LI Qianlong, FENG Qi, WANG Heqin, KANG Yunhai, ZHANG Conghe,, DU Ming, ZHANG Yunhu,WANG Hui,, CHEN Jinjie, HAN Bin, FANG Yu,,4, WANG Ahong

(1Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences,Shanghai 200032, China; 2Shanghai ZKW Molecular Breeding Technology Co. Ltd, Shanghai 200234, China; 3Win-all Hi-tech Seed Co. Ltd / Key Laboratory for New Variety Creative of Hybrid Rice, Ministry of Agriculture and Rural Affairs, Hefei 230088, China;4Institute of Crop Science & Institute of Bioinformatics, Zhejiang University, Hangzhou 310058, China)

Abstract: Germplasm resource innovation is a crucial factor for cultivar development, particularly within the context of hybrid rice breeding based on the three-line system. Quan 9311A, a cytoplasmic male sterile (CMS) line, has been successfully cultivated using rice restoration materials and extensively employed as a female parent in hybrid breeding program in China. This line was developed by crossing the CMS line Zhong 9A with a two-line restorer line 93-11, with the intention of eliminating the restoring ability of 93-11 while retaining the sterility gene WA352c from Zhong 9A. Quan 9311A effectively amalgamates the most favorable agronomic traits from both parental lines. In this study, the relationship between phenotypic characteristics and the known functional genes of Quan 9311A were analyzed using the rice genome navigation technology based on whole-genome sequencing. The findings revealed that Quan 9311A harbors multiple superior alleles from both 93-11 and Zhong 9A, providing exceptional agronomic traits that are unavailable in earlier CMS lines. Despite the removal of the fertility restorer gene Rf3 from 93-11, numerous chromosomal segments from 93-11 persist in the Quan 9311A genome.Furthermore, the hybrid rice Quanyousimiao (QYSM) and the restorer line Wushansimiao (WSSM) were used as examples to illustrate the important role of Quan 9311A as the female parent in heterosis. It was found that QYSM carries a great number of superior alleles, which accounts for its high grain yield and wide adaptability. These insights not only advanced the utilization of hybrid rice pairing groups but also provided guidance for future breeding endeavors. The study introduced innovative concepts to further integrate genomics with traditional breeding techniques. Ultimately, Quan 9311A signified a significant milestone in rice breeding technology, opening up novel avenues for hybrid rice development.

Key words: breeding; hybrid rice; phenotype; quantitative trait nucleotide locus; rice genome navigation system; whole-genome sequencing

Advances in plant breeding technology over the past few decades have revolutionized crop breeding, particularly for rice, the world’s primary staple food crop (Hickey et al, 2019; Varshney et al, 2021). Since the 1970s, a vast number of superior hybrid rice varieties have been bred in China using two-line and three-line hybrid systems, which have contributed to ensuring food security both in China and globally (Cheng et al,2007; Li and Yuan, 2010; Qian et al, 2016).

The two-line hybrid system primarily consists of an environment-sensitive genic male sterile (EGMS) line and a fertility restorer line. Under short-day conditions or when the ambient temperature is below approximately 24 °C, the EGMS line can self-fertilize (Yuan, 1994;Peng et al, 2006, 2010). Research indicates that EGMS mainly results from mutations in thetms5orpms3gene (Ding et al, 2012; Zhou et al, 2014).Commonindicarice can serve as a restorer line in the two-line hybrid system. The three-line hybrid system consists of a cytoplasmic male sterile (CMS) line, a sterility maintainer line and a fertility restorer line.The CMS line’s seed production depends on fertile pollen supplied by the maintainer line. The main CMS lines used in commercial hybrid seed production in China are wild abortive (WA) type, Honglian (HL)type, and Boro II (BT) type, which are classified based on various sterility genes located in the mitochondrial genome (Luan et al, 2013; Gu et al, 2021). The mitochondrial geneWA352ccauses WA-CMS lines(Luo et al, 2013), while genesorf79andorfH79are responsible for BT-CMS and HL-CMS, respectively(Wang et al, 2006; Wang K et al, 2013). Different sterility genes necessitate distinct restorer genes for fertility restoration. The fertility of WA-CMS lines is mainly governed by the dominant genes,Rf3andRf4,located on chromosomes 1 and 10, respectively (Qi et al,2008; Cai et al, 2013; Tang et al, 2014).Rf4has a more potent genetic effect, withRf3playing a synergistic role (Sattari et al, 2008). The BT-CMS line’s fertility restoration is controlled byRf1, which encompasses two closely linked restorer genes,Rf1aandRf1b(Wang et al, 2006). HL-CMS lines are restored by genesRf5(also known asRf1a) andRf6(Huang et al, 2003; Huang W C et al, 2015; Zhang et al,2017). Additionally, the restorer geneRf2restores the fertility of Lead Rice-type CMS (L-CMS), which is discovered in theindicarice variety ‘Lead Rice’(Itabashi et al, 2011).

The field of rice genomics and functional gene studies has progressed rapidly since the sequencing of the rice genome (Sasaki, 1998; Sasaki and Burr, 2000;Feng et al, 2002). Over the subsequent two decades,there have been significant advances in rice functional genomics research. Several functional genes related to essential agronomic traits in rice have been identified and utilized in rice breeding. These include genes such asGn1a(rice yield),Waxy(grain quality),Sd1(plant architecture),Pi2/Pi9(biotic stress),SKC1(abiotic stress), andNRT1.1B(nutrient-use efficiency) (Bai et al,2018; Guo et al, 2019; Chen et al, 2022). As quantitative genetics, computational biology, and genomics continue to develop, the genetic mechanisms of two-line, threeline, and inter-subspecies heterosis in rice are becoming clearer. Heterosis in rice is directly related to the presence of positively correlated dominant or semidominant genetic loci in either parent (Huang X H et al,2015, 2016). Based on these insights, a method for molecular design breeding of rice varieties was established.Researchers have constructed a vital variation map of quantitative trait genes/pathogenic genes in rice, and developed the rice genome navigation program,RiceNavi (Wei et al, 2021).

In traditional rice breeding, breeders select sterile lines, restorer lines, and elite hybrid rice combinations based on field phenotypic investigation. RiceNavi primarily analyzes known functional genes associated with these field phenotypic traits. In this study, we employed RiceNavi to dissect the relationship between the phenotypic characteristics and known functional genes of the CMS line Quan 9311A. We aimed to reconstruct the genetic basis of Quan 9311A based on conventional breeding phenotypic investigations and to explore the genetics and genomics reasons for its successful breeding. We also considered the hybrid rice Quanyousimiao (QYSM) and the restorer line Wushansimiao (WSSM) as cases to emphasize the important role of Quan 9311A in heterosis. The main purpose of this study was to enhance the utilization and refinement of Quan 9311A. We also aimed to assist researchers engaged in the field of genomics to better comprehend the rice breeding process. Meanwhile, we hope our work encourages a more harmonious integration of genomics with traditional breeding, offering insights for future rice design breeding endeavors.

RESULTS

Phenotypic characterization of Quan 9311A

Quan 9311A is a CMS line bred from the cross of 93-11, Zhong 9A, and 222B. Notably, 93-11 and Zhong 9A served as the core germplasm for its improvement(Fig. 1-A). The detail breeding process is described in the Methods section, but is briefly summarized here:93-11 was selected as the male parent for sexual cross with the three-line maintainer lines Zhong 9B and 222B. Subsequently, desirable individual plants were selected and continually backcrossed with sterile individuals. After multiple generations, the new CMS line, Quan 9311A, was chosen (Fig. 1-B). The selected Quan 9311A incorporated dominant agronomic traits from both 93-11 and Zhong 9A, such as moderate plant height, appropriate heading date, moderate 1000-grain weight (TGW), increased tiller number, and grains that were more transparent and polished (Figs.1-C and S1; Table S1). The grain quality met the second-class standards of the Ministry of Agriculture and Rural Affairs of China (Wang H Q et al, 2013), but the grain number per panicle was slightly less than that of both parents. Its panicle length and stigma exertion rate were similar to those of Zhong 9A.

Fig. 1. Phenotypic characteristics and breeding process of Quan 9311A.

Sequencing and genome-wide single nucleotide polymorphism (SNP) analysis

We obtained sequencing data for five cultivars (Quan 9311A, 93-11, Zhong 9A, WSSM, and QYSM) with coverage ranging from 37× to 140× of the rice genome. For each cultivar, the Q30 value exceeded 94%. Subsequently, the next-generation sequencing data for each cultivar were compared with the reference genome ofOryza sativassp.japonicacv. Nipponbare(MSU 7.0), leading to the identification of 2 × 106SNPs between each cultivar and Nipponbare (Table S2). The specific SNPs of 93-11 and Zhong 9A were compared with those of Quan 9311A, and the SNP loci consistent with Quan 9311A were tallied (Table S3). There were 74.9% and 67.8% similar SNPs between 93-11 and Quan 9311A, and Zhong 9A and Quan 9311A, respectively. Although Quan 9311A retained the genetic background of 93-11 as per the identification threshold (genetic similarity ＜ 96%)specified in the plant variety multiple nucleotide polymorphism marker method in GB/T 38551-2020,China (China National Institute of Standardization,2020), it can be classified as a new creation of germplasm resources. Furthermore, the SNPs obtained from the comparison between 93-11 and Quan 9311A,as well as Zhong 9A and Quan 9311A, were plotted across the entire genome to illustrate the differences in SNPs between the two parents on various chromosome segments (Fig. 2). These results revealed that Quan 9311A and 93-11 shared large similarity fragments on chromosomes 2, 3, 6, 7, 9, and 12 (Fig. 2-A), while Quan 9311A and Zhong 9A exhibited substantial differences on multiple chromosomes or across the entire genome (Fig. 2-B).

Fig. 2. Simple sequence polymorphism (SNP) discrepancy between two parents across entire genome.

Functional locus analysis of Quan 9311A, 93-11, and Zhong 9A

The QTNpick module in the RiceNavi method (Fig.S2) was utilized to conduct a genome-wide ‘physical examination’ analysis. The variations of 348 quantitative trait nucleotide (QTN) loci across the entire genome were assessed, and gene loci harboring functional trait variations were identified (Table S4). We have compiled the most significant genes associated with crucial agronomic traits in Zhong 9A, 93-11, and Quan 9311A in Table 1. These results demonstrated that Quan 9311A not only inherited superior alleles from 93-11,such asOsMADS51,SSG6,Waxy,Hd1,DLT,BG2,Sdr4,GW8,Sub1A,STV11,andRf2, but also acquired several from Zhong 9A, namelyXa1,Ghd7,Xa3,D2,BOC1, andRf3. However, a few superior genes likeBph3,PTB1,NOG1, andBph29in 93-11 were lost during the breeding process (Table 1 and Fig. 3).Importantly, in 93-11, certain genes that are unfavorable with respect to desirable rice crop traits were not passed down to Quan 9311A. Examples include restorative function geneRf3, theD2gene responsible for plant height dwarfism and increased tiller angle,and theBOC1gene, which tends to cause callus browning. Instead, Quan 9311A inherited the favorable alleles of these genes from Zhong 9A.Additionally,Pi21in 93-11 represented an unfavorable allele but was still inherited by Quan 9311A. However,in terms of common superior alleles carried by both parents, Quan 9311A undoubtedly inherited all of them (Table S5). Consequently, Quan 9311A, as a CMS line, possesses a greater number of superior allele genes compared with Zhong 9A.

Causal analysis of incomplete restoration of 93-11 to three-line male sterile lines (except HL-CMS) based on functional loci

It is well known that 93-11, serving as the male parent,functions as a strong restorer line in the two-line hybrid breeding system for rice (http://www.ricedata.cn/variety/varis/600611.html). However, it does not achieve complete restoration in the vast majority of WA-CMS lines (except HL-CMS). Based on genome sequencing information, we discovered that the WA- CMS restorer generf3in 93-11 possessed a mutant genotype with restorative function, whileRf4carried a wild-type genotype lacking restorative ability. It suggested that the weak restorative ability of 93-11 in the WA-CMS line might stem from the absence of theRf4gene. In the case of the BT-CMS restorer geneRf1b, it was of the wild-type in 93-11 and lacked restorative function(theRf1agenotype remains unknown). Meanwhile,the restorer generf6of the HL-CMS line in 93-11 also possessed restorative ability, explaining why 93-11 was capable of restoring HL-CMS in practical applications. In summary, this genotype information elucidated why the hybrid rice combinations involving WA-CMS lines with 93-11 as the male parent typically exhibit a split genetic pattern between recovery and sterility, resulting in hybrids displaying a state of half-fertility and half-infertility.

Table 1. Summary of genes associated with crucial agronomic traits in Zhong 9A, 93-11, Quan 9311A, WSSM, and QYSM.

Removal of restorer genes and retention of sterility genes in 93-11 genetic background

Fig. 3. Superior alleles inherited by Quan 9311A from both parents 93-11 and Zhong 9A.

Conventional breeding of male sterile lines typically involves the ‘maintainer begets maintainer’ method,which entails crossing maintainer lines with one another and selecting an outstanding and genetically stable individual plant from multiple generations’ progeny to establish a new maintainer line. In the historical breeding practices of maintainer lines, it posed a significant challenge to develop maintainer lines with stable fertility from rice cultivars containing restorer genes. However, through phenotypic investigations,breeders identified that 93-11 could only serve as a fertility restorer in the two-line system, not in the three-line system, making it a promising candidate for conversion to sterility. The RiceNavi analysis confirmed that 93-11 indeed contained only the restorer genesRf3andRf6. Upon scrutinizing the functional genes of the three cultivars, it was observed that the restorer genesrf3andrf6in 93-11 were absent in Quan 9311A,whereas the alleles ofRf3andRf6from Zhong 9A,which lack restoring ability, were retained. Simultaneously,the non-restorativeRf2genotype in 93-11 was inherited by Quan 9311A, and theRf2allele from Zhong 9A was eliminated. No other restorer genes underwent alteration in Quan 9311A (lacking restorative ability), resulting in the deficiency in restorative function for Quan 9311A. Furthermore, it was observed that the sterility gene of Quan 9311A originated fromWA352cof Zhong 9A. All rice cultivars containing the WA-CMS restorative genes have the capability to restore their fertility. Consequently, Quan 9311A was deemed suitable as a CMS line because it possessed the fundamental characteristics sought after in sterile lines: the absence of restorer genes and the presence of a sterility gene,WA352c.

Analysis of functional genes related to plant height,days to heading, grain number per panicle, 1000-grain weight, stigma exertion rate, and seed-setting rate in Quan 9311A

Quan 9311A was deliberately bred to serve as a vital material in the hybrid rice breeding program in southern China, particularly in regions adjacent to the Yangtze River. The geographical and ecological conditions in these regions have a positive impact on the plant height of Quan 9311A, which features a relative moderate height. However, it is also imperative to ensure ample biomass in rice plants to facilitate the production of hybrid seeds. As a result, when devising the breeding strategy for Quan 9311A, the primary objectives were established to encompass traits such as moderate plant height, appropriate heading date,increased tiller number, a high rate of stigma exertion,and elevated yield. The application of RiceNavi analysis revealed discernible differences in the genes linked to these agronomic traits between Zhong 9A,93-11, and Quan 9311A (Fig. 4). The phenotype of each trait was governed by an array of genes. For example, plant heightwas influenced bySd1,OsSPY,Hd1,Ghd7,andGhd7.1, grain number per panicle was associated withGn1a,Hd1,Ghd7, andGhd7.1,while heading date was tied toHd1,Ghd7,Ghd7.1,Hd3a, andDTH8. Similarly, seed morphology or 1000-grain weightwas controlled by genes likeGW5,GS3,GW7, andGS5, and grain qualitywas influenced byWaxy(Wei et al, 2021).

Fig. 4. Phenotypic characteristics and genetic loci in five cultivars.

Prominently,Ghd7andHd1are key genes within this spectrum, both exhibiting pleiotropy that affects plant height, days to heading, and grain number per panicle. Quan 9311A inherited theGhd7mutant genotype from Zhong 9A and theHd1allele from 93-11. Due to the pleiotropic nature ofGhd7andHd1,Quan 9311A not only possesses a shorter plant height than 93-11, but also flowers earlier (Fig. 4-A and -B).Given that genes likeD2,RFT1,Rf2, andHd3awere all wild-type in Quan 9311A, differing from its parents, they held no considerable sway over plant height. Other genes related to plant height showed uniform genotypes across all three cultivars. The RiceNavi analysis predicted that under long-day conditions in the middle and lower Yangtze River regions in China, Quan 9311A would have a plant height of 90 cm and a heading date of 78 d, which is consistent with the actual recorded plant height(83-90 cm) and heading date (74-80 d) during Quan 9311A’s breeding process. Consequently, the grain number per panicle was greatly reduced due to the inheritance of theGhd7mutant genotype from Zhong 9A by Quan 9311A. Meanwhile, theNOG1allele associated with increased grain number did not transfer to Quan 9311A (Fig. 4-C). These factors collectively accounted for the slightly diminished grain number per panicle observed in Quan 9311A compared with its parental sources. Concerning TGW,Quan 9311A carried the mutantGW5genotype,resulting in heightened grain weight. Several genes present in Zhong 9A, namelyTGW6,Rf2, andDTH8,failed to transfer to the resulting lines (Fig. 4-D).

The heightened stigma exertion rate endows the male sterile line with proficiency in outcrossing, which underpins both the reproduction of male sterile offspring and the robust yield of hybrid seed production. However,practical applications in China have revealed a recurrent negative correlation between a high stigma exertion rate and the closed-glume performance of hybrid seeds,resulting in lower germination rates, compromised storage capabilities, and weak resistance to glume closure. This contradiction poses a challenge to simultaneously enhance the grain yield and quality of hybrid rice seeds.Remarkably, Quan 9311A managed to address this issue. The hybrid seeds propagated from Quan 9311A exhibited traits including high seed-setting rate,effective glume closure, and robust germination rate.The RiceNavi analysis unveiled the presence of theGS3superior allele in Quan 9311A, originally found in both parents, which was capable of elevating the stigma exertion rate while also potentially increasing grain size (Fan et al, 2006; Zhou et al, 2017). In terms of germination rate, Quan 9311A carried the mutant gene typeSdr4inherited from 93-11, fostering improved seed germination success. Furthermore, it harbored the genesOsGSK2andOsTPP7, which respectively contribute to mesocotyl elongation and bolstered tolerance in rice plants for anaerobic germination.

Analysis of functional genes related to grain quality,pest/disease resistance, and other advantageous characteristics in Quan 9311A

The genome of Quan 9311A retained the mutant genotypesofSSG6,BG2, andGW8from 93-11, which can collectively increase grain size and decrease grain width. As a result, Quan 9311A exhibited a larger grain length-to-width ratio, resulting in a slender grain shape and reduced chalkiness. Additionally, the favorable allele ofWaxyfrom 93-11 was preserved in Quan 9311A, ensuring that the amylose content of Quan 9311A remains within an appropriate range.

In terms of pathogen resistance, Quan 9311A retained the bacterial blight resistance genesXa1andXa3/Xa26from Zhong 9B. Regarding rice blast resistance genes,Quan 9311A inherited several resistance alleles from both parents, includingPi25/Pid3,Pia, andPib, but it did not contain broad-spectrum rice blast resistance alleles such asPi2/Pi9andPigm. In addition, Quan 9311A also contains theSTV11gene, which enhances resistance to rice stripe virus. According to the RiceNavi analysis, although there was no risk of susceptibility to bacterial blight, a risk still existed for rice blast. It is recommended to improve rice blast resistance or select male parents with rice blast resistance for breeding.

Additionally, the genome of Quan 9311A also contained genes for higher nitrogen-use efficiency,includingNRT1.1BandOsNR2, as well as several other notable genes. These includeOsCERK1, which improves phosphorus absorption efficiency;Sub1A,which confers submergence tolerance;TOND1,which increases nutrient tolerance;NAL1, whichreduces plant height, narrows leaves, and increases yield;LAX1,which increases grain number;SKC1,which increases the sodium content of roots to improve salt tolerance; andSCM2/APO1, which enhances lodging resistance. The pleiotropic functions of some of these genes, the influence of gene interactions, and their commercial value still require further exploration and characterization.

Functional gene analysis of hybrid rice QYSM with Quan 9311A as female parent

We took QYSM as an example, a hybrid combination,with Quan 9311A as the female parent and WSSM as the male parent, to analyze the relationship between functional genes and phenotypic characteristics using RiceNavi. First, we evaluated the phenotypes of Quan 9311A, WSSM, and QYSM (Table S1 and Fig. S3).Then, we employed the RiceNavi technology to analyze the functional genes associated with these phenotypes.Clearly, both Quan 9311A and WSSM contained numerous alternative alleles, which were aggregated in the genome of QYSM (Table 1 and Fig. 5). Quan 9311A contributed several causative genes such asLAX1,Xa1,GW5,Sdr4,BG2,Ghd7.1,TIG1, andXa3,and the male parent WSSM carriedRf3,NOG1,Bph3,Pi21,PTB1,Bph29,GS6,Pi9,Ghd8,Pi5, andPi56, as well asRf4,Rf1b,andPi-ta.We observed that the fertility restorer genesRf3andRf4of the WA-CMS line were mutants in the WSSM genome, functioning to restore fertility, indicating that WSSM is a proficient restorer line. Additionally, Quan 9311A and WSSM shared a series of superior alleles (Table S6).

Fig. 5. Superior genes for Quanyousimiao (QYSM) aggregated from Quan 9311A and Wushansimiao (WSSM).

Based on the analyzed functional loci, we attempted to explain the reasons behind the high yield and disease resistance of QYSM. We identified several excellent mutant genes in the QYSM genome, including both heterozygous (inherited from one parent) and homozygous (present in both parents) genes. For instance, the rice blast resistance loci found in QYSM includedPib,Pi2/Pi9,Pi25/Pid3,Pid2,Pi21,Pia, and

Pi-ta. Among these, the most crucial broad-spectrum rice blast resistance locus,Pi2/Pi9, was inherited from the male parent, WSSM, while the other two rice blast resistance loci,Pi-taandPi21, also originated from WSSM. This conferred broad-spectrum resistance to rice blast and the potential for QYSM to thrive in various environments. This potential can compensate for the risk of Quan 9311A not being resistant to rice blast. Considering resistance to bacterial blight, Quan 9311A as the female parent, contributed more than the male parent, WSSM, due to Quan 9311A containing multiple bacterial blight resistance mutant loci, such asXa1,Xa4, andXa26, while WSSM only contained theXa4locus. In terms of resistance to the rice planthopper, an insect pest, WSSM contributed two resistance genes,Bph3andBph29, which offset the deficiency of rice planthopper resistance genes in Quan 9311A.

Regarding yield, QYSM inherited a series of superior alleles from both parents, including genes likeGn1a,NOG1, andGNP1, which increase grain number per panicle. Additionally, QYSM inherited the semi-dwarf geneSd1and theNAL1gene, which narrow rice leaves while increasing yield by reducing plant height. Other alleles consisted ofGS3, which affects grain shape,SLB1andSLB2, which increase tiller number,SCM2/APO1, which increases grain number while rendering the rice plant more resistant to lodging. Importantly, in this hybrid rice combination,Quan 9311A provided the superior genotype ofGW5,which increases TGW, the geneGhd7.1, which can alter heading date and yield, and the genePTB1,which increases seed-setting rate. In this way, the hybrid combination can achieve high and stable yields(Fig. 4).

In terms of abiotic stress, both parents also possessed excellent alleles, including theBET1gene for boron toxicity tolerance, theOsHKT1.1gene for improving salt tolerance, theSub1Agene conferring submergence tolerance, and theOsTPP7gene for enhancing tolerance to anaerobic germination. Regarding nutrient absorption, both parents contained the genotypesNRT1.1BandOsNR2, which enhance nitrogen-use efficiency, as well as the variant genotypeTOND1,which is more resistant to stress caused by low nitrogen availability. They also contained a variant genotype ofOsGSK2capable of increasing mesocotyl elongation,making seeds more likely to germinate.

In addition, Quan 9311A contained an excellent allele ofSdr4for seed germination tolerance on panicles, as well as theTIG1genotype for reduced tillering angle, but neither was present in WSSM.However, the unfavorable allelehbd2(weakening cold tolerance) found in Quan 9311A was absent in WSSM.Conversely, two other unfavorable genotypes,D2(resulting in diminished plant height and wider tiller angle after mutation), andOsSRO1c(associated with less drought tolerance), present in WSSM were absent in Quan 9311A. Hence, their hybrid rice combination,QYSM, gained complementarity in these prominent traits, fostering favorable phenotypes while mitigating unfavorable ones.

To validate the effectiveness of the aggregation of these superior alleles in QYSM for breeding, we collected phenotypic data related to plot yield, disease resistance, insect resistance and grain quality of QYSM during its participation in China’s National Variety Approval and Production Trials (https://www.ricedata.cn/variety/varis/615870.htm) (Table 2). QYSM participated in four ecological regions, including earlyseasonindicain South China, photosensitive late-seasonindicain South China, mid-seasonindicain the upper reaches of the Yangtze River, and mid-seasonindicain the middle and lower reaches of the Yangtze River.Compared with control cultivars from different ecological regions, QYSM demonstrated clear superiority in various agronomic traits, especially in grain quality.Additionally, QYSM exhibited significant advantages in plot yield and disease/insect resistance compared with control cultivars in the middle and lower reaches of the Yangtze River. QYSM also displayed apparent advantages in blast resistance, except in early-seasonindicain South China. Therefore, it can be inferred that the aggregation of multiple excellent genes in QYSM has a positive effect on breeding in practical production.

Table 2. Phenotypes of plot yield, blast resistance, bacterial blight resistance, planthopper resistance, and grain quality of Quanyousimiao and control cultivars.

Based on the results of this extensive genotype analysis, aside from a series of excellent genotypes shared by Quan 9311A and WSSM, there was strong evidence of achieved complementarity in multiple functional genes, including those related to resistance against rice blast, bacterial blight, and rice planthoppers,as well as bolstered yield, among others. The hybrid rice combination QYSM also harbored multiple heterozygous genotypes in its whole genome. This complementation and aggregation of dominant genes were also intrinsic causes of heterosis. Endowed with the desirable functions of these genes, QYSM is expected to exhibit extensive adaptability and high productivity when cultivated across various ecological regions in China.

DISCUSSION

The acquisition of heterosis, also known as the production of progeny with hybrid vigor through genetically distant crossing, has always been a central concept in hybrid crop breeding (Shull, 1952). However,due to the limitations of germplasm resources for sterile lines and the conventional breeding method of‘maintainer begets maintainer’, the development and breakthrough of CMS lines are greatly restricted. For example, most of the WA-CMS lines in commercial hybrid rice may be improved from one of the three core germplasms, Zhenshan 97A, Jin 23A, and V20A,while the EGMS lines might be derived from Guangzhan 63S (Gu et al, 2021). Therefore, finding innovative ways to breed excellent male sterile lines is an imperative task, which can expedite the development of adaptable, productive hybrid rice. Quan 9311A is a WA-CMS line improved by introducing the two-line restorer line 93-11 as the core germplasm in this situation. Consequently, we initiated an analysis to determine whether Quan 9311A exhibits the basic phenotypic characteristics of a sterile line, including no restorer genes but a sterile gene, lower plant height with strong tillering ability, appropriate heading date and high stigma exertion rate. We dissected the known genes associated with these phenotypic characteristics using the RiceNavi technology. These genes have been received support from numerous functional studies. Several genes associated with plant height,heading date, grain number per panicle, and TGW are also mentioned in Fig. 4. Furthermore, the pleiotropic effects and interactions among them are relatively clear, such as the interaction of the heading date genesHd1,Ghd7, andDTH8(Zhang et al, 2012; Fujino,2020; Sun et al, 2022); the phenotypic effects of genesGW8,GS5, andGS3on grain shape (Wang et al, 2012;Ngangkham et al, 2018; Zhong et al, 2020).GS3andGW5have been identified as major influences on stigma exertion levels through genome-wide association studies (Zhou et al, 2017). Based on the knowledge of these known genes, we were able to dissect the genetic loci in Quan 9311A in conjunction with the observed phenotypes. However, other excellent agronomic traits of Quan 9311A, such as strong stigma vigor, hightemperature tolerance, improved glume closure, and rapid yellowing of leaf color after maturity, were not analyzed due to the lack of corresponding known gene support.

Although we found that the vast majority of superior alleles in Quan 9311A were inherited from two parents, 93-11 and Zhong 9A, theNOG1,Bph29,andGW5alleles in Quan 9311A differed from both parents. It is speculated that these genes may have originated from a third parent, 222B (an inter-mediate material used in breeding). Since we did not obtain the sequencing data for 222B, we have not provided a description of it here. In addition, Quan 9311A also inherited some unfavorable alleles that are present in both parents (Table S4). This suggested that Quan 9311A still needs genetic and genomic improvement,which is consistent with the conclusions drawn from the observations made by its breeders in the field. The breeder has proposed three suggestions: improving its resistance to rice blast disease, reducing its tiller angle to make it more upright, and adjusting its grain shape or increasing its fragrance according to consumer preferences.

Quan 9311A can be paired with various types of restorer lines in hybrid rice breeding, showing excellent combining ability. The hybrid rice combinations obtained from Quan 9311A showed significant heterosis based on field phenotypic investigations. To date, 133 hybrid rice combinations with Quan 9311A as the female parent have received approval for planting at the national or multi-provincial level in China (https://www.ricedata.cn/variety). These hybrid rice combinations are widely distributed and cover the entire rice cultivation areas in southern China. One of these hybrid combinations is QYSM, which has been approved for planting as anindicarice cultivar in the regions along the Yangtze River and as double-cropped rice in southern China. It has shown yield increases ranging from 3.30% to 9.43% (https://www.ricedata.cn/variety/varis/615870.htm). These results indicate that the heterosis of QYSM may be attributed to the combination of multiple superior alleles from both parents. Beyond that, QYSM carries many heterozygous alleles, which contribute to complementary genotypes and result in a semi-dominant phenotype. As a result,QYSM partially exhibits the excellent phenotypes of the two parents while weakening the unfavorable phenotypes. Under the combined influence of multiple genes, QYSM features moderate tiller angles, improved leaf morphology, effective defense against diseases and pests, lodging resistance, high yield, and good adaptability traits. All these traits together constitute the remarkable expression of heterosis and the aggregation of dominant genes (Wang et al, 2016).

Obviously, there are some challenges in the current development of genomic-assisted breeding technology.However, with the advancement of modern biotechnology and the guidance of genomics techniques, it is expected to achieve a level of high efficiency that traditional breeding struggles to attain. This will allow for the exploration of more genetic backgrounds, the rapid accumulation of more superior genes, and the cultivation of breakthrough varieties, ultimately realizing the vision of targeted rice breeding.

In this study, we analyzed the relationship between phenotypic characteristics and functional genes in Quan 9311A using RiceNavi. We found that Quan 9311A has accumulated multiple superior alleles from 93-11 and Zhong 9A, which contributes to its excellent combining ability. The hybrid rice combinations developed from Quan 9311A exhibit significant heterosis. The breeding of Quan 9311A reflects the innovative approaches of Chinese breeders in hybrid rice development.

METHODS

Rice materials

The 93-11 is anindicacultivar that is often used as a two-line restorer line in hybrid rice breeding. Zhong 9A is anindicatype CMS line used in the early years of hybrid rice development. 222B is a maintainer line without restorer genes,but its genotype is not homozygous, which is only used as a transition material in the breeding process of Quan 9311A.Quan 9311A is anindica-type CMS line, which was bred by crossing 93-11, Zhong 9A, and 222B. As the female parent,222B was chosen to cross with 93-11 simply because it had desirable agronomic traits such as more tillers and high stigma exertion rate, which might improve the tillering ability and stigma exertion rate of 93-11 or Zhong 9A. After the initial cross, breeders only selected progenies with high tillering ability and high stigma exertion rate similar to 222B based on phenotypic observations but did not further breed 222B. Thus,there were no seeds or leaves available for sequencing and conducting RiceNavi analysis. WSSM is anindicacultivar,often used as a three-line restorer line in hybrid rice breeding.QYSM is anindica-type three-line hybrid rice combination with Quan 9311A as the female parent and WSSM as the male parent. All rice cultivars used in this study came from the Win-All High-Tech Seed Industry Co. Ltd, based in Anhui Province, China. These cultivars currently have accession numbers on the NCBI website, except QYSM, which corresponds to Quan 9311A, Zhong 9A, 93-11, and WSSM with accession numbers SRS7264958, SRS7201777, SRS14782548-551,and SRS7265224, respectively.

Phenotyping

A total of seven traits were evaluated, including plant height,days to heading, grain number per panicle, TGW, tiller number per plant, panicle length, and stigma exertion rate. All phenotypes,except stigma exertion rate, were investigated in Hefei city,Anhui Province, China, during the summer of 2022. The stigma exertion rates of Quan 9311A and Zhong 9A were cited from Wang H Q et al (2013) and Wang et al (2010), respectively.Twenty-four plants of each cultivar were randomly selected for phenotyping. Plant height was measured from ground level to the apex of the highest straightened panicle. Days to heading was recorded as the number of days from sowing to flowering.The grain number per panicle was the average value of each cultivar, with 24 plants selected from each cultivar and 3 spikes sampled from each plant. TGW was calculated by weighing the grain of each plant and then converting it according to the number of grains. At harvest, the total number of mature spikes per plant was recorded as the tiller number per plant. Panicle length was measured by taking five longest panicles per plant.

Breeding process of Quan 9311A/B

Quan 9311A was bred by cross-breeding among different rice cultivars and screening the hybrid offspring based on dominant agronomic characteristics. An important aspect of its breeding process is the selection of suitable female and male parents during the cross-breeding process. The female and male parents used in each generation of crosses were differed from those used in the previous generation (Fig. 1-B). The phenotypic requirements for screening the female parent (or sterile line) are:the hybrid progeny of sterile and maintainer lines differ only in fertility phenotype, while other agronomic traits are consistent with those of the maintainer line. However, the genomes of these lines are heterozygous, and their phenotypes cannot be stabilized. Therefore, these maintainer lines were planted every year for selfing, and individuals with desirable agronomic traits were selected from them (as the male parent). Sterile individuals were also selected from the offspring obtained by crossing the corresponding last generation maintainer lines with sterile individuals (as the female parent). Hybridization was carried out, and the hybrid seeds were sown to continue the process of selecting sterile individuals with desirable agronomic traits as the female parent. This process continued until Spring 2010. The maintainer line was inbred until the F13generation, and the sterile line was backcrossed until the BC7F1generation, with their dominant agronomic phenotypes being stable. Thus, the sterile line was named Quan 9311A, and the corresponding maintainer line was named Quan 9311B. Therefore, from Quan 9311B to Quan 9311A, it was also a purposeful improvement process. The sterile and maintainer lines based on the three-line hybrid system differed only in the fertility phenotype and were consistent in other agronomic traits. Their genomic origin and genomic background were basically the same. Therefore, we chose Quan 9311A to represent the set of Quan 9311A and Quan 9311B, and Zhong 9A to represent the set of Zhong 9A and Zhong 9B.

DNA extraction and genome sequencing

Genomic DNA was extracted from fresh young leaves of each individual by using the Hi-DNAsecure Plant Kit according to the manufacturer’s instructions (Tiangen Biotech, Beijing,China). All samples were fragmented into 500-bp lengths to construct Illumina sequencing libraries, utilizing the KAPA HyperPrep Kit (Kapa Biosystems, Roche, USA) as per the manufacturer’s recommendations. The libraries were sequenced on the Illumina HiSeq 4000 platform using its paired-end 150-bp mode, resulting in a total of 183 Gb of raw data for five cultivars.

High-throughput genotyping

Quan 9311A, 93-11, Zhong 9A, WSSM, and QYSM were each subjected to the whole-genome high-throughput sequencing technology. We used software tools such as Trimmomatic-0.38,Bwa-0.7.1, Samtools-1.9, and GATK v4.1.4.1 (The Genome Analysis Toolkit) to detect variants in the original sequencing data, aligning them to the reference genome Nipponbare(MSU7.0). This allowed us to obtain information about the variation sites in each cultivar relative to the Nipponbare reference genome. Next, we employed the ‘a(chǎn)wk’ command to link the respective SNP loci of 93-11 and Zhong 9A with the SNP loci of Quan 9311A to establish mutually consistent SNP loci. We then calculated the similarity between them. Meanwhile,the entire rice genome was divided into 3 740 intervals, each spanning 100 kb, and we counted all SNPs found in each interval. An interval was considered unrelated to breeding heritability if it had fewer than 200 SNPs. Based on this information, we created graphs illustrating the differential distribution of SNPs in the genome between the two parents(Fig. 2).

Functional locus analysis using RiceNavi

Utilizing the SNP information, we employed the QTNpick module in the RiceNavi software to genotype the variants present at each QTN site, obtaining the QTN genotype for each cultivar(https://github.com/xhhuanglab/RiceNavi or http://www.xhhuanglab.cn/tool/RiceNavi.html). We interpreted rice phenotypes based on the genetic effects of QTN. These QTN loci, totaling 348,represent genes where variations can lead to changes in gene functions and related phenotypic alterations. Among these loci,225 are reported QTLs/genes in rice. All of these genes involve naturally occurring mutations, with no artificially induced or reversed mutations in the data. Consequently, these genes,present in common cultivars, could potentially be transferred or acquired through crosses between different cultivars during rice breeding (Wei et al, 2021).

ACKNOWLEDGMENTS

This study was funded by the National Natural Science Foundation of China (Grant No. 32001516), Shanghai Agriculture Applied Technology Development Program, China (Grant No.X20190103), and Rice Industry of China Agriculture Research System (Grant No. CARS-01-03). The authors thank Ms. ZHANG Qin, president of the National Rice Commercial Molecular Breeding Technology Innovation Alliance, for supporting cooperation platform.

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.

Fig. S1. Grain shape and polished rice grains of Zhong 9A and Quan 9311A.

Fig. S2. QTNpick module in technical route of rice genome physical examination in RiceNavi.

Fig. S3. Plant morphologies of Quan 9311A, Quanyousimiao(QYSM), and Wushansimiao (WSSM).

Table S1. Phenotypes of Zhong 9A, 93-11, Quan 9311A,Wushansimiao (WSSM), and Quanyousimiao (QYSM).Table S2. Sequencing of five rice cultivars.

Table S3. Summary of simple sequence polymorphism (SNP)after comparison of three rice cultivars.

Table S4. Summary of quantitative trait nucleotide (QTN) maps in five cultivar genomes.

Table S5. Superior alleles co-existing in 93-11, Zhong 9A, and Quan 9311A.

Table S6. Superior alleles co-existing in Quan 9311A, QYSM,and WSSM.