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

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.

摘要采用生物信息学方法将大豆EST序列联配到大豆基因组序列上, 挖掘到大豆EST-SNP位点537个。对其靶向基因进行功能注释分析, 发现他们主要参与亚细胞定位、蛋白质结合与催化以及代谢等与大豆重要农艺性状形成相关的生物过程。同时开发了简便易行的SNP检测方法, 利用EMBOSS软件筛选导致酶切位点改变的EST-SNP, 分别以大豆绥农14、合丰25、Acher、Evans、Peking、PI209332、固新野生大豆、科丰1号、南农1138-2的DNA及其混合的DNA为模板, 设计引物进行PCR扩增, 发现44个PCR产物中有36个测序峰图在预期的EST-SNP位点表现出多态性。酶切分析发现26个PCR产物具有酶切多态性, 可以作为CAPS标记。结果表明, 该EST-SNP挖掘体系及其CAPS标记转化系统具有高效率、低成本等优点, 有利于促进大豆的遗传育种研究。