Allelic Variation Analysis Of Restorer Fertility Genes And Breeding By Design Based On SGRL In Rice

Rice is one of the most important crops in the world.Hybrid rice can significantly improve rice yield and play an important role in ensuring food security in China and all over the world.Cytoplasmic male sterility and restorer fertility system is the basis of the heterosis utilization and the core content of hybrid rice.The restoring ability of restorer gene directly affects the seed setting rate and yield of F₁ generation,so the allelic variation analysis of restorer gene is very important for digging and using excellent alleles for molecular breeding by design.The allelic variation of the restorer genes Rf3,Rf4 and Rf1 loci in rice were analyzed by constructing single gene restorer lines（SGRL）from a wide range of donors.Abundant allelic variation genes were identified at three loci,which laid a material foundation for molecular breeding of sterile lines and restorer lines,on which a number of excellent sterile lines and restorer lines were bred.The main results of this study are as follows:1.Screening,identification and developing of SGRL.In this study,21 Rf3-SGRLs with the genotypes Rf3^NRf3^Nrf4^XArf4^XA and 33 Rf4-SGRLs with the genotypes rf3^XArf3^XARf4^NRf4^N were obtained by using molecular marker-assisted selection.Among Rf3-SGRLs,there are 3,2,2,5,3,4 and 2 lines derived from the parent W0,W7,W10,W11,W14,W18 and W23,respectively.Among Rf4-SGRLs,there are 2,4,2,2,5,2,4,2,5,1 and 4 lines derived from the parent W0,W2,W4,W8,W11,W18,W20,W22,W23,W24 and W27,respectively.2.Allelic variation analysis of Rf3 and Rf4 loci.A number of alleles with different restorer ability were identified at both Rf3 and Rf4 loci by using testcross F₁ generations between SGRLs and HXA.Four alleles at the Rf3 locus,named Rf3¹,Rf3²,Rf3³and Rf3⁴,according to the restorer ability from weak to strong.Similarly,four alleles named Rf4¹,Rf4²,Rf4³ and Rf4⁴,according to their restorer ability at the Rf4 locus were identified.3.Cultivation of"three lines"rice in HJX74 background.In this study,16 male sterile lines and isonuclear alloplasmic male sterile lines were cultivated by using maintainer lines and polymeric maintainer lines of HJX74 background.8 male sterile lines,which their recessive restorer gene,rf,originated from non-sterile lines,were bred by SGRL.Two of these CMS lines,E5-HBA1 and E5-HBA2,swing of partially fertile-completely abortive under the influence of temperature in the spring and fall seasons.The other 14 male sterile lines showed stable and complete sterility phenotype of pollen and spikelet in successive generations.The abortive degree of pollen and spikelet meets the requirement of sterile lines in production.All of CMS lines cultivated in this study had good recoverability.Four restorer lines with HJX74 background,which their Rf genes originated from wild rice were developed.The fertility restoration ability of new cultivated restorer lines was equivalent to a strong restorer line,P46,and showed a high fertility restoration level.All of the CMS lines,maintainer lines and restorer lines developed in this research met the requirements for hybrid rice production.4.Allelic variation analysis of Rf1 locus.A total of 68 rice materials from all over the world were screened using functional marker of Rf1a locus,and a new allele of Rf1a was identified in CMS lines of Jiahe 212A and Chang11A.A comprehensive analysis of pollen fertility,spikelet fertility and relative expression of CMS gene orf79/orf H79 in 5 stages of F1 generation crossed between Rf1-SGRLs and Zhe04A（CMS-BT）,Gaohong A（CMS-HL）,found that there were abundant allelic variations at Rf1 locus.Four alleles from 6 donors with different fertility restoration ability named Rf1¹,Rf1²,Rf1³ and Rf1⁴ were identified at Rf1 locus.At the same time,the Rf1a and Rf1b genes at the Rf1 locus were sequenced,and found that the Rf1b locus of all the tested materials were recessive type（rf1b）.Also,there is no other Rf gene（Rf6）in the background of SGRL.Therefore,the difference fertility restoration ability of SGRLs is due to the difference of Rf1a gene locus.This study also analyzed the SNPs and amino acid variation sites in RF1A protein,the result showed that the No.235,357 and 511 amino acid residues may play important roles in RF1A protein function.In this study,SGRL was constructed to analyze the allelic variation of the restorer gene Rf3,Rf4,and Rf1 loci in rice,and then the excellent allelic types with different fertility restoration ability were obtained.On this basis,the male sterile line,maintainer line and restorer line of HJX74 background were cultivated,and the rice"three lines"molecular breeding platform was improved and developed.According to the specific route and requirement of rice molecular breeding technology,the rice"three lines"which meet the requirements of hybrid rice production and application are developed.This study provides an example for molecular breeding by design of hybrid rice.