中国科学院海洋研究所机构知识库

文蛤（Meretrix petechialis）是我国重要的经济养殖贝类之一。近年来，养殖过程中时有弧菌病和规模性死亡的发生，给文蛤养殖业带来了沉重的打击。这固然与养殖环境恶化、养殖密度增加等因素有关，但抗性优良品种的缺乏也是造成上述问题的重要原因。因此，通过遗传选育的方式培育抗病、抗逆的文蛤新品系具有重要的经济价值。然而，由于抗病性的遗传机制非常复杂，受环境影响较大以及抗性性状难以准确测量等原因，使得贝类抗性选育工作的进程十分缓慢。在抗性选育中，遗传参数的准确估计是制定有效选育策略的前提和基础，而与弧菌抗性显著相关的分子标记的应用，可以加速抗性品系的培育进程。本研究通过建立的具有抗性差异的全同胞家系材料，基于文蛤的转录组测序数据，对文蛤的弧菌抗性和免疫基因的表达性状进行了关联分析，并估计了基因表达性状的遗传参数，研究结果将有助于指导文蛤的弧菌抗性选育工作。本研究的主要结果如下：

1、本研究采用RNA-seq技术检测了文蛤感染弧菌后的肝胰腺中的基因表达变化。Illumina测序共获得了199,318,966条clean reads，进一步拼接得到214,577条转录本，其中，N50长度为1393 bp的147,255条unigenes用于后续分析。经过GO分析共得到21个生物学过程亚类、15个细胞组分亚类和12个分子功能亚类。KEGG分析发现有8,358条unigenes映射到267个生物信号通路，其中有16个信号通路与免疫系统相关。通过差异表达分析，共得到206个差异表达基因（DEGs），包括113个上调的DEGs和93个下调的DEGs。在这些DEGs中，至少在一个数据库中有注释的基因为96个，注释率为46.60%。为了验证转录组数据的可靠性，选取了15个DEGs进行qRT-PCR分析，结果表明有13个基因的表达模式与转录组分析的结果一致。在206个DEGs中，有14个被注释为与免疫相关的基因，我们进一步检测了其中4个基因（BIRC7、HSF、C1ql4和C1ql）在文蛤浸泡攻毒后的表达模式。这些研究结果丰富了文蛤的转录组数据库，并为研究文蛤感染弧菌后的免疫反应提供了基础数据。

2、基于基因表达数据，筛选了可用于指示文蛤抗性表型的相关标记。实验选取23个全同胞家系的1772只文蛤进行弧菌感染实验，结果表明不同家系间存在显著的弧菌病抗性差异。从表型稳定的抗性家系与敏感家系中各选取3个家系，用于抗性相关标记的筛选。对22个免疫相关基因进行弧菌感染分析，结果显示其中12个基因的表达水平在弧菌刺激后呈显著变化。进一步将这12个基因分别在3个抗性与3个敏感家系进行攻毒前后的表达定量分析，t检验和线性回归分析结果发现，在未攻毒的家系中有5个基因的基础表达水平与弧菌抗性具有显著相关性，分别为Big-Def、BIRC7、NFIL3、CTL9和Bax，表明其可作为抗性相关的候选标记。进一步选取了两个抗性不同的文蛤品系进行验证，结果证实了BIRC7和NFIL3的基础表达水平与弧菌抗性表型相关。本研究还监测了文蛤在副溶血弧菌（Vibrio parahaemolyticus）攻毒过程中体内病原菌含量的动态变化，结果发现，浸泡感染24 h后，文蛤肝胰腺内的副溶血弧菌载量达到了极高水平，随后载菌量开始下降，到第3 d后一直维持在一个较低的水平。我们对高、低载菌量组中的抗性相关基因的表达水平进行了分析，发现抗性相关基因在高载菌量组中的表达水平显著高于低载菌量组。本研究整合了弧菌抗性表型、基因表达数据和载菌量以用于抗性评价，为文蛤弧菌高抗品系的遗传选育提供了可靠的分子标记。

3、采用部分因子设计构建全同胞家系估计文蛤免疫相关基因表达性状的遗传参数和遗传力。研究选取了5个弧菌抗性相关基因（Big-Def、BIRC7、NFIL3、CTL9和Bax）在文蛤肝胰腺样品中进行基因表达分析，结果显示所有基因表达数据均呈正态分布（P > 0.05）。经亲本模型估计，文蛤中免疫相关的基因表达性状存在显著的加性效应。在这5个基因的表达性状中，有2个基因的表达性状（Big-Def和CTL9）表现出显著的父本效应，但所有5个基因表达性状都没有表现出显著的母本效应。5个基因表达性状的遗传力分布在0.113-0.530不等。同时，在每个基因的表达性状和生长性状之间没有检测到显著的表型相关和遗传相关。此外，选取了F1代家系中的抗性家系No. 47和No. 60构建了F2代家系，弧菌感染实验表明，F2代的弧菌抗性显著提高，感染后的存活率较F1代提高了10%-15%，获得了良好的抗性遗传进展。上述研究表明，基因表达变异是可遗传的，并且与之相关的抗性性状也可以通过遗传选育得到改良。因此，抗性相关基因表达标记的开发为文蛤弧菌高抗品系的间接选育提供了参考。

The clam Meretrix petechialis is one of the most important commercial clam species farmed in China. However, in rencent years, the outbreaks of vibriosis and mass mortality in the culture process inflicted serious financial losses to the clam farming industry. This is related to factors such as the deterioration of the breeding environment and the increase of the breeding density, but the lack of resistant strains is also an important reason for the above problems. Hence, it is of great significance to carry out genetic breeding for the selection of Vibrio resistance strains of the clam M. petechialis. However, the process of resistance breeding has been hindered by the complicated genetic mechanism of disease resistance and difficulty in measuring resistance phenotypes accurately. The accurate estimation of genetic parameters is a prerequisite for developing effective breeding strategy in resistance selection breeding. And the resistance-associated markers can accelerate the improvement of resistant strains. In this study, based on the M. petechialis transcriptome database and full sibling families exhibiting stable resistance level, we identified the Vibrio resistance related gene expression and estimated the genetic parameters of gene expression traits. These results provide a reference for Vibrio resistance enhancement in the genetic breeding of clam M. petechialis. The main results were as follows.

1. In this study, RNA-Seq was applied to explore global expression changes of hepatopancreas from this clam after Vibrio challenge. There were 199,318,966 clean reads obtained by Illumina sequencing, which were further assembled into 214,577 transcripts, and then 147,255 unigenes with an N50 of 1,393 bp were identified. Gene ontology (GO) analysis revealed 21 biological process subcategories, 15 cellular component subcategories and 12 molecular function subcategories. A total of 8,358 unigenes were mapped onto 267 biological signaling pathways by KEGG, among which there were 16 pathways related to the immune system. In total, 206 differentially expressed genes (DEGs) were identified, including 113 up-regulated unigenes and 93 down-regulated unigenes. In these DEGs, 96 DEGs were annotated in at least one database, accounting for 46.60% of all significant DEGs. To validate the transcriptome dataset, 15 DEGs were selected for real-time qPCR confirmation and the results showed that expression patterns of 13 genes (86.7%) agreed well with the RNA-Seq analysis. Fourteen of the 206 DEGs were annotated to be immune-related genes, and we examined the expression patterns of four immune-related DEGs (BIRC7, HSF, C1ql4 and C1ql) using clams post immersion challenge. This study enriched the M. petechialis transcriptome database and provided insight into the immune response of M. petechialis against Vibrio infection.

2. Gene expression data were used to select resistance-associated markers that indicate resistance phenotypes in the clam M. petechialis. A total of 1772 clams from 23 full-sibling families were subjected to a Vibrio challenge test, and the results showed significant variation in resistance to vibriosis among different families. Three of the most resistant families and three of the most susceptible families were selected for screening of resistance-associated markers. Then, among 22 genes, 12 demonstrated a significant change in expression, and their expression levels were further analysed by T-test and linear regression analyses, respectively, between resistant and susceptible families. Overall, the results showed that the basal expression levels of Big-Def, BIRC7, NFIL3, CTL9 and Bax were associated with Vibrio-resistant phenotypes and could be considered candidate resistance-associated markers. Further, two M. petechialis strains that differed in Vibrio resistance were used to examine the effectiveness of these five resistance-associated genes. The results further confirmed that the expression of BIRC7 and NFIL3 were associated with Vibrio-resistant phenotypes. Furthermore, the dynamics of bacterial load in hepatopancreas of the clams post Vibrio immersion infection were measured. The results showed that the bacterial load in hepatopancreas reached a peak at 24 h post immersion challenge, and then they dropped significantly and maintained at a lower level after 3 days. We further assessed the patterns of gene expression of resistance-related genes in high and low bacterial load groups, and that found that the gene expression levels were significantly higher in high bacterial load individuals, when compared with low bacterial load individuals. This study integrated the Vibrio-resistance phenotype, gene expression data and bacterial load for resistance evaluation and provided potential markers for the genetic breeding of high Vibrio-resistance strains in the clam M. petechialis.

3. 23 full sibling families which were produced using part factorial design and sire-dam model were used to estimate the genetic variation and heritability of gene transcription in the clam M. petechialis. Five Vibrio resistance-related genes (Big-Def, BIRC7, NFIL3, CTL9 and Bax) were selected for gene expression analysis in the hepatopancreas, and all the gene expression datasets exhibited normal distributions (P > 0.05). Significant additive genetic variations were observed for gene expression traits in the clam. Among those five genes, two genes (Big-Def and CTL9) showed significant sire effect, and no maternal effect variance was detected for any of the five genes. The heritabilities of the five gene expression traits varied from 0.113-0.530. And, no significant phenotypic and genetic correlations were detected between each gene transcription trait and body size. Additionally, the second generation (F2) was produced from the resistant families (No. 47 and No. 60). The Vibrio challenge test showed that the Vibrio resistance of the F2 was significantly improved, and the survival rate was approximately 10%-15% higher than that of their parents. This study highlights that gene expression level is heritable, and the resistance related to gene expression traits can also be improved through genetic breeding. So the discovery of gene expression markers provides references for indirect selection of M. petechialis with high Vibrio resistance.