Development of a molecular marker tightly linked to the C locus conferring a white bulb color in onion (Allium cepa L.) using bulked segregant analysis and RNA-Seq

摘要

To develop molecular markers linked to the C locus determining a white bulb color in onion (Allium cepa L.), a combination of bulked segregant analysis (BSA) and RNA-Seq was performed using bulked RNAs extracted from 12 plants each of yellow and white F-2 bulbs. Ninety-seven genes showed at least five-fold increased expression in the yellow bulk. Eleven previously isolated structural genes were identified from these genes. Two homologous genes coding for phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also identified. In addition, three glycosyltransferase genes and two acyltransferase genes showed increased transcription in the yellow bulk. Increased expression of 18 structural genes in the yellow F2 individuals were further confirmed by reverse transcription polymerase chain reaction (RT-PCR). Regarding regulatory genes, a gene encoding a WRKY transcription factor was most significantly down-regulated in the white bulk. In the MBW complex, known to control a flavonoid biosynthetic pathway, 47, four, and eight genes encoding putative MYB, bHLH, and WD40 proteins, respectively, were identified from transcriptome. Among them, two MYB-, one bHLH-, and one WD40-coding gene(s) showed up-regulation in the yellow bulk and high similarity with other known regulators of flavonoid biosynthesis. Among 1829 contigs containing single nucleotide polymorphisms (SNPs) or InDels, five contigs were shown to be linked to the C locus. In addition, SNPs in the gene encoding glutathione S-transferase (GST) also showed a linkage relationship. Six molecular markers were developed and a linkage map was constructed using 586 F2: 3 individuals. The GST1 marker based on the GST-coding gene showed perfect linkage to the C locus.