实验海洋生物学重点实验室知识库

裙带菜和海带同为海带目褐藻，两者均是我国具有重要经济价值的大型藻类。
在当前我国北方的养殖海区内，这两类海藻的自然群体和栽培群体通常在开放海
域内同域分布。 对于栽培群体， 若被来自自然群体的基因“污染”， 则有可能造成其农学性状改变，导致种质退化；另一方面，自然群体中含有适应了当地环境的基因型，因此是一类具有潜力的种质资源，应避免其受到栽培群体的影响。但目前，鲜有针对这两种藻类栽培群体和自然群体间遗传连通性（genetic connectivity）的研究，因此，选取位于中国大连的典型海藻养殖场，分析了裙带菜和海带自然和栽培群体的遗传多样性、遗传结构以及各群体间的基因交流状况。
裙带菜方面，利用前期开发的 10 对高多态性微卫星引物，对来自不同年份的 2 个栽培群体（F1&F2）、 2 个筏架自然群体（KWT&UWT）和 1 个潮下带自然群体进行分析（SW）。以等位基因数 Na 和期望杂合度 He 来衡量，遗传多样性
最高的群体为 UWT-18（Na： 14.6； He： 0.874），最低的群体为 F1-18（Na： 8.1；He： 0.765）；两两比较后的遗传距离最大值为 1.470，出现在 F1-18 和 SW-18 之间。基于遗传距离的 Neighbor-joining(NJ)聚类分析将栽培群体和潮下带群体分到了遗传距离较大的两个聚类中。基于贝叶斯模型的遗传结构分析将所有裙带菜个体分为 3 个聚类， SW-18、 KWT-18，和 W17 中的大部分个体属于聚类 1，所属成员比例分别为 0.98、 0.96 和 0.93； F1–18、 F1–17 和 F1–15 中的大部分个体被分配到聚类 2，所属成员比例分别为 0.98、 0.97 和 0.97； F2-15 中的大部分个体属于聚类 3，比例为 0.90。主成分判别分析（DAPC）也把栽培群体和潮下带自然群体归到两个不同的聚类中，与前文中两项聚类分析结果相符。以上结果表明养殖群体和潮下带自然群体之间存在显著的遗传差异；栽培群体和潮下带自然群体中都几乎不含有来自另一方的血统，这说明两者之间的基因流非常有限；而生长于裙带菜养殖筏架上的自然群体含有来自栽培和潮下带自然群体两者的血统。
海带方面，同样选用前期筛选的 10 对兼具多态性和高效性的微卫星引物，
对采集自 2018 年的 3 个栽培群体（FB、 FJ、 XS）、 1 个筏架自然群体（RW）和2 个潮下带自然群体（SE&SW）进行分析。 Na和 He 的最高值出现于 FJ（Na=3.8；He=0.496），最低值则出现于 SW（Na=2.6；He=0.382）。以 Na 和 He来衡量，海带栽培群体的遗传多样性要高于潮下带自然群体；遗传距离最大值出现在 XS 和SW 之间，为 0.159。除了 FJ 和 XS 之间的 Fst值均检测到了显著性。基于遗传距离的 NJ 聚类分析将栽培群体和潮下带群体分为了遗传距离较大的两个聚类，筏架野生群体 RW 处在两个聚类之间。基于贝叶斯模型的遗传结构分析将所有海带个体分为 2 个聚类，潮下带群体中 90%的个体都被分入聚类 1，而大多数栽培群体个体则被分入聚类 2，但 FB 和 FJ 中的部分个体中含有较高比例的来自潮下带群体的血统，潮下带血统在这两个群体中的占比分别为 0.28 和 0.19。主成分判别分析（DAPC）中的结果也与前两项分析结果相符，呈现栽培群体和潮下带自然群体分别聚类，筏架野生群体处于两者之间的模式。
以上结果显示，潮下带自然群体中几乎不含有来自栽培群体的血统，说明栽
培群体对潮下带群体的影响非常有限；而在栽培群体中却检测到少量来自潮下带
群体的血统，说明从潮下带群体向栽培群体的基因流更为显著，两者之间的基因
流呈现明显的不对称性。筏架野生群体包含来自栽培群体和潮下带自然群体两者
的血统。
本论文的结果为进一步理解同域分布的裙带菜和海带栽培群体和自然群体
间的相互作用提供了依据。
 

Undaria pinnatifida and Saccharina japonica belong to Laminariales,
Ochrophyta both of which are macroalgae with important economic value in China.
Under current farming system, the spontaneous and cultivated populations of these two species are usually sympatric in the open sea. For the cultivated populations, if they were "contaminated" by the genes from the spontaneous populations, their agronomic characters may be changed and degraded. In addition, there are specific genes that evolved under the local environment in spontaneous populations, and they
are potential germplasm resources and ought to be prevented from excess influence from farmed populations. But until now, there has been little research about genetic connectivity between cultivated and spontaneous populations of the two algae.
Therefore, we collected U. Pinnatifida and S. japonica on a typical kelp farm in Dalian, China, and analyzed the genetic diversity, genetic structure and gene connectivity between the spontaneous and cultivated populations.
For U. Pinnatifida, we employed 10 pairs of microsatellite primers developed and selected in previous study to analyze two cultivated populations (F1&F2), two spontaneous populations (UWT&KWT) occurring on rafts and one spontaneous population (SW) inhabiting the subtidal zone from different years. In terms of Na (numbers of alleles) and He(expected heterozygosity), the population with the highest genetic diversity was UWT-18 (Na: 14.6; He: 0.874), and the lowest population was
F1-18 (Na: 8.1; He: 0.765). The maximum pairwise genetic distance was 1.470 ( between F1-18 and SW-18). Neighbor-joining (NJ) clustering analysis based on genetic distance divided the cultivated populations and subtidal population into two clusters with large genetic distance. The genetic structure analysis based on Bayesian model conducted by STRUCTURE 2.3.4 divided all the individuals into 3 clusters.
SW-18, KWT-18, and W17 were basically assigned to cluster 1, and the proportions of membership were 0.98, 0.96, and 0.93, respectively. Most of the individuals of F1-18, F1-17 and F1-15 were assigned to cluster 2, and their proportions of membership were 0.98, 0.97 and 0.97, respectively. Most individuals of F2-15 belonged to cluster 3, with a proportion of membership of 0.90.
The principal component discriminant analysis (DAPC) also assigned the farmed and the subtidal spontaneous population into two different clusters, which was consistent with the results of above-mentioned two clustering analysis.
These results reveal significant genetic diversity in both cultivated and subtidal spontaneous population. Clustering analysis revealed that the gene flow between cultivated and subtidal spontaneous population is very limited. Spontaneous sporophytes on farmed rafts contain pedigree from both farmed and subtidal spontaneous populations.
For Saccharina japonica, 10 pairs of microsatellite primers with higher
polymorphism were used to analyze three cultivation populations（ FB、 FJ、 XS） , one wild raft population（ RW） and two subtidal spontaneous populations（ SE&SW） collected in 2018. The value of Na and He was highest in FJ（ Na=3.8； He=0.496），lowest in SW（ Na=2.6； He=0.382. In terms of Na and He, the genetic diversity of cultivated populations was higher than that of subtidal spontaneous population. The
maximum pairwise genetic distance was 0.159， and it appeared between XS and SW.
All Fst values revealed significance except FJ and XS. Pairwise genetic distance-based NJ clustering analysis divided the cultivated and subtidal populations into two clusters with large genetic distance, and RW was between the two clusters. The Bayesian-model based genetic structure analysis divided all individuals into two clusters. The subtidal spontaneous population and the cultivated populations were principally assigned into cluster 1and cluster2, respectively. FB and FJ contained relatively high proportions of membership originating fromthe subtidal population(0.28 and 0.19). The results of DAPC were consistent with the above two
analysis, revealing that the cultivated populations and the subtidal spontaneous populations were clustered separately, and the raft spontaneous population was in between. Above results suggested that there was almost no pedigree of the cultivated populations flowing to subtidal spontaneous populations, indicating that influence from the former to the latter was very limited; however, some pedigree of subtidal
populations were detected in the cultivated populations, indicating that the gene flow from the subtidal populations to the cultivated populations was more prominent, and thus the gene flow between them was obviously asymmetry. The raft spontaneous population contained pedigree from both the cultivated and the subtidal populations.
These results provide a foundation for further understanding of interaction between cultivated and spontaneous populations of U.pinnatifida and S. japonica.
 

第 1 章 引言...............................................................................................1
1.1 裙带菜的基本生物学特征及概述.....................................................................1
1.2 裙带菜的养殖历史、 养殖技术及当下的经济重要性.....................................3
1.3 海带的基本生物学特征及概述.........................................................................5
1.4 海带的养殖历史及当下的经济价值.................................................................7
1.5 遗传连通性在不同作物中的研究.....................................................................7
1.6 分子标记发展及 SSR 应用 ...............................................................................8
1.7 研究目的及意义...............................................................................................10
第 2 章 裙带菜同域分布的栽培和自然群体间的遗传连通性研究 ..... 12
2.1 引言...................................................................................................................12
2.2 材料与方法.......................................................................................................15
2.2.1 主要仪器与生化试剂................................................................................15
2.2.2 实验材料....................................................................................................15
2.2.3 样品基因组 DNA 的提取和检测.............................................................16
2.2.4 SSR 核心序列扩增及检测 ........................................................................17
2.2.5 SSR 核心序列长度获取 ............................................................................19
2.2.6 数据整理及分析........................................................................................19
2.3 实验结果...........................................................................................................21
2.3.1 裙带菜基因组 DNA 提取.........................................................................21
2.3.2 SSR 核心序列扩增及检测 ........................................................................21
2.3.3 扩增产物测序............................................................................................22
2.3.4 数据整理及分析........................................................................................23
2.3.4.1 数据整理汇总.....................................................................................23
2.3.4.2 群体基因多样性信息.........................................................................24
2.3.4.3 群体遗传距离和遗传分化指数（Fst） ............................................26
2.3.4.4 遗传距离树状图.................................................................................28
2.3.4.5 STRUCTURE 分析结果 .....................................................................29
2.3.4.6 DAPC 分析结果..................................................................................31
2.4 结论与讨论.......................................................................................................33
2.5 本章小结...........................................................................................................35
第 3 章 海带同域分布的栽培和自然群体间的遗传连通性研究........... 37
3.1 引言...................................................................................................................37
3.2 材料与方法.......................................................................................................40
3.2.1 主要仪器与生化试剂................................................................................40
3.2.2 实验材料....................................................................................................40
3.2.3 样品基因组 DNA 的提取和检测.............................................................41
3.2.4 SSR 核心序列扩增及检测 ........................................................................41
3.2.5 SSR 核心序列长度获取 ............................................................................42
3.2.6 数据整理及分析........................................................................................42
3.3 实验结果...........................................................................................................42
3.3.1 裙带菜基因组 DNA 提取.........................................................................42
3.3.2 SSR 核心序列扩增及检测 ........................................................................43
3.3.3 扩增产物测序............................................................................................43
3.3.4 数据整理及分析........................................................................................44
3.3.4.1 群体基因多样性信息..........................................................................44
3.3.4.2 群体遗传距离和遗传分化指数（Fst） .............................................45
3.3.4.3 遗传距离树状图..................................................................................46
3.3.4.4 STRUCTURE 分析结果 .....................................................................47
3.3.4.5 DAPC 分析结果..................................................................................49
3.4 结论与讨论.......................................................................................................50
3.5 本章小结...........................................................................................................52
第 4 章 结论与展望.......................................................................................................... 53
参考文献..................................................................................................................................... 55
致谢 .............................................................................................................................................. 61
作者简历及攻读学位期间发表的学术论文与研究成果............................................... 62