铁皮石斛EST-SSR引物的评价、甄选与应用研究

Evaluation,Selection and Application of Dendrobium officinale EST-SSR Primers

开发、评价已筛选的铁皮石斛EST-SSR引物,并探讨其在遗传多样性、物种鉴定、遗传图谱构建、亲缘关系鉴定等方面应用。利用前期筛选的20对多态性较好的引物用于16份材料(15份铁皮石斛与1份串珠石斛)的聚丙烯酰氨凝胶电泳,统计扩增条带。采用Pop Gen32软件进行Nei’s基因多样度和Simpson指数等多态性指标的统计,分析单个引物和组合引物的区分率,之后采用NTSYSpc2.1软件计算遗传相似系数聚类图的构建。结果表明,引物DN4等16对引物多态性较高,而DN13、DN23、DN60和DN67多态性较低。单个引物均不能将所有供试样品区分开来,其中DN4、DN10、DN105、DN81、DN39和DN71区分率相对较高;利用组合引物能够将所有样品进行有效区分,其中DN4+DN10+DN105+DN39引物组合可作为核心引物予以使用,可用于铁皮石斛指纹图谱的构建和遗传多样性分析。聚类分析结果表明,该20对引物可以较好地应用于铁皮石斛种内、种间亲缘关系的鉴定。此外,引物DN4、DN13、DN39、DN58、DN65、DN67、DN10和DN99可用于铁皮石斛与串珠石斛的鉴定。本研究开发的20对引物可较好地应用于铁皮石斛遗传多样性分析、种质鉴定、亲缘关系鉴定和遗传图谱构建等方面,具有广泛的应用性。

By developing and evaluating EST-SSR primers, we studied their application in genetic diversity, species identification, genetic map construction, genetic relationship identification of Dendrobium officinale. The early developed 20 pairs of primers with good polymorphism were used for the analysis of 16 samples（ 15 samples of D. officinale and 1 sample of D. falconeri） by polyacrylamide gel electrophoresis, and the amplified bands were calculated. Nei＇s gene diversity and Simpson index were tested using PopGen32 to analysis the differentiation rate of single primer and combination primers. Finally, the construction of genetic similarity coefficient clustering map was calculated by using NTSYSpc2.1. The 16 pairs of primers, such as DN4, were highly polymorphic, while lower in DN13, DN23, DN60 and DN67. Although single primer could not distinguish all the samples, DN4, DN10, DN105, DNS1, DN39 and DNT1 displayed relatively higher distinguish rate. All the samples could be distinguished effectively by combined primers, and the DN4 ＋ DN10 ＋ DN105 ＋ DN39 primer set could be used as the core primer for fingerprint construction and genetic diversity analysis of D. officinale. By cluster analysis, the 20 pairs of primers could be used for the identification of both intraspecific and interspecific phylogenetic relationships of D. officinale. The primers DN13, DN39, DN4, DN58, DN65, DN67, DN10 and DN99 could be used for the identification of D. officinale and D.falconeri. The 20 pairs of primers could be wildly used in the application of genetic diversity analysis, germplasm identification,