实验海洋生物学重点实验室知识库

　　自2007年起，我国黄海海域连续暴发大规模绿潮，对近海环境造成严重影响，已成为常态化的海洋生态灾害。前期研究表明，黄海绿潮是由不同于定生群体的浒苔（Ulva prolifera）漂浮生态型（floating ecotype）主导。黄海绿潮的发展包含了多个阶段，并经过长距离漂移。在紫菜筏架生长阶段，存在多个石莼属物种的混生；在早期漂浮阶段，会经历物种演替过程；只有到了暴发阶段，浒苔漂浮生态型才占据绝对优势，并一直保持到衰退阶段。因此，为了量化监控漂浮生态型在暴发全过程中的种群动力学特征，需要可靠的种间及种内特异分子标记作为工具。然而，目前区分浒苔与其近缘种——缘管浒苔（U. linza）的种间标记仍存在争议；另外，针对浒苔漂浮生态型的种内SCAR特异标记由于遗传背景不清楚，阳性检出率存在低估的可能，亟待开发遗传背景清晰、可靠的新标记。

　　叶绿体基因组携带丰富的遗传信息，常用作近缘种间和种内群体的遗传学研究。针对上述问题，本研究首先利用二代测序技术对一株定生浒苔叶绿体基因组进行了测序；其次，围绕浒苔与近缘种间、以及浒苔种内特异分子标记开发两个主要目标，利用生物信息学方法，与已有的缘管浒苔及浒苔漂浮生态型开展了叶绿体比较基因组分析；最后，利用获得的差异化遗传信息，分别开发种间及种内叶绿体分子标记，并在大量样本中进行了验证。上述研究主要取得以下结果：

　　1）完成了定生浒苔U161的叶绿体基因组测序，基本特征分析结果表明，基因组全长99,724 bp，共编码95个基因，含有100个简单重复序列（simple sequence repeat，SSR）位点和193条长重复序列。

　　2）将定生浒苔与缘管浒苔及浒苔漂浮生态型进行了叶绿体比较基因组分析，结果表明三者的密码子使用策略一致，共线性关系良好，亲缘关系近。但在浒苔与缘管浒苔种间以及浒苔种内仍存在差异化位点，通过叶绿体基因组序列比对分析，获得了591个浒苔与缘管浒苔的种间差异位点和168个浒苔种内差异位点。

　　3）根据获得的浒苔与缘管浒苔种间差异位点，分别利用浒苔petB和psaB基因的内含子片段开发了用于区分浒苔与缘管浒苔的种间分子标记，在不同地理群体的浒苔与缘管浒苔样本中进行验证，结果均符合预期。

　　4）通过获得的浒苔种内差异位点，开发了浒苔漂浮生态型叶绿体特异分子标记（Cp），在历年漂浮浒苔样本和广泛分布的岸基定生群体样本中进行了验证，结果均符合预期。

　　5）经生信分析和生活史跟踪检测，分别揭示了Cp标记与SCAR标记的不同遗传方式。证实Cp标记为母系遗传，而核编码的单拷贝SCAR标记存在杂合基因型，会随减数分裂产生阴性后代，从而导致对漂浮生态型阳性检出率的低估。

　　上述研究进展将为黄海绿潮的早期预警、过程监测及风险评估提供可靠的分子工具。

　　Large scale green tides, which have been recorded in the Yellow Sea since 2007, posed significant impact on ecological environment along the coast of the Yellow Sea, and have been considered as a routine marine ecological disaster in the Yellow Sea. Previous studies have shown that the Yellow Sea green tide was dominated by the floating ecotype of Ulva prolifera, which is different from the attached populations. The formation of the Yellow Sea green tide was consisted of several stages and undergone long distance drift. In the stage of grown in the nori rafts at Subei, the floating ecotype of U. prolifera was mixed with other species of Ulva; In the early floating stage, had a process of species succession; In the outbreak stage, the floating ecotype of U. prolifera took absolute advantage, and it remained until the decline stage. Therefore, in order to quantitatively monitor the population dynamics of floating ecotype throughout the outbreak process, reliable interspecific and intraspecific molecular markers are needed as tools. However, the interspecific markers to distinguish U. prolifera from its related species, U. linza, are still controversial; In addition, due to the unclear genetic background of the intraspecific SCAR marker which was developed to identify the floating ecotype of U. prolifera, and the positive detection rate may be underestimated. It is urgent to develop new markers with clear and reliable genetic backgrounds.

　　The chloroplast genome has rich genetic information, and often used for genetic research of related species and intraspecific populations. In view of the above background, this study used next-generation sequencing technology to obtained the chloroplast genome of a strain attached U. prolifera, then comparative genomics analyses were performed with existing chloroplast genomes from U. linza and the floating ecotype of U. prolifera in order to develop interspecific and intraspecific markers of U. prolifera. Last, the identified interspecific and intraspecific variations were used to develop interspecific and intraspecific markers, and the markers were validated in a number of samples. The main results are as follows:

　　1) The chloroplast genome sequencing and basic characteristic analysis of attached U. prolifera U161 were completed. The full length of chloroplast genome is 99,724 bp, encoding 95 genes, including 100 SSR sites and 193 long repeat sequences.

　　2) The comparative genomic analysis of the chloroplasts of U. linza, attached U. prolifera and floating ecotype of U. prolifera showed that the codon usage strategies were consistent and level of collinearity was high. However, there are still interspecific and intraspecific variations. Through the alignment and analysis of chloroplast genome sequences, 591 interspecific variations and 168 intraspecific variations were obtained.

　　3) According to the obtained interspecific variations, two pairs of markers were developed by using the introns of petB and psaB genes of U. prolifera and validation was carried out in the different populations of U. prolifera and U. linza, the results were all as expected.

　　4) Based on the obtained intraspecific variations of U. prolifera, a specific marker (Cp) for the floating ecotype of U. prolifera was developed and validation was carried out in the samples of floating ecotype and attached U. prolifera, the results were all as expected.

　　5) The inheritance patterns of Cp and SCAR markers were revealed through bioinformatics analysis and life history tracking detection. The Cp marker was inherited maternally and the SCAR marker located in the nuclear genome and has a heterozygous genotype, and allelic segregation happened during the genetic process so that a portion of samples amplification results were negative. It indicated that the SCAR marker underestimated the detection rate of floating ecotype of U. prolifera.

　　Our study provides reliable molecular tools for early warning, process monitoring and risk assessment of the Yellow Sea green tide.

 第1章 绪论... 1

1.1 黄海绿潮... 1

1.1.1 黄海绿潮简介... 1

1.1.2 黄海绿潮致灾种... 2

1.1.3 黄海绿潮的形成与暴发... 4

1.2 石莼属种间及浒苔种内分子标记概述... 6

1.2.1 石莼属种间分子标记的开发现状... 6

1.2.2 浒苔与缘管浒苔复合体的由来及分子标记开发现状... 6 

1.2.3 浒苔漂浮生态型特异分子标记开发现状... 7

1.3 石莼属绿藻叶绿体基因组研究... 7

1.3.1 叶绿体基因组概况... 7

1.3.2 石莼属绿藻叶绿体基因组研究现状... 8

1.4 研究目的与意义... 10

第2章 定生浒苔叶绿体基因组测序与分析... 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 实验方法... 12

2.3 结果... 15

2.3.1 叶绿体基因组测序与组装... 15

2.3.2 叶绿体基因组注释与系统发育分析... 16

2.3.3 叶绿体基因组特征分析... 19

2.4 讨论... 33

2.5 小结... 34

第3章 浒苔与缘管浒苔种间叶绿体分子标记开发... 35

3.1 引言... 35

3.2 材料与方法... 35

3.2.1 实验材料... 35

3.2.2 实验方法... 36

3.3 结果... 38

3.3.1 分子鉴定... 38

3.3.2 浒苔与缘管浒苔叶绿体基因组比较分析... 40

3.3.3 浒苔与缘管浒苔种间分子标记的开发与验证... 44

3.4 讨论... 46

3.5 小结... 47

第4章 浒苔漂浮生态型叶绿体特异分子标记开发... 49

4.1 引言... 49

4.2 材料与方法... 49

4.2.1 实验材料... 49

4.2.2 实验方法... 61

4.3 结果... 63

4.3.1 不同生态型浒苔叶绿体基因组比较分析... 63

4.3.2 浒苔漂浮生态型叶绿体特异分子标记的开发与验证... 66

4.3.3 浒苔漂浮生态型叶绿体特异分子标记的应用... 70

4.4 讨论... 72

4.5 小结... 73

第5章 结论与展望... 75

参考文献... 77

致 谢... 87

作者简历及攻读学位期间发表的学术论文与研究成果 89