实验海洋生物学重点实验室知识库

裙带菜是一种重要的经济褐藻，自二十世纪八十年代开始在我国进行商业化栽培，辽宁是我国的主产区，栽培群体最初从日本引进；我国有文献记载的裙带菜原生群体仅分布在浙江和福建两省，因人类活动和环境变化，野生裙带菜的分布范围向北收缩，近年在福建和浙江南部少见，仅在浙江的枸杞岛和渔山岛还有野生群体发现；辽东和山东半岛沿海的裙带菜都是从日本和韩国引进的，其中大连和青岛的野生种群起源于二十世纪初从韩国移植的群体，烟台和威海的野生群体是大连和青岛群体的衍生。在裙带菜育种中，种质资源库的建立和拓展是新品系培育的基础，栽培和野生群体均是进行种质资源收集的重要来源。在育苗和栽培过程中，北方同域分布的栽培群体和野生群体之间可能存在基因交流，二者的亲缘关系尚未可知，此外原产于我国南方的裙带菜群体也是重要的种质资源。为了有效开展裙带菜的种质资源收集与保存，构建核心种质库，需要首先系统研究我国野生与栽培裙带菜的遗传多样性和遗传结构。

前期的研究只包括了部分地区的部分群体，尚没有对我国裙带菜分布范围内的总体群体遗传学的系统研究，本研究利用SSR标记和线粒体DNA特定基因序列相结合，解析我国裙带菜主要野生与栽培群体的遗传多样性以及遗传结构，分析不同地理群体间的遗传关系，同时确定大连新发现种群的来源。

基于微卫星和线粒体序列的遗传分析显示，我国浙江枸杞岛和渔山岛的原产裙带菜群体具有较高的遗传多样性和独特的遗传组成，遗传结构分析显示出原产群体之间亲缘关系较近且与北方群体具有较大遗传差异，此外，种群近期并没有发生显著的瓶颈效应，表明其有效种群规模没有显著减少。推测它们有效种群规模较大，或与临近种群有足够的基因交流，抵消了遗传漂变对遗传多样性的负面影响。尽管枸杞岛和渔山岛裙带菜群体的杂合度较高，但渔山群体的F_is显著大于零，显示出杂合度缺失，表明群体内可能存在近交。

山东半岛潮下带的野生群体中，微卫星分析结果显示烟台长岛群体具有最高的遗传多样性（N_a=9.2；H_e=0.746），STRUCTURE聚类分析和PCoA分析显示山东半岛野生群体与大连栽培群体和南方野生群体之间具有高度遗传分化。除青岛群体外，山东半岛的野生群体经线粒体基因序列分析均体现出较高的遗传多样性。长岛野生群体与其他潮下带野生群体以及长岛的群体之间存在着不同程度的遗传分化，在小钦岛采集的样品中发现一个atp8-S 和W-I的组合序列的新单倍型。

通过线粒体cox3和tatC-tLeu串联序列分析，大连栽培裙带菜群体主要属于日本北部型，我国北方的潮下带野生群体主要属于大陆型，南方群体中两种类型同时存在但以大陆型为主，这与我国栽培和野生裙带菜群体来源的历史记录一致。

微卫星和线粒体分析显示大连育苗场排水口附近潮下带的野生种群（W22）具有丰富的遗传多样性。微卫星结果显示，与育苗场培育的栽培群体相比，W22与北方潮下带野生群体的亲缘关系更近；从线粒体序列来看，W22具有与我国北方潮下带野生种群相似、与栽培群体明显不同的单倍型组成，且cox3和tatC-tLeu串联序列以大陆型为主，由此确认该野生种群不是由育苗场排出的游孢子产生，而是起源于早期从韩国移植的种群，并且几乎没有受到附近育苗场培育的栽培群体的影响。

综上所述，本研究系统厘清了中国裙带菜群体的遗传多样性和遗传结构及其与日韩群体的遗传关系，研究结果不仅能为裙带菜种质资源的保存、发掘和合理利用提供理论依据，而且对于育种起始材料的选择与设计育种方案具有重要的指导意义，同时也为裙带菜群体的溯源研究提供了重要依据和参考。

Undaria pinnatifida is an economically important brown alga, which has been commercially farmed in China since the 1980s. In China, U. pinnatifida is mainly farmed in Dalian, Liaoning province, and the farmed stocks were originally introduced from Japan. Native populations of U. pinnatifida in China were documented to be distributed in the Zhejiang and Fujian provinces, but their distribution range has been declining northward and natural individuals have seldom been observed recently in Fujian and southern Zhejiang province, which is possibly a result of human activities and environmental changes. Populations of U. pinnatifida still prosper on small islands off the coast of Zhejiang province, such as Gouqi and Yushan Islands. Natural populations can also be observed on rocky shores in northern China, among which the populations in Dalian and Qingdao originated from intentional transplantation from Korea in 1930s, and those in Yantai and Weihai were secondary derivatives of individuals transplanted from Dalian and Qingdao.

The establishment and expansion of germplasm resource bank are the basis for the breeding of new U. pinnatifida cultivars, and both farmed and natural populations are important sources for germplasm collection. Besides, there may be gene flow between the farmed populations and the sympatric natural populations in northern China, and the genetic relationship between them remains uncertain. In addition, native populations in southern China are important potential germplasm resources that could be used for cultivar breeding. In order to effectively carry out the collection and preservation of germplasm resources of this economically important kelp and construct core germplasm bank, it is necessary to systematically study the genetic diversity and genetic structure of the farmed and natural U. pinnatifida populations in China.

The previous studies only contained some populations in partial areas of China, and systematic studies on the overall population genetics within the distribution range of U. pinnatifida in China have not been conducted. In the present study, informative mitochondrial sequences and nuclear microsatellites were used to analyze the genetic diversity and genetic structure of the main natural and farmed populations of U. pinnatifida in China, study their genetic relationships, and identify the origin of a newly discovered subtidal natural population in Dalian.

The analysis based on mitochondrial sequences and microsatellites showed high genetic diversity and unique genetic components of the native populations in Gouqi and Yushan Islands. We revealed shallow genetic structure among the southern native populations and great genetic differentiation between them and northern populations. Moreover, we did not detect signs of recent bottleneck in southern native populations, indicating that there had been no significant recent reduction of effective populations size in them. These findings suggested that the effective population size of U. pinnatifida from Gouqi and Yushan islands was either large, or otherwise, they had enough gene flow with other neighboring populations to counterbalance the negative impact of genetic drift on genetic diversity. Despite the high H_e values of southern native populations, the mean F_is value was significantly higher than zero across the 10 microsatellite loci in Yushan population, suggesting probable occurrence of inbreeding in this population.

Among the natural populations in the subtidal zone of the Shandong Peninsula, analysis based on microsatellites showed the highest genetic diversity（N_a=9.2；H_e=0.746）of U. pinnatifida sampled from Changdao. The result of STRUCTURE clustering and PCoA analysis revealed great genetic differentiation between the natural populations in Shandong Peninsula and the farmed populations from Dalian and the native U. pinnatifida in southern China. According to mitochondrial sequences, genetic diversity of natural populations was high in Shandong Peninsula except for that in Qingdao. Various degrees of genetic differentiation were revealed between samples from Changdao and other areas. One novel haplotype of concatenated sequences of atp8-S and W-I was detected in Xiaoqin Island samples.

According to the combined cox3 and tatC-tLeu sequences, almost all haplotypes in the farmed populations sampled from Dalian were classified as the Northern Japan type, and nearly all haplotypes detected in the subtidal natural populations in northern China belonged to the Continental type. Both the Northern Japan type and the Continental type were detected in southern native populations, with the Continental type being dominant in all populations. The classification of U. pinnatifida populations was consistent with the historical records of their origin.

We found a high level of genetic diversity in the newly discovered subtidal natural population of U. pinnatifida by the mitochondrial sequences and microsatellites. The results of microsatellites showed that W22 was genetically close to other subtidal natural populations in northern China, but highly differentiated from the farmed populations bred in the nearby hatchery. Analysis of mitochondrial sequences indicated that the haplotype component of W22 was similar to those detected in subtidal natural populations, but significantly distinct from those of farmed populations. Moreover, almost all haplotypes in W22 were classified as the Continental type for the combined cox3 and tatC-tLeu sequences. Therefore, we confirmed that this natural population was not founded by the zoospores discharged from the nearby hatchery and suggested that it originated from the early transplanted stocks from Korea. And it had almost not been contaminated by the pedigree of farmed populations bred in the nearby hatchery.

In conclusion, this study systematically clarified the genetic diversity and genetic structure of U. pinnatifida populations in China and the genetic relationship of Chinese populations with those from Japan and Kore. These results not only provide theoretical basis for conservation, exploration and rational utilization of stock resources of U. pinnatifida, but also have important guiding significance for the selection of breeding materials and the design of breeding programs, and also provide valuable reference for tracing the provenance of U. pinnatifida populations.

第1章 引言... 1

1.1 群体遗传学的研究内容和发展历程... 1

1.2 群体遗传学中常用的概念和参数... 2

1.3 分子标记的类型及应用... 3

1.4 大型藻类群体遗传学的研究进展... 5

1.5 裙带菜基本生物学特征和概述... 7

1.6 裙带菜的主要分布及在中国的栽培历史与产业技术的发展... 8

1.7 研究的目的和意义... 10

第2章 基于微卫星标记的中国主要野生与栽培裙带菜的群体遗传学研究... 11

2.1 引言... 11

2.2 材料与方法... 12

2.2.1 主要仪器与生化试剂... 12

2.2.2 实验材料... 13

2.2.3 裙带菜基因组DNA的提取... 14

2.2.4 微卫星核心序列扩增... 14

2.2.5 微卫星核心序列长度获取... 15

2.2.6 数据整理及分析... 15

2.3 实验结果... 16

2.3.1 DNA提取... 16

2.3.2 微卫星核心序列扩增及检测... 17

2.3.3 扩增产物测序... 17

2.3.4 结果分析... 17

2.4 结论与讨论... 24

2.5 小结... 26

第3章 基于线粒体基因序列的中国主要野生与栽培裙带菜群体遗传学研究... 27

3.1 引言... 27

3.2 材料与方法... 27

3.2.1 主要仪器与生化试剂... 27

3.2.2 实验材料... 28

3.2.3 裙带菜基因组DNA提取... 29

3.2.4 线粒体基因序列扩增及检测... 29

3.2.5 线粒体基因序列获取... 30

3.2.6 序列处理及分析... 30

3.3 实验结果... 33

3.3.1 DNA提取... 33

3.3.2 线粒体基因序列扩增及检测... 33

3.3.3 扩增产物测序... 33

3.3.4 序列处理及分析... 34

3.4 结论与讨论... 43

3.5 小结... 45

第4章 结论与展望... 46

参考文献... 48

致谢... 59

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 60