摘要
为了开发大豆基因靶向的功能分子标记,本研究采用生物信息学方法分析了大豆基因重测序数据,筛选出酶切位点突变的SNP位点,设计PCR引物163对,选用东北地区主栽品种绥农14的DNA为模板进行PCR扩增,其中139对引物获得大小为400～1200bp的特异片段。以大豆绥农14、合丰25、Acher、Evans、Peking、PI209332、固新野生大豆、科丰1号和南农1138-2的DNA为模板,采用筛选的139对引物进行PCR扩增,对扩增产物进行酶切分析,发现73对引物的PCR产物具有酶切多态性,开发出CAPS标记73个。通过功能注释分析发现,这73个CAPS标记靶向的基因主要参与细胞内亚细胞定位过程、蛋白质的结合与催化以及代谢过程等,与大豆重要农艺性状的形成相关,可以用于大豆品种的鉴定和分子系统进化的研究。
Single nucleotide polymorphism （SNP） is a kind of important molecular markers due to its high density and codominance. A quantity of resequencing information in the public database makes it possible to develop SNP markers by bioinformatics technique. Cleaved amplified polymorphism sequences （CAPSs）, also known as PCR-RFLP markers, can converts the mapped RFLP markers to PCR-based markers, and has been widely used in genotyping, map-based cloning, and molecular identification. To date, although more than 1 800 markers have been integrated to a soybean （Glycine max） genome map, the marker density cannot meet the requirements of genetic and breeding studies. To develop more applicable markers for the genetic linkage map of soybean, this study focused on developing gene-driven functional markers by means of using bioinformatics technique. A total of 163 pairs of primer were designed to detect SNPs, of which 139 pairs amplified single band in Suinong 14 genome with sequence length ranging from 400 to 1 200 bp. These primer pairs were further used for PCR amplification using the templates of genomic DNA from nine soybean varieties, i.e., 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, and 73 amplicons showed polymorphism among the nine varieties after digestion. Accordingly, 73 gene-specific CAPS markers were developed. According to Blast against the Arabidopsis proteins, the 73 CAPSs were classified into genes involved in subcellular localization, protein binding or catalyzing, metabolic process and cell rescue, defense and disease resistance, and so on. Most of these functions are in connection with important agronomic characteristics. Therefore, they have potential for marker-assisted selection in breeding programs of soybean. These newly developed CAPS markers are also valuable in studies on involution and genetic diversity of soybean.
出处
《作物学报》
CAS
CSCD
北大核心
2009年第11期2015-2021,共7页
Acta Agronomica Sinica
基金
国家高技术研究发展计划(863计划)项目(2006AA100104和2007AA10Z193)资助
