二倍体和四倍体泥鳅线粒体ND-5/6基因多态性的PCR-RFLP分析

PCR-RFLP Analysis on Mitochondrial ND-5/6 Gene Polymorphisms of Natural Diploid and Tetraploid Loach(Misgurnus anguillicaudatus)

采用PCR-RFLP分析方法,对洞庭湖、武汉两地的二倍体和四倍体泥鳅线粒体DNAND-5/6基因多态性及4个群体遗传变异和遗传关系进行了研究。结果表明,120尾泥鳅mtDNAND-5/6扩增片段长度均为2.2kb;从13种限制性内切核酸酶中筛选出6种多态性内切酶(HaeⅢ、DraⅠ、RsaⅠ、TaqⅠ、HinfⅠ、MspⅠ),对扩增产物进行酶切,共检测到66种单倍型。泥鳅群体内单倍型多样性和核苷酸多样性分别为0.7679～0.9385和0.01041～0.03212;其中,洞庭湖二倍体核苷酸多样性最高(0.03212),武汉二倍体其次(0.02452),二倍体群体内核苷酸多样性高于四倍体。群体间的核苷酸多样性(π)大小为0.02588～0.04144,平均值为(0.031737±0.000005)。群体间的核苷酸歧化距离(δ)大小为0.00462～0.01617,平均值为(0.010659±0.000003);其中,武汉二倍体和四倍体之间的核苷酸歧化距离最大(0.01617),武汉四倍体和洞庭湖二倍体之间的核苷酸歧化距离最小(0.00462)。MonteCarlo模拟x2检验表明,4个群体间的单倍型频率存在极显著差异(P<0.0001)。UPGMA聚类分析表明,武汉四倍体、洞庭湖二倍体和四倍体聚为一支,亲缘关系较近;武汉二倍体为单独一支。

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism（PCR-RFLP）analysis was used to investigate mitochondrial ND-5/6 gene polymorphisms,genetic variations and relationships of four natural diploid and tetraploid loach（Misgurnus anguillicaudatus）populations from Dongting Lake（DT）and Wuhan city（WH）in China.The results showed that the length of mtDNA ND-5/6 regions amplified in 120 samples was 2.2 kb.66 haplotypes were detected using six polymorphic restriction enzymes（Hae Ⅲ、Dra Ⅰ、Rsa Ⅰ、Taq Ⅰ、Hinf Ⅰ、Msp Ⅰ）,which were screened from 13 enzymes.The haplotypic and nucleotide diversities within populations ranged from 0.7679 to 0.9385 and from 0.01041 to 0.03212,respectively.The nucleotide diversity of DT diploid was the largest（0.03212）,and that of WH diploid was the second biggest（0.02452）.Of the four loach populations,the nucleotide diversities of two diploid populations were larger than those of two tetraploid populations.The nucleotide diversity（π）among four populations ranged from 0.02588 to 0.04144,with a mean of（0.031737±0.000005）and nucleotide divergence（δ）ranged from 0.00462 to 0.01617,with a mean of（0.010659±0.000003）.Of the four populations,the nucleotide divergence between the WH diploid and WH tetraploid populations was the highest（0.01617）,and the nucleotide divergence between the WH tetraploid and DT diploid populations was the lowest（0.00462）.The contingency x2 test with Monte Carlo simulations revealed a highly significant heterogeneity in the haplotype frequencies for four populations（P0.0001）.The UPGMA dendrogram tree,which was constructed based on the nucleotide divergences,showed that WH tetraploid,DT diploid and DT tetraploid populations were clustered into one branch and their relationships were nearer,and the other branch contained only WH diploid population.