Identification of a variant of CMS-T cytoplasm and development of high resolution melting markers for distinguishing cytoplasm types and genotyping a restorer-of-fertility locus in onion (Allium cepa L.)

Abstract

Cytoplasmic male-sterility (CMS) has been used in F1 hybrid seed production in onion (Allium cepa L.). Two types of CMS (CMS-S and CMS-T) have been reported in onions. Almost complete mitochondrial genome sequences of normal, CMS-T, and CMS-S cytoplasm types have been obtained in previous studies. Unlike highly divergent CMS-S mitochondrial genome, there were only three single nucleotide polymorphisms (SNPs) between normal and CMS-T mitochondrial genomes except for orf725, the causal gene for CMS induction. Cleaved amplified polymorphic sequence (CAPS) and high resolution melting (HRM) markers were developed based on one of these SNPs. When these markers were tested for 243 diverse breeding lines, four of them containing CMS-T cytoplasm showed SNP genotypes of the normal cytoplasm. Although mitochondrial genome organizations of these four accessions were assumed to be similar to those of normal and CMS-T, the copy number of orf725 was less than half of that of general CMS-T, suggesting that male-sterility of this variant of CMS-T might be induced by an independent event of substoichiometric shifting of orf725. To distinguish normal and all types of CMS cytoplasms, an HRM marker was developed on the basis of cox1 and orf725. In addition, for genotyping the Ms locus, a nuclear restorer-of-fertility (Rf) gene, approximately 15 kb full-length genomic DNA sequences of AcPMS1, a candidate Rf gene, were obtained. Two reproducible HRM markers were developed among ten candidate HRM markers designed on the basis of more than 800 SNPs or InDels between AcPMS1 alleles.