缢蛏新品种“申浙1号”ScHsc70基因SNPs筛查与耐高温性状的关联性分析

ScHsc70 gene SNP markers screening and correlation analysis for high temperature resistance of Sinonovacula constricta new variety “Shenzhe NO.1”

为获取缢蛏ScHsc70基因单核苷酸多态性位点与耐高温性状的相关性,通过直接测序方法克隆了ScHsc70基因全长序列,长度为4048 bp,包括6个内含子和7个外显子,编码区1950 bp,从其外显子区域筛查到6个潜在的SNPs,分别命名为Rs1(g.588 C>T)、Rs2(g.840 C>T)、Rs3(g.885 T>A)、Rs4(g.1233 A>G)、Rs5(g.1467 T>G)、Rs6(g.1482 T>C)。利用Sanger测序方法,对缢蛏新品种“申浙1号”耐高温群体和对照组群体潜在SNPs进行基因分型,遗传多样性分析结果显示,两个群体的多态信息含量(PIC)值在0.1112~0.3718之间,对照组群体的平均多态信息含量(0.2670)高于耐高温群体的平均多态信息含量(0.2365)。ScHSc70基因SNPs与耐热性状关联分析结果显示,Rs1、Rs3和Rs4的基因型频率和等位基因频率在对照组和耐高温群体之间存在显著性差异;单倍型连锁不平衡分析结果显示,ScHsc70基因SNPs可形成2个单倍体块,7种单倍型,其中CCT单倍型与耐高温性状显著相关;本实验发现,Rs2和Rs3处于连锁状态(r2=0.86,LOD=25.56,Dx=1.0),可以作为缢蛏与耐高温遗传育种的SNP标签。综上所述,Rs1、Rs3、Rs4和单倍型CCT均可作为缢蛏耐高温遗传育种的候选辅助分子标记,为后续的抗逆性相关SNPs筛选与功能验证奠定了理论基础。

To obtain single nucleotide polymorphism (SNP) markers of heat shock protein 70 (ScHsc70) gene related to heat tolerance traits in Sinonovacula constricta, the full-length sequence of ScHsc70 gene was cloned by direct sequencing. The results showed that the ScHsc 70 gene was 4 048 bp, including 6 introns and 7 exons, and the coding region was 1 950 bp. The high temperature resistance group and control group from a new variety of razor clam were established, and six potential SNPs Rs1 (g.588 C > T), Rs2 (g.840 C > T), Rs3 (g.885 T > A), Rs4 (g.1233 A > G), Rs5 (g.1467 T > G), Rs6 (g.1482 T > C) were screened from the exon region of ScHSc70 gene by PCR product direct sequencing. SNPs genotyping was performed on the high temperature tolerant population and the control population of the new variety “Shenzhe No.1” by Sanger sequencing. The results of genetic diversity analysis showed that the polymorphism information content (PIC) of the two populations was between 0.111 2 and 0.371 8, and the average polymorphism information content of the control population was 0.267 0, which is higher than that of the high temperature resistant populations (0.236 5). Association analysis results showed that the genotype frequency and allele frequency of Rs1, Rs3 and Rs4 were significantly different between control group and high temperature tolerant group. Haploid linkage disequilibrium analysis showed that SNPs markers of ScHsc70 gene could form 2 haploid blocks and 7 haplotypes, among which CCT haplotypes were significantly correlated with high temperature tolerance traits. It was found that Rs2 and Rs3 were in a linkage state (r2 = 0.86, LOD = 25.56, Dx = 1.0), and could be used as SNP markers for the Genetic breeding of S. constricta for high temperature tolerance. In summary, Rs1, Rs3, Rs4 and haplotype CCT can be used as candidate auxiliary molecular markers for high temperature tolerance breeding of razor clam, which lays a theoretical foundation for the subsequent functional verification of resistance-related SNPs.