实验海洋生物学重点实验室知识库

鹿角菜（Silvetia siliquosa）分类上隶属于Orchorophyta门，褐藻纲（Phaeophyceae），墨角藻目（Fucales），墨角藻科（Fucaceae），鹿角菜属（Silvetia）。其生态、经济价值大，属潮间带多年生大型褐藻，该种仅分布于黄渤海及朝鲜半岛南岸和西岸。自20世纪90年代以来，受气候变化和人类活动影响，鹿角菜的地理分布范围不断萎缩，种群数量也急剧下降，在中国黄渤海生态区已被处于濒危状态。目前，关于鹿角菜种群遗传结构和多样性研究较为缺乏。本文就黄渤海地区的鹿角菜种群遗传开展了分析，在此基础上对其叶绿体、线粒体基因组进行了分析，相关结果为鹿角菜保护和遗传学研究提供理论参考，主要结果如下：

（1）利用细胞核内转录间隔区II（ITS2）、线粒体细胞色素氧化酶Ⅰ与线粒体23s/trnK基因间区的串联标记（cox1+mtIGS），对采自中国和韩国的6个鹿角菜种群的遗传多样性及其种群连通性进行了分析。结果表明，中、韩两国的鹿角菜种群单倍型多样性和核苷酸多样性均较低，仅分别检测到3个单倍型和核糖型。方差分析（AMOVA）表明，86-95%的遗传变异发生在种群间，5-14%的遗传变异发生在种群内。F_ST和基因流分析表明，韩国南部的两个种群（JIN和GWA）间以及它们与其它种群之间，都有较高的遗传分化，且基因流较低。除这2个种群外，其它4个种群间无明显的遗传分化。据此，我们建议将这6个种群划分为4个管理单元（MUs），即中国2个种群（LII和RUS）、韩国西海岸2个种群（YEO和RUS）、韩国南部GWA种群以及韩国南部的JIN种群。

（2）线粒体基因组测序分析表明，其全长为36,036 bp，GC含量为38.84%。共编码67个基因，包括35个蛋白编码基因，3个核糖体RNA基因（rRNA），26个转运RNA基因（tRNA）以及3个开放阅读框（ORF），所有编码基因均无内含子。鹿角菜线粒体基因中存在43条简单重复序列（SSR）和2对散在重复序列。基于线粒体基因组35个蛋白编码基因的系统发育分析，据此，可以得出鹿角菜与墨角藻的亲缘关系更近；其在褐藻纲与其它种类对比发现，仅在网地藻（Dictyota dichotoma）中发现1处重排。

鹿角菜叶绿体基因组大小为124,991 bp，属于典型的四段式结构。GC含量为28.84%，共编码173个基因，包括139个蛋白质编码基因，28个tRNA和6个rRNA，在tRNA^Leu-2基因内存在一个保守的内含子。鹿角菜叶绿体基因组中存在131个SSR位点和23对散在重复序列。另外，褐藻纲叶绿体基因组在基因组结构和组成上保守性较差，不同目之间发生了较多的基因丢失和重排，而同一分目内的叶绿体基因组则高度保守。

（3）保护策略。针对当前我国黄渤海潮间带环境的现状，我们建议要大力加强鹿角菜异地移植和人工增植，逐渐恢复其种群资源。在鹿角菜种群分布区，如在威海乳山、荣成俚岛等地，建立其保护区，进行原地鹿角菜资源的保护，减缓其灭绝的程度。

本研究首次对黄渤海海域褐藻—鹿角菜种群遗传多样性特征进行了分析，发现其处于濒危状态；另外开展了鹿角菜线粒体、叶绿体的基因组测序分析，提出了鹿角菜资源保护和恢复的建议，为黄渤海潮间带经济褐藻的资源保护和管理提供依据。

Silvetia siliquosa taxonomically belongs to Silvetia, Fucaceae, Fucales, Phaeophyceae, Orchorophyta, and is an ecologically and commercially vital perennial brown alga that uniquely distributed in the Yellow-Bohai Sea and along the southwest coast of Korean peninsula. Population biomass and distribution range of S. siliquosa in East Asia have declined dramatically since the 1990s due to climate change and anthropogenic impacts. Thus, this species has been listed as endangered in the ecological region of Yellow-Bohai Sea, with a high extinction risk. However, the knowledge of population genetic structure and diversity characteristics across the entire range are still scarce. In the present study, we conducted the population genetic analysis of S. siliquosa in the Yellow-Bohai Sea, and on this basis, we sequenced and characterized the mitochondrial and chloroplast genomes genomes of S. siliquosa. The relevant results provide theoretical reference for the conservation and genetic research of S. siliquosa .The main findings are as following:

(i) We used nuclear internal transcribed spacer-2 (ITS2) and concatenated mitochondrial cytochrome oxidase I subunit and intergenic spacer (cox1+mtIGS) to estimate genetic diversity and population connectivity of six S. siliquosa populations in China and Korea. Molecular results exhibited strikingly low levels of haplotype/ribotype and nucleotide diversity in S. siliquosa, with only three mitochondrial haplotypes and nuclear ribotypes detected, respectively. The analysis of molecular variance (AMOVA) revealed 85-95% of genetic variance among populations and 5-14% of genetic variance within populations. Population differentiation coefficient (F_ST) and gene flow (Nm) suggested that the two populations (JIN and GWA) along the southern coast of Korea are highly divergent from others. No significant genetic differentiation was observed among populations either in China or along the geographically proximate west coast of Korea. Thus, four independent management units (MU) were designated for sustainable management: the LII and RUS populations in China, the YEO and CHA populations from western of Korea, and each of the GWA and JIN populations from southern Korea.

(ii) Sequencing analysis of the mitochondrial genome (mtDNA) show that its whole length is 36,036 bp, with GC content of 38.84%. There are 67 genes in the mtDNA of S. siliquosa, including 35 protein-coding genes (PCGs), 3 ribosomal RNA genes (rRNA), 26 transport RNA genes (tRNA) and 3 open reading frames (ORFs). All these genes do not comprise introns. Forty-three simple sequence repeats (SSR) and 2 pairs of interspersed sequence repeats are detected in S. siliquosa mtDNA. Phylogenetic analysis based on 35 shared protein-coding genes in the mtDNA of Phaeophyceae demonstrated a close genetic relationship between S. siliquosa and F. vesiculosus. In addition, comparing the mtDNA of other species in Phaeophyceae, only one rearrangement is found in Dictyota dichotoma.

The chloroplast genome (cpDNA) of S. siliquosa consists of a 124,991 bp circular DNA molecule, exhibiting a typical quadripartite structure with a GC content of 28.84%. It comprises 173 genes, including 139 PCGs, 28 tRNA genes and 6 rRNA genes. One intron is identified in the tRNA^Leu-2 gene. We detect 131 SSRs and 23 pairs of interspersed sequence repeats in S. siliquosa cpDNA. In addition, cpDNA in Phaeophyceae is less conservative than mtDNA in gene composition, order and content. More gene losses and rearrangements have been identified between different orders in the Phaeophyceae.

(iii) Conservation strategy. In view of the current situation of the environment in the intertidal zone of the Yellow-Bohai Sea in China, we propose to gradually restore the population resources of S. siliquosa by artificial cultivation and transplantation of S. siliquosa. Furthermore, we suggest to establish conservation areas in Weihai Rushan, Rongcheng Li Island and other habitats with extremely low genetic diversity of S. siliquosa for in situ conservation to avoid extinction.

This study first described the genetic diversity characteristics of the brown seaweed S. siliquosa populations in the Yellow and Bohai Sea and found that it is in an endangered state; in addition, we analyzed the characteristics of its mitochondrial and chloroplast genomes, and put forward suggestions for the conservation and recovery of S. siliquosa resources. These results lay a foundation for guiding conservation and management of economic brown seaweed resources in the intertidal zone of the Yellow-Bohai Sea.

第1章 引言

1.1 鹿角菜的生物学特征

1.1.1 鹿角菜的分类及命名

1.1.2 鹿角菜的形态、特性与生活史

1.1.3 鹿角菜地理分布和应用价值

1.1.4 鹿角菜的资源现状

1.2 遗传学及其在海藻资源保护中的应用

1.2.1 保护遗传学及其应用

1.2.2 种群遗传学研究及应用

1.2.3 海藻资源衰退及保护措施

1.3 海藻细胞器基因组

1.3.1 线粒体基因组

1.3.2 叶绿体基因组

1.4 本研究的目的和意义

第2章 鹿角菜种群遗传多样性分析和保护

2.1 材料与方法

2.1.1 样品采集

2.1.2 实验器材及试剂

2.1.3 基因组DNA提取

2.1.4 基因片段的扩增和测序

2.2 数据分析

2.2.1 序列编辑与比对

2.2.2 种群遗传多样性和遗传分化

2.2.3 种群连通性

2.3 结果

2.3.1 遗传多样性

2.3.2 种群遗传分化

2.3.3 种群连通性

2.4 讨论

2.4.1 鹿角菜种群遗传多样性

2.4.2 种群连通性和保护

第3章 鹿角菜的细胞器基因组测序及分子进化分析

3.1 材料与方法

3.1.1 样品采集与鉴定

3.1.2 实验器材和试剂

3.1.3 基因组DNA提取和检测

3.1.4 文库构建及测序

3.1.5 基因组的组装和注释

3.1.6 重复序列和密码子使用分析

3.1.7 IR边界和叶绿体基因组比较分析

3.1.8 褐藻线粒体基因组系统发育分析

3.2 结果

3.2.1 鹿角菜线粒体基因组结构

3.2.2 鹿角菜叶绿体基因组结构特征

3.2.3 重复序列分析

3.2.4 密码子偏好性

3.2.5 IR区的收缩和扩张

3.2.6 细胞器基因组共线性分析

3.2.7 褐藻纲分子系统发育

3.3 讨论

3.3.1 褐藻纲线粒体基因组进化

3.3.2 褐藻纲叶绿体基因组进化

第4章 结论

参考文献

附录

致  谢

作者简历及攻读学位期间发表学术论文与成果