During seed production of onion (Allium cepa L.) and Welsh onion (A. fistulosum L.) cultivars, seeds are inadvertently cross-contaminated with each other. However, it is difficult to identify cross-contaminated seeds by visual examination since seed and seedling morphologies of onion and Welsh onion are almost identical. To develop molecular markers for distinguishing onion and Welsh onion at early seedling stages, polymorphic mitochondrial genome sequences between two species were isolated. Using complete mitochondrial genome sequences of onions as references, genome walking was performed to isolate polymorphic Welsh onion sequences. Unlike conserved 3′ sequences flanking the atp9 gene, the 5′ flanking sequences were completely different between onion and Welsh onion mitochondrial genomes. A simple PCR marker was developed on the basis of polymorphic 5′ flanking regions of atp9, and a high resolution melting (HRM) marker was developed based on one of single nucleotide polymorphisms (SNPs) in the 3′ flanking regions. A total of 41 onion and 19 Welsh onion cultivars were analyzed using these two molecular markers. Results showed that the onion-specific marker genotype was detected only in onion cultivars, and vice versa. To estimate distribution of onion-specific and Welsh onion-specific organizations of atp9 among Allium species, 14 Allium species related to onion and Welsh onion were analyzed. Results showed that specific organizations were conserved among closely related species of onion and Welsh onion, respectively, implying that there might be no intraspecific variation in the atp9 organizations.

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