Identification of allelic variation in the atp6 gene and its use in developing molecular markers for distinguishing two cytoplasmic types of bunching onion (Allium fistulosum L.)

The atp6 is a significant candidate gene that may be related to cytoplasmic male sterility (CMS) in higher plants. In this study, atp6 gene fragments were cloned from the CMS line and its maintainer line in bunching onion. The primers were designed according to sequences obtained from GenBank accession no. JQ283733. An important variation between the CMS and maintainer line was observed at the 171T and 171A position, respectively. The atp6 genes obtained from the CMS line had 171T mutations designated as T-atp6 (GenBank accession no. KR973431), and the maintainer line had 171A mutations designated as A-atp6 (GenBank accession no. KR973430). Furthermore, some plants in the maintainer line possessed both 171A and 171T mutations designated as heteroplasmic genotype A/T-atp6. To confirm this point mutation site, the derived cleaved amplified polymorphic sequence (dCAPS) method was used. Consequently, we developed a competitive allele-specific PCR (KASP) marker based on the observed single nucleotide polymorphisms (SNPs) in the atp6 genes. This marker will allow breeders to identify male-sterile or fertile cytoplasmic types on a large scale and may significantly contribute to the breeding of bunching onion F₁ hybrid cultivars.