton, which are supported by primary metabolic pathways ( Bolton, 2009; Rojas et al., 2014).

Numerous studies have reported that photosynthesis is impaired when plants shift towards non-assimilatory metabolism in response to pathogen attack ( Kangasjärvi et al., 2012; Major et al., 2010; Roberts and Paul, 2006). In the present study, 59 of 63 genes involved in light reaction of photosynthesis, 18 of 19 Calvin cycle genes and 14 of 18 tetrapyrrole biosynthesis genes were down-regulated by MSP1 treatment (, , Supplementary Table 3), indicating broad down-regulation of photosynthesis in response to MSP1 treatment. These results are in line with those of previous reports that biotic stress causes uniform down regulation of the transcription of genes encoding the major components of photosynthesis ( Bilgin et al., 2010). Thirty of 45 genes (e.g., fatty-acid desaturase OsSSI2, Acyl-CoA-binding protein OsACBP1 and OsLACS1) involved in lipid synthesis and 73 of 83 genes related to protein synthesis were down-regulated in MSP1 treated samples, suggesting the activation of non-assimilatory metabolism in response to MSP1 treatment. Previously, it was observed that OsSSI2-knock down plants exhibit markedly enhanced resistance to blast and leaf blight diseases in rice and Arabidopsis ( Liu et al., 2014a; Shah et al., 2001), indicating it is a negative regulator of stress tolerance in plants. Here, we observed down-regulation of OsSSI2 in response to MSP1 treatments, indicating an enhanced resistance by MSP1.

Inhibition of photosynthesis activity and collapse of assimilatory metabolism results in a metabolic transition in which the demand for carbohydrates and energy is achieved through increased activities of respiratory metabolism and other types of metabolism (Smedegaard-Petersen and Stolen, 1981). In the present study, three main pathways of respiration were activated during the resistance response, including glycolysis, the TCA cycle, and the electron transport chain. Three of eight genes encoding glycolysis-related proteins, ten of 18 genes involved in the TCA cycle, and seven of 16 genes involved in mitochondrial electron transport were up-regulated by MSP1 treatment, revealing a transcriptional change in the three main pathways of respiration in response to MSP1. Totally, 23 of 28 genes involved in lipid degradation and 115 of 198 genes involved in protein degradation were up-regulated by MSP1 treatment, providing an alternative respiratory substrate for MSP1-treated rice. Moreover, the up-regulation of protein and lipid degradation indicates that senescence processes in rice leaves may be highly elevated and nutrients released are mobilized from the leaves to other parts of the plants to support the immune response.

Defense signal transduction pathways were activated by MSP1 treatments

PTI is a form of basal defense that confers resistance to broad-spectrum pathogens in plants ( Boller and Felix, 2009; Niks et al., 2015). Pattern recognition receptors (PRRs) trigger PTI, a relatively weak immune response restricting pathogen growth by recognizing highly conserved PAMPs. In this study, most of the 31 up-regulated RLK genes were leucine-rich repeat (LRR) RLKs and S-locus glycoprotein like RLKs, indicating potential RLKs for MSP1 triggered immunity. In FLS2, EFR, and Xa21, there is a large ectodomain including 28 LRRs, 21 LRRs, and 23 LRRs, respectively ( Gómez-Gómez and Boller, 2000; Song et al., 1995; Zipfel et al., 2006), which are directly involved in elicitor binding ( Antolín-Llovera et al., 2012). The DUF26 RLKs, which are cysteine-rich RLKs, comprise a large subgroup of RLKs involved in plant defense and programmed cell death ( Chen et al., 2004). In the present study, the gene encoding OsDUF26 (also named OsRLK) was highly up-regulated by MSP1 treatment, which is in agreement with the results of a previous study in which OsDUF26 was induced by M. oryzae infection and fungal elicitor at both the RNA and protein levels ( Kim et al., 2004, 2009b).

Following RLKs recognition, MAPK cascades are activated. In the present study, 14 MAPKKK, one MEK, and two MPK genes were up-regulated by MSP1 (e.g., OsMPK5 gene and MAPKKK1 gene). OsMPK5, also known as OsBIMK1 and OsMPK3, has been reported to be involved in disease resistance responses ( Song and Goodman, 2002) and to positively regulate the JA signaling pathway and plant resistance to a chewing herbivore in rice ( Wang et al., 2013). Moreover, MAPKKK1 (also known as OsEDR1 and OsACDR1) is a potential positive regulator of fungal disease resistance ( Kim et al., 2009a) and a negative regulator of rice bacterial resistance via activation of ethylene biosynthesis ( Shen et al., 2011). These results indicate that MAPKs play a vital role in the transduction of MSP1 induced signal to disease resistance in rice.

As a downstream signal of MAPKs, numerous WRKY transcription factors are involved in the development and initiation of stress and defense responses. Among 30 up-regulated genes encoding WRKYs, OsWRKY23, OsWRKY24, OsWRKY28, OsWRKY45, OsWRKY51, OsWRKY53, OsWRKY62, OsWRKY70, OsWRKY71, OsWRKY72, and OsWRKY76 have been thoroughly investigated for their roles in rice innate immune responses. Several previous transcriptional analyses showed expression of WRKYs were increased in response to attack by M. oryzae (e.g., OsWRKY45, OsWRKY47, OsWRKY53, OsWRKY55, OsWRKY62 and OsWRKY71), and over-expression of some of these genes enhanced resistance to rice blast infection ( Bagnaresi et al., 2012; Chujo et al., 2007; Ryu et al., 2006; Shimono et al., 2012; Wei et al., 2013). Moreover, four WRKYs ( OsWRKY51, OsWRKY62, OsWRKY71, and OsWRKY76) were reported to be involved in resistance against X. oryzae pv. oryzae infection in rice ( Hwang et al., 2016; Liu et al., 2016, 2007; Peng et al., 2008). For instance, OsWRKY45 plays important roles in rice resistance to both M. oryzae and X. oryzae pv. oryzae via SA hormone signaling ( Shimono et al., 2012) and negatively modulates resistance against the brown planthopper, Nilaparvata lugens ( Huangfu et al., 2016). Moreover, OsWRKY45 and Pb1 interaction contributes to blast resistance through protection of OsWRKY45 from ubiquitin-proteasome system degradation ( Matsushita et al., 2013). These results indicate that complicated signaling of WRKY transcription factors could be initialized by the MSP1 treatment that can be transduced to defense responses.

Phytohormones are central regulators of plant defense. In the present study, the JA, ET, and ABA signaling pathways were significantly upregulated by MSP1 treatment, while only three genes involved in the SA signaling pathway were detected in MSP1 treated samples, which is in accordance with our previous finding that JA and ABA positively regulate MSP1 induced cell death ( Wang et al., 2016). It is well known that JA regulates growth development and stress responses, especially defense responses to herbivores and necrotrophic pathogens ( Browse, 2009). Previous studies in rice have revealed that JA signaling was involved in rice basal defense against bacterial and fungal pathogens ( Yamada et al., 2012; Yang et al., 2012, 2013). Moreover, ABA was reported to be both a positive and negative regulator of rice disease resistance, depending on the pathogen type and infection stage ( Yang et al., 2013). There are also several lines of evidence that suggest ET confers broad-spectrum resistance against the fungal infection ( Helliwell et al., 2013). Additionally, numerous studies have shown that SA acts as a positive regulator of immunity against biotrophic pathogens and hemibiotrophic pathogens ( Berens et al., 2017; Glazebrook, 2005). However, rice plants did not show any increase in SA after inoculation with M. oryzae ( Silverman et al., 1995). Limitation of the microarray may be one of the reasons why only three SA signaling genes were identified. Moreover, a recent study revealed that overexpression of MSP1 in rice confers broad-spectrum resistance through modulation of the SA- and JA-mediated signaling pathways ( Hong et al., 2017). In addition, a study of the newly identified CP protein, SsCP1, revealed that the SA signaling pathway is involved in CP-mediated plant defense ( Yang et al., 2018). Together, these results indicate that intricate networks of phytohormone signaling are activated by MSP1 treatment, leading to increased rice immunity.

Overall, our study provides a broader picture of the major physiological reactions underlying MSP1-induced PTI signaling at the transcriptional level. Upon treatment with MSP1, elicitor perception and a series of signal transductions (including MAPKs, WRKY transcription factors, hormone signaling and PR proteins) were activated. Our results suggest MSP1 triggers the activation of plant defense responses against various pathogens.

# data[].attributes.attachment_link.title
Transcriptomic Analysis of Oryza sativa Leaves Reveals Key Changes in Response to Magnaporthe oryzae MSP1

# data[].attributes.attachment_link.description
In this study, we employed a DNA-microarray-based transcriptomic approach to investigate the gene regulation in response to M. oryzae MSP1 protein. Rice leaves were treated with PBS (control) and MSP1 recombinant protein and harvested at 6 hpt and 24 hpt. A total of 4386 DEGs were detected in response to MSP1 treatment.

# data[].attributes.attachment_link.readable_text
The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant ("Nona Bokra", "Pokkali", and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron. Typical characteristics of salinity tolerance in rice were found to be Na⁺ exclusion and an increased absorption of K⁺ to maintain a good Na-K balance in the shoot. Genetic component analysis (GCA) revealed that a low Na-K ratio is governed by both additive and dominance gene effects. The trait exhibited overdominance, and two groups of genes were detected. Environmental effects were large, and the heritability of the trait was low. Our findings suggest that when breeding for salt tolerance, selection must be done in a later generation and under controlled conditions in order to minimize environmental effects. Modified bulk and single-seed descent would be the suitable breeding methods. Combining ability analysis revealed that both GCA and specific combining ability (SCA) effects were important in the genetics of salt tolerance. Moderately tolerant parents - e.g., IR4595-4-1-13 and IR9884-54-3-1E-P1 - were the best general combiners. Most of the best combinations had susceptible parents crossed either to moderate or tolerant parents. The presence of reciprocal effects among crosses necessitates the use of susceptible parents as males in hybridization programs. Large heterotic effects suggest the potential of hybrid rice for salt-affected lands.

# data[].attributes.attachment_link.title
Genetic analysis of salinity tolerance in rice (Oryza sativa L.) - Theoretical and Applied Genetics

# data[].attributes.attachment_link.description
The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant ("Nona Bokra", "Pokkali", and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron.

# data[].attributes.attachment_link.readable_text
Rice 6, Article number: 4 (2013) Cite this article

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Abstract

Background

Rice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa ( japonica group).

Results

The Nipponbare genome assembly was updated by revising and validating the minimal tiling path of clones with the optical map for rice. Sequencing errors in the revised genome assembly were identified by re-sequencing the genome of two different Nipponbare individuals using the Illumina Genome Analyzer II/IIx platform. A total of 4,886 sequencing errors were identified in 321 Mb of the assembled genome indicating an error rate in the original IRGSP assembly of only 0.15 per 10,000 nucleotides. A small number (five) of insertions/deletions were identified using longer reads generated using the Roche 454 pyrosequencing platform. As the re-sequencing data were generated from two different individuals, we were able to identify a number of allelic differences between the original individual used in the IRGSP effort and the two individuals used in the re-sequencing effort. The revised assembly, termed Os-Nipponbare-Reference-IRGSP-1.0, is now being used in updated releases of the Rice Annotation Project and the Michigan State University Rice Genome Annotation Project, thereby providing a unified set of pseudomolecules for the rice community.

Conclusions

A revised, error-corrected, and validated assembly of the Nipponbare cultivar of rice was generated using optical map data, re-sequencing data, and manual curation that will facilitate on-going and future research in rice. Detection of polymorphisms between three different Nipponbare individuals highlights that allelic differences between individuals should be considered in diversity studies.

Background

The International Rice Genome Sequencing Project (IRGSP) completed the sequencing of the japonica rice cultivar Nipponbare in 2005 (International Rice Genome Sequencing Project 2005). In this project, the consortium employed a clone-by-clone sequencing strategy after construction of a minimum tiling path (MTP) for each chromosome. Subsequently, two genome assemblies were independently produced. One by the Rice Genome Annotation Project initially located at The Institute for Genomic Research and now at Michigan State University (MSU) and another by the Rice Annotation Project (RAP) (Ouyang et al. 2007; Tanaka et al. 2008). The two sets of pseudomolecules differed slightly due to differences in selection of the clones underlying the MTP and the lengths of gap insertions used by the two projects. The two genome assemblies have made it difficult for the rice community to move between the resources produced by the two annotation groups. To facilitate genomic-enabled research, a unified, single genome assembly of the Nipponbare rice reference genome was constructed by updating the MTP, validating the final MTP with optical mapping data, and error-correcting the unified assembly using next generation re-sequencing data.

The finished quality of the reference genome sequence of Nipponbare rice in 2005 was estimated to be less than one error in 10 kb (International Rice Genome Sequencing Project 2005). Recent advancements in sequencing technologies have enabled re-sequencing of multiple rice genomes. To date, several groups have performed genome-wide genetic diversity analyses among rice cultivars. The genome of an elite japonica rice cultivar, Koshihikari, which is closely related to Nipponbare, was sequenced using the Illumina platform and single nucleotide polymorphisms (SNPs) between Nipponbare and Koshihikari were estimated on an average to be at least one per 5.7 kb (i.e., 1.8 × 10⁻⁴ per site) (Yamamoto et al. 2010). Sequencing of 517 rice landraces identified approximately 3.6 million SNPs, a frequency of 9.32 per kb (Huang et al. 2010). Since the polymorphism differences between rice cultivars are limited, a high quality reference genome sequence is essential for the comparison of closely related rice cultivars (Zhang et al. 2010; Lu et al. 2010; Huang et al. 2012; Xu et al. 2012; Yang et al. 2012) so that errors can be minimized. Here, we report a single high-quality reference genome sequence for rice from the japonica cultivar Nipponbare (Os-Nipponbare-Reference-IRGSP-1.0) in which the MTP and bacterial artificial chromosome (BAC)/P1 artificial chromosome (PAC) assemblies were validated using optical map data. Fine-scale validation was performed using error correction and whole genome re-sequencing data obtained from two next generation platforms, the Illumina Genome Analyzer II/IIx and Roche GS FLX.

Results and discussion

Construction of a minimum tiling path validated by an optical map

Detection of small sequencing errors using Illumina-platform generated reads

To correct sequencing errors in the original reference sequence, the genomes of two different individuals of Nipponbare rice were re-sequenced at the National Institute of Agrobiological Sciences (NIAS) and Cold Spring Harbor Laboratory (CSHL). For the NIAS dataset, ~70 million 36-bp and 60 million 51-bp single-end reads, corresponding to a total of 5.6 Gbp genomic sequence, were generated (Table 1). The CSHL dataset included more than ~269 million 76-bp paired-end reads, corresponding to a total of 20.4 Gbp of sequence (Table 1). After preprocessing of these reads to filter out low quality reads, ~110 million single-end reads from NIAS, and ~247 million paired-end and ~10 million unpaired reads from CSHL remained (Table 1). To identify sequencing errors in the reference sequence as well as potential allelic sites in the NIAS and/or CSHL individuals relative to the reference sequence, all reads were mapped to the pseudomolecules using the Burrows-Wheeler Aligner (BWA) (Li and Durbin 2009). In total, ~72 million of the NIAS single-end reads and ~7 million of CSHL unpaired reads mapped uniquely to the pseudomolecules (Table 2). For the CSHL paired-end reads, ~192 million reads uniquely mapped within the estimated insert size of the paired end reads (Table 2).

Table 1 Assessment and processing of re-sequencing datasets used in this study

Table 2 Statistics of mapping results by BWA

Depth of coverage of Illumina reads on the assembled genome. The depth of coverage of the Illumina reads at ≥1 read, ≥5 reads, and ≥10 reads are shown for the ( A) NIAS, ( B) CSHL, and ( C) NIAS + CSHL datasets.

Table 3 Coverage and number of effective sites for detection of variation

We classified all nucleotides into five categories: "reference type," "sequencing error," "allele (within an individual)," "allelic difference between individuals," and "low depth" (see Methods and Additional file 7). The classification is based on the type and frequency of nucleotide at each site for the three datasets. Our survey of SNP-type sequencing errors detected 3,447 sites that showed high frequencies of non-reference type bases and were classified into "sequencing error" (Additional file 8 and Additional file 9). Since BWA generates gapped alignments, small insertion/deletion (indel) type variants (1-4 bp gaps) could also be detected: 642 sites were insertions and 797 were deletions. Thus, 4,886 errors were found in the 321 Mbp of the reassembled reference genome. The IRGSP estimated that the error rate of the reference genome sequence was less than one per 10,000 nucleotides (International Rice Genome Sequencing Project 2005). In fact, the average frequency of sequencing errors estimated by our Illumina re-sequencing data was 0.15 errors per 10,000 nucleotides, substantially lower than the estimation by the IRGSP. While this may be an under-estimation of the error rate due to our inability to map reads to all nucleotides in the assembled rice genome and the lack of the requisite read depth at every nucleotide, we were able to assess 321 of the 373 Mb assembly for errors.

The re-sequencing analysis by Illumina reads also revealed that a number (6,433) of the sites had allelic differences in the datasets. The frequency of the allelic sites was on average 0.20 in 10,000 nucleotides (Figure 2), which may not be negligible for genome sequence comparison between cultivars. Chromosomes 4 and 10 are the most repetitive chromosomes in the rice genome (International Rice Genome Sequencing Project 2005) and have larger sequencing error rates than other chromosomes (Figure 2). Thus, the higher allele frequency detected for these two chromosomes may be attributable in part to sequencing error. In this analysis, the same rice cultivar (Nipponbare) was used in the NIAS and CSHL re-sequencing as was used in the original IRGSP sequencing effort. However, the Nipponbare rice NIAS and CSHL individuals were derived from different Nipponbare populations preserved in different laboratories and have been propagated independently for an unknown number of generations. Thus, these two individuals may have accumulated mutations after multiple generations as described previously in Arabidopsis thaliana (Ossowski et al. 2010). Comparison between the CSHL and NIAS datasets indicated that 250 sites differed between the NIAS and CSHL individuals and were not allelic to each other (Additional file 10). Hence, the occurrence frequency of the allelic sites is 0.0078 per 10,000 nucleotides. It should also be noted that some of these differences may be derived from somatic mutations as the DNA used for sequencing was derived from young leaves two weeks after germination. Residual heterozygosity in recombinant inbred lines has been reported in maize (McMullen et al. 2009; Eichten et al. 2011) and as the IRGSP employed a clone-by-clone approach, only a single clone (and homolog) was selected for sequencing. Thus, if residual heterozygosity were present in the original individual used in the IRGSP sequencing effort, only a single allele would be represented in the reference assembly. Indeed, we were able to identify a small subset of allelic sites within individuals (Additional file 11). Therefore, caution regarding the origin of allelic differences should be considered when conducting re-sequencing studies. Allelic SNPs within an individual (5,412) and between individuals (232) were classified into six groups (Nonsense, Missense, Silent, UTR, Intron and Intergenic) based on the RAP-DB gene models. The majority (79.4%) of allelic sites were located in intergenic regions (Additional file 12) with only 3.6% of allelic variations with the potential for affecting the encoded amino acid.

Frequency of allelic sites per 10 kb among the 12 rice chromosomes.

Detection of misassembly

Long read sequences were generated using the Roche GS FLX platform and used to identify misassembly of BAC/PAC clones as well as large indel sequencing errors. A total of 1.0 Gbp from 2,706,353 reads was produced. The reads were filtered to remove low quality reads, yielding 2,705,634 reads with an average read length of 377 bp. To detect large gaps in the reference assembly, these reads were aligned to the pseudomolecules using Megablast (Zhang et al. 2000). We selected reads that mapped unambiguously to two different chromosomal positions within a 1 Mbp interval and where the total of two high scoring pairs reported by Megablast covered >90% of the original read. From this initial set of alignments, 205 reads were found to meet these criteria. A total of 200 of these reads were removed through manual inspection and five large indel gaps were retained. Three were insertions, indicating that additional nucleotides should be inserted into the reference genome, while the other two were deletions. The two deletion errors were further validated by mapping the Illumina reads to the long reads that matched these regions. In one case, a physical gap that had originally been inserted into the reference was found to be unnecessary based on our long read validation, and this gap was removed. For the other four cases, the erroneous indels were located near tandem repetitive sequences.

Nomenclature, data availability and annotation of the Os-Nipponbare-Reference-IRGSP-1.0 genome

We have named this updated assembly "Os-Nipponbare-Reference-IRGSP-1.0" to signify that it is from rice ( Oryza sativa), the Nipponbare cultivar, a high quality reference assembly, from the IRGSP, and version 1.0. We envision that future assemblies of rice will be of draft quality and from other entities, and as is the case with the Nipponbare rice genome, will be updated as new sequencing datasets become available in the future. We are proposing this nomenclature for other rice genome assemblies as an informative way for the community to readily interpret the origin, quality, and iteration of rice genome sequences. One objective in this study was to provide a single unified set of pseudomolecules for two parallel annotation efforts, the RAP (Tanaka et al. 2008, http://rapdb.dna.affrc.go.jp/) and the MSU Rice Genome Annotation Project (Ouyang et al. 2007, http://rice.plantbiology.msu.edu/) in which both annotation projects have now updated their annotation with the underlying Os-Nipponbare-Reference-IRGSP-1.0 pseudomolecules and provided this via their project websites.

The annotation of the Rice Annotation Project Database (RAP-DB) was updated for Os-Nipponbare-Reference-IRGSP-1.0. A total of 4,993 loci from the RAP annotation on the IRGSP build 5 genome were deprecated in this release. Of those, 4,766 (95%) loci became obsolete due to the overlaps with repetitive sequences on the new pseudomolecules. The exon-intron structures of genes and their splicing variants were identified or predicted as described previously (Tanaka et al. 2008). As a result, 37,872 loci, of which 35,681 have protein-coding potential, were determined. In addition, 6,642 variants of alternative splicing were found. Furthermore, a number of functional annotations were carefully examined through literature surveys. Manual curation efforts improved the gene structures and functional descriptions of 97 loci. All the information including the repeat-masked genome assembly can be downloaded from the RAP-DB ( http://rapdb.dna.affrc.go.jp/download/irgsp1.html). For users' convenience, previous versions based on obsolete genome assemblies are also available at the RAP-DB Legacy database ( http://rapdblegacy.dna.affrc.go.jp/).

Comparison of the RAP and MSU annotation datasets show a high degree of concordance between the two annotations with 33,708 loci overlapping by at least 1bp between the two annotation sets. However, as the two approaches weight ab initio gene predictions and transcript/protein evidence differently (Ouyang et al. 2007, Tanaka et al. 2008), there are differences in the two datasets with more protein-coding genes predicted in the MSU than the RAP annotation.

Conclusions

The genome assembly of Oryza sativa (cv. Nipponbare), which had been constructed and provided independently by two groups, was unified. Sequencing errors were thoroughly examined so that in-depth analyses among rice cultivars will be possible. Furthermore, our survey of alleles found variations within the Nipponbare cultivar that are mostly attributable to outcrossing, residual heterozygosity, and/or somatic mutations through the standard process of propagation in individual laboratories. Our ability to detect a number of allelic differences in the three Nipponbare rice individuals surveyed suggests that allelic differences should not be readily dismissed in SNP assessments of rice diversity. The high-quality reference genome assembly presented here is an invaluable resource for studies of emerging re-sequencing data and is available in the RAP-DB ( http://rapdb.dna.affrc.go.jp/) and the MSU Rice Genome Annotation Project ( http://rice.plantbiology.msu.edu/) websites.

Methods

Sequence data sources

The IRGSP clone and PCR sequences of the O. sativa ( japonica group, cultivar Nipponbare) genome deposited in the International Nucleotide Sequence Databases as of 25 February 2010 were used in construction of the MTP. In addition, sequence reads generated by the Syngenta rice genome sequencing project (Goff et al. 2002) were assembled and used to extend contigs.

For the next-generation DNA sequencing of an NIAS individual, total genomic DNA was prepared from nuclei isolated from Nipponbare rice young leaves (two weeks after germination) using the CTAB method (Murray and Thompson 1980). The DNA samples were fragmented by a nebulizer or Branson Sonifier 250 (Danbury, CT). Sequencing libraries were constructed following the protocols with Illumina Genomic DNA Sample Preparation Kit and Roche GS DNA Library Preparation Kit, respectively. Illumina genome sequencing was performed by Illumina Genome Analyzer II/IIx with the Illumina version 2 sequencing kit. GS-FLX genome sequencing was performed using the Roche GS LR70 Sequencing Kit. The sequence reads are available at the DDBJ Sequence Read Archive (DRA000651).

For the CSHL individual, ~5 μg of Nipponbare rice genomic DNA was used as input for standard Illumina libraries. The DNA was sheared by adaptive focused acoustics using the Covaris (Woburn, MA) instrument and end-repaired using T4 DNA polymerase, Klenow fragment, and T4 polynucleotide kinase. Fragments were then treated with Klenow fragment (3' - 5' exonuclease) to add a single 3' deoxyA overhang and ligated to standard paired-end Illumina adapters. Qiagen (Valencia, CA) columns were used for purification between steps. The fragments were size-selected at ~225 bp (including adapters) using agarose gel electrophoresis. The actual insert size excluding adapters was ~150 bp. The library was then PCR amplified using Phusion DNA polymerase in HF buffer for 14 cycles and quantified using the Agilent BioAnalyzer (Santa Clara, CA). All libraries were normalized to 10 nM before loading on the Illumina sequencers. Production sequencing was performed using Illumina GAIIx instruments with paired-end modules using the Illumina version 3 sequencing kits. The library was sequenced with 76 bp paired-end read lengths. Sequence data was processed using the Illumina GAPipeline v1.1 and v1.3.2 (Firecrest/Bustard v1.9.6 and Firecrest/Bustard v1.3.2). The sequence reads are available at the Sequence Read Archive of NCBI (SRX032913).

Syngenta rice genome sequences (Goff et al. 2002) were filtered by using IRGSP rice genomic sequences with similarity searches. The filtered sequences were then assembled; 50 large Syngenta contigs (between 4 kb and 40 kb), a total of 748 kb were used for potential gap filling.

Construction of the genome assembly based on a minimum tiling path and validation with the optical map

The MTP of the IRGSP clones for each of the 12 pseudomolecules were updated prior to validation with the optical map. We manually checked information on the clone order and clone overlaps in the IRGSP physical map. For the overlapping regions, phase 3 BAC/PAC sequences, which have finished, quality sequences without gaps (see http://www.ncbi.nlm.nih.gov/projects/genome/glossary.shtml), were preferentially chosen. Any ambiguous nucleotides, such as Rs and Ys, were converted to Ns. The lengths of physical gap between contigs were estimated by FISH (International Rice Genome Sequencing Project 2005). A total of 1,000 Ns were inserted in at each physical gap. Other gaps in the original entries were left unchanged.

Sequences of 4,005 rice BACs/PACs, 50 Syngenta contigs, and the 12 pseudomolecules based on the aforementioned MTP were digested in silico by SwaI. The sequences were then aligned (Valouev et al. 2006) against the Nipponbare rice optical map (Zhou et al. 2007) to automatically reveal any map-sequence discordances. The alignments were graphically viewed using GnomSpace (Teague et al., 2010). The alignments of all 12 pseudomolecules, along with the BACs/PACs, were manually examined. In general, the pseudomolecules were in good concordance with the optical map. There were 169 discordances between the optical map and the pseudomolecules, including missing SwaI sites, extra SwaI sites, missing sequence, extra sequence, complex events, and potential misassembly. When there was a discordance, a modification (changing BAC/PAC/Syngenta contig, or adding/eliminating a gap) was made on the MTP when:

1. 1)
There was another BAC/PAC sequence that aligned better against the optical map;

2. 2)
A BAC/PAC/Syngenta contig extended the sequence into a physical gap;

3. 3)
A reverse orientation of a BAC/PAC was aligned better against the optical map;

4. 4)
A BAC/PAC/Syngenta contig was aligned perfectly against the optical map within a physical gap of a pseudomolecule;

5. 5)
A new gap was clearly identified between two neighboring clones;

6. 6)
A gap was eliminated when an overlap of two clones that were supposed to flank a gap was found.

In total, there were 23 modifications made in the tiling paths of the 12 chromosomes. Additional discordances were derived from the sequences of the BACs/PACs, rather than from the pseudomolecule construction; no attempt was made to correct the assembly of the BAC/PAC sequences to match the optical maps.

The lengths of rDNA regions were estimated to span 0.2 Mbp for 5S rDNA on chromosome 11 (Ohmido et al. 2000) and 3.5 Mbp for 17S and 25S rDNA on chromosome 9 (620 daltons/bp; Oono and Sugiura 1980) in a haploid set.

Error corrections with Illumina and 454 reads

To detect small sequencing errors (single nucleotides and indels of 1-4 bases) in the newly assembled reference genome, Illumina Genome Analyzer II/IIx generated reads were used. The genomes of two different individuals of Nipponbare were independently re-sequenced at NIAS and CSHL. Low quality bases (<Q20) were trimmed from both 5'- and 3' ends of the read until two or more consecutive bases with a high quality score (≥Q20) were observed. Next, the Illumina adapter sequences (5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG) and (5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT) were removed using the fastx_clipper, which is part of the FASTX-Toolkit ( http://hannonlab.cshl.edu/fastx_toolkit/). Reads that were <32 bp in length were discarded for further analyses. If only one read of a paired-end read set was discarded in these preprocessing steps, the other read was regarded as a single-end read and named "unpaired." All qualified reads were aligned to the reference genome using BWA v0.5.8a with default options (Li and Durbin 2009). The NIAS single-end reads and CSHL unpaired reads were aligned in the single-end mode using the BWA command "samse". The CSHL paired-end reads were aligned in the paired-end mode using the BWA command "sampe". The reads that matched to multiple genomic positions were discarded. A pile up alignment file of all uniquely mapped reads with a mapping quality value of ≥20 was generated using SAMtools v1.8 (Li et al. 2009). To avoid erroneous detection of variants, only sites with a read depth of 10 or more were selected.

By comparing the Illumina reads with the reference genome, each aligned site was first classified into four categories: "reference type (R)," "non-reference type (N)," "allelic (A)," and "low depth (L)" for each of three sets (NIAS, CSHL and NIAS + CSHL) (Additional file 7). If a site had less than 10 reads, the site was "low depth (L)," which means we were unable to assess the site due to low sampling. If ≥80% of the reads were identical to the reference base, the site was classified as "reference type (R)". If ≥80% of the reads were discordant with the reference base, the site was classified as "non-reference type (N)". If there were two alleles with ≥40% read support, the site was classified as "allelic (A)". Since we have two data sets from NIAS and CSHL, the classifications of the three sets (NIAS, CSHL and NIAS + CSHL) were combined and reexamined to decide the genotype for each site (Additional file 7): "reference type", "sequencing error (Additional file 9)", "alleles between individuals" (Additional file 10), "alleles within individuals" (Additional file 11), and "low depth". SNPs classified as allelic variations were annotated based on the RAP-DB gene models using SnpEff v. 3.1 (Cingolani et al. 2012) (Additional file 12).

Re-validation and annotation of final assembly

The final, error-corrected pseudomolecules were virtually digested with SwaI and aligned against the rice optical map. A total of 144 major discordances were annotated in the current build of rice pseudomolecules (Additional file 5). Among these discordances, there were 53 physical gaps, including 19 telomeric gaps. The sizes of the gaps range from 0.6 kb to 2.4 Mb, measured by adding up the sizes of the un-matched optical fragments at the gap locations. The discordances were grouped into 5 classes:

Class 1: Physical gaps in the pseudomolecule;

Class 2: Missed fragment or extra fragment: only the discordances with ≥5 kb missed/extra fragment(s) were annotated;

Class 3: Significant size difference: mostly the size differences were ≥5 kb;

Class 4: Multiple different SwaI sites in the same area;

Class 5: Multiple different SwaI sites and multiple un-matched SwaI fragments. The total sizes of the pseudomolecule and the optical map at the location were not comparable, indicating possible misassembly).

Authors' information

Nipponbare Genome Re- sequencing Project

Genome sequencing and assembly team

Yoshihiro Kawahara, Melissa de la Bastide, John P Hamilton, Hiroyuki Kanamori, W Richard McCombie, Shu Ouyang, David C Schwartz, Tsuyoshi Tanaka, Jianzhong Wu and Shiguo Zhou.

MSU annotation team

Kevin L Childs, Rebecca M Davidson, Haining Lin, Lina Quesada-Ocampo and Brieanne Vaillancourt.

RAP annotation team

Hiroaki Sakai, Sung Shin Lee, Jungsok Kim and Hisataka Numa.

Coordinators

Takeshi Itoh, C Robin Buell and Takashi Matsumoto.

Abbreviations

BAC:

Bacterial Artificial Chromosome

BWA:

Burrows-Wheeler Aligner

CSHL:

Cold Spring Harbor Laboratory

DDBJ:

DNA Data Bank of Japan

IRGSP:

International Rice Genome Sequencing Project

MSU:

Michigan State University

MTP:

Minimum Tiling Path

NIAS:

National Institute of Agrobiological Sciences

NCBI:

National Center for Biotechnology Information

PAC:

P1 Artificial Chromosome

RAP:

Rice Annotation Project

SNP:

Single Nucleotide Polymorphism.

References

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Acknowledgments

Funding for this work was provided in part by a grant from the U. S. National Science Foundation (DBI-0834043) to CRB, and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation, GIR1001).

Author information

Affiliations

1. Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

Yoshihiro Kawahara, Hiroyuki Kanamori, Tsuyoshi Tanaka, Jianzhong Wu, Hiroaki Sakai, Sung Shin Lee, Jungsok Kim, Hisataka Numa, Takeshi Itoh & Takashi Matsumoto

2. Cold Spring Harbor Laboratory (CSHL), Cold Spring Harbor, NY, 11723, USA

Melissa de la Bastide & W Richard McCombie

3. Department of Plant Biology, Michigan State University, Plant Biology Laboratories, 612 Wilson Rd, East Lansing, MI, 48824, USA

John P Hamilton, Kevin L Childs, Rebecca M Davidson, Haining Lin, Lina Quesada-Ocampo, Brieanne Vaillancourt & C Robin Buell

4. Perkin Elmer, Room 4096, 8490 Progress Drive, Frederick, MD, 21701, USA

Shu Ouyang

5. Laboratory for Molecular and Computational Genomics, University of Wisconsin-Madison, UW-Biotechnology Center, 425 Henry Mall, Madison, WI, 53706, USA

David C Schwartz & Shiguo Zhou

6. Integrated Center for Genes, Environment and Health, National Jewish Health, Denver, CO, USA

Rebecca M Davidson

7. Dupont Pioneer, 7200 NW 62nd Ave, Johnston, IA, 50131, USA

Haining Lin

Corresponding author

Correspondence to Takeshi Itoh.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CRB, TI, and TM, designed the study. DCS, MB, SZ and WRM generated data. BV, HK, HL, HN, HS, JK, JPH, JW, KLC, LQO, RMD, SO, SSL, SZ, TM, TT and YK performed data analyses. CRB, SO, TI and YK wrote the manuscript. All authors read and approved the final manuscript.

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Kawahara, Y., de la Bastide, M., Hamilton, J.P. et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice 6, 4 (2013). https://doi.org/10.1186/1939-8433-6-4
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Keywords

Oryza sativa

Nipponbare

Unified rice reference genome

Pseudomolecules

Minimum tiling path

Optical mapping

Genome re-sequencing

Next-generation sequencing

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Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data - Rice

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Background Rice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa (japonica group).

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- kaisie to huhu

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Rice fragrance is one of the most highly valued traits of rice, commanding higher prices on the global market than non-fragrant varieties.

(PhysOrg.com) -- A new Cornell study reports that the gene that gives rice its highly valued fragrance stems from an ancestor of basmati rice and dispels other long-held assumptions about the origins of basmati.

Rice is classified into two major varietal groups: Japonica and Indica, both of which were grown in China some 8,000 years ago and are believed to have originated from there. The new study, published Aug. 25 in the Proceedings of the National Academy of Sciences, confirms that basmati rice, long assumed to be an Indica variety, is actually more closely related genetically to Japonica rice.

Basmati, which is endemic to northern India, Pakistan and Iran, has been falsely assumed to be in the Indica group due to its characteristic long, thin grains and because it is grown in India, where Indica varieties are widespread. Japonica varieties, which include sushi rice, are widely grown in East and Southeast Asia and tend to have shorter, stickier grains.

When the gene, called BADH2, loses its function through the natural process of mutation, rice becomes fragrant. This study reports eight novel mutations in BADH2 associated with fragrance and found that a previously discovered mutation, or allele, is shared by the vast majority of fragrant rice varieties today, including the fragrant Japonica varieties known as basmati and the fragrant Indica variety known as Thai jasmine.

Through genetic analysis of the DNA flanking BADH2, the researchers determined that the major fragrance allele originated in a Japonica-ancestor of basmati rice and was later transferred to Indica varieties, including Thai jasmine rice.

"People think that all rice [varieties] in India are from the Indica varietal group, but that's not true," said Susan McCouch, professor of plant breeding and genetics and the paper's senior author. Michael Kovach, a doctoral student in McCouch's lab, is the paper's first author.

The new study supports findings from a 2005 paper by McCouch that showed the close genetic relationship of basmati rice to the Japonica varietal group.

"India has both Indica and Japonica rice," McCouch added. "Basmati is a unique type of rice but it is genetically more closely related to sushi rice from Japan than to many of the long grained Indica rices grown elsewhere in India. It is intriguing to think about what these relationships tell us about human migration and cultural exchange."

The findings have important implications for claims of ownership of rice varieties and traits, said Kovach. Rice fragrance is one of the most highly valued traits of rice, and it can command higher prices on the global market.

Thai scientists recently patented the use of a genetic engineering strategy to knock out the BADH2 gene while claiming the fragrance trait was part of their national heritage -- through Thai jasmine rice -- and "belonged to the Thai people," Kovach said.

"They would like to use this approach to impart this characteristic fragrance on other crops like wheat and maize," Kovach added. "There was no proof that the common BADH2 allele causing fragrance in Thai jasmine rice actually did not originate in jasmine varieties, until this study."

"The results suggest something profound and interesting about human culture, and that is, we are all hybrids," said McCouch. "Claims of ownership of rice are important for national identity, but people's concepts of national identity are often over-simplified. Humans continuously exchange ideas, technology and everything that is valuable, and in the exchange, they become something new. The lesson is that while each culture and each rice variety represents something unique, much of what we value most is shared by all."

Provided by Cornell University ( news : web)

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Researchers uncover genetic origins of rice fragrance

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(PhysOrg.com) -- A new Cornell study reports that the gene that gives rice its highly valued fragrance stems from an ancestor of basmati rice and dispels other long-held assumptions about the origins of basmati. Rice is classified into two major varietal groups: Japonica and Indica, both of which were grown in China some 8,000 years ago and are believed to have originated from there.

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Genetic Structure and Diversity in Oryza sativa L.

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The population structure of domesticated species is influenced by the natural history of the populations of predomesticated ancestors, as well as by the breeding system and complexity of the breeding practices exercised by humans. Within Oryza sativa, there is an ancient and well-established divergence between the two major subspecies, indica and japonica, but finer levels of genetic structure are suggested by the breeding history.

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Abstract

The genetic basis of character association related to differentiation found in the primary gene pool of rice was investigated based on the genomic distribution of quantitative trait loci (QTLs). Major evolutionary trends in cultivated rice of Asiatic origin ( Oryza sativa) and its wild progenitor ( O. rufipogon) are: (1) differentiation from wild to domesticated types (domestication), (2) ecotype differentiation between the perennial and annual types in wild races, and (3) the Indica versus Japonica type differentiation in cultivated races. Using 125 recombinant inbred lines (RILs) derived from a cross between an Indica cultivar of O. sativa and a strain of O. rufipogon carrying some Japonica-like characteristics, we mapped 147 markers, mostly RFLPs, on 12 chromosomes. Thirty-seven morphological and physiological quantitative traits were evaluated, and QTLs for 24 traits were detected. The mapped loci showed a tendency to form clusters that are composed of QTLs of the domestication-related traits as well as Indica/Japonica diagnostic traits. QTLs for perennial/annual type differences did not cluster. This cluster phenomenon could be considered "multifactorial linkages" followed by natural selection favoring co-adapted traits. Further, it is possible that the clustering phenomenon is partly due to pleiotropy of some unknown key factor(s) controlling various traits through diverse metabolic pathways. Chromosomal regions where QTL clusters were found coincided with the regions harboring genes or gene blocks where the frequency of cultivar-derived alleles in RILs is higher than expected. This distortion may be partly due to unconscious selection favoring cultivated plant type during the establishment of RILs.

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SPECIAL ISSUE

This article is part of the Safety of GM Crops: Compositional Analysis special issue.

Plant breeding can be considered a coevolutionary process between humans and edible plants. People caused changes in the plants that were used for agriculture and, in turn, those new plant types allowed changes in human populations to take place. Plants yielding more generous harvests freed some of the people's time for developing art, handcrafting, and science, eventually leading to modern human life as we know it. Civilization could not exist without agriculture, and agriculture could not sustain the civilized world without modern crop varieties.(1) From this point of view, it becomes clear that plant breeding is one of the main foundations of civilization.

In industrialized countries, only a small portion of the population is engaged in agriculture. The vast majority of people rely on a tacit social pact for their survival, which assures that someone will provide food in exchange for some service or good. This pact is so basic to modern life that people take for granted that food is available in the nearest supermarket. However, agriculture failure could cause a disruption of this pact, leaving people in a situation of food insecurity. Thus, protecting agriculture means warranting the foundation pact of modern civilization.

The core of plant breeding is the selection of better types among variants, in terms of yield and quality of edible parts; ease of cultivation, harvest, and processing; tolerance to environmental stresses; and resistance against pests. Each of these aspects of agronomic or food value can be dissected in many specific traits, each presenting its own range of variation. Manipulating a single trait, disregarding all others, is relatively straightforward; however, this is unlikely to result in a useful variety.

The challenge of plant breeding resides in improving all of the traits of interest simultaneously, a task made more difficult by the genetic correlations between different traits, which may be due to genes with pleiotropic effects, to physical linkage between genes in the chromosomes, or to population genetic structure.(2) Selecting for one trait will change correlated traits, sometimes in the desired direction, other times in an unfavorable way.(3) For this reason, selection can lead to unanticipated changes, which are normally within the range that is normally observed in the crop and thus assumed to pose no risk to consumers or the environment. Whether this assumption is reasonable or not is a matter of debate.(4)

The objective of this paper is to discuss plant breeding methods as an evolving technology, considering the increasing levels of knowledge of the underlying mechanisms and the control of the process of generating and selecting superior plant types. In this context, three main eras of plant breeding can be identified: (i) plant breeding based on the selection of observed variants, disregarding their origin; (ii) generation and selection of expanded variation by controlled mating; and (iii) monitoring the inheritance of within-genome variation and selection of specific recombinants. The fourth stage of plant breeding, which is not discussed in this paper, can be considered the creation and introduction of novel variation into genomes through genetic engineering. The varieties resulting from the methods presented in this paper can be considered a reference against which transgenic plants are compared with regard to their food safety.

The most primitive form of plant breeding was the selection of naturally occurring variants in the wild and, later, in cultivated fields. Genetic variation was continuously submitted to the selection pressure of food gathering or planting-harvesting cycles. In some cases, this process resulted in deep changes in plant phenotypes, as exemplified by the derivation of maize from teosinte.(5) This early phase of plant breeding spans the period from the origin of agriculture until the first hybridization experiments carried out by Kölreuter in the 1760s.(6) With the discovery of the laws of heredity, in the turn from the 19th to the 20th century, the importance of hybridization in plant breeding became widely recognized.(7) Today, almost all plant breeding programs involve some use of hybridization.(8)

Plant Domestication: The Origin of Crops

For a given gene, mutations are rare events, but considering the large numbers of plants in a field and of genes in a plant, mutations are quite frequent events in a population.(9) Most mutations are unfavorable for survival in the wild, being eliminated from the population in a few generations, as a consequence of natural selection. However, some of these mutations may result in more favorable phenotypes either in terms of cultivation or in terms of food quality. Some of those mutants were rescued by ancient farmers, who protected them against competition and established with those otherwise disabled plants a relationship of symbiosis. Unlike wild habitats, cultivated fields were environments in which those mutations conferred a selective advantage, thus becoming the predominant type through human selection. The accumulation of this type of mutation is the major cause of the domestication syndrome, a set of characteristics that made many cultivated species irreversibly dependent on humans for their survival.(10)

The molecular variability in domesticated plants tends to be smaller than in related wild species, as a consequence of the founder effect during domestication. By strongly selecting for the rare mutant plants adapted to cultivation, early farmers dropped most of the variation present in the wild populations from which cultivated forms arose. It is now clear that many valuable genes, especially those related to resistance to pests, were left out of the cultivated gene pool.(11) Incorporating those genes into modern cultivars, without losing ground in terms of yield and product quality, is one of the challenges of modern plant breeding and one of the most relevant applications of molecular tools in breeding programs.

Intuitive Farmer Selection: The Origin of Landraces

Landraces are populations of plants that have been cultivated for many generations in a certain region, being shaped by biotic and abiotic stresses, crop management, seed handling, and eating preferences. They are dynamic genetic entities: continuously changing as a consequence of intentional and unintentional selection, seed mixture, and pollen exchange. Landraces are shaped by a balance between stabilizing selection, which keeps the identity of the landrace in a given region, and mild directional selection, leading to slow adjustments to environmental changes. In some cases, quick changes can take place, especially when the landrace is taken to a different region or when new materials are cultivated in close proximity with the original landrace. Landraces can still nowadays derive from modern cultivars, if certified seed production is discontinued and farmer-saved seeds are planted recurrently, without care for isolation against seed or pollen contamination.

The major characteristics of landraces are(12) (i) high levels of genetic diversity within populations, characterized by a limited range of variation between individuals, with distinctive traits that make the landrace identifiable; (ii) adaptation to soil and climate conditions typical of the region, combined with resistance to common pests; (iii) edible parts that are valued by local people, normally shaping and being shaped by the local cuisine; and (iv) modest but stable yield, conferring food security to the local community under normal environmental variation.

Intuitive farmer selection has the virtue of shaping varieties for the actual and specific environment of use and for the local food preferences, serving well the case of subsistence agriculture, where most of the production is locally consumed. However, when farmers select for one trait, genetic correlations may result in undesirable changes in other traits. For example, cereal landraces are normally tall plants, prone to lodging and presenting low harvest index, probably as a result of human selection for large edible parts (panicles, ears, spikes).

Nevertheless, for their wealth in genetic variability and adaptability to different environments, landraces are the most valuable genetic resources for long-term plant breeding programs and also prime targets for germplasm collections. Seed banks around the world keep thousands of samples of landraces under "ex situ" conservation. In some countries, there are efforts to devise regulatory mechanisms and financial incentives for traditional communities to keep growing their heritage varieties, aiming for their "in situ" conservation. New systems of germplasm conservation have been built on social networks connecting people interested in the subject as a hobby (e.g., seedsavers.org). Those networks take advantage of modern communication tools to replicate on a global scale what used to happen through personal contact in traditional communities.

Notwithstanding, a large part of the variability that once existed in cultivated fields of annual plants may have been irreversibly lost during the introduction of modern, high-yielding cultivars. In this sense, the same modern cultivars that saved millions from starving may have wiped out varieties that were the result of centuries of local intuitive selection by farmers and a valuable resource for future genetic improvement.

Pure Line Selection and Mass Selection: The Origin of Cultivars

The earliest method of plant breeding based on an elementary knowledge of the laws of inheritance has been the selection of plants within landraces, based on the assumption that the progenies of the best individuals are expected to be superior to the progeny of a random sample of the population. This method was formally proposed by Louis de Vilmorin in 1856, although there are mentions of the use of its principles by some farmers earlier in the 19th century.(13) This realization can be considered as the origin of the paradigm of homogeneity that dominates breeding, and agriculture as a whole, until today. From this point on, within-field heterogeneity was considered to be undesirable and both plant breeding and agronomy developed methods to achieve maximum spatial homogeneity (e.g., "precision agriculture").

In self-pollinating species, such as rice and wheat, landraces can be thought of as a mixture of pure lines, including some heterozygous individuals derived from a low frequency of cross-pollination. In this type of population, selecting single plants and deriving inbred progenies invariably result in some lines that outperform the original landrace for a given growing condition. However, this superiority comes at a cost, because pure lines are normally less stable than diverse populations in the face of stresses, especially diseases, and have no capacity for long-term adaptation, because it is monomorphic for most genes.

In the case of open-pollinated species, such as maize, landraces are populations of random mating individuals, approximating the Hardy-Weinberg equilibrium (HWE), with some deviations due to mild selection. Mass selection and recombination of the top-performing portion of the population result in a gradual increase in the frequency of favorable alleles. Successive generations of selection on maize landraces resulted in improved open-pollinated varieties, which were the basis of corn production until the advent of hybrid maize.

Despite the great spontaneous diversity that can be found in the landraces, simply applying selection on preexisting diversity is an eroding process that eventually comes to a limit. The true creative power of plant breeding resides in promoting recombination for shuffling favorable alleles.(14) The combination of different alleles in many loci results in a virtually infinite number of genotypes. One could conceivably start a commercial breeding program from a dozen well-adapted founding parents, with a clear focus on a specific target environment and evaluating large segregating progenies. Injection of novel variability might become necessary in the case of a significant change of the target environment, such as the emergence of new pests for which the founder materials had no resistance.

Given the myriad of possible genotypes resulting from crossing diverse parents, the limitation for genetic gains becomes the capacity of the breeding program to evaluate a large number of plants, derived from a large number of crosses. For this reason, plant breeding is frequently dubbed a numbers game, and large competitive programs in commodities invest heavily in high-throughput methods for seed handling, planting, evaluating, and harvesting. As genetic gains accumulate, the bar is gradually raised, and increasingly higher investments are required to keep a steady rate of genetic progress. The limit of this escalation is the financial viability of returns in the seed market and associated business. The main methods developed for efficient use of resources in breeding programs are discussed next.

Pedigree Breeding: Playing with Parents

The vast majority of the released cultivars of self-pollinating species have been developed through the pedigree method. Pedigree breeding consists of crossing parents and generating segregating populations, which are conducted through generations of self-pollination and selection, until a set of derived lines that combines the good characteristics of both parents is obtained. Because it is based on the complementation of traits, this method is efficient for breeding for qualitative traits, such as disease resistance, or easily classifiable traits, such as plant architecture or the color or shape of plant parts. The pedigree method is appealing to breeders because it allows building better varieties by putting together, in the same plant, good characteristics that were present in different materials. Because all crossings are controlled, it is possible to know the genealogy of each cultivar.

The main weakness of the pedigree method resides in the fact that yield is evaluated efficiently only at the end of the process, on inbred lines, when seed is available for replicated trials. At this point, though, unless a large number of lines have been advanced, there is little room for improvement of yield potential. Consequently, the rate of progress for yield resulting from the pedigree method is normally modest, rarely exceeding 1% per year.(15)

Ideotype Breeding: Playing with Traits

The ideotype breeding approach can be regarded as a strategy to improve the capacity of the pedigree method to promote gains for quantitative traits, especially yield. It is based on the hypothesis that one can improve complex traits by changing simpler traits that are positively correlated with them.(16) The advantage of this method is that, if the underlying hypothesis proves correct, one could promote significant gain for yield, even with a small breeding program, taking the "smart" approach, as opposed to the "numbers game" approach of large-scale breeding programs. Additionally, it is scientifically attractive to breeders, because they have a chance of changing paradigms in their favorite crop. However, it is important to keep in mind that unfavorable genetic correlations can offset the advantage brought by the traits that make up the ideotype.(17)

Changing the ideotype of a crop often requires looking for variation beyond the boundaries of elite germplasm, which is normally of the current plant type. However, using landraces as parents in breeding programs normally results in marked reduction in yield. For this reason, backcross toward the elite materials is necessary to recover a competitive progeny.(18) If the donor of the new trait is a wild relative, problems of sexual compatibility, flower sterility, grain quality, or seed dispersal (i.e., grain shattering) may persist for several generations.

To avoid undesirable effects of direct introduction of exotic materials into elite breeding populations, those materials are normally used first in a phase of prebreeding, when breeders try to break the association between useful and undesirable traits. Once the new trait needed to assemble the ideotype is inserted into an elite background, those vector lines can be transferred to the elite breeding program.

Population Breeding: Playing with Genetic Variance

Although all breeding methods imply the application of selection pressure on a variable population, the term "population breeding" indicates a method designed to improve the phenotypic performance of an intermating population by increasing the frequency of favorable alleles controlling traits of interest. The simplest version of population breeding is the mass selection method applied to cross-pollinated species, in which the improved population is directly used as a cultivar. Later, more sophisticated schemes of population breeding have been designed, providing the framework for the development of the quantitative genetics theory. In modern population breeding, the objective is to increase the value of the population as a source of elite lines. Improving the mean quality of the population, while preserving the variation within it, results in top individuals that outperform previously existing lines. Those lines can be used as cultivars, in the case of self-pollinated species, or as parents of hybrids, in the case of cross-pollinated species.

Population breeding is an open-ended scheme of consecutive rounds of selection and recombination, thus being also known as recurrent selection breeding. Recurrent selection requires an efficient crossing system, which can be a limitation for self-pollinating species. This scheme, when applied repeatedly on small populations, leads to depletion of genetic variation and slowing of genetic gains.(19) For this reason, a large effective population size must be kept throughout or else genetic drift will override the other forces acting on the population, hindering genetic improvement.(20)

However, even moderate population sizes (e.g., 50 plants) appear to be sufficient to avoid rapid depletion of genetic variation.(21) This is probably due to the fact that, whereas selection leads to the loss of diversity in some genes, recombination breaks linkage blocks into smaller pieces, allowing many more combinations of genes than was possible with large DNA segments. This phenomenon releases hidden variation in latter generations down the process of recurrent selection, compensating for the loss of variation due to selection.(22)

Breeders create new populations by intercrossing several lines, chosen as sources of favorable alleles for one or more traits. These synthetic populations under recurrent selection mimic the genetic events that used to take place in the landraces in the hands of traditional farmers, with the difference that the whole process is monitored and controlled, and selection pressure is intensified for faster gains. The rate of genetic gain per unit of time can be increased by speeding up the selection-recombination cycles, by intensifying the selection pressure, by improving the evaluation precision (thus increasing the heritability), or by any combination of those.

The general scheme of population breeding is very flexible, allowing customization to specific needs and objectives of different species and breeding programs. The system can be formatted for rapid, short-term results, normally by applying strong selection pressure on genetically narrow-based populations, or for sustained, long-term results, by applying moderate selection pressure on genetically broad-based populations. Population breeding can also be used as a prebreeding scheme, because its frequent crossing events promote recombination between exotic and elite genomes, purging unfavorable exotic genes from the population.

Hybrid Breeding: Playing with Heterosis

Heterosis is the superiority of hybrid individuals compared to inbred individuals.(23) Within certain limits, the more divergent are the parents, the higher is the heterosis on their offspring.(24, 25) Hybrid vigor decays rapidly through generations of inbreeding, indicating that, whatever is the mechanism underlying heterosis, it is due to the presence of heterozygous loci. For this reason, maize breeding programs nowadays are focused on developing competitive F1 hybrids, in which heterozygosis is at its maximum.

Two challenges are present in hybrid breeding programs: (i) the need to improve at least two populations toward agronomic adaptation, while keeping them genetically distant enough to express strong heterosis, and (ii) developing efficient seed production of selected hybrids, such that the cost of seed production does not offset the value of the additional yield resulting from heterosis. In maize, the first problem led to the concept of heterotic groups,(26) splitting the elite gene pool into subsets, within which population breeding is applied. The second problem was solved by the improvement of parental lines, resulting in higher yield in seed production fields, and by the use of male sterility genes and mechanical emasculation ("detasselling") to avoid self-pollination.

On the other hand, hybrids present great advantages from the business perspective. The seeds produced by hybrid plants are genetically heterogeneous due to the segregation of thousands of genes. If planted, the resulting crop would present a large variation in agronomic traits, plant architecture, and cycle duration, thus reducing yield and grain quality. Hence, farmers must buy new seeds every year, resulting in a constant demand for hybrid seeds. Additionally, with the advent of the transgenics, hybrid seeds became preferential carriers of those valuable proprietary traits because they allow a better control of the event by the owner.

Traditional breeding methods were based on the complementarity between parental characteristics. However, little or nothing was known about which part of the genome came from each parent. This situation changed with the advent and dissemination of molecular marker technologies, which made it possible to monitor the transmission of chromosome segments in the progeny.(27)

Virtually any sequence variation between individuals can be used to design a marker that will allow the identification of the parent that contributed a specific segment of the chromosome in a recombinant line. Until recently, the most popular markers were the simple sequence repeats (SSRs), also known as microsatellites. Those markers were superseded by the single nucleotide polymorphisms (SNPs), which are more abundant in the genome and more amenable to high-throughput genotyping.

Molecular markers are essential tools for studying the genetic control of any trait of interest, eventually leading to the identification of the genes underlying the trait and the metabolic chains involved. This venue can be broadly defined as molecular biology, which dominated the field of biological sciences in the past decades. However, in this paper we limit the discussion to the application of molecular markers as tools for plant breeding.

Mapping Genes of Interest: Finding Needles in the Haystack

When many molecular markers are genotyped in a set of plants derived from a single cross, the frequency of recombination between them can be used to infer their order and relative distance in the chromosomes, resulting in a genetic map. If those plants, or their progeny, are evaluated for a quantitative trait, a statistical model can be built in which part of the phenotypic variance can be explained by some of the markers, which implies that those markers should be linked to the genes underlying the trait. This approach results in quantitative trait loci (QTL) maps, which are normally the first step toward understanding the genetic control of a quantitative trait.(28) From a QTL map, a plant breeder can get an insight into the number of loci controlling a trait, their relative importance, and their approximate position in the genome. In the case of large-effect QTLs, it may be possible to jump directly into marker-assisted selection, using the closest markers available in the map. However, in most cases a step of fine mapping, involving a larger population and denser marker spacing in the target genomic region, is necessary for developing useful selection tools. The ultimate result of this approach is the identification of the gene, and the polymorphism in its nucleotide sequence, responsible for the observed phenotypic differences.

The QTL mapping approach is effective for explaining contrasts between two parents, but is inefficient for exploring the wider genetic diversity for a trait in the germplasm. The association mapping approach offers a shortcut in this path and for this reason has strong appeal to breeders.(29, 30) In this approach, a panel of genetically diverse lines is densely genotyped and carefully evaluated for a phenotypic character, with the aim of identifying associations between marker alleles and traits. Because the genealogical distance between the materials in the panel is large, those associations will remain significant only if the marker is tightly linked to the causative gene or if factors related to population genetics (population structure) create associations between unlinked loci. For the latter case, estimating population structure and taking it into account explicitly in the statistical model can avoid the detection of false associations.(31) When correctly applied, association analysis can result in the detection of genomic regions related to the trait of interest and, simultaneously, in the identification of donor lines of favorable alleles from a wider germplasm.(32)

Gene banks harbor thousands of accessions that are potentially useful for plant breeding. Those accessions include wild crop relatives and obsolete landraces. Although presenting poor agronomic value, compared to modern cultivars, those materials are believed to have useful genes(33) that have not been captured from wild species in the process of domestication or from landraces in the early phases of scientific breeding. Rescuing those useful genes that were "left behind" is a difficult task, because the genes of interest may be tightly linked to unfavorable genes, which would be dragged along into the breeding population. Understanding the genetic diversity in germplasm collections is the first step toward better use of a broader gene pool in breeding programs. Marker-assisted backcrossing allows identifying the rare recombinants in the vicinity of the introduced gene, trimming the chromosome segment as close as possible to the target gene.(34) Additionally, by identifying individuals with the cleanest genome (with the least residue from the donor parent), it is possible to reduce the number of backcrosses needed for full recovery of the elite phenotype.(35)

Collections of chromosome segment substitution lines (CSSL libraries) are a set of lines derived from an elite variety, in which each line has one chromosome segment replaced by the corresponding segment in the wild species of interest.(11) The CSSL library carries the whole genome of the wild relative split in bins. The phenotypic effect of the set of genes in each bin can be evaluated against the original elite line, used as a check. Those genetic stocks can facilitate the identification and introgression of genes from wild relatives into elite germplasm. Superior CSSLs can be used for fine mapping and simultaneous development of an improved version of the elite variety.(36)

Marker-Assisted Selection: Building Tailored Genotypes

Whereas in the case of marker-assisted gene introgression the breeder will normally tag only one gene, a full-featured marker-assisted selection scheme would monitor several genes simultaneously. Knowledge of the allele borne by each plant in each major locus of interest creates the means for building specific allele combinations that would maximize the agronomic value of the line. However, when one is dealing with several genes, it is important to keep in mind that the interaction between genes (epistatic effect), and not only the additive effects of the genes, defines the expression of a trait.

A typical application of marker-assisted selection is resistance gene pyramiding. When one is dealing with diseases caused by pathogens presenting high variability, a single resistance gene may confer complete resistance for a few plant generations until it is overcome by strains of the pathogen. Accumulating several resistance genes in a variety can confer durable resistance, because the pathogen would have to beat all of the genes simultaneously, which drastically reduces the odds of resistance breakdown.(37) Resistance gene pyramiding by classical breeding methods is almost impossible, because the effect of a gene could be visible only in the absence of other resistance genes. Marker-tagged resistance genes can be easily combined without the need for phenotypic screening. A similar strategy can be used for preventively breeding resistance to pathogens that are currently absent from a region, because in the absence of the pathogens no phenotypic screening of resistance would be possible.

Molecular markers of interest for breeders of a given species can be combined in optimized sets for simultaneous genotyping ("multiplexed"), allowing high-throughput application in applied breeding programs, especially those dealing with a broader genetic base.(38) The implementation of such a marker set depends on a large body of previous research. For each of the traits of interest, several steps of research are required, normally starting with mapping quantitative trait loci (QTL), followed by fine mapping and eventually positional cloning. Once the causative gene is identified, based on a pair of contrasting alleles, "mining" novel alleles in diverse germplasm may expand the range of variation of the trait or the toolbox to deal with specific stresses.(39)

Genomic Selection: Speeding up Genetic Progress

Recent advancements in genotyping technology sharply reduced the cost of genotyping, creating the possibility of scoring thousands of markers in populations of plants under selection. The several steps of genetic analysis required to identify the relationship of each of those markers with the phenotype could not keep pace with that. In this scenario, the breeder can have a large amount of genotypic information with unknown relationship with the traits under selection. The genomic selection approach(40) proposes that knowledge of the relationship between specific markers and specific genes is not necessary in the breeding context. Instead, the breeder can use the information available for all markers in a plant to predict its breeding value, without effectively evaluating its phenotype, based on previous statistical models built for those markers using a "training population" for which all genotypes and phenotypes have been scored. Markers are considered as random factors, in the framework of mixed model analysis, because the number of markers used in genomic selection is normally superior to the number of individuals in the training population, such that estimating the effect of each marker would not be possible due to the lack of degrees of freedom. Simulation studies demonstrated that genomic selection can accelerate the genetic progress of quantitative traits in population breeding.(41)

Genomic selection can be seen as a method to maximize the return from the phenotypic evaluation step in the breeding cycle.(42) The information drawn from one round of proper phenotypic evaluation can be propagated to subsequent generations. Once models are built for different environments, the unique marker profile for a given individual can be used to predict its performance in each of these environments, improving considerably the inference made on each individual performance and largely reducing evaluation costs. The potential use of this approach in choosing parents of new breeding populations is also promising. However, the predictability of the phenotype from the marker profile fades out along generations, and new rounds of phenotypic data must be input to update the statistical model. This approach will be especially useful in cases when phenotypic evaluation is difficult, expensive, or conditioned to uncertain events, such as the occurrence of a pest or specific weather condition.

Genomic selection can be used to accelerate breeding for yield potential, which is frequently considered as the most difficult trait for marker-assisted selection. In highly competitive seed industries, such as the cases of maize and soybean, any increase in the rate of genetic gain could make a significant difference in market share. For this reason, large seed companies are investing heavily in high-throughput DNA analysis and bioinformatics for seamless integration of genomic selection in breeding programs.(43) Seeds can be genotyped before planting, such that only preselected individuals will use expensive plots in the field. Full application of genomic selection should happen more easily in large private companies, when compared to small seed companies, public institutions, and universities, considering the amount of investment required for setting and keeping up-to-date the genotyping pipeline, the short turnaround time for DNA analysis, data processing and selection, and the well-trained team required.

The raw material of natural genetic diversity was carved by human selection during plant domestication, resulting in profound changes in plant phenotypes. Intentional or unintentional selection through millennia of traditional farming resulted in a wealth of genetic diversity, adapted to different human needs. However, this process worked at a speed that no longer matches the demands of modern society. The advent of scientific plant breeding accelerated the rhythm of varietal improvement, and at this point it is difficult to predict the limits of this approach. Molecular tools now permit monitoring the dynamics of genomic recombination, making possible a gene-by-gene breeding approach. The impact of those new methods in farmer's fields is just in the beginning, but the expectations for plant breeding to help meet the challenges related to food supply, environmental sustainability, and even fossil fuel replacement are huge. The most modern methods will be necessary for going back to what remains from the raw material where our ancestors found their sources of food, to find the genes that will be the building blocks of the cultivars that will solve the problems of the 21st century.

Rice Domestication

There are two species of cultivated rice: Oryza sativa, originated from Oryza rufipogon in Asia, and Oryza glaberrima, originated from Oryza barthii in Africa.(44) The South American wild species Oryza glumaepatula, although similar to O. rufipogon, has not been domesticated (Figure ).

O. sativa accounts for almost the entirety of global rice production, whereas O. glaberrima is planted only in some parts of Africa, but is being displaced by more productive O. sativa varieties, some of them with introgressions of O. glaberrima genes.(45) The history of rice domestication is highly complex and still not completely understood. O. rufipogon is the most widespread and diverse of those species, which makes it the most likely source of the original domestication event. However, unlike cultivated rice, this species is perennial and predominantly outcrossing, whereas Oryza nivara is annual and self-pollinating, so it would take a lesser change to originate the cultivated type.(46) It is probable that both species contributed genes for the early forms of cultivated rice.(47)

A suite of differences are observed between wild and cultivated rice.(44) Wild rice presents seed dispersal mechanisms, including seed shattering, long awns, and hairy hulls. Cultivated rice lost those characteristics, partially or completely, with nonshattering being the key trait of domestication. Seeds of wild rice present long dormancy, which increases the odds of perpetuation in uncertain wild environments, but is disadvantageous in cultivated fields, where the farmer provides adequate conditions for germination in every growing season. The change in those traits may have resulted simply from harvesting, when the majority of the seeds of shattering plants were lost, and from sowing, when plants derived from nondormant seeds dominated the field. Dark hulls and red pericarp are also typical traits of wild rice that have been eliminated in cultivated materials, possibly by intentional human selection of straw-colored hulls and white pericarp.

O. sativa presents two subspecies, indica and japonica. It is uncertain if each type derives from an independent domestication event or if a single event later split into two subtypes.(48) In favor of the independent domestication hypothesis is the fact that the level of divergence observed between those two groups projects the separation before the origin of agriculture;(49) furthermore, wild rice populations present some degree of population structure in line with the differences between indica and japonica rice.(50) Nevertheless, the fact that the mutation causing the loss of shattering in both indica and japonica is the same(51) is strong evidence in favor of a single domestication event. Trying to reconcile those two conflicting pieces of evidence, Vaughan et al.(52) offered an interesting explanation for the observation of similar differentiation in wild rice populations: the two rice types under cultivation would have frequently hybridized with wild rice populations, transferring part of the genetic structuring to natural populations. If this hypothesis holds true, what we see today in the wild would be a consequence of cultivated types, as much as the opposite. This implies that plant breeding reshapes not only the plant under cultivation but also, to some degree, its relatives in the wild.

Understanding the domestication history of a species is important to estimate the potential of wild relatives in breeding. QTL mapping studies showed that O. rufipogon can be used to improve the agronomic performance of O. sativa.(53, 54) Understanding the genetic relationship between germplasm accessions, through high-throughput marker technology, will allow deep understanding of thousands of rice accessions in seed banks around the world.(55)

Rice Ideotype Breeding

Since the semidwarf rice plant, grown under higher N fertilization, brought a dramatic increase in yield in the period of the Green Revolution (1960s), rice breeders search for other traits, or set of traits, that could promote another leap forward in rice productivity. Although there have been yield gains, conventional breeding was more successful in improving disease resistance, grain quality, and earliness than yield potential.(56)

On the basis of simulation studies,(57) scientists from the International Rice Research Institute (IRRI) designed a hypothetical ideotype that should push the limits of rice grain yield upward. This model plant, known as the rice "new plant type", had few tillers, but all of them bore a large panicle with more than 200 grains on top of thick, lodge-resistant stems. Leaves were thick and erect, with intense green color.(58) Those traits, which are relatively easy to select in the breeding program, when combined should result in increased harvest index and yield (Figure ).

Although based on reasonable premises, the new plant type failed to yield more than the best varieties with the typical, modern plant type. Reduced tillering resulted in low canopy biomass, which, combined with incomplete grain filling, offset the gain in harvest index on a plant basis. A second generation of new plant type lines, with larger proportion of indica genotype, performed better but still failed to beat the best checks.(58)

China's national breeding program also pursued an ideotype for achieving unprecedented rice productivity. The Chinese ideotype is similar to IRRI's ideotype, with the main difference of having the panicles lower in the canopy, below the height of the top three leaves, which should remain productive until the grain-filling period. By adding those key traits and taking the hybrid breeding approach, the Chinese program had greater success.(59) Hybrid breeding represented a shortcut toward construction of improved ideotypes, because instead of having to introduce all of the ideotype traits into a single inbred line, it was possible to breed two complementary parents that, when crossed, completed the ideotype, on top of which, heterosis added additional yield potential.(60)

Direct Selection of Cloned Genes in Rice

With the recent advances of molecular knowledge of genes and trait control, the possibility of doing plant breeding by picking specific genes and building planned combinations is gradually becoming real. Soon it may be possible to model complete molecular ideotypes, which would be hypothesized optimal combinations of alleles at many major genes, according to the target environment of the crop.

With a small genome size, rich genetic diversity, and other experimental conveniences, rice became the model plant among crops. Reference genome sequencing has been completed, both for japonica(61) and for indica(62) types, an invaluable resource for gene cloning. Upon this body of information, rice genes are being cloned in laboratories around the world.(63) Classical breeding programs will benefit from the knowledge of major genes with significant effect on important traits. Specific primers can be made to detect the favorable alleles, making sure they are present in the elite lines.

One of the genes of high interest to breeders is Ghd7, which simultaneously affects grain number per panicle, plant height, and heading date.(64) This locus alone has a large influence on adaptation to long growing seasons, with high yield potential, or short growing seasons, with lower yield. Controlling this locus can help to bring about the use of exotic germplasm in breeding programs in different geographic regions.

Submergence tolerance is a trait better handled molecularly than phenotypically. As an erratic natural disaster, testing plants for flooding tolerance in the field is difficult and expensive. Scientists from IRRI identified a large effect QTL for flooding tolerance (Figure ), in which the favorable allele conferred tolerance to several days of complete submergence.(65) Further investigation led to the underlying gene, named Sub1A, which played a role in the response of the plant to ethylene.(66) Marker-assisted selection was used for developing submergence-tolerant versions of important rice cultivars.(67)

Soils with low P content are common in the tropics and limit rice yield. Rice varieties differ significantly for adaptation to low-P soils, and part of those differences is due to a major QTL on chromosome 12, named Pup1.(68) Several traditional upland varieties present the favorable allele at this locus, whereas modern varieties appear to lack it, probably because selection is often done in well-fertilized experimental fields. Additional studies identified the underlying gene, named Pstol1, which promotes root growth.(69) Larger roots explain not only the better P uptake of some traditional rice varieties but also their superior drought tolerance. Marker-assisted selection of the Pup1 locus will facilitate reintroducing the favorable allele into modern cultivars, without yield penalty.(70)

This article is cited by 106 publications.

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# data[].attributes.attachment_link.title
Traditional and Modern Plant Breeding Methods with Examples in Rice (Oryza sativa L.)

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Plant breeding can be broadly defined as alterations caused in plants as a result of their use by humans, ranging from unintentional changes resulting from the advent of agriculture to the application of molecular tools for precision breeding.

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Received: 10 April 2020 / Revised: 8 June 2020 / Accepted: 19 June 2020 / Published: 22 June 2020

Rice-based intensive cropping systems require high input levels making them less profitable and vulnerable to the reduced availability of labor and water in Asia. With continuous conventional puddled rice transplanting, the situation is exacerbated by damaged soil structure, declining underground water and decreasing land and water productivity. To minimize these negative effects a range of new crop establishment practices have been developed (zero tillage, dry direct seeding, wet direct seeding, water seeding, strip planting, bed planting, non-puddled transplanting of rice, mechanical transplanting of rice crop and combinations thereof) with varying effects on soil health, crop productivity, resource saving and global warming mitigation potential. Some of these allow Conservation Agriculture (CA) to be practiced in the rice-based mono-, double- and triple cropping systems. Innovations in machinery especially for smallholder farms have supported the adoption of the new establishment techniques. Non-puddling establishment of rice together with increased crop residue retention increased soil organic carbon by 79% and total N (TN) in soil by 62% relative to conventional puddling practice. Rice establishment methods (direct seeding of rice, system of rice intensification and non-puddled transplanting of rice) improve soil health by improving the physical (reduced bulk density, increased porosity, available water content), chemical (increased phosphorus, potassium and sulphur in their available forms) and biological properties (microbiome structure, microbial biomass C and N) of the soil. Even in the first year of its practice, the non-puddled transplanting method of rice establishment and CA practices for other crops increase the productivity of the rice-based cropping systems. Estimates suggest global warming potential (GWP) (the overall net effect) can be reduced by a quarter by replacing conventional puddling of rice by direct-seeded rice in the Indo-Gangetic Plains for the rice-based cropping system. Moreover, non-puddled transplanting of rice saves 35% of the net life cycle greenhouse gases (GHGs) compared with the conventional practice by a combination of decreasing greenhouse gases emissions from soil and increasing soil organic carbon (SOC). Though the system of rice intensification decreases net GHG emission, the practice releases 1.5 times greater N ₂O due to the increased soil aeration. There is no single rice establishment technology that is superior to others in all circumstances, rather a range of effective technologies that can be applied to different agro-climates, demography and farm typologies. View Full-Text

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Alam, M.K.; Bell, R.W.; Hasanuzzaman, M.; Salahin, N.; Rashid, M.H.; Akter, N.; Akhter, S.; Islam, M.S.; Islam, S.; Naznin, S.; Anik, M.F.A.; Apu, M.M.R.B.; Saif, H.B.; Alam, M.J.; Khatun, M.F. Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields. Agronomy 2020, 10, 888. https://doi.org/10.3390/agronomy10060888

AMA Style

Alam MK, Bell RW, Hasanuzzaman M, Salahin N, Rashid MH, Akter N, Akhter S, Islam MS, Islam S, Naznin S, Anik MFA, Apu MMRB, Saif HB, Alam MJ, Khatun MF. Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields. Agronomy. 2020; 10(6):888. https://doi.org/10.3390/agronomy10060888

Chicago/Turabian Style

Alam, Md. K., Richard W. Bell, Mirza Hasanuzzaman, N. Salahin, M.H. Rashid, Nadia Akter, S. Akhter, Mahammad S. Islam, S. Islam, S. Naznin, M.F.A. Anik, Md. M.R.B. Apu, Hasib B. Saif, M.J. Alam, and Mst. F. Khatun 2020. "Rice ( Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields" Agronomy 10, no. 6: 888. https://doi.org/10.3390/agronomy10060888

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# data[].attributes.attachment_link.title
Rice (Oryza sativa L.) Establishment Techniques and Their Implications for Soil Properties, Global Warming Potential Mitigation and Crop Yields

# data[].attributes.attachment_link.description
Rice-based intensive cropping systems require high input levels making them less profitable and vulnerable to the reduced availability of labor and water in Asia. With continuous conventional puddled rice transplanting, the situation is exacerbated by damaged soil structure, declining underground water and decreasing land and water productivity.

# data[].attributes.attachment_link.title
Arsenic Accumulation and Metabolism in Rice (Oryza sativa L.)

# data[].attributes.attachment_link.description
The use of arsenic (As) contaminated groundwater for irrigation of crops has resulted in elevated concentrations of arsenic in agricultural soils in Bangladesh, West Bengal (India), and elsewhere. Paddy rice (Oryza sativa L.) is the main agricultural crop grown in the arsenic-affected areas of Bangladesh.

# data[].attributes.attachment_link.title
Physiological evaluation of responses of rice (Oryza sativa L.) to water deficit

# data[].attributes.attachment_link.description
In view of the need to increase rice yields in rainfed areas, an experiment was done to (1) determine physiological traits that contribute to tolerance for water deficit at the seedling stage, (2) identify quick but reliable indices for selection for tolerance for water deficit, and (3) characterize rice cultivars

# data[].attributes.attachment_link.title
Non-destructive Quality Analysis of Kamod Oryza Sativa SSP Indica (Indian Rice) Using Machine Learning Technique

# data[].attributes.attachment_link.description
Rice is one of the most important cereal grains. The paper presents a solution for quality evaluation and grading of Krishna Kamod rice using image processing and soft computing technique. In this paper basic problem of rice industry for quality assessment is defined which is traditionally done manually by human inspector.

# data[].attributes.attachment_link.title
Changes in Grain Size and Grain Storage Protein of Rice (Oryza Sativa L.) in Response to Elevated UV-B Radiation under Outdoor Conditions

# data[].attributes.attachment_link.description
Variation in growth, grain size and grain storage protein content of rice (Oryza sativa L.) in response to elevated UV-B radiation under sunlight was examined in a cool rice-growing region of Miyagi Prefecture, Japan, in 1999, 2001 and 2002. Tiller number, dry mass, panicle number, grain yield and grain size significantly decreased under elevated UV-B radiation in 2001 and 2002.

# data[].attributes.attachment_link.title
Effects of Inorganic and Organic Nitrogen Nutrients on Drought Resistance of Rice (Oryza sativa L.)

# data[].attributes.attachment_link.description
Using hydroponic culture method, the effects of inorganic and organic nitrogen nutrients on drought resistance of rice (Oryza sativa L.) grown under drought stressful conditions induced by polyethylene glycol (PEG) were studied. The results were as followed, drought stress made leaf water potential and root vigor decreased, in addition, the activities of SOD, POD and CAT enzyme were restrained by drought.