Devolopment And Analysis Of SSCP Markers In Candidate Region Of Restorer Gene For Cytoplasmic Male Sterility Cotton

The cytoplasmic male sterility（CMS） system is convenient and efficient for hybrid seed production in Upland cotton. Molecular Molecular mapping and analysis of restore gene is of great significance for better understanding the molecular mechanism and improving restorer lines. In this study, the cytoplasmic male sterile lines and restorer lines were used to establish a F2 population containing 2500 individuals, then the genetic model of fertility restoring genes was determined through the fertility investigation of each plant. The previously identified polymorphic 13 SSR marker sequences were then used to perform a BLAST search against the D5 genome sequence of G. raimondii to determine their distribution. Those PPR and not-PPR genes were then used to development the SSCP markers, the bulked segregant analysis（BSA） was used to search SSCP markers linked to the Rf1 gene. According to the phenotype and genotype of each plant in the F2 populations, genetic distances between Rf1 and associated markers were calculated and a genetic linkage map was constructed using Join Map 4.0. On the other hand, quantitative RT-PCR was performed to determine the expression profile of 17 candidate genes in candidate region in different stage flower buds about 1-4 mm in length between CMS, maintainer and restorer line. The main results are as follows:1. 13 previously identified markers were distributed in 9 different scaffolds, nine PPR-like genes were clustered in a region of about 160 kb on Scaffold 333, in which with the nearest marker BNL3535 located between two PPR genes Cotton_Dgene₁0013437 and Cotton_Dgene₁0013446, the corresponding physical distance is 40.89 kb and 53.66 kb respectively.2. 155 pairs of SSCP primers covered all 18 genes were designed, 15 of them were identified as polymorphism primers. Combining with the previously published 3 SSR markers, the 18 markers were mapped in a region of 4.8 c M. BNL3535 was the nearest SSR maker, with a genetic distance of 2.9 c M, 343401-1 and 344805-2 were the nearest SSCP makers, with a genetic distance of 3.2 c M.3. The results of q RT-PCR of 17 genes in candidate region showed that no obvious difference could be detected at the early stage（1-2 mm） buds for 9 PPR-like genes, but at the stage of 3-4 mm, the expression profiles of these 9 genes were quite different. As for those not-PPR genes, obvious differences could be detected for most of them.