Assigning the sex-specific markers via genotyping-by-sequencing onto the Y chromosome for a torrent frog Amolops mantzorum

We use a genotyping-by-sequencing (GBS) approach to identify sex-linked markers in a torrent frog (Amolops mantzorum) using wild-caught individuals of 21 males and 19 females from the same population. A total of 141 putatively sex-linked markers were screened from 1,015,964 GBS tags through three approaches, respectively based on sex differences in allele frequencies, sex difference in heterozygosity, and sex-limited occurrence. With validations, 69 sex-linked markers were confirmed, all of which point to male heterogamety. The male specificity of eight sex markers was further verified by PCR amplifications with a large number of additional individuals covering the whole geographic distribution of the species. Y chromosome (No. 5) was microdissected under a light microscope, amplified by whole-genome amplification, and assembled a draft Y genome. 55 out of 69 sex-linked markers could be mapped to the Y chromosome assembly (i.e 79.7 %). Thus the chromosome 5 could be added as candidate chromosomes that particularly favored to recruit for sex determination than others among frogs. Three sex-linked markers that mapped on Y chromosome were aligned to three different promoter regions of Rana rugosa CYP19A1 gene, which might be considered as a candidate gene to trigger sex determination in A. mantzorum.本研究采用测序分型技术（genotyping-by-sequencing, GBS），以采自同一种群的21只雄性与19只野生捕获的曼氏湍蛙（Amolops mantzorum）个体为实验材料，开展性别连锁标记筛选工作。研究人员从1,015,964条GBS标签中，通过三种分别基于等位基因频率性别差异、杂合度性别差异及性别限制性存在的分析策略，共筛选得到141个候选性别连锁标记。经验证后，最终确认69个性别连锁标记，所有标记均指向雄性异配性别决定模式。研究人员进一步通过PCR扩增实验，对覆盖该物种全地理分布范围的大量额外个体进行验证，确认了8个性别标记的雄性特异性。此外，研究人员在光学显微镜下显微切割第5号染色体（即Y染色体），经全基因组扩增后完成序列扩增，并组装得到Y染色体草图基因组。69个已确认的性别连锁标记中，有55个可定位至该Y染色体组装序列（占比79.7%），由此可见，在蛙类中，第5号染色体相较于其他染色体更易参与性别决定过程，可作为候选性别决定染色体纳入研究范畴。定位至Y染色体的3个性别连锁标记，可比对至皱皮蛙（Rana rugosa）CYP19A1基因的三个不同启动子区域，该基因或可作为曼氏湍蛙（Amolops mantzorum）性别决定过程的候选触发基因。