Inheritance And Candidate Genes Identification For The Fertility Restorer Of Cytoplasmic Male Sterility In Pepper

Cytoplasmic male sterility?CMS?and it's restoration system is the important way to produce hybrid seeds and it can not only reduce production cost,but also ensure the purity of hybrid seeds.However,there are few examples of using CMS/Rf system to produce hybrid seeds in pepper,the reason is that it is difficult to transfer cytoplasmic male sterile gene to mother parent and restorer gene to father parent.To solve these problems,it is necessary to understand the genetic and molecular mechanism of cytoplasmic male sterility and the resotorer of fertility in Pepper.Previous studies have shown that CMS is jointly controlled by cytoplasmic male sterility gene?S-?and cytoplasmic male sterile gene?msms?,and CMS is mainly caused by rearrangement of mitochondrial genome which lead to disorders of mitochondrial energy metabolism and substance metabolism.At present,some sterility genes have been cloned.However,the mechanism of the fertility restorer is unclear.It is unknown that how many Rf genes do regulate the fertility,which biological step do regulate,on DNA,transcription or translation level? So,it will have important theoretical significance for the breeding and transfer breeding of the restorer to discover the genetic and molecular mechanism of the fertility restorer,which will accelerate the production of hybrid seeds using CMS/Rf system.In this study,pepper cytoplasmic male sterile line 8A,restorer line R₁ and R₂,and their derived populations were used as materials to study the mechanism of the fertility restorer.First,the mechanism of the fertility restorer was analysed by six generations joint analysis using mixed model of major gene plus polygene.Second,the quantitative trait locies of the fertility restorer were mapped using a molecular genetic map.Third,to explore the molecular mechanism of the fertility restorer of CMS in pepper,transcriptome of male sterile line 8A and restorer line R₁,and their extreme sterile pool SP and restore pool RP were analized by transcriptome sequencing technology.The different expressed genes were selected,which were annotated on GO,KEGG and nr databases.Meanwhile,the Rf genes were also mapped by BSR analysis,and the candidate genes were identificated.The main results are displayed as follows.Firstly,the results of six generations joint analysis indicated that the fertility restorer of CMS was controlled by two major additive-dominant epistatic genes and additive-dominant epistatic polygenes in pepper,and the heritability of genes was very high with a value over 0.9388.In the cross of 8A×R₁,the first pairs of major genes took on additive effect and dominant effect with the value of-0.9540 and 1.2490 respectively,while the second pairs of major genes mainly took on additive effect with a value of-0.9540,and the dominant effect value was only-0.0447.In the cross of 8A×R₂,two major genes showed additive effect and dominant effect,the additive effects between two pairs of major genes were equal with a value of-0.8734,while the dominant effect of the first pairs of major gene was higher than that of the second pairs of major genes,and the value were 0.8959 and 0.3512 respectively.Secondly,a molecular genetic map has been constructed,which was composed of 12 linkage groups and covered 553.45 c M.Using this molecular genetic map,two major QTLs and seven minor QTLs related to the fertility restorer for CMS were mapped in pepper.q IF-3-1 was the first major QTL,which could explain 46.68% of phenotypic variation,with the dominant effect value and additive effect value of 1.28 and 0.84 respectively.q IF-5-1 was the second major QTL,which could explain 47.10% of phenotypic variation,with the dominant effect value and additive effect value of 1.76 and-0.02 respectively.Among seven minor QTLs,three QTLs?q IF-4-1,q IF-5-2andq IF-8-1?mainly showed additive effects,while another four QTLs?q IF-1-1,q IF-2-1,q IF-6-1 andq IF-12-1?mainly showed dominant effects.Thirdly,the RNA of R₁,8A,RP and SP had been sequenced by Illumina Hiseq2500.In 8A vs R₁,3790 different expressed genes were selected and among them 2274 genes appeared up-regulated expression and 1516 genes appeared down-regulated expression in R₁.In SP vs RP,1762 different expressed genes were selected and among them 1490 genes appeared up-regulated expression and 272 genes appeared down-regulated expression in RP.There were 944 co-different expressed genes between 8A vs R₁ and SP vs RP,amomg them 891 genes appeared up-rugulated expression in both R₁ and RP,and 53 genes appeared down-regulated expression in both 8A and SP.Fourthly,the KEGG pathway analysis of the 891 up-rugulated genes indicated that many pathways were closely related to the fertility restorer,such as Citrate cycle?TCA cycle?,Pentose phosphate pathway,Glycolysis/Gluconeogenesis,Oxidative phosphorylation,Starch and sucrose metabolism,Ubiquitin mediated proteolysis,beta-Alanine metabolism,Phenylalanine metabolism,Tyrosine metabolism,Cysteine and methionine metabolism,ABC transporters,Phosphatidylinositol signaling system and Inositol phosphate metabolism.Fifthly,nine genes related to the fertility restorer were selected,such as Capana00g002348 ? Capana00g004424 ? Capana02g000930 ? Capana00g003267 ?Capana01g002849 ? Capana05g002270 ? Capana01g004065 ? Capana06g002131 and Capana02g001595 so on,which encode putative fructokinase,phosphatidylinositol 4-phosphate 5-kinase,pectate lyase,exopolygalacturonase,pectinesterase,cellulose synthase,fasciclin-like arabinogalactan protein,phosphoinositide phospholipase C?PLC?and ABC transporter B family member 9,respectively.Sixthly,the fertility restorer genes were mapped onto Chromosome 03 and Chromosome 06 using BSR methord.The region on Chromosome 03 covered 0.24 Mb and were composed of 19 reference genes.The region on Chromosome 06 covered 16.47 Mb and were composed of 231 reference genes.The total region covered 16.71 Mb and were composed of 250 reference genes.Seventhly,according to the co-different expressed genes and function annotation between the mapping region,two candidate genes,Capana06g002866 and Capana06g002871 were selected,both of which encode NEDD8-conjugating enzyme and are related to the fertility restorer.The results of q RT-PCR showed that the expression of two candidate genes was obviously higher in every stage of F1 than that of 8A,and the changes of expression among four stage were not obviously in 8A.While the changes of expression between the first two stage were basically unchanged in F1,but the expression improved rapidly at stage ?,and peaked at stage ? which the expression level were far higher than that of first two stage.These indicated that the expression of two candicate genes were significantly correlated to the normal developmental process of anther.