Development of Functional Markers for Detection of Inactive DFR-A Alleles Responsible for Failure of Anthocyanin Production in Onions (Allium cepa L.)

Abstract

Inactivation of the gene coding for dihydroflavonol 4-reductase (DFR) is responsible for the color difference between red and yellow onions (Allium cepa L.). Two inactive DFR-A alleles, DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX>, were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-<TEX>$A^{PS}$</TEX> allele. A derived cleaved amplified polymorphic sequences (dCAPS) primer was designed to detect the single nucleotide polymorphism, an A/T transition, which produced the premature stop codon. Digested PCR products clearly distinguished the homozygous and heterozygous red <TEX>$F_2$</TEX> individuals. Meanwhile, to develop a molecular marker for detection of the DFR-<TEX>$A^{DEL}$</TEX> allele in which entire DFR-A gene was deleted, genome walking was performed and approximately 3 kb 5' and 3' flanking sequences of the DFR-<TEX>$A^R$</TEX> coding region were obtained. PCR amplification using multiple primers binding to the extended flanking regions showed that more of the extended region of the DFR-A gene was deleted in the DFR-<TEX>$A^{DEL}$</TEX> allele. A dominant simple PCR marker was developed to identify the DFR-<TEX>$A^{DEL}$</TEX> allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX> alleles in yellow onion cultivars bred in Korea and Japan was surveyed using molecular makers developed in this study. Results showed predominant existence of the DFR-<TEX>$A^{PS}$</TEX> allele in yellow onion cultivars.