一种适用于太平洋牡蛎高多态基因组单核苷酸突变验证的两步式高分辨率熔解曲线方法(英文)

Two-step High-resolution Melting Method for SNP validation in the Highly Polymorphic Crassostrea gigas Genome

长牡蛎(Crassostrea gigas)基因组具有高度的多态性。因此,导致传统的高分辨率熔解曲线(HRM)方法在长牡蛎基因组SNP分型验证中表现出很低的效率,并且传统HRM方法花费比较高。本文提出一种经过改良的适合高度多态性基因组SNP验证的HRM方法。这种two-step HRM方法在传统的HRM方法基础上进行了许多改进;利用这种改进的方法,使用一对引物即可准确完成一个特定SNP位点基因型的确定和区分,并且相应的花费较传统方法大大降低。本实验中共使用56组引物对长牡蛎SNP进行验证,有8组引物因为特异性较差在引物筛选中被排除。最终有30组引物成功验证SNP,成功率约为62.5%。超过63%的SNP位点为转换类型(C/T:30%,A/G:33.3%),只有大约36.7%的SNP位点为颠换类型(A/C:10%,G/T:10%,A/T:10%,C/G:6.7%)。随即选取5组引物的扩增产物进行Sanger测序,通过序列对比后获得与该方法完全相同的分型结果,这也证明了本方法的准确性。以上数据表明本文提出的two-step HRM方法是一种可使用于高多态性牡蛎基因组的高效、经济并且有高通量潜力的SNP验证方法。

The genome of oyster （ Crassostrea gigas ） was certified highly polymorphic, and the traditional high resolution melting （HRM） a- nalysis presented low eflSciency and costly expense for single nuclcotida polymorphisms （SNP） genotyping in C. gigas genome. An improved two-step HRM method for SNP validation in highly polymorphie genome was introduced in this article. Several improvements based on the traditional HRM protocol were carried out in the two-step HRM method. With this method, genotypes of a certain SNP were accurate｜y detec- ted and easily distinguished using only one pair of primers; furthermore, the operation cost was dramatically reduced. A total ti56 pairs of primers were tested in this experiment, and 8 pairs of primers were rejected by primer screening because of bed specificity. Ultimately, 30 SNP markers were successfully validated in C. gigas, for a success ratio of approximately 62.5 %. More than 63 % of the validated SNPs were transitions （C/T： 30 % and A/G： 33.3 %）, while transversions were only accounting for 36.7 % （A/C： 10 %, G/T： 10 %, A/ T： 10 % and C/G： 6.7 % ）. PCR amplicon of rive genotyped SNP primers were selected by random to performed Sanger sequencing, and the same genotype result was obtained by the sequence alignment, which validated the accuracy tithe HRM. These data indicated that this method is an efficient, economical and high-threughpet possible method for SNP validation in highly pelymorphic genome of C. gigas.