Abstract
The DFR-A gene, which encodes dihydroflavonol 4-reductase, an enzyme involved in the anthocyanin biosynthesis pathway, was previously identified as the causal gene responsible for yellow bulb color in onion (Allium cepa L.). Among the various DFR-A alleles, no PCR product corresponding to the DFR-A sequence could be amplified from the DFR-A (DEL) allele. However, the precise boundaries of the suspected deletion had not been determined. Guided by the onion whole genome sequence, the approximate boundaries of the deletion were identified through serial PCR amplifications and sequence analysis of the PCR products. The 5' and 3' boundaries were connected via long PCR amplification in the DFR-A (DEL) allele, revealing that a 982,099 bp region was likely deleted. Based on the junction sequence and the conserved 5' upstream region of DFR-A, a codominant marker was developed for detection of the DFR-A (DEL) allele. A perfect correlation between bulb color phenotypes and marker genotypes was observed in a segregating population. Analysis of 197 onion accessions revealed that 15 accessions previously believed to be homozygous for other DFR-A alleles were, in fact, heterozygous and carried the DFR-A (DEL) allele. The codominant marker was also used to identify a novel DFR-A allele in an accession previously thought to be homozygous for the DFR-A (DEL) allele. In this newly designated allele, DFR-A (DTP2) , a 5,479-bp DNA transposon belonging to the CACTA superfamily was inserted into exon 1, introducing a premature stop codon. Based on the structures of the DFR-A (DEL) and DFR-A (DTP2) alleles, the stepwise process for identifying specific DFR-A alleles was improved. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-026-01637-w.