Mining and Identification of SNPs from EST Sequences in Soybean and Converting SNP markers into CAPS

Abstract

Abstract Single nucleotide polymorphism (SNP) is a type of ideal markers distributed widely throughout genomes. The objectives of this study were to develop novel SNPs in soybean [ Glycine max (L.) Merr.] and convert them into cleaved amplified polymorphic sequence (CAPS) markers. A total of 62,939 expressed sequence tags (ESTs) were aligned with the whole genome sequences in different soybean varieties and 537 EST-SNPs were identified. These EST-SNPs participated in various physiological and biochemical processes that influence important agronomic traits, such as subcellular localization, protein binding or catalyzing, metabolic process, and cell rescue, defense and disease resistance. Using the Restrict program in EMBOSS software, 48 EST-SNPs were identified with alteration of the restrict enzyme recognition site, and 48 pairs of primers were designed accordingly to detect these EST-SNPs. Forty-four pairs of primers amplified single band (400–800 bp) from genomic DNA of Suinong 14, which is widely planted in Northeast China. The SNP polymorphisms of these primer pairs were verified with genomic DNAs of Suinong 14, Hefeng 25, Acher, Evans, Peking, PI209332, Guxin wild soybean, Kefeng 1, Nannong 1138-2, and pooled DNA of the 9 varieties. The PCR amplicons were sequenced, and the traces of 36 discordant ones were detected as candidate SNPs, which were further validated by re-sequencing the individuals. SNPs were identified using restriction enzymes, and the products of 26 pair primers with unequivocal restriction patterns were identified as CAPS markers. The SNP mining and CAPS conversion system developed is a fast, economical, and efficient method to be used in molecular marker-assisted breeding of soybean.