合浦珠母贝基因组微卫星富集文库的构建与分析

摘要

Microsatellite-enhanced genomic library of thePinctada 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 con-tained 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 inP. fucata genome. This study provides a base for molecular assistant selective breeding and assessment of germplasm resources ofP. fucata.%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。