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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 nucleotide repeat characteristics of Artemisia annua EST-SSRs and develop microsatellites markers to provide theoretical basis and technical support for the utilization of germplasm resources of Artemisia annua. METHODS: 94 923 EST sequences of Artemisia annua were cloned and sequenced. The sequences of EST-SSRs were obtained by MISA. The composition and distribution of EST-SSRs were analyzed. Pfam2go annotation Artemisia annua SSR-ESTs dataset, GOSlim program to annotate results. Primer3 program designed 18 pairs of SSR primers amplified genomic DNA of Artemisia annua, polyacrylamide gel electrophoresis to detect polymorphisms. Results: 24 601 sequences were obtained after splicing of Artemisia annua, including 2 110 SSRs with frequency of 8.6%. Among them, 28% and 50.4% of them were duplicated by two and three nucleotides, respectively. ACC / GGT As the main type, accounting for 9.8%. 312 SSR-ESTs of A. annua were functionally annotated. 15 pairs of primers to establish a suitable PCR reaction system in 36 samples of Artemisia annua amplified good polymorphism. Conclusion: There are abundant EST-SSR motifs in Artemisia annua L., which are highly effective in 18 pairs of primers and SSR markers are developed according to the size of EST resources. In order to further develop the germplasm resources of Artemisia annua Research laid the foundation.