Fine-Mapping Of The Fertility Restoration Gene Rf10 For Maize CMS-S And The Pyramiding Effect Analysis Of This Gene With The Main Restoration Gene Rf3

Since 2009,the planting area of maize has exceeded that of rice and become the largest crop.In rice,rape,sorghum and other crops,the use of heterosis is one of the important ways to improve yield.Maize is a kind of crop which used CMS/Rf system for hybrid breeding very early.The utilization of heterosis has greatly improved the yield of maize.S-type CMS is the largest group of the three types of cytoplasmic male sterility.Its fertility restoration genes have not been cloned yet,and its mechanism is not clear.Therefore,fine mapping and cloning of the fertility restoration genes for maize CMS and clarifying its mechanism can accelerate its utilization in heterosis.Previous genome-wide association analysis revealed that CMS-S fertility restoration mechanism is very complex,and it was controlled by multiple major and minor loci.In this study,one of the loci was fine-mapped by genetic linkage population in combination with BSR-Seq technique,and the interaction between the locus and the main restorer gene Rf3 was analyzed.The main results are as follows:1.The fertility in the BC2/BC3 populations which derived from a fertile plant in a BC1 population was observed.There were two types of phenotypes in populations:in field sown in spring the anthers normally exsertion with partially pollen shedding or completely non-exsertion;while in the autumn,anthers exsertion with non-shedding or completely non-exposed.Chi-square test revealed the segregation of the two types fit to1:1,indicating that CMS-S fertility restoration was controlled by a single gene in the populations.Fertility performance combined with molecular marker analysis revealed that it is a new restorer gene,it is named Rf10.2.Molecular marker polymorphism screening and primary location analysis of the restoration gene in the BC2 population were carried out.Six SSR markers were screened out by BSA method.Genotype analysis and local linkage map construction were carried out.Rf10 was initially located in the interval of a143-M326 markers on chr3 with a genetic distance of 10.1 cM.Thirty spikelets of sterile and fertile plants in the population were randomly screened for BSR-Seq analysis.The location of the most associated SNP marker were consistent with the results of QTL mapping.3.According to the SNP information provided by BSR-Seq analysis,PCR markers were designed in the initial mapping interval to screen recombinants in the BC3/BC4populations and carry out fine-mapping.Finally,Rf10 was located in a range of 1.7Mb,which contains 37 genes.In addition,differentially expressed analysis of the genes were conducted with the data of BSR-Seq,and expression of some genes were verified by qPCR.Finally,a gene encoding a protein of RING/Ubox superfamily protein,was considered as the candidate gene of Rf10.4.The pollen fertility of homozygous Rf10 under CMS-S background is about50%-60%.The pollen fertility in an F₂ population constructed by crossing with restorer lines carrying Rf3 shows that there is a significant pyramiding effect between Rf10 and Rf3,which can improve the pollen fertility in the hybrids to more than 60%.In this study it was found that Rf10 could restore CMS-S anther exsertion,but its ability to restore pollen shedding was related to environmental conditions.It could shed pollen in Wuhan sown in spring,but not shedding in Wuhan sown in autumn.In addition,Rf10 was not linked to the main restorer gene Rf3,and the pyramiding with Rf3 could significantly improve the pollen fertility of the hybrids.Therefore,further genetic research on this gene would enrich the theory of fertility restoration of maize and provide useful information for the utilization of this gene in hybrid production.