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 non-invasive 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 dataset (411,446 RADtags) was more successful than searches from a filtered dataset (33,073 RADtags) 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 R. 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.

本研究以田野林蛙（Rana arvalis）为对象，基于野生个体的非侵入式采样策略，开发了一套测序分型（genotyping-by-sequencing）方法，用于识别同形性染色体及其在远缘参考基因组中的同源序列。研究人员从同一种群的30只雄性与21只雌性个体的颊拭子中提取样本，构建了双酶切RADseq（double-digest RADseq）文库。针对杂合度或等位基因频率存在性别差异的标记，从未过滤数据集（含411,446个RADtags）中筛选性别限制性标记的效果，优于从过滤后数据集（含33,073个RADtags）中开展的同类筛选。最终共获得292个推定的性别连锁RAD位点，其中98%的位点指向雄性异配性别决定系统。其中15个位点可比对到热带爪蟾（Xenopus tropicalis）基因组，除1个位点外，其余均定位于1号染色体对；该染色体在两栖类的性别决定过程中似乎常被共招募为性别决定相关功能位点。最高效的比对策略为三步分层法：首先将田野林蛙的测序读段比对到林蛙（Rana temporaria）的低覆盖度基因组（分化时间约17百万年），随后将林蛙的基因组支架（scaffold）比对到高原蛙（Nanorana parkeri）基因组（分化时间约90百万年），最后将高原蛙的基因组支架比对到热带爪蟾基因组（分化时间约210百万年）。本研究针对定位于1号染色体的候选性别决定基因Dmrt1设计PCR引物扩增其部分片段，以此验证研究结论：性别诊断性等位基因仅存在于全部30只雄性个体中，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，具备良好的应用潜力。