中国科学院海洋研究所机构知识库

牡蛎是全球性分布的重要养殖经济物种之一，然而在养殖过程中，牡蛎大规模的死亡事件频发，对养殖产业造成巨大的经济损失。高温被认为是导致夏季大规模死亡的重要原因之一。因此，对牡蛎温度适应性和热耐受性机制的研究成为迫切需要。2017年，我们启动了一个人工选育高温抗性长牡蛎的育种项目，旨在提高长牡蛎的热耐受性，从而提高长牡蛎的度夏存活率。通过从表型和基因组层面比较选育子一代群体和对照群体的差异，评估基于急性应激的选育方法对其提高热耐受性是否有效，并探讨急性应激选择对其遗传结构的影响并进一步解析耐热性机制。

1.选育群体与自然群体的耐热性及生理响应差异

我们利用42℃，热激1小时的方式控制选择强度（37%）选育出热耐受牡蛎亲本，比较亲本繁育的子一代选育群体与自然群体热耐受性的差异。具体内容是评估6月龄的F₁代选育群体与自然群体在生长、热耐受性以及呼吸率和酶活等生理指标的差异；并且统计海上养殖到19月龄时，选育群体与自然群体的度夏生长和存活率。结果表明：经一代选育后，6月龄的选育群体与自然群体在生长上没有显著差异。高温胁迫下，选育群体的存活率显著高于自然群体，这个结果与野外实验的结果一致，度夏后，19月龄的选育群体的死亡率显著低于自然群体，表明我们的选育方法对提高其度夏存活率是有效的。选育群体在38℃时呼吸率相比于20℃显著升高，而自然群体的呼吸率在35℃时便显著提高了，表明自然群体对于高温胁迫更加敏感，选育群体更强的供氧能力。酶活结果显示选育牡蛎在热激过程中与代谢和抗氧化相关的酶（PK，SOD）活性更高。利用多种手段证实经过一代高温急性应激选择后，牡蛎的耐热性显著提升，说明了选育策略的有效性。

2.热耐受群体与热敏感群体的遗传结构差异分析

为解析一代应激选择对牡蛎遗传结构的影响并进一步揭示热耐受性的遗传机制,我们比较了选育群体F₁代高温热激后存活个体与未选育群体热激后死亡个体的遗传结构。利用简化基因组测序的方法对两群体（每个群体各50只牡蛎）进行遗传结构分析，并且定位到受选择区域及候选基因，并检测候选基因在热激下的表达分化差异。测序和质控后得到379002个SNP位点。基于高质量的SNP位点，系统发育树将两群体分为3个聚类，群体结构分析也将100个个体分为3个群组。PCA的结果与系统发育树和群体结构分析结果一致。这些结果表明选育群体与对照群体的遗传结构出现了分化，但鉴于只经过一代选育，因此分化是很有限的。根据F_ST和θπ值确定了115个受选择区域，包含472个基因。对472个基因进行GO富集，得到7个显著的GO注释（p < 0.05），其包含了18个受选择基因（实验室已有研究表明BAG4在温度适应中可能具有重要作用，因此BAG4也被选为候选基因），候选基因的表达结果显示，在两群体中，有8个基因（IF4A2, IF6, EIF3A, MANBA, DDX43, RECS, CAT2, BAG4）在热激过程中表达量发生了分化，说明这些基因的表达分化参与了选育群体与对照群体的分化，而且其中6个基因在选育群体中表现出低的基础表达和高的诱导表达，表明大部分选育群体的基因表达具有更高的可塑性。生物的热耐受性对预测生物响应气候变化的潜在适应能力起着重要作用，我们的结果为牡蛎的热耐受研究提供了分子基础。

Oyster is one of the most important worldwide economic species. Mass mortality was happened during summer and has become a serious threat to the global oyster aquaculture industry. Elevating temperature is considered as one of the most important abiotic factors causing the summer mortality of oysters. Therefore, it is necessary to explore the mechanism for thermal adaptation and thermotolerance of oysters. We initiated an artificial selective breeding program aimed at increasing survival rate of C. gigas during the summer since 2017. we collected phenomics and genomics evidence to identify discrepant responses to temperature elevation between thermal tolerance oysters after one-generation of selection, and to elucidate the potential molecular mechanisms for heat tolerance in oysters.

1. Differences in thermotolerance and physiological responses between selected F₁ population and natural population.

Parental selection is based on acute thermal tolerance under controlled heat stress (42 ℃, 1h) to ensure selection intensity (37%). We compared the difference in ability of thermal adaptation between the selected F₁ and natural population. Specifically, the growth, respiration rate, heat tolerance and physiological indicators was measured between 6-month-old selected F₁ and natural population. The summer mortality and growth was evaluated to verify practical significance for aquaculture industry of artificial selection breeding in thermotolerance when the oysters was 19-month-old. The results showed that there was no significant difference in growth after one-generation selection between selected and control population. The thermal tolerance of the progeny post exposure to acute heat stress in the selected population was significantly higher than that in the natural population even only after one-generation selection, suggesting the thermotolerance of selected oysters was improved. This result was in line with the results under field conditions. The respiration rate of the selected population at 38 ℃ was significantly higher than that of 20 ℃, while the respiration rate of the natural population was significantly increased at 35 ℃, indicating that the natural population was more sensitive to high temperature stress, selected population has a greater capacity of oxygen supply. The results of physiological indicators showed that the selected oysters had higher enzyme activities related to metabolism and anti-oxidation (PK, SOD) during the heat stress, indicating that selected population had stronger antioxidant capacity. It has been proved by various means that the thermotolerance of oyster has been significantly improved after one generation selection of acute heat stress, which proves that the breeding strategy is effective.

2. Genetic evolution analysis of thermotolerant population and heat-sensitive population

In order to analyze the molecular mechanism of oysters’ thermal adaptability, the survival individuals of selected population were considered as thermotolerant population, the dead individuals of natural population after heat stress were considered as heat-sensitive population (control). Specific-locus amplified fragment sequencing (SLAF-seq) was used to analysis the genetic structure of the two population (50 individuals per population). We also identified the selected region and candidate genes. The gene expression under heat shock was analyzed. After sequencing and quality control, 379002 SNPs were obtained. Based on high quality SNPs, phylogenetic tree divides the two populations into 3 clusters. The results of PCA verified the results of phylogenetic tree and population structure analysis. These results suggested that the restricted genetic divergence between the oysters with one-generation selection and un selected oysters. The 115 selected region and 472 genes was determined based on F_ST and θπ values, and 18 candidate genes were obtained by GO enrichment (BAG4 was selected according previous studies that suggests it may play important role in temperature adaptation). Eight genes (IF4A2, IF6, EIF3A, MANBA, DDX43, RECS, CAT2, and BAG4) diverged in transcriptional expression response to heat stress. Six of genes showed lower basal expression and higher induced expression under heat stress in thermotolerant population compared with control population. which suggested higher plasticity of thermotolerant population. The heat tolerance of organisms plays an important role in prediction the potential ability to adapt climate change. Our results provide a molecular basis for researches on thermotolerance of oysters.

第1章 绪论... 1

1.1 长牡蛎的产业现状... 1

1.1.1 长牡蛎简介... 1

1.1.2 牡蛎的夏季大规模死亡与高温... 1

1.2 生物的温度适应性研究... 3

1.2.1 生物适应环境的两种形式... 3

1.2.2 温度对海洋生物的生理影响... 4

1.2.3 温度对海洋生物遗传结构的影响... 6

1.3 本研究的目的及意义... 8

第2章 选育群体与自然群体在热激下的生理响应差异... 11

2.1 引言... 11

2.2 材料与方法... 11

2.2.1 实验材料... 11

2.2.2 生长数据测定... 13

2.2.3 热耐受性... 13

2.2.4 不同温度下呼吸率测定... 13

2.2.5 高温急性应激... 14

2.2.6 生理指标测定... 14

2.2.7 野外度夏实验... 15

2.2.8 数据分析... 15

2.3 实验结果... 15

2.3.1 生长... 15

2.3.2 热耐受性... 16

2.3.3 呼吸率... 17

2.3.4 生理指标... 18

2.3.5 野外度夏后生长数据与存活率... 19

2.4 讨论... 21

2.4.1 经一代选育后生长无差异... 21

2.4. 2 经一代选育后热耐受性的分化... 21

2.4.3 热激胁迫下呼吸率升高... 22

2.4.4 热激胁迫下的能量代谢和抗氧化调节... 22

2.5 本章小结... 23

第3章 热耐受群体与热敏感群体的遗传进化分析... 24

3.1 引言... 24

3.2 材料与方法... 24

3.2.1 实验材料... 24

3.2.2 基因组DNA提取... 24

3.2.3 SLAF测序技术应用于牡蛎SNP开发... 25

3.2.4 遗传进化分析... 26

3.2.5 受选择区域确定及功能富集分析... 27

3.2.6 候选基因表达水平... 28

3.3 实验结果... 29

3.3.1 测序结果及SNP分型... 29

3.3.2 遗传进化分析结果... 30

3.3.3 受选择区域和候选基因的确定... 33

3.3.4 基因表达... 35

3.4 讨论... 38

3.5 本章小结... 40

第4章 结论与展望... 41

4.1 主要结论... 41

4.2 不足与展望... 41

参考文献... 43

致 谢... 53

作者简历及攻读学位期间发表的学术论文与研究成果    55