实验海洋生物学重点实验室知识库

　　紫贻贝自引入中国后，由于人工养殖的兴起，从辽宁大连快速扩张至浙江、福建等南方沿海，南方于2000年前后放弃紫贻贝养殖，而以养殖厚壳贻贝为主，在南方海区形成了零星分布的紫贻贝“孑遗”群体，为研究紫贻贝群体遗传结构提供了独特的研究材料。另一方面，中国沿海紫贻贝与厚壳贻贝的重叠分布也有利于开展贻贝基因渐渗、物种隔离机制研究。

　　中国沿海紫贻贝群体遗传学研究

　　为评估中国沿海紫贻贝的遗传多样性与遗传结构，采用COI基因对中国沿海从北至南11个紫贻贝群体（秦皇岛、庄河、大连、长岛、崆峒岛、荣成、乳山、鳌山卫、胶南、赣榆和平潭）进行了分析，并使用简化基因组2b-RAD方法对其中具有地理代表性的5个群体（庄河、大连、崆峒岛、荣成和平潭）予以进一步验证。

　　基于COI基因的遗传结构分析表明，乳山与秦皇岛群体、乳山与崆峒岛群体及乳山与平潭群体间出现了遗传分化（F_st > 0.05），其他群体间均无分化。对COI数据进行的Mantel检验并不能证明地理隔离的存在。11个群体的COI基因共获得13个单倍型，单倍型网络分析将13个单倍型分为4个单倍群，其中的3个单倍群包含了12个单倍型，这3个主要的单倍群在11个地理群体间的分布是比较均匀的，缺乏明确的群体遗传结构。将本研究获得的中国沿海紫贻贝的13个单倍型与大西洋东北部群体与地中海群体进行系统发生分析，发现中国紫贻贝的所有单倍型均聚到地中海一支，证明了中国的紫贻贝起源于地中海群体。

　　采用2b-RAD技术对其中5个群体的进一步验证发现，群体间均无分化。Mantel检验分析发现遗传距离与地理距离之间有比较明显的正相关关系。采用2b-RAD 得到的遗传多样性参数（H_e、H_o及PIC）表明，紫贻贝5个群体的遗传多样性均较低，但大致呈现由北向南略微升高的趋势。差异SNP聚类分析发现95个紫贻贝个体与5个厚壳贻贝之间的亲缘关系是不同的，特别是庄河与平潭群体存在部分个体与厚壳贻贝亲缘关系更近，这暗示着可能存在厚壳贻贝向紫贻贝的基因渐渗。

　　紫贻贝与厚壳贻贝杂交研究

　　为了进一步评估中国沿海紫贻贝的群体遗传多样性与群体遗传结构是否会受到土著厚壳贻贝的影响，以及为良种选育提供理论与实践基础，本研究系统开展了紫贻贝与厚壳贻贝的种间杂交实验研究。使用双列杂交构建四个组别：正交组GC（紫贻贝♀×厚壳贻贝♂）、反交组CG（厚壳贻贝♀ × 紫贻贝♂）以及两个对照组GG（紫贻贝♀ × 紫贻贝♂）和CC (厚壳贻贝♀ ×厚壳贻贝♂ )，发现紫贻贝与厚壳贻贝可以双向受精，但受精强度具有不对称性，即紫贻贝的卵子很容易与厚壳贻贝的精子结合，且具有较高的受精率、孵化率；而厚壳贻贝的卵子很难与紫贻贝的精子结合，只有少数能够受精，且孵化率极低。正交组GC从第9天开始呈现较快的生长速度，壳长显著大于两个自交组（P < 0.05），且该生长优势保持至13日龄。正交组GC与自交组GG在整个幼虫阶段存活率均无显著差异（P > 0.05），自交组CC的存活率显著低于自交组GG与正交组（P < 0.05），仅GG和GC组获得附着变态幼体。使用ITS1可以鉴定反交组CG个体，但无法有效鉴定正交组GG。

    Mediterranean mussel ( Mytilus galloprovincialis ) is an introduced species for China and appeared only in Dalian, Liaoning province in 1950s. With the development of M. galloprovincialis aquaculture, the distribution of this species rapidly expanded to the coast of Zhejiang and Fujian province. However, M. galloprovincialis aquaculture declined sharply in southeast coast of China. In contrast, Mytilus coruscus became the major aquaculture species. “Relic” M. galloprovincialis still can be found in fragmentary areas along southeast coast of China, which provides a unique material for genetic structure study. On the other hand, the sympatry of M. galloprovincialis and M. coruscus facilitates the demonstration of introgression and species isolation mechanism.

Population Genetic Study on Mytilus galloprovincialis in China Coast

    Our research analyzed the genetic diversity and population genetic structure of  M. galloprovincialis in China coast. 11 populations of M. galloprovincialis (Qinhuangdao, Zhuanghe, Dalian, Changdao, Kongtongdao, Rongcheng, Rushan, Aoshanwei, Jiaonan, Ganyu and Pingtan) were analyzed based on COI gene. 5 typical populations of them (Zhuanghe, Dalian, Kongtongdao, Rongcheng and Pingtan) were further analyzed by means of 2b-RAD.

    According to the COI genetic differentiation coefficient for 11 populations, there are differentiations between Rushan and Qinhuangdao, Rushan and Kongtongdao, Rushan and Pingtan (F_st > 0.05), respectively. We used Mantel test to find the correlation between geographic distance and genetic distance of 11 populations. COI data could not prove the existence of isolation by distance. The COI sequences of 11 populations can be attributed to 13 haplotypes and then divided into 4 haplogroups according to their relationships. The ratios of 3 main haplogroups are similar　in 11 populations, revealing homogenous genetic structures for Chinese populations of M. galloprovincialis. We compared 13 COI haplotypes acquired with haplotypes from Mediterranean and northeastern Atlantic Ocean. The result showed that Chinese populations of M. galloprovincialis might be introduced from the Mediterranean Sea.

    We used 2b-RAD to test 5 typical populations and no differentiation was found. Mantel test showed a significant positive correlation between geographic distance and genetic distance. The genetic diversity parameters (H_o, H_e and PIC) calculated using 2b-RAD data showed a low genetic diversity of 5 populations, however，there is a slightly increasing trend from north to south, especially that of the southernmost Pingtan population. Heatmap of differential SNPs clustering showed discrepant genetic relationships between 95 M. galloprovincialis and 5 M. coruscus, this might be an evidence of hybridization between the two mytilus species and introgression from native M. coruscus to M. galloprovincialis.

Hybridization between Mytilus galloprovincialis and Mytilus coruscus

    In order to clarify the feasibility of interspecific hybridization between the two commercially important mussel species, a diallel cross was conducted with 3 replicates using the broodstocks of M. galloprovincialis from Dalian, Liaoning province and M. coruscus from Ningde, Fujian province respectively. Two cross groups (M. galloprovincialis ♀ × M. coruscus ♂ and M. coruscus ♀ × M. galloprovincialis ♂) were established, along with two control groups (M. galloprovincialis ♀ × M. galloprovincialis ♂ and M. coruscus ♀ × M. coruscus ♂) as controls. The development and growth were compared by monitoring of fertilization, hatching, survival and growth of the offspring from the four groups. Results showed that the fertilization rate exhibited notably asymmetric between the two cross groups. Mean fertilization rate of 88.87% was observed in the cross-group of M. galloprovincialis ♀ × M. coruscus ♂, which was not significantly different from the two control groups. The fertilization and hatching rate of cross-group M. coruscus ♀ × M. galloprovincialis ♂ were significantly lower than all the other groups (P < 0.05), resulting in no offspring obtained eventually. The offspring from cross-group M. galloprovincialis ♀ × M. coruscus ♂ showed notable advantage in growth, as evidenced by the shell length of the planktonic larvae on day 9, 11, 13 and the juvenile on day 78 were significantly larger than the control groups (P < 0.05). The survival rate of cross-group M. galloprovincialis ♀ × M. coruscus ♂ had no significant difference from the control group M. galloprovincialis ♀ × M. galloprovincialis ♂ during the planktonic larvae stage, but significantly higher than the control group M. coruscus ♀ × M. coruscus ♂ (P < 0.05). The results of this study provided valuable information for mussels breeding program in the future.

第1章　引言... 1

1.1　贻贝的研究现状... 1

1.1.1　贻贝的分类地位和我国主要种类... 1

1.1.2　形态特征... 2

1.1.3　生态习性... 4

1.1.4　繁殖... 4

1.1.5　生长发育... 5

1.1.6　贻贝养殖... 5

1.2　遗传多样性及其研究方法... 9

1.2.1　遗传多样性的含义... 9

1.2.2　遗传多样性的研究方法... 10

1.3　全球范围紫贻贝群体遗传结构研究现状... 17

1.3.1　利用线粒体基因开展的紫贻贝群体遗传结构研究现状... 17

1.3.2　利用核基因开展的贻贝群体遗传结构研究现状... 18

1.4　贻贝的杂交研究现状... 19

1.4.1　自然杂交... 19

1.4.2　人工杂交... 20

1.5　本研究的目的及意义... 20

第2章　中国沿海紫贻贝群体遗传学研究... 23

2.1　研究背景... 23

2.2　基于线粒体COI基因的紫贻贝群体遗传学研究... 24

2.2.1　材料与方法... 24

2.2.2　结果与讨论... 27

2.3　利用简化基因组2b-RAD技术开展紫贻贝群体遗传多样性研究... 34

2.3.1　材料与方法... 34

2.3.2　结果与讨论... 35

2.4　小结....................................................................................................... 41

第3章　紫贻贝与厚壳贻贝杂交研究... 43

3.1　研究背景... 43

3.2　紫贻贝与厚壳贻贝杂交子代早期发育与生长存活... 44

3.2.1　材料与方法... 44

3.2.2　结果与讨论... 45

3.3　紫贻贝与厚壳贻贝杂交鉴定... 49

3.3.1　材料与方法... 49

3.3.2　结果与讨论... 50

3.4　小结... 52

第4章　结论与展望... 55

参考文献... 57

致谢... 65

作者简历及攻读学位期间发表的学术论文与研究成果... 67