珠母贝属6个种的ITS 2分子标记研究

Study on ITS 2 molecular markers of six pearl oyster species in the genus Pinctada

对珠母贝属的大珠母贝、珠母贝、白珠母贝、黑珠母贝、长耳珠母贝、黑珠母贝和合浦珠母贝6个种的内部转录间隔区2(ITS2)序列及其两侧的5.8S和28S的部分序列进行了比较分析。其中黑珠母贝的序列来自GenBank。PCR扩增片段大小为600bp左右,测序结果表明,ITS2长211～254bp,两端的5.8S和28S分别长84bp和272bp(均含引物)。序列比对分析结果表明,5.8S和28S序列高度保守,不适合于种类鉴定,而ITS2序列高度变异,270个比对位点中有146个位点发生突变,其中72个位点发生插入/缺失突变。除白珠母贝和黑珠母贝之间的遗传距离较小外,其余种类之间的遗传距离远远大于种内遗传距离。基因型分析表明,每个种具有各自特有的基因型。基因型和序列变异分析表明ITS2序列可作为珍珠贝种类鉴定的分子标记。可用于种间、杂交育种、幼体和珍珠贝肉等材料的种类鉴定与遗传分析。

The ITS 2 complete sequences and flanked 5.8S and 28S rDNA partial sequences of six pearl oysters, namely, Pinctada maxima, P. margaritifera , P. albina , P. nigra , P. chemnitzi and P. fucata in the genus Pinctada were analyzed, among which the sequences of P. nigra were obtained from GenBank. The PCR product is about 600 bp, which contains 84 bp of 5.8S and 272 bp of 28S rDNA fragments （both including primer sequence） . The full length of ITS 2 ranges from 211 to 254 bp. The sequence variation analysis indicated that 5. 8S and 28S rDNA fragments are highly conservative and not suitable for species identification whereas ITS 2 is highly variable with 146 mutation sites （including 72 insertion/deletions） within 270 alignment positions. The interspecific genetic distances are much greater than the intraspecific distances except that between P. albina and P. nigra. The genotype analysis also demonstrated that each species has its own genotypes. These findings suggested that ITS 2 is a high-resolution molecular marker useful in identification of species, hybrids, larvae and tissue samples of the pearl oysters.