Identification of a Novel DFR-A Mutant Allele Determining the Bulb Color Difference between Red and Yellow Onions (Allium cepa L.)

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-AR4-like allele. Sequencing of 4,830-bp full-length sequences of this DFR-AR4-like allele revealed that the nucleotide sequences of the DFR-AR4 and DFR-AR4-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-APS2. The RT-PCR results showed that transcripts of the DFR-APS2 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-APS2 allele among 16 different DFR-A alleles. No additional accession was found to contain the DFR-APS2 allele from 155 diverse onion germplasm, indicating very limited distribution of this new DFR-APS2 allele.