关键词: 大豆, 重测序数据, 单核苷酸多态性, 酶切扩增多态性序列, 基因靶向功能标记

Abstract:

Soybean (Glycine max) is an important global crop and main source of oil and protein, so the research on economic traits of soybean is valuable. Single nucleotide polymorphism (SNP) has become important genetic markers according to its high density, co-dominance and so on. Gene-driven SNPs affecting traits relevant to host gene’s function are important type of molecular marker. Large numbers of re-sequencing data in database make it possible to easily develop SNP markers by bioinformatics technology. Cleaved amplified polymorphism sequence (CAPS) are also known as PCR-RFLP markers, provides a way to utilize the DNA sequences of mapped RFLP markers to develop PCR based markers thereby eliminating the tedious DNA blotting. The CAPS markers are co-dominant, locus specific, easily to use, lower cost and have been used to distinguish between plants that are homozygous or heterozygous for alleles. Thus, CAPS proves useful for genotyping, positional or map based cloning and molecular identification studies where sequence-based identification is not feasible. Many SNPs localize in restrict enzyme sites, so they could be identified with CAPS. A software was compiled and implemented, and the soybean genes’ re-sequencing data were analyzed by the bioinformatics method, and SNP sites which alter the restrict enzyme recognition sites were identified to develop the gene-deriven functional markers of soybean. Then 163 pair primers were designed to detect SNPs. 139 pair primersamplified single bands (400–1 200 bp) from genomic DNA of Suinong 14, which was widely planted in the Northeast China. To verify the SNP polymorphisms, we used these primer pairs for PCR amplification from genomic DNA of the nine soybean genotypes Suinong 14, Hefeng 25, Acher, Evans, Peking, PI209332, Guxin wild soybean, Kefeng 1 and Nannong 1138-2. The PCR amplicons were digested with different restriction endonucleases, 73 amplicons had polymorphism among the nine genotypes after digested, and 73 gene specific CAPS markers were developed. And then the functions of these genes, which these markers were driven by, were annotated by blast against the Arabidopsis proteins, and the soybean genes’ functions were grouped and analyzed. Result indicated that these genes participate in subcellular localization, protein binding or catalyzing, metabolic process and cell rescue, defense and disease resistance, etc. Most of these functions are relevant to important agronomic characters of soybean, so they have more possibility to be the ideal tools for marker-assisted breeding of soybean, and to be valuable for soybean genetic diversity analysis and molecular phylogeny analysis..The result means that these SNPs have more potential to be used in soybean breeding.

Key words: Soybean, Re-sequencing data, SNP, CAPS, Gene-driven functional marker