磁珠富集法开发北柴胡多态性简单序列重复标记

目的:利用磁珠富集法分离北柴胡微卫星序列,以开发北柴胡微卫星引物,获得有多态性的简单序列重复(SSR)标记。方法:用生物素标记的混合探针(AC)15、(AG)15、(MAB)12和两端连接已知序列人工接头的北柴胡基因组DNA酶切片段混和后与磁珠杂交,构建微卫星序列富集的小片段插入文库;利用接头引物分别与生物素探针引物Biotin-(AC)15、Biotin-(AG)15、Biontin-(MAB)12形成3个组合,用PCR方法对文库进行初步筛选;对可能的阳性克隆子进行测序复筛,选取微卫星侧翼序列足够长的序列设计引物,用荧光标记的基因分型技术以栽培柴胡种质为材料分析其多态性。结果:开发了5对多态性SSR标记,它们在5份柴胡栽培种质中共扩增出30.70个多态性等位基因,平均每条引物可以扩增出6.14个多态性等位基因;观察等位基因数最多13个,最少3个;有效等位基因数最多11.4个,最少1.6个。同时分析了4对EST-SSR引物,比较了2种SSR标记扩增结果。结论:磁珠富集法是开发柴胡多态性SSR标记的有效方法。

Development of Polymorphic Simple Sequence Repeats Markers in Bupleurum chinense DC.by Magnesphere

Objective：To develop simple sequence repeats（SSR） primer pairs with the method of isolation of genomic microsatellite in Bupleurum chinense DC.by magnesphere and to obtain the polymorphic SSR markers.Methods：To construct a microsatellite sequence enriched library,the DNA fractions with 200～1500 bp containing microsatellite sequences of B.chinense were captured by hybridizing the digested genomic DNA fragments with the oligonucleotide probes Biotin-（AC）15,Biotin-（AG）15 and Biontin-（MAB）12 attached to streptavadin coated magnetic beads.PCR screening using adaptor primer and one of Biotin-（AC）15,Biotin-（AG）15 and Biontin-（MAB）12 as primer pairs was conducted separately.Probable positive clones were selected and sequenced.Then flanking primers were designed.Finally,polymorphism of the primers was detected by fluorescence labeled genotyping of Bupleurum cultivated germplasms.Results：Five polymorphic SSR markers were created.An analysis of five Bupleurum cultivated germplasms with these SSR markers detected 30.70 polymorphic alleles,with an average of 6.14.Observed number of alleles is up to 13 and low to 3;while the effective number of alleles is up to 11.4 and low to 1.6.Another four EST-SSR markers were used to compare the polymorphism with these five genome SSR markers in these materials as well.Conclusion：The results demonstrated that magnesphere was an efficient method to develop polymorphic SSR markers in Bupleurum spp.