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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To introduce downy mildew resistance from a yellow-colored resistant cultivar, ‘Santero’, into a yellow breeding line, OT803, the F₁ hybrid was produced by crossing Santero and OT803. The bulb color of the F₁ hybrids became light pink, suggesting involvement of complementation between the DFR-A and ANS genes in the onion anthocyanin biosynthesis pathway. Since Santero contained active DFR-A and inactive ANS alleles, OT803 was assumed to harbor active ANS and inactive DFR-A alleles. However, some yellow-colored individuals of OT803 were shown to contain the homozygous genotype of the active DFR-A^R4-like allele. Sequencing of 4,830-bp full-length sequences of this DFR-A^R4-like allele revealed that the nucleotide sequences of the DFR-A^R4 and DFR-A^R4-like alleles were identical except for a single nucleotide deletion in the last exon. This single base-pair deletion resulted in creation of a premature stop codon at 2-bp downstream of the deletion mutation. This new DFR-A mutant allele was designated DFR-A^PS2. The RT-PCR results showed that transcripts of the DFR-A^PS2 allele were significantly reduced, suggesting involvement of nonsense-mediated mRNA decay (NMD) mechanism. The systematic process consisting of PCR amplification and sequencing of the PCR products was modified to identify the DFR-A^PS2 allele among 16 different DFR-A alleles. No additional accession was found to contain the DFR-A^PS2 allele from 155 diverse onion germplasm, indicating very limited distribution of this new DFR-A^PS2 allele.

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