大豆EST-SNP的挖掘、鉴定及其CAPS标记的开发

Mining and Identification of SNP from EST Sequences and Convertion of CAPS Markers in Soybean

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。

SNPs widely distribute throughout genomes from non-coding regions to coding regions,constituting the most abundant molecular markers used in animal and plant genetic breeding. With the rapidly growing genome sequencing projects,a large amount of genomic and EST sequences has become available to the public. Many SNPs are identified by comparing genome sequences or ESTs obtained from genetically diverse lines or individuals in plants. However the SNP assay always relies on expensive equipments or reagent,which has limited the application of SNPs in genetics and breeding especially in plants. The CAPS marker,also known as PCR-RFLP marker,is the technique combining PCR and restriction enzymes digestion to detect the restriction fragment length polymorphisms. With the development of high-throughput sequencing technology,more and more SNPs are identified,among them many mutations have altered the restriction enzymes recognition sites. This provides an opportunity for high-through development of CAPS markers. With soybean genome sequences becoming available,high-throughput SNP marker development will significantly improve genetic mapping,map based cloning in soybean. To discover new SNPs in soybean,we aligned the ESTs with whole genome sequences in different soybean varieties and identified 537 EST-SNPs. The function of genes targeted by these EST-SNPs was analysed,the results showed that these genes participated in subcellular localization,protein binding or catalyzing,metabolic process and cell rescue,defense and disease resistance,etc. Most of these functions are involving in various physiological and biochemical processes influencing important agronomic traits. To develop easy assay method for these EST-SNPs,we identified the EST-SNPs which alter the restrict enzyme recognition sites by software EMBOSS,and 48 pair primers were designed to detect these EST-SNPs. forty-four pair primers amplified single bands （400–800 bp） from genomic DNA of Suinong 14 widely planted in the Northeast China. To verify the SNP polymorphisms,we used these primer pairs for PCR am-plification from genomic DNA of Suinong 14,Hefeng 25,Acher,Evans,Peking,PI209332,Guxin wild soybean,Kefeng 1,Nan-nong 1138-2 and pool DNA of the nine soybean varieties. The PCR amplicons were sequenced,the traces of the 36 discordant ones were detected as candidate SNPs,which were then validated by re-sequencing the individuals. SNPs were identified using restriction enzymes,and the products of 26 pair primers with unequivocal restriction pattern were identified as CAPS markers. The SNPs discovery and CAPS markers conversion system developed in this study is fast,low cost and efficient,and holds great promise for molecular assisted breeding of soybean.