Chromosome-level assembly and annotation of Lates japonicus

It is known that some endangered species have persisted for thousands of years despite their very small effective population sizes and low levels of genetic polymorphisms. To understand the genetic mechanisms of long-term persistence in threatened species, we determined the whole genome sequences of akame (Lates japonicus), which has survived for a long time with extremely low genetic variations. Genome-wide heterozygosity in akame was estimated to be 3.3–3.4 × 10-4 /bp, one of the smallest values in teleost fishes. Analysis of demographic history revealed that the effective population size in akame was around 1,000 from 30,000 years ago to the recent past. The relatively high ratio of nonsynonymous to synonymous heterozygosity in akame indicated an increased genetic load. However, a detailed analysis of genetic diversity in the akame genome revealed that multiple genomic regions, including genes involved in immunity, synaptic development, and olfactory sensory systems, have retained relatively high nucleotide polymorphisms. This implies that the akame genome has preserved the functional genetic variations by balancing selection, to avoid a reduction in viability and loss of adaptive potential. Analysis of synonymous and nonsynonymous nucleotide substitution rates has detected signs of positive selection in many akame genes, suggesting adaptive evolution to temperate waters after the speciation of akame and its close relative, barramundi (L. calcarifer). Our results indicate that the functional genetic diversity likely contributed to the long-term persistence of this species by avoiding the harmful effects of the population size reduction.

Methods
The chromosome-level assembly of Lates japonicus was generated by ordering the scaffolds using mScaffolder (Chakraborty et al. 2018) guided by their alignments to the chromosome-level genome assembly of L. calcarifer (Vij et al. 2016) using Nucmer implemented in MUMmer (Kurtz et al. 2004). Gene predictions in the assembly were carried out using the masked genome and RNA-Seq spliced alignment information by GeneMark-ET (Lomsadze et al. 2014) and AUGUSTUS (Keller et al. 2011) implemented in the BRAKER1 pipeline (Hoff et al. 2019).

已知部分濒危物种虽有效种群规模（effective population size）极小、遗传多态性（genetic polymorphism）水平极低，却已存续数千年。为解析受威胁物种长期存续的遗传机制，我们对眼斑尖吻鲈（akame, *Lates japonicus*）开展全基因组测序——该物种凭借极低的遗传变异实现了长期存活。经估算，眼斑尖吻鲈的全基因组杂合度（heterozygosity）为3.3~3.4×10⁻⁴/碱基对，是硬骨鱼类（teleost fishes）中最低的杂合度水平之一。种群历史动态分析显示，自3万年前至今，眼斑尖吻鲈的有效种群规模始终维持在1000左右。其非同义与同义杂合性比率偏高，提示遗传负荷（genetic load）有所升高。然而对眼斑尖吻鲈基因组的遗传多样性进行精细分析后发现，包括免疫相关基因、突触发育相关基因以及嗅觉感知系统相关基因在内的多个基因组区域，仍保留了相对较高的核苷酸多态性。这意味着眼斑尖吻鲈基因组通过平衡选择（balancing selection）保留了功能性遗传变异，从而避免了生存能力下降与适应潜力丧失。对同义与非同义核苷酸替换速率的分析检测到眼斑尖吻鲈众多基因存在正选择（positive selection）信号，表明在其与近缘物种澳洲肺鱼（barramundi, *L. calcarifer*）分化后，该物种经历了针对温带水域的适应性演化（adaptive evolution）。本研究结果表明，功能性遗传多样性通过规避种群规模缩减带来的有害效应，助力该物种实现了长期存续。

## 研究方法
本研究通过将眼斑尖吻鲈的scaffold序列与澳洲肺鱼（*L. calcarifer*）的染色体级基因组组装结果（Vij et al. 2016）进行比对，借助MUMmer工具包中的Nucmer软件（Kurtz et al. 2004）完成比对流程，再以mScaffolder（Chakraborty et al. 2018）为指导对scaffold进行排序，最终获得眼斑尖吻鲈的染色体级基因组组装结果。
针对该组装结果的基因预测工作，采用了BRAKER1流程（Hoff et al. 2019）中整合的GeneMark-ET（Lomsadze et al. 2014）与AUGUSTUS（Keller et al. 2011）工具，同时使用了重复序列屏蔽后的基因组序列与RNA-seq剪接比对信息。