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Abstract Background Hybrid rice has significant yield advantage and stress tolerance compared with inbred rice. However, production of hybrid rice seeds requires extensive manual labors. Currently, hybrid rice seeds are produced by crosspollination of male sterile lines by fertile paternal lines. Because seeds from paternal lines can contaminate the hybrid seeds, mechanized production by mixed-seeding and mixed-harvesting is difficult. This problem can be solved if the paternal line is female sterile. Results Here we identified a female infertile mutant named h569 carrying a novel mutation (A1106G) in the MEL2 gene that was previously reported to regulate meiosis entry both in male and female organs. h569 mutant is female infertile but male normal, suggesting that MEL2 regulates meiosis entry in male and female organs through distinct pathways. The MEL2 gene and h569 mutant gave us tools to construct female sterility maintaining systems that can be used for propagation of female sterile lines. We connected the wild-type MEL2 gene with pollen-killer gene ZmAA1 and seed-marker gene DsRed2 in one T-DNA cassette and transformed it into ZZH1607, a widely used restorer line. Transgenic line carrying a single transgene inserted in an intergenic region was selected to cross with h569 mutant. F2 progeny carrying homozygous A1106G mutation and hemizygous transgene displayed 1:1 segregation of fertile and infertile pollen grains and 1:1 segregation of fluorescent and non-fluorescent seeds upon self-fertilization. All of the non-fluorescent seeds generated female infertile plants, while the fluorescent seeds generated fertile plants that reproduced in the way as their previous generation. Conclusions These results indicated that the female sterility maintaining system constructed in the study can be used to breed and propagate paternal lines that are female infertile. The application of this system will enable mechanized production of hybrid rice seed by using the mixed-seeding and mixed harvesting approach, which will significantly reduce the cost in hybrid rice seed production.

Background: Hybrid rice has significant yield advantage and stress tolerance compared with inbred rice. However, production of hybrid rice seeds requires extensive manual labors. Currently, hybrid rice seeds are produced by crosspollination of male sterile lines by fertile paternal lines. Because seeds from paternal lines can contaminate the hybrid seeds, mechanized production by mixed-seeding and mixed-harvesting is difficult. This problem can be solved if the paternal line is female sterile. Results: Here we identified a female infertile mutant named h569 carrying a novel mutation (A1106G) in the MEL2 gene that was previously reported to regulate meiosis entry both in male and female organs. h569 mutant is female infertile but male normal, suggesting that MEL2 regulates meiosis entry in male and female organs through distinct pathways. The MEL2 gene and h569 mutant gave us tools to construct female sterility maintaining systems that can be used for propagation of female sterile lines. We connected the wild-type MEL2 gene with pollen-killer gene ZmAA1 and seed-marker gene DsRed2 in one T-DNA cassette and transformed it into ZZH1607, a widely used restorer line. Transgenic line carrying a single transgene inserted in an intergenic region was selected to cross with h569 mutant. F2 progeny carrying homozygous A1106G mutation and hemizygous transgene displayed 1:1 segregation of fertile and infertile pollen grains and 1:1 segregation of fluorescent and non-fluorescent seeds upon self-fertilization. All of the non-fluorescent seeds generated female infertile plants, while the fluorescent seeds generated fertile plants that reproduced in the way as their previous generation. Conclusions: These results indicated that the female sterility maintaining system constructed in the study can be used to breed and propagate paternal lines that are female infertile. The application of this system will enable mechanized production of hybrid rice seed by using the mixed-seeding and mixed harvesting approach, which will significantly reduce the cost in hybrid rice seed production. [ABSTRACT FROM AUTHOR]

Considering the male sterile line has the phenomenon of panicle enclosure, panicle elongation length (PEL) plays an important role in hybrid rice seed production. However, the molecular mechanism underlying this process is poorly understood. In this study, we investigated the PEL phenotypic values of 353 rice accessions across six environments, which shows abundant phenotypic variation. Combining the 1.3 million single-nucleotide polymorphisms, we performed a genome-wide association study on PEL. Three quantitative trait loci (QTL) qPEL4, qPEL6, and qPEL9 were identified as significantly associated with PEL, of which qPEL4 and qPEL6 were previously reported QTLs and qPEL9 was novel. One causal gene locus, PEL9, was identified and validated. The PEL of accessions carrying allele PEL9GG was significantly longer than that of those carrying allele PEL9TT. We also demonstrated that the outcrossing rate of female parents carrying allele PEL9GG increased by 14.81% compared with that of the isogenic line carrying allele PEL9TT in an F1 hybrid seed production field. The allele frequency of PEL9GG increased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement of the PEL of the female parent of hybrid rice. [ABSTRACT FROM AUTHOR]

Pollen shedding and dispersal processes are inextricably linked to the movement state of hybrid rice panicles. The purpose of this work was to study the effect of the working parameters of the pneumatic pollinator on the kinematic characteristics of hybrid rice panicles during flowering and to further optimize the working parameters of the pollinator. An experimental platform was built to characterize the movement of the panicle. Orthogonal experiments were conducted with airflow velocity, action position, and walking speed as experimental factors and kinematic parameters such as displacement, velocity, and acceleration of the rice panicle as experimental indicators. A quadratic regression model between the working parameters and the motion characteristics of the rice panicle was constructed and analyzed by analysis of variance (ANOVA). Matrix analysis was used for multi-objective optimization of the working parameter combinations. Field pollination trials were conducted for the optimized combination of parameters. The results showed that the coefficients of determination of the regression models were all above 0.85, which indicated good accuracy. The motion characteristics of the panicle, including the displacement maximum, velocity maximum, and acceleration maximum, can be maintained at a high level when the airflow velocity is 24 m/s, the action position is 120 mm from the top of the panicle, and the walking speed is 0.4 m/s. The field experiments showed that the optimization results could obtain the desired pollen density, distribution, and uniformity. This study can serve as a reference for the development of pneumatic hybrid rice pollination theory and the optimal design of the pollinator structure. [ABSTRACT FROM AUTHOR]

Considering the male sterile line has the phenomenon of panicle enclosure, panicle elongation length (PEL) plays an important role in hybrid rice seed production. However, the molecular mechanism underlying this process is poorly understood. In this study, we investigated the PEL phenotypic values of 353 rice accessions across six environments, which shows abundant phenotypic variation. Combining the 1.3 million single-nucleotide polymorphisms, we performed a genome-wide association study on PEL. Three quantitative trait loci (QTL) qPEL4, qPEL6, and qPEL9 were identified as significantly associated with PEL, of which qPEL4 and qPEL6 were previously reported QTLs and qPEL9 was novel. One causal gene locus, PEL9, was identified and validated. The PEL of accessions carrying allele PEL9GG was significantly longer than that of those carrying allele PEL9TT. We also demonstrated that the outcrossing rate of female parents carrying allele PEL9GG increased by 14.81% compared with that of the isogenic line carrying allele PEL9TT in an F1 hybrid seed production field. The allele frequency of PEL9GG increased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement of the PEL of the female parent of hybrid rice.

Conventional production of hybrid rice seeds is based on sowing and transplanting parental lines separately (‘separate seeding and transplanting’, SST). To decrease the labour inputs of field management, a labour-saving model was implemented under a strategy of directly seeding the two parents as a mixture (‘mixed direct seeding’, MDS) to produce hybrid seeds. The strategy utilized a ‘yellow-hull/red-hull’ pair, ensuring that the female parent could be easily separated from the male parent after mixed harvesting. To assess the effects of MDS on seed production, two pairs, P88S/G-4 and Y58S/G-15, were tested and compared with those under SST at the same female/male seed ratio of 5:1 in 2015 and 2016. The parental lines for two pairs flowered synchronously, and the male sterile lines presented a high total stigma exertion rate in SST that ranged from 75.82% to 93.27%. At maturity, the hybrid seeds generated on seed parents were harvested together with those from pollen parents, and later, the hybrids were successfully colour sorted at a correctness rate of more than 99.90% on a machine. In MDS, the heading date of tested lines was advanced by 2~6 days. The outcrossing rate was improved by 8.59% and 7.61%, and the total panicle number increased by 4.78% and 5.80%, respectively. Ultimately, the actual yield in MDS significantly exceeded that in SST by 12.56% for P88S/G-4 and 8.95% for Y58S/G-15. This model succeeded in recycling hybrid seeds via color sorting and seems promising for decreasing labour inputs in hybrid seed production.

Conventional production of hybrid rice seeds is based on sowing and transplanting parental lines separately ('separate seeding and transplanting', SST). To decrease the labour inputs of field management, a labour-saving model was implemented under a strategy of directly seeding the two parents as a mixture ('mixed direct seeding', MDS) to produce hybrid seeds. The strategy utilized a 'yellow-hull/red-hull' pair, ensuring that the female parent could be easily separated from the male parent after mixed harvesting. To assess the effects of MDS on seed production, two pairs, P88S/G-4 and Y58S/G-15, were tested and compared with those under SST at the same female/male seed ratio of 5:1 in 2015 and 2016. The parental lines for two pairs flowered synchronously, and the male sterile lines presented a high total stigma exertion rate in SST that ranged from 75.82% to 93.27%. At maturity, the hybrid seeds generated on seed parents were harvested together with those from pollen parents, and later, the hybrids were successfully colour sorted at a correctness rate of more than 99.90% on a machine. In MDS, the heading date of tested lines was advanced by 2~6 days. The outcrossing rate was improved by 8.59% and 7.61%, and the total panicle number increased by 4.78% and 5.80%, respectively. Ultimately, the actual yield in MDS significantly exceeded that in SST by 12.56% for P88S/G-4 and 8.95% for Y58S/G-15. This model succeeded in recycling hybrid seeds via color sorting and seems promising for decreasing labour inputs in hybrid seed production. [ABSTRACT FROM AUTHOR]

Anther indehiscence in certain wide crosses combines male sterility with stigma exertion, a phenomenon that is desirable for hybrid rice seed production. This study sought to identify chromosomal region(s) that combine anther indehiscence with exerted stigmas. A mapping population consisting of 189 BC1F1 plants was derived from a cross between CRI-48 and Jasmine 85 and backcrossing the resulting F1 to Jasmine 85. Contrary to the three complementary genes mode of inheritance reported earlier, a single locus (AI6-1) was mapped on chromosome 6 at 27.4 cM for anther indehiscence with exerted stigmas through a mixed model-based composite interval mapping (MCIM). This locus was flanked by two single nucleotide polymorphism (SNP) markers, K_ID6002884 and K_ID6003341 within a range of 23.1–28.9 cM. The allele at the locus was contributed by the CRI-48 parent which has Oryza glaberrima ancestry. This locus is suggested to control anther indehiscence and stigma exertion through pleiotropic gene action or cluster of genes.

SUMMARY: Stigma characteristics are important factors affecting the seed yield of hybrid rice per unit area. Natural variation of stigma characteristics has been reported in rice, but the genetic basis for this variation is largely unknown. We performed a genome‐wide association study on three stigma characteristics in six environments using 1.3 million single‐nucleotide polymorphism (SNPs) characterized in 353 diverse accessions of Oryza sativa. An abundance of phenotypic variation was present in the three stigma characteristics of these collections. We identified four significant SNPs associated with stigma length, 20 SNPs with style length (SYL), and 17 SNPs with the sum of stigma and style length, which were detected repeatedly in more than four environments. Of these SNPs, 28 were novel. We identified two causal gene loci for SYL, OsSYL3 and OsSYL2; OsSYL3 was co‐localized with the grain size gene GS3. The SYL of accessions carrying allele OsSYL3AA was significantly longer than that of those carrying allele OsSYL3CC. We also demonstrated that the outcrossing rate of female parents carrying allele OsSYL2AA increased by 5.71% compared with that of the isogenic line carrying allele OsSYL2CC in an F1 hybrid seed production field. The allele frequencies of OsSYL3AA and OsSYL2AA decreased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement in stigma characteristics of parents of hybrid rice. Significance Statement: The utilization of heterosis in rice is an effective strategy to enhance the grain yield. However, this entails the production of F1 hybrid seeds every year. The yield of F1 hybrid seeds is affected by stigma characteristics. The cloning of OsSYL2AA filled a gap in the genetic study of style length and could be used for improving stigma characteristics to increase the yield of F1 seeds. [ABSTRACT FROM AUTHOR]

Considering the male sterile line has the phenomenon of panicle enclosure, panicle elongation length (PEL) plays an important role in hybrid rice seed production. However, the molecular mechanism underlying this process is poorly understood. In this study, we investigated the PEL phenotypic values of 353 rice accessions across six environments, which shows abundant phenotypic variation. Combining the 1.3 million single-nucleotide polymorphisms, we performed a genome-wide association study on PEL. Three quantitative trait loci (QTL) qPEL4 , qPEL6 , and qPEL9 were identified as significantly associated with PEL, of which qPEL4 and qPEL6 were previously reported QTLs and qPEL9 was novel. One causal gene locus, PEL9 , was identified and validated. The PEL of accessions carrying allele PEL9 GG was significantly longer than that of those carrying allele PEL9 TT . We also demonstrated that the outcrossing rate of female parents carrying allele PEL9 GG increased by 14.81% compared with that of the isogenic line carrying allele PEL9 TT in an F 1 hybrid seed production field. The allele frequency of PEL9 GG increased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement of the PEL of the female parent of hybrid rice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dang, Jing, Zhang, Li, Xu, Hu, Li, Zhang, Wang, Hong and Jiang.)

Abstract: The great progress in super rice breeding both in China and other countries has been made in recent years. However, there were three main problems in super rice breeding: 1) the super rice varieties were still rare; 2) most super rice varieties exhibited narrow adaptability; and 3) current breeding theories emphasized too much on the rice growth model, but they were unpractical in guidance for rice breeding. According to the authors’ experience on the rice breeding, the breeding strategies including three steps (super parent breeding, super hybrid rice breeding and super hybrid rice seed production) were proposed, and the objectives of each step and the key technologies to achieve the goals were elucidated in detail. The super parent of hybrid rice should exhibit excellent performance in all agronomic traits, with the yield or sink capacity reached the level of the hybrid rice control in regional trials. The super hybrid rice combination should meet the following criteria: good rice quality, wide adaptation, lodging resistance, resistance to main insects and diseases, and the yield exceeded above 8% over the control varieties in the national and provincial regional trials. To achieve the goal, the technical strategies, such as selecting optimal combination of the parents, increasing selection pressure, paying more attention to harmony of ideal plant type, excellent physiological traits and all the agronomic traits, should be emphasized. The yield of seed production should reach 3.75 t/ha and 5.25 t/ha for the super hybrid rice combinations derived from early-season and middle-season types of male sterile lines, respectively. The main technologies for raising seed production yield included selecting optimum seed production site, using the male sterile line with large sink capacity and good outcrossing characteristics, and improving the amount of the pollen by intensive cultivation of the male parent. According to the technologies of the three-step breeding on super hybrid rice, two super rice parents, including a male parent 996 and a thermo(photo)-genic male sterile [T(P)GMS] line C815S, were bred. Furthermore, a super early hybrid rice combination, Luliangyou 996, which could be used as a double-season early rice variety in middle and lower reaches of the Yangtze River, China, was bred by using the super rice variety 996 as the male parent, and several hybrid rice combinations with higher yield than control variety in regional trials both of Hunan Province and state were bred with the T(P)GMS line C815S as the female parent. [Copyright &y& Elsevier]

Anther indehiscence in certain wide crosses combines male sterility with stigma exertion, a phenomenon that is desirable for hybrid rice seed production. This study sought to identify chromosomal region(s) that combine anther indehiscence with exerted stigmas. A mapping population consisting of 189 BC 1 F 1 plants was derived from a cross between CRI-48 and Jasmine 85 and backcrossing the resulting F 1 to Jasmine 85. Contrary to the three complementary genes mode of inheritance reported earlier, a single locus ( AI6-1 ) was mapped on chromosome 6 at 27.4 cM for anther indehiscence with exerted stigmas through a mixed model-based composite interval mapping (MCIM). This locus was flanked by two single nucleotide polymorphism (SNP) markers, K_ID6002884 and K_ID6003341 within a range of 23.1-28.9 cM. The allele at the locus was contributed by the CRI-48 parent which has Oryza glaberrima ancestry. This locus is suggested to control anther indehiscence and stigma exertion through pleiotropic gene action or cluster of genes., (© 2021 The Author(s).)

Stigma characteristics are important factors affecting the seed yield of hybrid rice per unit area. Natural variation of stigma characteristics has been reported in rice, but the genetic basis for this variation is largely unknown. We performed a genome-wide association study on three stigma characteristics in six environments using 1.3 million single-nucleotide polymorphism (SNPs) characterized in 353 diverse accessions of Oryza sativa. An abundance of phenotypic variation was present in the three stigma characteristics of these collections. We identified four significant SNPs associated with stigma length, 20 SNPs with style length (SYL), and 17 SNPs with the sum of stigma and style length, which were detected repeatedly in more than four environments. Of these SNPs, 28 were novel. We identified two causal gene loci for SYL, OsSYL3 and OsSYL2; OsSYL3 was co-localized with the grain size gene GS3. The SYL of accessions carrying allele OsSYL3 AA was significantly longer than that of those carrying allele OsSYL3 CC . We also demonstrated that the outcrossing rate of female parents carrying allele OsSYL2 AA increased by 5.71% compared with that of the isogenic line carrying allele OsSYL2 CC in an F 1 hybrid seed production field. The allele frequencies of OsSYL3 AA and OsSYL2 AA decreased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement in stigma characteristics of parents of hybrid rice., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)