海洋生态与环境科学重点实验室知识库

中国近海小黄鱼SNP标记开发及种群基因组学研究
中文摘要
对遗传组成有差异的生物群体进行鉴定、区分并制定相应合理的管理单元是保护生物学以及渔业资源管理的重要内容。由于海洋环境缺少相对明显的地理屏障，同时海洋鱼类具有较大的种群大小，海洋鱼类不同群体间存在较高的连通性，因此海洋鱼类群体间具有较低的遗传分化水平。近年来不断涌现和完善的多种高通量测序方法，对于精确解析高基因流海洋鱼类的群体遗传结构、获取更多准确的种群动态参数以及揭示潜在的适应性分化等方面具有前所未有的优势，为高基因流海洋鱼类的种群基因组学研究提供了重要的技术平台。RAD-seq （Restriction-site associated DNA-sequnencing），即基于酶切的简化基因组测序技术，该技术通过Illumina二代测序平台，能够对成百上千的个体同时进行SNP位点的鉴定，获得基因组水平上成千上万的SNP标记，为种群基因组学研究带来了前所未有的革新。
小黄鱼（Larimichthys polyactis）是中国乃至东亚地区最重要的海洋经济鱼类之一，然而受过度捕捞及其他人类活动等因素的影响，中国沿海的小黄鱼渔业资源正面临全面衰退的严峻现状。前期的小黄鱼群体遗传学研究由于受到遗传标记种类及数目的限制，基于不同类型的遗传标记所获得的研究结果存在一定的分歧甚至相互矛盾。在本研究中，我们基于简化基因组测序技术（RAD-seq）成功对中国近海不同地理群体的小黄鱼个体进行RAD文库构建和双末端测序，并获得了基因组层面高密度的SNP位点。基于所获得的SNP位点对我国沿海各群体进行群体遗传学分析，首次从基因组水平上精确解析了小黄鱼的群体遗传结构及本地适应性特征，相关研究结果将为我国小黄鱼资源的合理管理和保护提供重要的理论基础。
主要的研究结果如下：
1） 采用RAD-seq技术对我国丹东东港和霞浦三沙2个地理群体的24个小黄鱼个体进行全基因组扫描，首次从基因组层面开发出了高密度的SNP位点（共27, 556SNP），并组装了高质量的参考序列，研究结果为今后小黄鱼基因的功能注释、引物设计以及相关的群体遗传学研究提供了重要的资源。
2）比较并探讨了不同SNP筛选标准（每个contig保留F_ST最大值的SNP以及每个contig保留质量得分最高的SNP）在种群基因组学研究中对群体结构分析的影响。研究结果表明，不同的SNP筛选标准对于小黄鱼的群体遗传结构分析带来较大偏差，原因可能是由于小黄鱼具有较短的种群历史和较高的群体间基因流，因此本研究结果对相似遗传背景的海洋生物的种群基因组研究具有重要的借鉴意义。
3）基于全部36,221个SNP位点对我国沿海7个地理群体小黄鱼进行群体遗传结构分析，研究结果表明群体间的遗传分化水平较低（0.00117≤ Pairwise F_ST ≤ 0.00723），研究证明了我国近海小黄鱼存在隐存的群体遗传结构且不符合地理隔离的分布模式。
4）通过LOSITAN和Bayescan两种方法对36,221SNP进行离散位点的筛选，共得到171个SNP位点可能受到自然选择的作用。通过软件BLAST2GO对离散位点所在的contig序列进行直系同源注释，共有12条序列注释成功。候选基因功能注释结果显示这些基因与肾小管发育以及抗原呈递等多种生物学功能相关，研究结果证明了中国近海小黄鱼可能形成了与水盐代谢以及病原体免疫相关的本地适应性现象。
5）基于171个离散位点对我国近海小黄鱼的适应性分化格局进行了分析和探讨。该分析方法极大地提高了对于小黄鱼群体遗传结构的解析能力，研究结果表明我国近海小黄鱼主要存在三大类群，其中长江口小黄鱼群体具有独特的遗传分化特征。研究结果表明我国近海小黄鱼存在适应性分化且不符合地理隔离的分布模式，其可能原因是由于对不同产卵栖息地的定向选择以及海流等海洋环境因子共同作用而形成的。
本文的研究结果揭示了小黄鱼的群体遗传结构及适应性分化的遗传学特征，研究结果为我国小黄鱼渔业资源的科学管理和保护提供了重要的理论基础。
 
关键词：小黄鱼，单核苷酸多态性标记，RAD-seq，群体遗传结构，本地适应性，种群基因组学

Development of single nucleotide polymorphisms resource and population genomic analysis for the small yellow croaker （Larimichthys polyactis）in offshore China
ABSTRACT
Determining genetically distinct populations and establishing appropriate management units are primary goals of conservation biology and fishery management. Generally in the past, the general pattern of population structure in marine fishes were prevalently believed to be low levels of genetic differentiation, which might be attributed to the lack of obvious dispersal barriers as well as large population sizes, resulting in a relatively high connectivity among populations. Recent advances in high-throughput sequencing technologies have offered the possibility to generate genome-wide sequence data to delineate previously unidentified genetic structure, obtain more accurate estimates of demographic parameters, and to evaluate potential adaptive divergence in marine fish with high gene flow. Restriction site-associated DNA tags sequencing (RAD-seq) uses Illumina next-generation sequencing platform to simultaneously discover and score tens to hundreds of thousands of SNP markers in hundreds of individuals.
The small yellow croaker, Larimichthys polyactis is one of the most economically important marine fishery species in China and East Asian countries, however, the abundance of L. polyactis in China had severely decreased due to overfishing as well as other anthropogenic factors. Limited source of markers has somehow constrained genetic studies of L. polyactis, and previous studies based on various types of genetic markers have obtained discrepant or even conflicting results. Here, we identified a genome-wide single-nucleotide polymorphisms resource for L. polyactis using restriction-site-associated DNA (RAD) library construction and pair-end sequencing. In this study, high-precision genetic structure and local adaptation of L. polyactis were characterized at the genomic level for the first time, which provides an important theoretical basis for the management and conservation of L. polyactis in China. The main results are as follows:
1） RAD-seq approach was used to developed a genome-wide high-density single-nucleotide polymorphisms resource of 27,556 SNP based on 24 individual of L. polyactis from Dandong Donggang and Xiapu Sansha in offshore China. High-quality contigs were locally assembled for L. polyactis for the first time, which is crucial for homology searching, high-throughput assay primer design and relevant population genetic studies in the future.
2） We tested possible effects of different SNP filtering criteria (keeping the SNP with the highest F_ST per contig and keeping the SNP with the highest quality score per contig) on population genetic structure inference. The results indicated that potential biases resulting from different marker filtering criteria for each contig could be obtained for following population genetic analysis. Possible causes for the systematical biases might be attributed to the relatively short demographic history and high-level gene flow among L. polyactis populations. Therefore, the results should increase the quality and comparability of population genomic studies of other marine organmism with the similar genetic background.
3） Population genetic study of L. polyactis from 7 geographical locations based on the 36,221 SNP revealed shollow genetic differentiation among L. polyactis populations (0.00117≤ Pairwise F_ST ≤ 0.00723). In the present study, the pattern of isolation by distance was not observed and the results suggest the existence of hitherto unrecognized cryptic population structuring within this species
4） Both LOSITAN and Bayescan were used to detect outlier from 36,221SNP and a total of 171 outliers were finally obtained. Contigs containing the outlier were used for orthology annotation and 12 significant hits were obtained and functional annotation of the candidate genes involved in pronephric duct development and antigen presentation, etc. The results indicated the existence of local adaptation associated with salt metabolism and pathogen immune among geographical populations of L. polyactis in China.
5）The adaptive differentiation pattern of L. polyactis was investigated based on 171 outliers, which greatly improved the precision and accuracy of population genetic structure of L. polyactis. The results indicated that there were three groups of L. polyactis in offshore China and Yangtze Estuary population was observed to be differentiated from the other two groups. Deviation from pattern of isolation by distance was found, which might be attributed to spawning habitat choice and ecological factors such as see currents.