Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq.

A combination of BSA and RNA-seq was performed to identify candidates for the restorer-of-fertility gene in onion. The AcPMS1 involved in DNA mismatch repair was identified as the best candidate. To identify candidate genes of the restorer-of-fertility gene (Ms) responsible for fertility restoration of onion cytoplasmic male-sterility, a combined approach of bulked segregant analysis and RNA-seq was employed. From 32,674 de novo assembled contigs, 430 perfectly homozygous SNPs between male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. After verifying the homozygosity of the SNPs by PCR amplification and sequencing, the SNPs on 139 of the contigs were genotypes for the two recombinants which contained crossover events between the Ms locus and two tightly linked molecular markers. As a result, 30 contigs showing perfect linkage with the Ms locus in the large-sized segregating population were identified. Among them, 14 showed perfect linkage disequilibrium (LD) with the Ms locus, as determined by genotyping 251 domestic breeding lines. Furthermore, molecular markers tagging the 14 contigs also showed almost perfect LD with each other in 124 exotic accessions introduced from 21 countries, except for one accession which contained a crossover event by which the 14 markers were divided into two groups. After sequencing of the full-length cDNA of the 14 contigs showing perfect LD, the deduced amino acids sequences of the MF and MS alleles were compared. Four genes were shown to harbor putative critical amino acid changes in the known domains. Among them, the gene encoding PMS1, involved in the DNA mismatch repair pathway, was assumed to be the best candidate gene responsible for fertility restoration of male-sterility in onion.