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| 合浦珠母贝基因组微卫星富集文库的构建与分析 |
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油九菊1,2, 范嗣刚1, 黄桂菊1, 郝博飞1,2, 陈飞飞1,2, 喻达辉1
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1.农业部南海渔业资源开发与利用重点实验室, 南海生物资源与开发利用协同创新中心, 中国水产科学研究院 南海水产研究所;2.上海海洋大学 水产与生命学院
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| 摘要: |
| 采用生物素标记的(AC)15探针和磁珠富集法构建合浦珠母贝(Pinctada fucata)基因组DNA 微卫星富集文库。随机挑选2097 个克隆进行筛选, 得到483 个候选克隆(23.03%), 对其中135 个阳性克隆测序分析发现122 个克隆含有微卫星重复单元(90.37%)。进一步通过序列比对, 最终得到65 个具有特异微卫星序列的阳性克隆(53.28%), 其中包含85 个微卫星 DNA 结构域, 其中完美型(perfect)70 个, 占82.36%; 非完美型(imperfect)7 个, 占 8.24%; 混合型(compound)8 个, 占9.41%, 重复次数主要分布在5～20(95.74%), 平均重复次数为7.83, (AC/GT)n 重复所占比例最高(75.53%)。基于微卫星两端的侧翼序列, 利用Primer Premier 5.0 设计引物, 获得11 对具有多态性的微卫星引物。本研究为开展合浦珠母贝分子育种及资源评价分析提供了基础资料。 |
| 关键词: 合浦珠母贝(Pinctada fucata) 微卫星 磁珠富集 |
| DOI：10.11759/hykx20120107001 |
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| 基金项目:国家贝类产业技术体系项目(CARS-48); 国家自然科学基金项目(31372525); 广东省海洋渔业科技推广专项项目(A201301A02) |
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| Construction and identification of enriched microsatellite library from Pinctada fucata genome |
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| Abstract: |
| Microsatellite-enhanced genomic library of the Pinctada fucata was constructed using a repeat-enrichment method with biotin-labeled oligos (AC)15 and streptavidin magnetic beads. From randomly selected 2097 clones, 483 clones (23.03%) containing microsatellite motifs were obtained by PCR screening. 135 positive clones were sequenced and 122 microsatellite loci (90.37%) were identified. By alignment, 65 microsatellite clones were unique. Sequence analyses of repeat motifs indicate that the microsatellites can be divided into three types: 70 perfect types (82.36%), 7 imperfect types (8.24%) and 8 compounds (9.41%). In addition, most microsatellite sequences contained 5-20 repeat units (95.74%) and the average was 7.83. The proportion of (AC/GT)n repeat was the highest (75.53%). Based on the flanking sequences at both ends of the microsatellite, 11 pairs of microsatellite polymorphic primers were obtained at last, which were effective for PCR amplification in P. fucata genome. This study provides a base for molecular assistant selective breeding and assessment of germplasm resources of P. fucata. |
| Key words: Pinctada fucata microsatellite enrichment with magnetic beads |