Rational Design of Grain Size to Improve Rice Yield and Quality

Tao Yajun, Wang Jun, Xu Yang, Wang Fangquan, Li Wenqi, Jiang Yanjie, Chen Zhihui, Fan Fangjun, Zhu Jianping, Li Xia, Yang Jie

Letter

Rational Design of Grain Size to Improve Rice Yield and Quality

Tao Yajun1, 2, Wang Jun1, 2, Xu Yang1, 2, Wang Fangquan1, 2, Li Wenqi1, 2, Jiang Yanjie1, 2, Chen Zhihui1, 2, Fan Fangjun1, 2, Zhu Jianping1, 2, Li Xia1, 2, Yang Jie1, 2

(Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Branch of Chinese National Center for RiceImprovement, Nanjing 210014, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China)

Grain size, determined by grain length, grain width and grain thickness, is associated with grain yield and quality. Many genes controlling grain size were cloned and their related regulatory mechanisms were clearly clarified. However, whether these genes can be directly introduced intorice for grain size improvement is unknown. We edited,andin the background ofrice JXY1 and created a series of single, double and triple mutants by the CRISPR/Cas9 genome editing system. Grain quality and yield component analysis showed that all the mutants can significantly increase the grain length of JXY1, while mutatedhad a negative effect on grain weight and yield.anddouble mutants can improve grain yield and eating and cooking quality (ECQ). Therefore, we also introducedandinto a breeding line, NJ0378, which is fragrance rice with high chalkiness. NJ0378-showed a decreased chalkiness degree and increased yield and ECQ. These results suggested that we can generate combinational genes for grain yield and quality improvement in rice.

Rice is one of the most important foods and is consumed by half of the world population (Cheng et al, 2007). With increasing population and improving living standards, rice breeders have become increasingly concentrated on the balance of grain quality and food production. Grain size, determined by grain length, grain width and grain thickness, is associated with grain yield and quality. The grain size of rice varies greatly. Grain length can be as short as 6 mm or longer than 15 mm, and 1000-grain weight (TGW) can vary from 10 to 70 g (Huang et al, 2013). To date, several genes and majorQTLs controlling grain size have been cloned and characterized, forming complex regulatory networks of seed growth, which include G-protein signaling (,and) (Takano-Kai et al, 2009; Miao et al, 2019; Tao et al, 2020), mitogen-activated protein kinase (MAPK) signaling () (Xu et al, 2018), ubiquitin-proteasome pathway () (Hao et al, 2021), and transcriptional regulatory factors (and) (Wang et al, 2012, 2015). Among them,, encoding an unknown protein, negatively regulates grain length and appearance quality in rice (Zhao et al, 2018). Additionally, introducinginto rice varieties significantly improves grain quality (Zhao et al, 2018). A major grain length QTL,, plays vital roles in regulating grain length, filling and weight (Qi et al, 2012; Zhang et al, 2012). The TGW genecontrols indole-3-acetic acid supply and consequently influences grain weight and length (Ishimaru et al, 2013). However, these genes are relatively low in distribution inrice. Conversely, thegene, which encodes a G protein γ subunit, is widely used inrice in China (Huang et al, 2009; Zhou et al, 2009). Thegene has positive effects on population characteristics and results in high-yieldingrice (Huang et al, 2009; Liu et al, 2021). However, thegene also leads to short grain length and low TGW (Liu et al, 2018; Sun et al, 2018; Li et al, 2019). Therefore, breeding long-grainrice requires the introduction of elite grain length genes, which can be incorporated with.

In the present study, we edited,andin the background ofrice JXY1 and NJ0378, and created a series of single, double and triple mutants using the CRISPR/Cas9 technology. Furthermore, the grain yield and quality of each line were compared. The objectives of this study included: (1) evaluating the breeding values of,andin yield and quality genetic improvement of erect panicle rice, (2) creating newrice germplasms, and (3) providing a theoretical basis for breeding long-grain shaperice.

First, we analyzed the genotypes of JXY1 at the,andloci, which are widely distributed inrice. The results showed that JXY1 had the ‘Nipponbare’ type at all the abovementioned loci. Therefore, we selected,andas candidate genes for breeding long-grain shaperice. Additionally, CRISPR/Cas9 can function as a useful tool for quickly pyramiding these genes. According to the principle of target design, we constructed a triple mutant vector (Fig. S1) and transformed it into JXY1 by themediated method. After sequencing, a series of single, double, triple and marker-free T3generation lines were selected for further analysis (Fig. S1).

Fig. 1. Effects of mutant,andon grain shape and quality.

A, Plant morphologies of JXY1 and mutants JXY1#1?JXY1#8. Scale bars, 10 cm. B, Grain shapes of JXY1 and mutants JXY1#1?JXY1#8. The gray square indicates the homozygous Nipponbare genotype, the blue square indicates the homozygous long-grain genotype, and the green square indicates the heterozygous long-grain genotype. Scale bars, 1 cm. C, Rapid Visco Analyzer spectra of JXY1 and mutants JXY1#1?JXY1#8. D?F, Comparisons of grain length (D), grain width (E) and grain length to width ratio (F) in JXY1 and mutants JXY1#1?JXY1#8. Data are Mean ± SD (= 7). Different lowercase letters above the bars denote significant differences (< 0.05).

Theanddouble mutants (JXY1#1?JXY1#3) andorsingle mutants (JXY1#4 and JXY1#5) had small differences from JXY1 when comparing the plant architecture (Fig. 1-A and Table S1). However, when the three genes were mutated (JXY1#6?JXY1#8), the plant heights and tiller numbersper plant were slightly reduced compared with JXY1 (Fig. 1-A and Table S1). Yield-related traits were determined at the maturity stage. The grain length of JXY1 was 6.83 mm, and the grain width was 3.23 mm, showing a short and round grain shape (Fig. 1-B, -D and -E). The grain lengths of JXY1#1?JXY1#3 were approximately 7.66?7.75 mm, increase of 12.13%?13.47%, suggesting that simultaneously mutatedandcan significantly increase grain length. We also observed the grain shape of single mutants, including JXY1#4 and JXY1#5, and the results showed that JXY1#4-increased grain length by 9.54% but had no influence on grain width. The grain length of JXY1#5-was 7.55 mm, increasing 10.50% compared with JXY1 (Fig. 1-B and-D). We successfully obtained several triple mutants (JXY1#6?JXY1#8). JXY1#6 had homozygousand, while thelocus was heterozygous (Figs. 1-B and S1). The genotype of both JXY1#7 and JXY1#8 was. Statistical analysis showed that the grain length of JXY1#6 was not significantly different from that of JXY1#1, while the average grain lengths of JXY1#7 and JXY1#8 were longer than those of the double mutants (Fig. 1-B and-D).

The average grain length to width ratio of these mutants was 2.65, which was significantly higher than that of JXY1 (2.11) (Fig. 1-F). Grain size plays a vital role in regulating grain weight and subsequently affects grain yield. Therefore, we also compared the TGW and grain yield per plant (GYPP) of the mutants with those of JXY1 (Table S1). The results showed that-knockout can significantly increase grain weight in the background of JXY1 or JXY1-. However, when the three genes were mutated, the average TGW of JXY1#6?JXY1#8 was 22.27 g, which was significantly decreased by 1.63% relative to that of JXY1 (Table S1). Consequently, the average GYPP of JXY1#1?JXY1#4 was 30.66 g, significantly increased by 16.93% compared with JXY1, and the average GYPP of JXY1#6?JXY1#8 was 20.58 g, significantly decreased by 21.51% (Table S1).

Rice varieties with moderately low amylose content (～10%), that is, ‘soft rice’, have become increasingly popular commercially in China (Li and Gilbert, 2018). JXY1 is also classified as a soft rice variety, with an amylose content of 11.0%. We further examined the ECQs of JXY1 and its related mutants. A Rapid Visco Analyzer (RVA) assay was performed to evaluate the texture of cooked rice (Fig. 1-C and Table S2). The results showed that the values for peak viscosity (PKV) ofanddouble mutants JXY1#1 and JXY1#3 were elevated, while there was no change in JXY1#4, and slightly decreased in JXY1#5?JXY1#8 (Fig. 1-C and Table S2). The breakdown viscosity (BDV) values of all theandsingle or double mutants were increased, but decreased in JXY1#7 and JXY1#8 (Fig. 1-C and Table S2). Most importantly, the setback viscosity (SBV) was obviously decreased in JXY1#1?JXY1#5 and increased in JXY1#7 and JXY1#8, suggesting that the ECQs of the rice from JXY1#1?JXY1#5 were improved, while those from JXY1#7 and JXY1#8 were somewhat decreased (Fig. 1-C and Table S2). Taken together, these results suggested that we can generate combinational genes for grain yield and quality improvement in rice.

Fig. 2. Effects of mutantandon grain shape and quality in NJ0378.

A, Plant morphologies of NJ0378 and mutants NJ0378#1?NJ0378#3. Scale bars, 10 cm.B and C, Grain yield per plant (B) and white rice appearance (C) of NJ0378 and mutants NJ0378#1?NJ0378#3. Scale bars, 2 cm. D, Scanning electron micrographs of endosperms of mature seeds. Scale bars, 10 μm. E, Grain lengths and widths of NJ0378 and mutants NJ0378#1?NJ0378#3. Scale bars, 1 cm. F?I, Comparisons of 1000-grain weight (F), grain yield per plant (G), chalkiness rate (H) and chalkiness degree (I) in NJ0378 and mutants NJ0378#1?NJ0378#3. Data are Mean ± SD (in F and G,= 7; and in H and I,= 3). Different lowercase letters above the bars denote significant differences (< 0.05). J, Rapid Visco Analyzer spectra of NJ0378 and mutants NJ0378#1?NJ0378#3.

Based on these results, we introduced mutantandinto a breeding line, NJ0378, which is a fragrance rice with high chalkiness (Fig. 2-A to -D). Compared with NJ0378, the average grain length of transgenic lines NJ0378#1?NJ0378#3 was 8.10 mm, with an increase of 6.16%, and the average grain width was 2.88 mm, decreased by 15.54% (Fig. 2-E and Table S3). Consequently, the grain length to width ratios of NJ0378#1?NJ0378#3 were obviously decreased, showing a slender grain shape. The average TGW and GYPP of NJ0378#1?NJ0378#3 were increased by 4.41% and 19.48%, respectively (Fig. 2-B, -F and-G). We also investigated the grain chalky characteristics of NJ0378#1?NJ0378#3. The chalkiness rate and degree ofNJ0378 were 95.56% and 32.97%, respectively, showing a poor appearance quality (Fig. 2-C, -H and-I). The lowest chalkiness rate and degree were observed in NJ0378#1, with values of 58.38% and 11.38%, respectively. The two indices of NJ0378#2 and NJ0378#3 were nearly the same, with average values of 66.21% and 16.29%, respectively. Then, we compared the cross-sectioned endosperm and the morphology of starch granules of NJ0378#1?NJ0378#3 with those of wild type (NJ0378) using a scanning electron microscope. The cross-sections of NJ0378 were irregular, and their starch granules were round spheres, irregularly arranged and loose, while the cross-sections of the edited lines were neat and smooth, and their starch granules were uniform polyhedron, arranged regularly and densely (Fig. 2-D). Additionally, the PKV and BDV values of NJ0378#1?NJ0378#3 were all elevated, and the SBV values of NJ0378#1?NJ0378#3 were obviously decreasedcompared with those of NJ0378 (Fig. 2-J and Table S4). Finally, we investigated potential off-target mutations in these mutants by analyzing all the predicted off-target sites (http://skl.scau.edu.cn/) through amplifying and sequencing of the potential off-targets. For all the 11 individuals, we did not find mutation in any of the 22 potential off-target sites (Table S5), suggesting the specificity of the sgRNA used in this study. Therefore, pyramiding the long-grain type alleles ofandshould be an effective way to improverice appearance and eating quality and grain yield.

In summary, our results demonstrated that gene editing can provide a convenient and high-efficiency tool for rice genetic improvement.,andare all cloned as grain shape regulation genes, but they have not been proven whether they can be applied inrice, especially in the middle and lower reaches of the Yangtze River in China. Our results suggested thatandcan be directly introduced into soft and commonrice for quality and yield improvement. The germplasms created in this study provide a reference for breeding high-quality and -yieldrice varieties.

ACKNOWLEDGEMENTS

This study was funded by the National Natural Science Foundation of China (Grant No. 31901525), the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20190255), the International Cooperation and Exchange of the National Natural Science Foundation of China (Grant No. 31861143011), the Revitalization of Jiangsu Seed Industry in China (Grant No. JBGS[2021]039).We thank all our colleagues at the Jiangsu Academy of Agricultural Sciences and Yangzhou University, China for reading and participating in discussion relating to the preparation of this manuscript.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Vector construction and genotype of each line.

Table S1. Comparisons of yield traits in JXY1 and mutants.

Table S2. Rapid Visco Analyzer profile characteristics of JXY1 and mutants.

Table S3. Comparisons of yield traits in NJ0378 and mutants.

Table S4. Rapid Visco Analyzer profile characteristics of NJ0378 and mutants.

Table S5. Analysis of potential off-target sites.

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Yang Jie (yangjie168@aliyun.com)

24 February 2022;

13 April 2022

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http://dx.doi.org/10.1016/j.rsci.2022.04.003