中国科学院昆明动物研究所机构知识库

随着越来越多的动物基因组测序完成，以表型筛选为主的正向遗传学方法正逐步被反向遗传学方法替代而成为功能基因组学研究的主要方法。而测序成本的降低和拼接算法的研发以及高性能计算资源的普及，使得单个实验室也能够独立的完成基因组的测序和拼接，进行物种的基因组学研究。本研究关注的物种是眼斑双锯鱼，为鲈形目雀鲷科双锯鱼属的一种，具有独特的与海葵共生和性逆转等生物学特性。我们应用第二代高通量测序技术完成了眼斑双锯鱼(Amphiprion Ocellaris)基因组序列图谱的绘制工作，并通过与其它7种已知基因组序列的硬骨鱼类进行比较，分析了在基因组水平上与双锯鱼属特殊形状相关的序列变化。组装产生的基因组大小为902.28Mbp，序列的Contig N50长度为56.17Kb，Scaffold长度为1.06Mb，接近于之前报道的硬骨鱼的基因组大小。双锯鱼基因组测序深度约192X，超过98%的碱基测序深度大于20X，具有非常高的单碱基正确性。用CEGMA和转录组数据对组装结果进行评估，发现96.37%的核心基因能够在基因组中覆盖，95%以上的转录本能够比较完整地比对到基因组。在双锯鱼基因组中识别到超过一百四十九万个杂合SNP位点，杂合度约为0.17%。同时，我们在眼斑双锯鱼基因组中共注释出22420个蛋白编码基因，平均编码序列长度为1733bp，平均外显子长度为230.58bp。基因集中92.4%的基因在公共数据中能找到相似的同源序列，17594个基因被注释上基因名。在双锯鱼和其它几种硬骨鱼这八个物种中共鉴定出8518个高质量同源基因，对其进行物种树构建，认为双锯鱼与青鳉鱼和新月鱼的共同祖先互为姐妹群，并且在距今约103.7个百万年发生分歧。观察发现双锯鱼具有较低的蛋白进化速率，同时利用四重简并位点进行分析也得到相同结果，使用斑马鱼作为外群，双锯鱼中的同义突变率（dS）显著低于其它硬骨鱼类。为了评估双锯鱼枝中所发生的物种特异的适应性进化，我们使用PAML进行了正选择分析，筛选出了308个基因发生了相关位点的变化。进一步对发生适应性进化的基因进行功能富集分析发现，双锯鱼中许多与离子转运和生长发育相关的基因发生了更多的适应性进化，并且相比于其近缘种青鳉鱼有明显差异的富集功能。这些基因的变化与双锯鱼共生海葵的能力相关，调控生长发育的基因，使得双锯鱼在性逆转过程中快速发生体型变化。

With the increasing number of animal genome projects, reverse genetics is becoming a main research approach instead of forward genetics which based on phenotype. The cost of generating sequencing data has dramatically declined. Rapid advances in bioinformatics tools and accessible computational resources have democratized the field. It is now within reach for individual research group to generate de novo draft genome sequences for any organism of choice. Here we de novo assemble a reference genome of ocellaris clownfish which is a species in the family Pomacentridae of order Perciformes and comparative genomics analysis identify candidate gene associated with its specialized morphology, such as symbiosis with sea anemone and sex change. We obtain a 902.28Mb genome assembly with a contig N50 of 56.17kb and a scaffold N50 of 1.06Mb. The sequencing depth is about 192X accroding to the size of genome assembly and over 98% bases in genome can be covered over 20X depth. We do assessment of completeness of genome assembly using CEGMA and transcriptome data. 96.37% core eukaryotic genes can be found in clownfish genome and over 95% transcripts can be aligned to genome with a good quality. Over 1.49 million SNP site are uncovered in genome and the heterozygosity is about 0.17%. We identify 22420 protein coding genes with average coding region length of 1733bp and average exon length of 230.58bp. 92.4% of gene set can find homologous sequences in public database and 17594 genes can be assigned with gene symbols. 8518 high quality ortholog families were selected from eight teleost fish genomes to construct a phylogenomic tree. It shows A.ocellaris is a sister group of common ancestor of medaka and platyfish and diverged about 103.7 Mya. Intriguingly, we found the branch length of clownfish is shorter than of other teleosts, suggesting a lower protein evolutionary rate compare to other teleosts analysed in this study. It comes out the same result with two-cluster test and relative rate test with fourfold degenerate sites. To identify genes in clownfish lineage that are under positive selection, we use PAML to evaluate the adaptive selection which led to 308 genes with positive selected site. To further evaluate the function of PSGs, GO enrichment analysis for the stringent list of 308 genes is performed. We found more genes with functions of iron transportation and body growth are under adaptive selection compare to its close related species medaka. These changes in clownfish coding sequence may be associated with its ability of symbiosis with sea anemone. PSGs which regulate the body development and growth enable clownfish to response quickly to brain signal during sexual change process.