大黄鱼(Larimichthys crocea)ISG15基因单核苷酸多态性及与抗病性状的相关性研究

SINGLE NUCLEOTIDE POLYMORPHISMS AND ITS ASSOCIATION WITH DISEASE RESISTANT TRAIT OF ISG15 GENES IN LARIMICHTHYS CROCEA

由哈维氏弧菌(Vibrioharveyi)等细菌感染引起的弧菌病对我国大黄鱼(Larimichthyscrocea)的养殖造成了严重危害。通过哈维氏弧菌人工感染大黄鱼建立易感组和抗病组,采用PCR扩增和直接测序法对大黄鱼干扰素刺激基因ISG15双拷贝(ISG15-1和ISG15-2)进行单核苷酸多态性(SNPs)检测和分型,并与其哈维氏弧菌抗性进行关联分析。结果表明,从大黄鱼ISG15-1和ISG15-2基因中分别筛选到10个和4个SNP位点并进行了成功分型。经统计分析, ISG15-1基因的186 G/C和318 C/T位点以及ISG15-2基因的297G/T位点的基因型频率和等位基因频率在易感群体和抗病群体中均存在极显著差异,表明这3个SNP位点与大黄鱼哈维氏弧菌抗性显著相关。连锁不平衡分析结果显示,ISG15-1的SNPs可形成1个单倍块和11种单倍型,而ISG15-2的SNPs可形成1个单倍块和5种单倍型。其中, ISG15-1基因的单倍型H2(CCCCGGTACC)、H6(TCCCACTGTC)和H9(TCCCAGTGCC)与大黄鱼哈维氏弧菌抗性显著相关;ISG15-2基因的单倍型H1(CCCG)和H4(TCCG)与大黄鱼哈维氏弧菌抗性极显著相关。这些ISG15-1和ISG15-2基因的SNP位点以及单倍型可以作为抗哈维氏弧菌病大黄鱼选育的候选分子标记。

Individuals of large yellow croaker(Larimichthys crocea) were infected by bacterial Vibrio harveyi, from which susceptible and resistant ones were grouped. The single nucleotide polymorphisms(SNPs) of interferon-stimulated gene ISG15 copies(ISG15-1 and ISG15-2) in large yellow croaker were screened and genotyped by PCR amplification and direct DNA sequencing, and the association of SNPs with the resistance of large yellow croaker to V. harveyi was analyzed.Results show that, 10 and 4 SNPs were screed and successfully genotyped from the ISG15-1 and ISG15-2 gene,respectively. The statistical analyses showed that the differences of genotype and allele frequencies of 186 G/C and 318C/T in ISG15-1 gene and 297 G/T in ISG15-2 gene were extremely significant between the susceptible and resistant groups,indicating that the three SNPs were significantly associated with the resistance of large yellow croaker to V. harveyi infection. In addition, the linkage disequilibrium analyses showed that the 10 SNPs of ISG15-1 gene formed 1 haplotype block and 11 haplotypes, and the 4 SNPs of ISG15-2 gene formed 1 haplotype and 5 haplotypes. Thereinto, haplotypes of H2(CCCCGGTACC), H6(TCCCACTGTC), and H9(TCCCAGTGCC) of ISG15-1 gene, and haplotypes of H1(CCCG)and H4(TCCG) of ISG15-2 gene were all significantly associated with the resistance of large yellow croaker to V. harveyi infection. These SNPs and haplotypes of ISG15-1 and ISG15-2 genes could be used as candidate molecular markers for selective breeding of disease resistant varieties of large yellow croaker.