三疣梭子蟹线粒体基因组单核苷酸多态性

Analysis of single nucleotide polymorphism(SNP) in mitochondrial genome of Portunus trituberculatus

为获得三疣梭子蟹线粒体全基因组(mt DNA)SNP突变位点,本研究针对其mt DNA全序列设计了179对引物,采用高分辨率熔解曲线(HRM)检测技术对SNP进行了筛查。在179对引物中,有89对引物具有特异性扩增结果;进一步研究表明,89对引物中共有22对引物的扩增区域含有SNP突变位点,共包含24个SNPs。统计结果显示,三疣梭子蟹线粒体基因组中的SNP分布频率为0.15/100 bp,其中转换突变比例为79.1%、颠换突变比例为16.6%;C/T(G/A)突变比率为79.1%、G/T(C/A)突变比率为8.3%、A/T突变与G/C突变的比率均各占4.16%。本研究中所获得的SNP位点分布于三疣梭子蟹线粒体基因组的11个区域,且分布不均。其中COX1基因区域的SNP数目最多,其次在D-LOOP、ND1基因区域分别为3和4个SNP位点;而t RNA、12S r RNA等其他区域尚未发现突变位点。本研究所建立的三疣梭子蟹线粒体全基因组SNP的快速筛查方法及发现的SNPs,为进一步开展三疣梭子蟹种质遗传资源多样性、增殖放流标记等方面的研究提供了分析工具。

To obtain SNP mutations of mitochondrial genome in Portunus trituberculatus, a total of 179 primer pairs were designed, nearly covering the whole mitochondrial genome in this study. Of the 179 primer pairs, 89 got the expected specific amplification fragments by regular PCR. Then, 22 of 89 amplification fragments were found to contain the SNPs, by testing of HRM technology（High resolution melting curve）. Though sequencing, 24 SNPs were obtained in the 22 amplification fragments. The results by statistical analyzing demonstrated that SNP frequency in mitochondrial genome of P. trituberculatus was 0.15/100 bp, in which the conversion mutation rate was79.1%, 16.6% for transvesion mutation rate, 79.1% for mutation rate of C to T（or G to A）, 8.3% for the mutation rate of G to T（or C to A）, and 4.16 % for the mutation rate of G to C and A to T, respectively. Thus, the mutation of C to T（containing the type of G to A） is the most common mutation type. Additionally, the distribution of 24 SNP mutation loci was not even in the whole mitochondrial genome. The number of SNPs was the greatest in COX1 gene region, and the second is in D-LOOP and ND1 genes. However, no SNPs were found in some regions,such as t RNA, 12 S r RNA. Our findings will provide an analysis tool that may help further researches in genetic diversity of germplasm resources and enhancement in P. trituberculatus.