澳洲鳗鲡微卫星分子标记的筛选与检测

Isolation and characterization of microsatellite markers in Australia short-finned eel Anguilla australis

采用小片段克隆法构建了澳洲鳗鲡(Anguilla australis)的部分基因组文库,并用地高辛标记的(CA)15作探针筛选阳性克隆,共获得76条微卫星序列,其中10次以下CA连续重复序列占83.67%,没有检测到30次以上的连续重复序列。根据微卫星序列两端足够长的侧翼序列,设计引物55对,选择合成引物26对,用澳洲鳗鲡3个个体的混合基因组进行引物筛选,其中的18对具有清晰的扩增条带。将筛选出的18对引物对澳洲鳗鲡1个群体的40个个体进行了遗传多样性分析,其中1对引物扩增产物为单态,17对扩增产物呈现多态;17对扩增多态的引物在40个个体中扩增出等位基因数目为5～14,平均为9个。该澳洲鳗鲡群体的PIC、Ho、He的平均值分别为0.7157、0.6779、0.7374,所有位点均符合哈迪-温伯格平衡(P=0.05),结果证明这17个微卫星位点适于澳洲鳗鲡群体结构的研究分析。

Partial genome library of Anguilla australis was constructed by the method of small fragments DNA cloning and the positive clones were screened with the probes（CA）15 labeled with DIG. Seventy-six sequences containing microsatellites were obtained. The percentage of repeat sequences with continuum repeat numbers of CA less than 10 times was 83.67%, while no continuum repeat number was over 30 times. Fiftyfive sets of primers were designed and 26 primers were synthesized and tested with the compound DNA of 3 individuals. Eighteen loci were produced with clear bands. A population of 40 individuals were tested with the 18 loci. Seventeen loci revealed polymorphic, while one loci was monomorphic. The primers amplified the loci with relatively high numbers of alleles ranging from 5 to 14 with an average of 9 per locus among 17 ploymorphic loci. The observed heterozygosity（Ho）, expected heterozygosity（He）and polymorphism information content （PIC）was 0.677 9,0.737 4 and 0.715 7 respectively. No loci deviated significantly from Hardy-Weinberg equilibrium（P=0.05）. These indicated that 17 ploymorphic loci could be useful for the analysis of population structure.