Data from: Identifying homomorphic sex chromosomes from wild-caught adults with limited genomic resources

We demonstrate a genotyping-by-sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild-caught individuals, in the moor frog Rana arvalis. Double-digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex-limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex-linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co-opted for sex determination among amphibians. The most efficient mapping strategy was a three-step hierarchical approach, where R. arvalis reads were first mapped to a low-coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex-diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.

本研究以林蛙（Rana arvalis）为研究对象，基于野生捕获个体的非侵入式采样，开发了一种测序分型法（genotyping-by-sequencing），用于鉴定同形性染色体及其在远缘参考基因组中的同源序列。本研究采集同一种群的30只雄性和21只雌性林蛙的颊拭子样本，构建了双酶切RADseq（double-digest RADseq）文库。针对未过滤数据集（含411446个RAD标签）的性别限制性标记筛选，相较于过滤后数据集（含33073个RAD标签），在基于杂合度或等位基因频率的性别差异标记筛选中效果更优。最终我们共获得292个推定的性连锁RAD位点，其中98%的位点支持雄性异配性别系统。我们可将其中15个位点定位至热带爪蟾（Xenopus tropicalis）基因组，除1个位点外其余均位于1号染色体对；而1号染色体在两栖动物的性别决定中似乎常被招募为性别决定相关区域。本研究采用的最高效的定位策略为三步分层法：首先将林蛙（R. arvalis）的测序读段比对至欧洲林蛙（Rana temporaria）的低覆盖度基因组（二者分化时间约17百万年），随后将欧洲林蛙的基因组支架比对至高原蛙（Nanorana parkeri）基因组（二者分化时间约90百万年），最终将高原蛙的基因组支架比对至热带爪蟾（Xenopus tropicalis）基因组（二者分化时间约210百万年）。我们采用靶向扩增候选性别决定基因Dmrt1（定位于1号染色体）部分序列的PCR引物对研究结论进行了验证：性别诊断等位基因仅存在于全部30只雄性个体中，而21只雌性个体均未携带该等位基因。该方法在生物样本匮乏或基因组资源有限的诸多研究场景中均具有良好的应用潜力。