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藏鸡微卫星文库的构建与微卫星标记筛选
Construction of a Microsatellite DNA Library and Isolation of Microsatellite Loci in Tibetan Chicken
于颖1,2, 杨孔1,2, 王永1,徐亚欧1,郑玉才1,2,陈智华1,江晓霞1,2, 张斌1,2
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DOI:
作者单位:1.青藏高原动物遗传资源保护与利用四川省重点实验室;2.西南民族大学生命科学与技术学院
中文关键字:藏鸡;微卫星;富集文库;筛选
英文关键字:Tibetan chicken; microsatellite; enriched-library; screening
中文摘要:通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析。将藏鸡基因组DNASau3AI酶切后纯化回收,连接特定接头。用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200900bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库。从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性。实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率。
英文摘要:
A microsatellite-enriched genomic library for the Tibetan chickenwas constructed by the method of enrichment by magnetic beads, and some Tibetan chicken microsatellite sequences were isolated and analyzed. Genomic DNA was extracted from the muscle, digested with restriction enzyme Sau3AI, and connected with the specific joint. The 200~900 bp DNA fragments were captured by hybridization with the biotin-probe of (CA)12. The enriched DNA fragments were ligated into a pMD-18 T-vector and then transformed into E. coli JM109 cells. Finally, the microsatellite enriched library was constructed. A total of 353 positive clones were screened from 1200 transformants, and 53 positive clones were randomly selected and then sequenced. According to sequencing results, 18 pairs of primers were designed and 6 markers with high polymorphism were selected, of which the polymorphism information content (PIC) values were more than 0.5. These primers would be helpful in further analysis of genetic diversity in Tibetan chicken. The results obtained in this study show that the method of enrichment by magnetic beads could improve the efficiency of screening of microsatellite markers.
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国内统一连续出版物号:51-1193/Q |国际标准出版物号:1000-7083
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