大黄鱼细胞遗传图谱的构建和初步应用

Large yellow croaker (Larimichthys crocea, LYC) is one of most important marine culture fish species in China. In recent years, the researches on genetics and breeding of LYC have advanced rapidly. For example, two new varieties have been bred, three generations of genetic maps have been constructed, and two versions of draft map of whole genome were published. In addition, there were new findings in the cytogenetic of LYC and its related species as well: the sex chromosomes were identified in LYC and Collichthys lucidus; high variable regions existing on the chromosome of Sciaenids were proposed on the basis of FISH signals of rDNA. However, the studies on the chromosomal level are still the “short slab” in the genetics of LYC, due to the lack of effective cytogenetic markers. Therefore, the major objectives of this project are to construct the cytogenetic map of LYC and obtain the linkage group specific BACs, and then initially applied to solve the conundrum of chromosome recognition and to investigate the possible rearrangement in the sex chromosome of LYC. We will use the recent genomic and cytogenetic tools to achieve the objectives, which includes the following steps: i) screening linkage group specific BACs in a library with PCR; ii) anchoring the linkage map specific BACs on the chromosome with BAC-FISH to integrate the linkage map and the cellular maps of chromosome; iii) developing new method to identify the whole set of chromosome of LYC based on the linkage group specific BACs; iv) analyzing the fine structure of X and Y chromosomes comparatively with sex linkage group specific BAC; v) evaluating the compatibility of the linkage map specific BACs of LYC in other Sciaenids. The expected achievements of this project include the first cytogenetic map and first panel of chromosome specific FISH reagents of LYC, and the possible chromosome rearrangement in the sex chromosome of LYC. These expected achievements will provide a bridge to exchange the findings between cytogenetics and genomics of LYC, push a jumbo advance in cytogentics of LYC and other Sciaenids.

本项目拟利用大黄鱼基因组的最新研究成果，构建大黄鱼细胞遗传图谱，筛选连锁群特异的BAC克隆，并初步应用于解决大黄鱼染色体识别难题与性染色体精细结构分析中。研究内容包括：1) PCR法筛选连锁群特异BAC克隆，双末端测序验证；2) 利用FISH将连锁群特异BAC克隆锚定在染色体上，整合连锁图和染色体细胞图；3) 利用连锁群特异BAC克隆开发识别全套染色体的方法；4) 利用性连锁群特异BAC克隆比较分析X和Y染色体的精细结构；5) 初步评估连锁群特异BAC克隆作为其他石首鱼FISH探针的可行性。本项目将构建首个大黄鱼细胞遗传图谱，预期结果将推动大黄鱼和其他石首鱼的染色体研究实现跨越式发展，并提供沟通大黄鱼细胞遗传学与基因组学信息的桥梁，为大黄鱼及其他石首鱼的遗传育种和进化研究提供重要工具。

本项目基于大黄鱼参考基因组，结合生物信息学和分子细胞遗传学方法，筛选了大黄鱼连锁群特异的BAC克隆，构建其细胞遗传图谱。主要研究内容与结果如下：（1）染色体候选特异BAC克隆的筛选。从大黄鱼BAC文库中随机取500个BAC克隆进行双末端测序，再比对到大黄鱼参考基因组上，共获得双末端序列均在同一条染色体的BAC克隆435个（平均每条染色体18个）。统计BAC克隆对应的基因组区段的重复序列含量，并选出各条染色体重复序列含量最低的5个作为候选BAC克隆。（2）细胞遗传图谱的构建。候选BAC经过PCR和FISH验证，全部BAC扩增出预期长度的单一条带，通过FISH验证的BAC共67个，平均每对染色体2~3个。以染色体特异BAC为媒介，整合染色体图与基因组信息，构建了细胞遗传图谱。（3）大黄鱼全套染色体识别方法的开发。用染色体特异BAC制备探针池，通过FISH获得染色体彩色条形，实现全套染色体的辨识。（4）大黄鱼性染色体识别与特征分析。筛选获得Dmrt1基因阳性的BAC，利用FISH定位识别性染色体，证明X和Y染色体没有明显的细胞学差异；但序列分析显示，Y染色体存在小范围的重复。（5）大黄鱼染色体特异BAC克隆在物种间通用性的初步评估。比对到了棘头梅童鱼的参考基因组的大黄鱼染色全特异BAC共219个，其中5号染色体上的BAC克隆101-J21、101-K12通过FISH验证。本项目开发了大黄鱼全套染色体的FISH标记，首次实现鱼类全套染色体的准确识别，将推动大黄鱼和其他石首鱼的染色体研究的发展，并提供沟通大黄鱼细胞遗传学与基因组学信息的桥梁，为大黄鱼及其他石首鱼的遗传育种和进化研究提供重要工具。