The development of a co-dominant marker for the β-conglycinin α′ subunit in soybeans

Abstract

The development of soybean varieties that lack the b-conglycinin a 0 subunit is an attractive goal because the b-conglycinin a 0 subunit exerts a negative influence on nutrition and tofu gelation, and is also a major allergen. We sought to develop a codominant DNA marker for the b-conglycinin a 0 subunit (Cgy-1) gene for use in marker-assisted selection (MAS). We identified a deleted sequence responsible for the null allele of Cgy-1 in a soybean variety lacking the b-conglycinin a 0 subunit known as 'Keburi'. The deletion spanned 12,998 bp and included Cgy-1 and its incomplete tandem duplicate on chromosome 10. Based on the Cgy-1 sequence from both the Williams 82 soybean reference sequence and the Keburi variety, a set of three allele-specific primers was designed for multiplex PCR assay. These primers enabled allelic discrimination by the sizes of PCR products. This amplified two distinct DNA fragments of 913 and 649 bp in Williams 82 and Keburi, respectively. The practicality of the developed co-dominant marker for Cgy-1 was also confirmed by amplification in five other soybean varieties including three wild types and two mutants. The heterozygosity of the F 1 plants at the Cgy-1 locus was ascertained using our novel co-dominant marker. This PCR-based co-dominant marker is capable of detecting the presence or absence of b-conglycinin a 0 subunit for soybean marker assisted breeding system.Keywords b-Conglycinin a 0 subunit Á Cgy-1 Á Co-dominant marker Á Seed storage protein Á Soybean

Identification of Gy4 nulls and development of multiplex PCR-based co-dominant marker for Gy4 and α’ subunit of β-conglycinin in soybean

“…The screening of soybean lines by MAS could be efficient using multiplex PCR. In this study, the developed co-dominant marker for β-conglycinin α' subunit (Kim et al, 2011) was used for multiplex PCR along with our primers for Gy4 SNAP marker.…”

“…For Gy4, both 'G' and 'A' allele-specific primer sets were used. Also, three primers designed based on the genomic sequences of Williams 82 and Keburi were used for β-conglycinin α' subunit, corresponding to each allele of the Cgy-1 locus (Kim et al, 2011). To optimize multiplex PCR conditions, various ratios of the primer concentrations (data not shown) and genomic DNA concentrations (30, 50, and 70 ng) were tested.…”

“…Two pairs of primers ('G' and 'A' allele specific) were used to amplify the genomic region of Gy4 mutant (Table 1) and three other primers were also included for amplifying the genomic region between 5' upstream and 3' downstream of the deletion region of Cgy-1 in Keburi and full Cgy-1 gene in wild-type (Kim et al, 2011).…”

“…β-conglycinin is a trimetric protein composed of α (~67 kD), α' (~71 kD) and β (~50 kD) subunits (Thanh and Shibasaki, 1978) and all three subunits of β-conglycinin have potentials for food allergens (Krishnan et al, 2009). Since α' subunit has a greater negative effect on tofu gel formation than whole β-conglycinin (Salleh et al, 2004), molecular markers were developed for identifying α' subunit-lacking soybean lines (Ishikawa et al, 2006;Kim et al, 2011).…”