Allele-specific hybridization markers for soybean

Abstract

Soybean (Glycine max) is one of the world’s most important crop plants due to extensive genetic improvements using traditional breeding approaches. Recently, marker-assisted selection has enhanced the ability of traditional breeding programs to improve soybeans. Most methods of assessing molecular markers involve electrophoretic techniques that constrain the ability to perform high-throughput analyses on breeding populations and germplasm. In order to develop a high-capacity system, we have developed allele-specific hybridization (ASH) markers for soybean. As one example, restriction fragment length polymorphism (RFLP) locus A519-1 (linkage group B) was converted into an ASH marker by (1) sequencing the pA519 cloned insert, (2) designing locus-specific PCR amplification primers, (3) comparative sequencing of A519-1 amplicons from important soybean ancestors, and (4) designing allele-specific oligonucleotide probes around single nucleotide polymorphisms (SNPs) among soybean genotypes. Two SNPs were identified within approximately 400 bp of the sequence. Allele-specific probes generated a 100-fold greater signal to target amplicons than to targets that differed by only a single nucleotide. The A519-1 ASH marker is shown to cosegregate with the A519-1 RFLP locus. In order to determine ASH usefulness, we genotyped 570 soybean lines from the Pioneer Hi-Bred soybean improvement using both A519-1 SNPs. Combined haplotype diversity (D) was 0.43 in this adapted germplasm set. These results demonstrate that ASH markers can allow for high-throughput screening of germplasm and breeding populations, greatly enhancing breeders’ capabilities to do marker-assisted selection.