黄花蒿EST资源的SSR信息分析及标记开发

Analysis of SSR information in EST resource of sweet wormwood(Artemisia annua) and development of EST-SSR marker

目的:研究黄花蒿Artemisia annua EST-SSRs的分布频率及核苷酸重复特征,开发微卫星(microsatellites)标记,为黄花蒿种质资源利用提供理论依据与技术支持。方法:NCBI下载黄花蒿94 923条EST序列经组装后去除冗余,MISA获得EST-SSRs序列,分析EST-SSRs组成特点和分布规律。Pfam2go注释黄花蒿SSR-ESTs数据集,GOSlim程序对注释结果进行分类。Primer3程序设计18对SSR引物扩增黄花蒿基因组DNA,聚丙烯酰胺凝胶电泳检测多态性。结果:黄花蒿拼接组装后获得24 601条序列,含2 110个SSR,出现频率为8.6%,其中二、三核苷酸重复分别占28%,50.4%,三核苷酸重复中ACC/GGT为主要类型,占9.8%。312条黄花蒿SSR-ESTs序列被功能注释。15对引物建立了合适的PCR反应体系,在36个黄花蒿样品中扩增呈现良好多态性。结论:黄花蒿EST-SSR基元类型丰富,在检测的18对引物中有效扩增和多态性均较高,根据EST资源规模开发SSR标记是可行的,为进一步开展黄花蒿种质资源的研究奠定了基础。

Objective： To study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers,providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood（Artemisia annua）.Method： EST-SSR detection was performed using Perl program MISA.Gene Ontology（GO） annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category.Primer 3 software was used to design 18 pairs primers,amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining.Result： By searching with Active Perl,totally 2 110 SSRs were detected,accounting for 8.6%.The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%,respectively.The most common repeat motifs of trinucleotide were ACC/GGT,accounting for 9.8%.Three hundred and twelve SSR-ESTs were annotated using GO terms.The suitable PCR system of 15 pairs primers was established,and revealed microsatellite polymorphism in 36 individuals.Conclusion： There are a variety of motifs at EST-SSR locus in sweet wormwood,and more effective amplification and polymorphism in 18 pairs detected primers.Therefore,EST resource is an effective and feasible approach to develop SSR markers,and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.