Rational design of high-yield and superior-qualityriceDali Zeng1†, Zhixi Tian2†, Yuchun Rao1, Guojun Dong1, Yaolong Yang1, Lichao Huang1, Yujia Leng1,Jie Xu1, Chuan Sun1, Guangheng Zhang1, Jiang Hu1, Li Zhu1, Zhenyu Gao1, Xingming Hu1,Longbiao Guo1, Guosheng Xiong3, Yonghong Wang4, Jiayang Li4* and Qian Qian1,3*

Rice (Oryza sativa L.) is a staple food for more than half of theworld’s population. To meet the ever-increasing demand forfood, because of population growth and improved living stan-dards, world rice production needs to double by 20301. Thedevelopment of new elite rice varieties with high yield andsuperior quality is challenging for traditional breedingapproaches, and new strategies need to be developed. Here,we report the successful development of new elite varietiesby pyramiding major genes that significantly contribute tograin quality and yield from three parents over five years.The new varieties exhibit higher yield potential and bettergrain quality than their parental varieties and the China’sleading super-hybrid rice, Liang-you-pai-jiu (LYP9 or Pei-ai64S/93-11). Our results demonstrate that rational design is apowerful strategy for meeting the challenges of future cropbreeding, particularly in pyramiding multiple complex traits.

Rice (Oryza sativa L.), one of the most important staple cereals,feeds over 60% of China’s population and contributes nearly 40% ofthe nation’s total calorie intake2. A continuous increase in rice pro-duction is required to meet the demands of increasing populationand food consumption3. High-yield indica rice and its derivedhybrid rice varieties have played an essential role in innovatingnew rice varieties4. However, because of their poor eating andcooking quality (ECQ), most Chinese customers find these rice var-ieties undesirable5,6 and are rejected during commercial extension,even though they have an extremely high yield potential.Therefore, it is a priority for rice breeders to develop ‘super-rice’varieties that have both high yield and superior quality7,8.

Enormous effort has been devoted for the development of super-rice over the past several decades; however, the breeding process hasnot advanced as rapidly as expected, and the achievements have notmet consumer demand9. This outcome has primarily occurredbecause most yield- or quality-related traits are quantitative.Traditional breeding alone is ineffective in simultaneously improv-ing multiple quantitative complex traits because of low heritability,genotype–environment interaction and linkage drag8. Furthermore,abundant work on marker-assisted selection (MAS) has been per-formed to improve these traits, but successful cases were mainlyfrom resistance to biotic or abiotic stresses and were rare incomplex traits10–19. The breeding of super-rice with high yield andsuperior quality is a considerable challenge for rice breedersbecause yield and quality are usually negatively related. Recently, arational design approach, which is based on the extensive accumu-lated knowledge about the genes that regulate important agronomic

traits, has been reproposed to increase the accuracy and effectivenessof selection and to shorten the time required for pyramidingmultiple desirable traits20. In this study, we prove this concept byexploring the breeding of high-yield, superior-quality super-rice.

Rice yield is an extremely complex trait that is determined bymultiple components, wherein each component is a typical quanti-tative trait21. To date, several hundred quantitative trait loci(QTLs) for rice yield have been identified. However, only a few ofthese QTLs have been clearly characterized18,19,21; unlike yield, theregulating network that determines grain quality has been wellcharacterized18,22. Therefore, breeding high-yield superior-qualityelite varieties by increasing the grain quality of high-yield, poor-quality varieties will be much easier than the opposite practice.Teqing, a well-known super-high-yield indica variety23, wasdeveloped in China in 1984, and its hybrid, Liang-You-Te-Qing(Pei-ai 64S/Teqing), displayed a recorded high yield potentialup to 17.11 tons ha−1, which is substantially higher than that ofother hybrid or conventional varieties23,24 (Supplementary Fig. 1a).Teqing was widely cultivated in China in the 1980s, but its cultiva-tion area quickly decreased (Supplementary Fig. 1b) because of itspoor grain quality25. If the grain quality of Teqing can be improved,its rice production is expected to rebound considerably. Therefore,we decided to develop super-rice varieties with both high yieldand superior quality by crossing Teqing with both Nipponbare(NPB), which is a variety with superior ECQ but a low yield, and93-11, which is a variety with excellent appearance quality and isthe parental line for the hybrid Pei-ai 64S/93-11 (also called LYP9)that was widely cultivated during the last decade because of itsmoderately high yield and grain quality26 (Supplementary Fig. 1c).

Starch synthesis-related genes (SSRGs) play important roles incontrolling ECQ in rice22,27; GS3 and qSW5 are two major QTLsthat control grain shape and grain weight28,29. Genotype identifi-cation revealed that 21 of these genes, which are distributed acrossdifferent chromosomes (Fig. 1a and Supplementary Table 1), exhib-ited polymorphism between Teqing and NPB or 93-11 (Fig. 1b).These genes were determined as the target genes for introducinginto Teqing from NPB or 93-11. In addition to the genes controllinggrain quality, the characterized major genes related to yield werealso considered in the rational design. Through genotyping, sevenyield-related genes (Fig. 1a and Supplementary Table 1) werefound to exhibit polymorphisms between Teqing and NPB(Fig. 1b): Gn1a and SCM2 control grain number30,31; SCM2 isuseful for improving lodging resistance31; TAC1 and sd1 determineplant architecture32,33; the loss-of-function Hd1 increases the

1State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China. 2StateKey Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,China. 3Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China. 4State Key Laboratory of PlantGenomics and National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,China. †These authors contributed equally to this work. *e-mail: qianqian188@hotmail.com; jyli@genetics.ac.cn

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biomass of most indica varieties under short-day conditions34,35;and Ghd7 and Ghd8 regulate grain yield, heading date and plantheight36,37. Interestingly, both Teqing and 93-11 contained thesame genotypes for these genes (Fig. 1b), but Teqing displayed ahigher yield potential than 93-11 in hybrid breeding (SupplementaryFig. 1a), suggesting that unidentified loci exist that regulate grainyield in Teqing. Therefore, to achieve a yield potential as high as thatof Teqing and quality equivalent to that of NPB or 93-11, it is essentialto retain as much as of the Teqing genetic background as possible whilepyramiding the designed genes in the rational design lines.

We successfully developed the super-rice using rational designover five years. First, we separately introduced the beneficialalleles that control the fine ECQ traits from NPB and the allelesthat govern good appearance quality from 93-11 into Teqing; then,we combined these beneficial alleles by crossing (SupplementaryFig. 2). In detail, Teqing was crossed with NPB and 93-11 separatelyin 2009. Then, the F1 plants were backcrossed with Teqing inHangzhou in the summer of 2009. The BC1F1 seeds were sowedin Hainan in the spring of 2010. From this generation, we screenedfor the genotypes at each rational design step, and the linescarrying the SSRGs from NPB were identified to backcross withTeqing to select lines that contained both the favourable ECQand the high-yield genes in the Teqing background. Similarly, wedeveloped lines that carried the good appearance quality genes,GS3 and qSW5, from 93-11 in the Teqing background. Afterfour successive backcrosses, the lines carrying ECQ and good

appearance quality genes were further crossed in the BC4F1 andBC5F1 pools, respectively, and then selfed to generate homozygotes.Finally, three rational design lines that pyramid the desired traits(RD1, RD2 and RD3) were obtained (Supplementary Figs 3–5).All three lines contained GS3 and qSW5 from 93-11 and thetwo major ECQ genes (Wx and ALK) from NPB but exhibitedslight differences in certain minor ECQ genes (see Fig. 1c andSupplementary Figs 3–5).

To determine whether the rational design rice achieved thedesired characteristics, we first compared their grain appearancequality to that of their parents (Fig. 2a–d and SupplementaryTable 2). The results demonstrated that, similar to 93-11, thethree rational design lines all exhibited better grain appearancethan Teqing and NPB. The improved appearance included signifi-cantly longer milled grain length (Fig. 2b), slender grain shape(length-to-width ratio) (Fig. 2c) and a significantly decreased per-centage of chalky kernels (Fig. 2d). Then, we compared the ECQof the rational design rice to that of their parents (see Fig. 2e–hand Supplementary Table 2). ECQ can be characterized by threemain physicochemical properties: amylose content, gel consistencyand gelatinization temperature22. In addition, the taste and palat-ability score (called the taste index in this study) is considered tobe another important factor for the ECQ of cooked rice38,39. NPBand 93-11 both exhibited significantly better ECQ than Teqingfor the three physicochemical properties and the taste index, andNPB had a higher taste index than 93-11 (Fig. 2e–h). Comparingthe ECQs of the rational design rice to that of their parents revealedthat the three rational design lines exhibited ECQ properties equiv-alent to those of NPB. For instance, they showed significantlydecreased amylose content values (Fig. 2e), significantly improvedalkali spreading values (ASVs, the index for the gelatinization temp-erature) (Fig. 2f ) and significantly increased gel consistency levels(Fig. 2g) compared with Teqing. Moreover, their taste indiceswere significantly higher than that of Teqing and even surpassedthat of 93-11 (Fig. 2h). In summary, the rational design ricepyramided the good appearance from 93-11 and the superiorquality from NPB and showed significantly improved grainquality compared with Teqing.

We then investigated the yield-related performance of the rationaldesign lines and their parents (Fig. 3a–c and Supplementary Table 3).Overall, Teqing exhibited slightly better yield performance than 93-11and significantly better performance than NPB (Fig. 3). The com-parison revealed that the grain number per panicle (GNPP) valueof the RD2 line was similar to that of Teqing, whereas the RD1and RD3 lines exhibited slightly less GNPP values than that ofTeqing, similar to that of 93-11 and much higher than that ofNPB (Fig. 3a,d). Nevertheless, the rational design lines showedhigher kilo-grain weights than Teqing (Supplementary Table 3),which may result from their longer grain lengths (Fig. 2g). Theculm diameter of the fourth internode (DFI), which is an importantindex for the lodging resistance in high-yield rice31, was similar forthe rational design lines and both Teqing and 93-11 and was signifi-cantly larger than that of NPB (Fig. 3b,e). No significant differencesin the tiller numbers were detected among the rational design linesand their parents (Fig. 3c,f ). Furthermore, we investigated the grainyield in a field experiment. The results demonstrated that althoughthe rational design lines, Teqing and 93-11 displayed similar grainyield values (Fig. 3g and Supplementary Table 3), the rationaldesign lines and Teqing exhibited a higher rate of dry matteraccumulation than 93-11 (Fig. 3h and Supplementary Table 3).Therefore, our investigations determined that the rational designlines mostly maintained the high-yield characteristic of Teqing.However, the three rational design lines showed slight differencesin their yield performances (Supplementary Table 4), suggestingthat these lines may have different combinations of minor genesin different genomic regions although they shared the same seven

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Figure 1 | Gene and marker survey for the rational design in this study.a, The distribution of designed genes on the chromosomes. Solid triangles,genes related to starch synthesis; solid squares, genes related to grain yield;solid circles, genes related to grain appearance; pink, favourable alleles fromNPB; blue, favourable alleles from 93-11; lavender, favourable alleles fromTeqing. The position of targeted genes on chromosomes refer to the linkagemap released by IRGSP (International Rice Genome Sequencing Project) andRAP-DB (Rice Annotation Project Database). Detailed information about thegenes can be found in Supplementary Table 1. b, Polymorphism of designedmarkers among NPB, 93-11 and Teqing, where N, 9 and T denote NPB, 93-11and Teqing, respectively. c, Genotype of the target genes in RD1, RD2 andRD3; the graphical genotypes are shown in Supplementary Figs 3, 4 and 5,respectively. Red, major ECQ genes; blue, minor ECQ genes.

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major yield-related genes (Supplementary Figs 3–5). The resultsfurther demonstrate the existence of unidentified loci or regulatoryelements for yield in Teqing.

Hybrid rice plays an important role in rice production in China.Pei-ai 64S is a photoperiod-thermosensitive genic male sterile linethat has been widely used as a maternal parent for two-line hybridrice breeding in China. LYP9, which is a hybrid rice that was devel-oped using Pei-ai 64S as the maternal parent, was a pioneer hybridrice40,41. To explore the application of the rational design rice in

hybrid rice breeding, we crossed them with Pei-ai 64S.Compared with LYP9, the yield potentials of the hybrid ricederived from the rational design lines and Pei-ai 64 s (Pei-ai 64S/RD1,Pei-ai 64S/RD2 and Pei-ai 64S/RD3) were increased 15.4, 13.1and 12.7%, respectively (Supplementary Fig. 6a and SupplementaryTable 5). Their taste indices were also higher than that of LYP9(Supplementary Fig. 6b and Supplementary Table 5). Nevertheless,the taste index of each hybrid was lower than that of the correspond-ing rational design line (Fig. 2h and Supplementary Fig. 6b), which

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Figure 3 | The performance of rational design lines and their parents. a, Main panicles. Scale bar, 10 cm. b, Cross-sections of the fourth internode of themain culm. Scale bar, 2 mm. c, Plant morphology. Scale bar, 20 cm. In a–c, the samples are, from left to right, NPB, 93-11, Teqing, RD1, RD2 and RD3.d, Grain number per panicle (n = 50). e, The DFI (n = 50). f, Tiller number (n = 50). g, Grain yield (n = 500). h, Grain yield per day (days from heading toharvest). In d–h, the bars indicate the standard error of the mean.

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might be caused by the lower quality of the sterile maternal parentPei-ai 64S (Supplementary Fig. 6b). Genotype investigationrevealed that only 7 of the 19 detected SSRGs in Pei-ai 64S exhib-ited the same haplotypes as that in NPB. In particular, the twomajor ECQ genes, Wx and ALK, were the same in both Pei-ai64S and Teqing (Supplementary Fig. 7). Therefore, to furtherimprove the ECQ quality of hybrid rice in the future, we need toimprove the maternal parent of Pei-ai 64S by changing theirSSRGs combinations.

The eternal goal of crop breeding is to develop super-varieties byassembling multiple desirable traits, such as high yield, superiorquality, pest resistance and tolerance to environmental stresses,into a single variety. Rational design was proposed as a potentiallypowerful and efficient approach for super-variety breeding16,19 toconquer the low efficiency of traditional breeding by phenotypicselection8,15. In practice, rational design will essentially rely onprecise genetic dissection of agronomic traits and high-resolutionchromosome haplotyping16,20. Because the networks underlyingcomplex traits has been poorly understood, the application ofrational design in improving complex traits has moved forwardslowly in the past several decades. Recently, rapid advancementsin functional genomics makes the implementation of rationaldesign to be possible15,18. However, the practical strategy to carryout the rational design is critical and is still a challenge. Becausethe network controlling yield is much more complicated than thatcontrolling grain quality, in this study, we correctly chose toimprove the grain quality of the Teqing variety that was high yieldbut poor quality. This strategy facilitated us to successfullydevelop super-rice lines with both high yield and superior qualitywithin only five years, a period much shorter than traditionalbreeding practice.

Although our study demonstrated that rational design is a power-ful tool to improve complex traits and for breeding super-varieties,much more research is needed. For instance, if more parents (bothfor high yield and high grain quality) are selected, the strategy willbe more complex and ‘trade-offs’ of different traits will need to becarefully considered. Another aspect that needs to be consideredis balancing the selection population size and the number oftarget genes. In this study, because of the low genotyping efficiencyat present, not all of the SSRG alleles from the high-quality parentswere pyramided in the recurrent rational design lines, resulting ingrain quality of rational design lines that need to pyramid moredesirable alleles from their both parents. With the development offunctional genomics and high throughput genotyping and pheno-typing technologies, rational design will be a powerful breedingstrategy in the post-genome era.

MethodsField plot experiment. Field trials were raised in a rice paddy during the standardgrowing season at the experimental farm of China National Rice Research Institutein Hainan (110°00′ E, 18°31′ N) and Hangzhou (119°54′ E, 30°04′ N) in 2009–2014.Similar cultivation practices were performed each year. The cultivation methods forthe field plot experiment in 2014 are briefly described here. All the seeds were soakedin water at 30 °C in the dark for 2 days and were germinated at 35 °C for 12 h.The germinated seeds were then sown in a seedbed located in the paddy field.Five replications of each line and variety were arranged in a randomized blockdesign. The 25-day-old seedlings were transplanted into a plot that consisted of40 rows × 13 lines, and the spacing for each plant was approximately 20 cm × 20 cmin a grid pattern (approximately 12 m2 for each plot). The field plots weresupplemented with 100 kg nitrogen ha−1 (in the form of urea), 80 kg K2O ha−1

(in the form of KCl) and 70 kg P2O5 ha−1 (in the form of calcium superphosphate)

as a basal dressing. Then, the fields were supplemented with 60 kg nitrogen at thetillering stage and with 40 kg nitrogen and 80 kg K2O ha−1 at the heading stage.The field management was performed as described in a previous report42.

Measurements of the major agronomy traits. The measurements of the plantheight, tiller number, panicle length and other panicle traits were performed at30 days after heading. For all those traits, ten plants of each line or variety weresampled from each plot, and the main culm of each plant was chosen for trait

measurement. The DFIs of the main culms were measured using a vernier calliper.The number of fully filled grains per panicle and the total number of grains perpanicle were used to calculate the setting percentage. At maturity, the grain harvestedfrom 100 plants in each plot was naturally dried and used to estimate the grain yield.The grain weight was calculated based on 200 grains and converted to a kilo-grainweight. Five replications were performed for each trait.

Measurements of the grain quality. Evaluation of the grain width, grain length,length-to-width ratio and chalky kernels was performed according to the NationalStandards of the People’s Republic of China (NSPRC) (1999)43. Ten fully filledgrains were randomly selected to measure the grain length, grain width and grainthickness using a vernier calliper. The length-to-width ratio was calculated usinggrain length divided by grain width. One hundred fully filled grains were randomlyselected to determine chalky kernels using a chalkiness visualizer constructed at theChina National Rice Research Institute (NSPRC 1999)43. Approximately 150 g ofgrains was de-husked using a huller (SDL-A; CNRRI, Hangzhou, China) and milledusing a JMJ-100 rice miller (CNRRI, Hangzhou, China) for ECQ measurement. TheECQ was measured as previously described22. All traits were measured withfive replications.

Evaluation of taste and palatability in cooked rice. The assay of taste andpalatability in cooked rice was conducted on an STA1A rice taste analyser accordingto the methods reported by previously44 with slight modifications. Freshly harvestedraw rice was naturally dried to a final moisture content of 14.5% and then milled to90% of the initial weight using an auto-milling detector. The head rice was selectedfor taste evaluation on the STA1A rice taste analyser. Each 30 g sample was placedinto a special stainless steel tin and washed thoroughly with flowing water. Then,36 g of water was added into the tin to soak the rice for 30 min; next, the tin wastightly sealed and transferred to a steam rice cooker. Ten minutes after the cookerswitched from the ‘cook’ to the ‘warm’ setting, the cooked rice along the top, bottomand side inside the tin was discarded. Then, 7 g of cooked rice for the indica varietyor 8 g of cooked rice for the japonica variety from the middle of the tin wastransferred to a mould to form a cooked rice pie. This pie was used for evaluating thetaste and palatability using an STA1A rice taste analyser.

Development of markers. The markers with names starting with ‘RM’ were SSRmarkers and were selected from the Gramene database (http://archive.gramene.org/markers/). The markers with names starting with ‘STS’ were designed based on thegenomic DNA sequence differences among NPB, 93-11 and Teqing at the targetedgenes. Detailed information about the RM and STS markers is listed inSupplementary Tables 6 and 7, respectively.

Rational design breeding procedure. Teqing was selected as a recurrent parent;NPB and 93-11 were chosen as donor parents. The crossing, selecting andbackcrossing procedure was performed from 2009 to 2014, as shown inSupplementary Fig. 2. The materials developed from this study have been depositedin the Chinese Academy of Natural Sciences (CAAS) and International RiceResearch Institute (IRRI) Joint Laboratory under access numbers AGIR-10112,AGIR-10113 and AGIR-10114.

Statistical analysis. Statistical analysis was performed using SAS 9.0.

Data availability. All data generated or analysed during this study are included inthis published article and the Supplementary Information.

Received 21 December 2015; accepted 14 February 2017;published 20 March 2017

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AcknowledgementsThis work was supported by the National Natural Science Foundation of China (grantnos. 91535205, 91435105 and 31521064), the National Key Basic Research Program (grantno. 2013CBA014) and the ‘Strategic Priority Research Program’ of the Chinese Academy ofSciences (grant no. XDA08000000).

Author contributionsQ.Q., J.L. and Y.W. designed the project; D.Z. and Y.R. performed the experiments in thisstudy; D.Z., G.D., Y.Y., L.H. and Y.L. performed the molecular assistant selection; D.Z., C.S.and G.Z. contributed to measuring the grain ECQ; J.X., J.H., L.Z. and Z.G. evaluated thetaste and palatability of the cooked rice; Z.T. and G.X. were responsible for the developmentof the gene markers; Z.T., L.G. and X.H. performed the statistical analysis; and D.Z., Z.T.,Q.Q. and J.L. wrote the manuscript.

Additional informationSupplementary information is available for this paper.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to J.L. and Q.Q.

How to cite this article: Zeng, D. et al. Rational design of high-yield and superior-quality rice.Nat. Plants 3, 17031 (2017).

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Competing interestsThe authors declare no competing financial interests.

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