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

Abstract

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-A PS and DFR-A DEL , were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-A PS 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 F 2 individuals. Meanwhile, to develop a molecular marker for detection of the DFR-A DEL 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-A R 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-A DEL allele. A dominant simple PCR marker was developed to identify the DFR-A DEL allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-A PS and DFR-A DEL 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-A PS allele in yellow onion cultivars.

Molecular markers in the improvement of Allium crops

“…Furthermore, RT-PCR results showed that no DFR-A transcript was detected in any yellow F 2 individuals. Further, Park et al (2013b) …”

Enhancing onion breeding using molecular tools

“…Four alleles encoding anthocyanidin synthase (ANS) have been proposed to condition red, pink and yellow colour in bulbs (Kim et al 2006). DFR and ANS mapped on chromosomes 7 and 4, respectively, and functional markers from these candidates have been developed, which could be used in marker-assisted selection for bulb colour (Masuzaki et al 2006, Khar et al 2008, Kim et al 2009, Park et al 2013, Duangjit et al 2014.…”

Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.)

“…1 positive control containing the functional DFR-A R1 allele, 2 430C, 3 PI276324, 4 control containing the DFR-A DEL allele in which the entire DFR-A gene was deleted. The 898-bp PCR products amplified using the primer combinations of DFR-DEL-F, DFR-DEL-R1, and DFR-DEL-R2 were amplified from the DFR-B gene, an inactive pseudogene (Park et al 2013) (DFR-LF1 and DFR-DEL-R1) binding to the 5′ and 3′ flanking sequences of the DFR-A gene (Fig. 1a), resulting in the amplification of single large-sized PCR products in both accessions ( Supplementary Fig.…”