Data from: High-throughput identification of informative nuclear loci for shallow-scale phylogenetics and phylogeography

One of the major challenges for researchers studying phylogeography and shallow-scale phylogenetics is the identification of highly-variable and informative nuclear loci for the question of interest. Previous approaches to marker generation have generally required extensive testing of loci of unknown utility from other systems or development of markers from the nearest model organism with genomic resources. Here, we present a fast and economical approach to generating thousands of variable, single-copy nuclear loci for any system using next-generation sequencing. We performed Illumina paired-end sequencing of three reduced-representation libraries (RRLs) in chorus frogs (Pseudacris) to identify orthologous, single-copy loci across libraries and to estimate sequence divergence at multiple taxonomic levels. We also conducted PCR testing of these loci across the genus Pseudacris and outgroups to determine whether loci developed for phylogeography can be extended to deeper phylogenetic levels. Prior to sequencing, we conducted in silico digestion of the most closely-related reference genome (Xenopus tropicalis) to generate expectations for the number of loci and degree of coverage for a particular experimental design. Using the RRL approach, we: (1) identified >100,000 single-copy nuclear loci, 6,339 of which were shared across individuals within species and 904 of which were shared between species, (2) estimated average nuclear sequence divergence at 0.1% between alleles within an individual, 1.1% between conspecific individuals, and 1.8% between species, and (3) determined from PCR testing that 53% of the loci successfully amplify within-species and also many amplify to the genus-level and beyond (16%). Our study effectively identified nuclear loci present in the genome that have levels of sequence divergence on par with mitochondrial markers commonly used in phylogeography. Specifically, we estimated that ~7% of loci in the chorus frog genome are >3% divergent within species; this translates to ~50,000 single-copy loci in the genome with >3% divergence. Moreover, successful amplification of many loci at deeper phylogenetic levels indicates that the RRL approach represents an efficient method for rapid generation of informative markers for both phylogenetics and phylogeography. We conclude by making recommendations for minimizing the cost and maximizing the efficiency of marker development for future studies in this field.

从事系统地理学（phylogeography）与浅尺度系统发育学（shallow-scale phylogenetics）研究的学者所面临的核心难题之一，是针对目标研究问题筛选出高变异且信息丰富的核基因位点。过往的分子标记开发方法，通常需要对其他研究体系中功能未知的基因座进行大量验证，或是从拥有基因组资源的近缘模式生物中开发标记。本研究提出一种快速且经济的方法，可借助下一代测序（next-generation sequencing）为任意研究体系生成数千个变异型单拷贝核基因位点。本研究以拟蝗蛙属（Pseudacris）为研究对象，对3个简化基因组文库（reduced-representation libraries, RRLs）进行Illumina双端测序，以鉴定不同文库间的直系同源单拷贝基因位点，并估算不同分类层级下的序列分歧度。此外，本研究针对拟蝗蛙属及其外类群开展了聚合酶链式反应（polymerase chain reaction, PCR）检测，以验证针对系统地理学开发的基因座能否拓展应用至更深的系统发育层级。测序前，我们对亲缘关系最近的参考基因组——热带爪蟾（Xenopus tropicalis）进行了计算机模拟酶切（in silico digestion），以针对特定实验设计预估基因座数量与覆盖度水平。借助简化基因组文库方法，本研究达成以下成果：(1) 筛选得到超过10万个单拷贝核基因位点，其中6339个在物种内个体间共享，904个在物种间共享；(2) 估算得到平均核序列分歧度：个体内等位基因间为0.1%，同种个体间为1.1%，物种间为1.8%；(3) 通过PCR检测发现，53%的位点可在物种内成功扩增，且有16%的位点可在属级及以上分类层级成功扩增。本研究成功筛选出基因组中序列分歧度与系统地理学研究中常用线粒体标记相当的核基因位点。具体而言，我们估算得到拟蝗蛙属基因组中约7%的基因座在物种内分歧度超过3%，对应基因组内约5万个序列分歧度超过3%的单拷贝核基因位点。此外，多个位点在更深系统发育层级的成功扩增表明，简化基因组文库方法可高效快速地为系统发育学与系统地理学研究生成信息丰富的分子标记。本研究最后针对该领域未来研究中如何降低标记开发成本、提升开发效率提出了相关建议。