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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