半滑舌鳎抗哈维氏弧菌病相关微卫星标记筛选及QTL定位

为了筛选出与抗哈维氏弧菌病相关的微卫星标记并进行QTL定位,以2014年感染实验中存活率为52.22%的半滑舌鳎(Cynoglossus semilaevis)家系F1412(Family+年份+家系号)为材料,采用混合分离子分析法(bulked segregant analysis,BSA)对169个微卫星标记(SSR)进行筛选,得到1个可能与哈维氏弧菌病相关的微卫星标记scaffold479_23523。用scaffold479_23523所在的连锁群LG18上的37个SSR对94尾抗病、感病个体进行基因分型,得到3个图谱:整合图谱(LG18)、雌性图谱(LG18F)和雄性图谱(LG18M),并用两种不同的模式进行单标记分析和QTL定位。模式一得到3个显著性相关标记(P0.05)和1个极显著性相关标记(P0.01),图谱LG18F定位出一个区间qE-F1;模式二得到4个显著相关标记(P0.05)和1个极显著相关标记(P0.01),图谱LG18M定位出两个区间qE-M1、qE-M2。在全基因组序列的分布范围上,区间qE-F1包含了qE-M1及qE-M2,因此qE-F1更可能与抗病性状相关。本研究开发出了抗哈维氏弧菌病性状相关微卫星标记及QTL区间,为抗病新品种的培育奠定了基础。

Detection of SSR and a QTL analysis of Vibrio harveyi resistance in Cynoglossus semilaevis

The bacterium Vibrio harveyi has caused tremendous losses in the Cynoglossus semilaevis aquaculture industry.The fundamental approach has been to cultivate a new disease-resistant strain by combining traditional breeding methods with molecular techniques.In this study,bulked segregant analysis and quantitative trait loci (QTL) mapping were used to screen for disease-resistance markers.A total of 100 individuals were selected to form the F1412 family (nomenclature rule:F + year + family number:survival rate,52.22％),which was challenged with V.harveyi,and 169 microsatellite loci were detected across all chromosomes.Following the genomic scan,the scaffold479_23523 marker in the DNA pool was significantly different between the resistant and susceptible groups (P=0.000006).Ninety-four individuals were genotyped using all 32 simple sequence repeat markers on LG18,where scaffold479_23523 was located.Three new linkage groups (LG18,LG18F,and LG18M) were identified.Furthermore,two different analytical models were applied to perform a single marker analysis and composite interval mapping with different levels of significance in LG18,LG18F,and LG18M,respectively.In model 1,three significant markers (scaffold4475_71287,scx9-1,and cyse80) and one very significant marker (scaffold080437) were identified,and the qE-F1 resistance-related QTL was detected.The scaffold479_23523 marker was the left LG18F marker with a p-value of 0.0516 in model 1.Model 2 detected four significant markers (hncysell0,scaffold414_19940,scaffold4475_71287,and cyse80),one very significant marker (scaffold08043),and the qE-M1 and qE-M2 QTLs.Both scaffold080437 markers were significantly different (P＜0.001) in the two models.Four markers (scaffold080437,scaffold479_23523,scaffold4475_71287,and cyse80) may be closely associated with resistance to V.harveyi infection in C.semilaevis.qE-F1 explained 87.36％ of the phenotypic variance and contained G18M qE-M1 and qE-M2.Thus,qE-F1 was considered a major candidate region for V.harveyi resistance.After scanning the C.semilaevis genome,three immunity-related genes,such as meteorin-like,the WD repeat domain phosphoinositide interacting 2,and Toni beta-propeller repeat containing 1,were detected inside qE-F1.This is the first study to identify V.harveyi resistance-related markers and conduct a related QTL analysis in C.semilaevis.These results provide a foundation for selective breeding of disease-resistant C.semilaevis.