A new co-dominant PCR-based marker to identify the high-molecular-weight glutenin subunit combination “ 5 + 10 ” of common wheat

Glutenin, which is one of the major storage proteins of wheat, is composed of high and low molecular weight subunits. The high-molecular-weight subunits encoded by the Glu-A1, -B1 and -D1 genes are closely related to bread-making quality. Each locus has several alleles, among which the Glu-D1d allele, responsible for producing the subunit combination defined as "5+10", has the greatest positive effect on bread-making properties. We developed a new PCR-based co-dominant marker to identify this allele. This marker can also be used for multiplex PCR along with previously developed markers for the selection of partial waxy wheat lines. The bread-making quality of wheat flour depends on the quantity and quality of storage proteins. In particular, high-molecular-weight glutenin (HMWG) has a large effect on bread-making quality, and several types of HMWG subunits have been identified (Shewry 2003). The "5+10" subunit combination is known to have a positive effect on dough properties and most cultivars possessing this subunit profile show good bread-making quality (Shewry 2003). Since most of the cultivars produced in Japan's wheat breeding programs were developed for "Udon" noodles, the strong dough properties required for bread-making were not a major focus. Only two cultivars with the "5+10" subunit combination were identified when the HMWG subunits of Japanese cultivars were analyzed twenty years ago. However, there is a high demand for new cultivars with good bread-making quality due to increasing interest in food safety and domestic food products. Therefore, in the last decade, breeders have made efforts to introduce the "5+10" subunit combination into Japanese wheat lines using the SDS-PAGE screening method. During the same time period, DNA marker assisted selection (MAS) has been introduced into several public breeding programs, including rice, vegetable, orchard plant, and wheat programs. In particular, MAS for partial waxy lines with low amylose has been widely using in breeding programs for noodle-making cultivars. This success has induced demand for the development of a DNA marker for MAS of the "5+10" subunit combination. The HMWG subunits are encoded at three homoeologous complex Glu-1 loci, Glu-A1, -B1 and -D1. Each locus has two tightly linked structural genes encoding a larger x-type subunit and a smaller y-type subunit (Payne et al. 1981). Various alleles occur at each locus, and the "5+10" subunit combination is derived from the Glu-D1d allele (Payne and Lawrence 1983). Although PCR-based markers for a number of subunits have been developed (e.g. Ahmad 2000; de Bustos et al. 2001; Ma et al. 2003; Moczulski and Salmanowicz 2003), most of these, including the markers for the "5+10" subunit combination, are dominant markers that amplify target products only when the targeted allele exists. When we tested the markers designed for the "5+10" subunit combination, we could not get clear amplified target fragments of the reported sizes. Unfortunately, a non-specific amplification product of the same size as the expected fragment from the "5+10" allele was identified in lines which do not carry this allele. In addition, reproducibility was poor, although we carefully conducted PCR using the same procedure and solutions as outlined in the original reports. All markers were dominant, and as such, were not suitable for continuous backcross procedures because they could not distinguish heterozygous from wild type F2 plants. To solve these problems, we attempted suitable