Development of a candidate gene marker for Rf 1 based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton

Abstract

The cytoplasmic male sterility (CMS) system is convenient and efficient for hybrid seed production in Upland cotton (Gossypium hirsutum L.). However, it has not been widely used because of limited restorer lines carrying the restorer gene Rf1 in the CMS-D2 system. 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 was determined by one dominant restorer gene (Rf1). A sequence alignment showed that 13 Rf1-linked simple sequence repeat marker sequences were distributed on nine scaffolds of chromosome 9 in the sequenced D5 genome of G. raimondii Ulbrich. Ten pentotricopeptide repeat (PPR)-like genes were identified on two scaffolds, including Scaffold 333 where nine PPR-like genes were clustered in a region of about 160 kb. Among them, PPR-like gene Cotton_D_gene_10013437 was identified as the candidate for the Rf1 gene through a comparative sequence analysis of the homologous gene among sterile (A), maintainer (B) and restorer (R) lines, and co-segregation analysis. 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. A cleaved amplified polymorphic sequence (CAPS) marker named CAPS-R was developed from the SNP site using the restriction enzyme DraI, and was further used to track the restorer gene and its homozygous or heterozygous status in molecular breeding for restorer lines. 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.