| 摘要: |
| [目的]分离柿属植物的微卫星标记,为柿种质资源的分类、进化等遗传研究奠定基础.[方法]提取磨盘柿(Diospyros kaki)基因组DNA后,用EcoRⅠ和MesⅠ 2种内切酶酶切并连接接头,再用接头特异引物进行PCR扩增.扩增产物与生物素标记的(GA)15和(AC)15探针杂交,杂交复合物用链亲和素包裹磁珠进行结合,得到单链DNA目标片段,经PCR扩增.连接pGEM-T载体,转化入感受态大肠杆菌,得到微卫星富集小插入片段DNA文库.用Colony-PCR法筛选阳性克隆,进行测序分析.[结果]测序96个克隆,54个含微卫星序列,24个为有效微卫星序列,其中完美型(perfect)9个,占37.5%;非完美型(imperfect)7个,占29.2%;混合型(compound)8个,占33.3%.(GA)n重复最为常见.21条含微卫星序列已登录GenBank(Accession Number:EF567396～EF567416).成功设计21对引物,经初步筛选,14对表现出多态性.[结论]试验方法分离柿基因组微卫星简便有效,得到的多态性引物可用于柿种质资源的遗传研究. |
| 关键词: 柿 微卫星 磁珠富集 |
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| 基金项目:科技部国家科技基础条件平台项目（2005DKA21002-22） |
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| Isolation of microsatellite in Diospyros kaki by magnetic beads |
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| Abstract: |
| 【Objective】 Isolating microsatellite markers of persimmon is important for germplasm resources,the classification and evolution of persimmon's genetic study.【Method】 Genomic DNA of Diospyros kaki cv Mopanshi was extracted and digested with restriction enzyme Eco RⅠ and MesⅠ.Adapters were ligated to the end of restriction fragments.Ligated products were amplified by PCR with primers complementary to adapters.The PCR products were hybridized with biotin-labelled Oligo (GA)15 and (AC)15 probes,hybridized complex was fished by magnetic beads coated with streptavidin.Then selected fragments were amplified by using adapter specific primers and clonedinto pGEM-T vectors and transformed into E.coli hosts,SSR enriched genomic DNA library was constructed.96 clones were selected by colony-PCR method and then sequenced.【Result】 24 distinct microsatellite loci were detected.Of these sequences,9 (37.5%) were perfect repeat motif,7 (29.2%) imperfect and 8 (33.3%) compound repeat motifs.(GA)n motif was the most common repeat.PCR primers were designed for 21 loci.GenBank Accession Number was EF567396-EF567416.【Conclusion】 14 of the primer pairs were characterized with polymorphism by this simple and effective method,and could be used for genetic studies on Diospyros spp. |
| Key words: Diospyros kaki microsatellite magnetic beads enriched library |
