Development of a CAPS marker for Rf1 based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton

Germplasm and Genetic Stocks

poster

Wu, Jianyong; Xing, Chaozhu; Cao, Xiuxia; Guo, Liping; Qi, Tingxiang; Wang, Hailing; Tang, Huini; Zhang, Jinfa

Wu, Jianyong

Xing, Chaozhu

The cytoplasmic male sterility (CMS) system is convenient and efficient for cotton hybrid seed production. However, because of the limited restorer lines carrying the restorer gene Rf1, it has not been widely used. So, to improve the efficiency of restore line breeding, we want to develop some new tightly markers related to Rf1 gene. In this study, the fertility segregation in a backcross (BC8F1) population of 409 individuals and an F2 population of 695 plants confirmed that the fertility restoration of CMS-D2 was determined by one dominant restorer gene (Rf1). Subsequently, 13 Rf1-linked simple sequence repeat (SSR) markers were identified from 8248 pairs of SSR primers. A combined map showed that the 13 polymorphic SSR markers were mapped in a region of 0.279 cM encompassing the Rf1 gene. Sequence alignment showed that they were distributed on nine scaffolds of chromosome 9 in the sequenced D5 genome of G. raimondii. Then, ten pentotricopeptide repeat (PPR)-like genes were identified on two of the nine scaffolds, including Scaffold 333 where nine PPR-like genes were clustered in a region of about 160 kb with marker BNL3535 in. Among them, PPR-like gene Cotton_D_gene_10013437 was the nearest gene with BNL3535, comparative sequence analysis of the homologous gene among sterile (A), maintainer (B) and restorer (R) lines, and co-segregation analysis further confirmed that this gene maybe the candidate for the Rf1 gene. Compared with the non-restoring lines, the restorer had a 9-nucleotide (nt) insertion and a single nucleotide polymorphism (SNP) 8 nt upstream of the insertion at the 3' untranslated regions (3' UTRs) in this gene. And this SNP leads to the deficiency of a cleavage site for restriction enzyme DraI in the restorer line, so then a cleaved amplified polymorphic sequence (CAPS) marker named CAPS-R was developed from the SNP site, and was further used to track the restorer gene and its homozygous or heterozygous status in molecular breeding for restorer lines. In our previous study, a CMS-D2 cytoplasm-specific SCAR marker has beed developed, and can be used to identify the CMS-D2 cytoplasm. So, here a marker-assisted selection system using the Rf1-specific CAPS-R marker and a CMS-D2 cytoplasm-specific SCAR marker was established to distinguish the three-line hybrids from other genotypes and artificial emasculation and pollination.