Data from: Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for non-model organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next we test the utility of our markers. BAPS allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. NewHybrids, a hybrid index and HIest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

下一代测序（Next-generation sequencing）是一种快速且经济高效的技术，可针对多个标记位点与多个个体获取非模式生物的序列数据。本研究报道了一套实验方案，可用于获取冠脊螈（两栖纲：蝾螈科：肋突螈属，Triturus）的同源标记，适用于种间杂交分析。我们利用单个肋突螈物种的转录组数据，设计了96对引物，可扩增位于3'非翻译区（3-prime untranslated regions）的约180 bp片段。随后，针对涵盖肋突螈属全部分类类群的一组物种，采用单重PCR对这些标记进行测试。最终筛选出52个符合要求的标记：在所有受试冠脊螈物种的凝胶电泳中，这些标记均可稳定扩增出单一条带且片段长度与预期一致。接下来，我们针对132只螈类个体，通过5个多重PCR体系（每个体系含10~11对引物）对这些标记进行扩增，其中84只个体代表7个（候选）物种，剩余48只个体来自假定的杂交种群。将每个个体的多重PCR产物混合后，连接独特标签以实现扩增子与个体的一一对应。随后将所有个体的产物按等摩尔浓度混合，在Ion Torrent下一代测序平台上完成测序。通过生物信息学流程识别等位基因，并将其重构为基因型格式。随后我们对该套标记的实用性进行了验证：BAPS软件将代表（候选）物种的84只冠脊螈个体分配至其对应的（候选）物种类群中，证实该套标记可用于物种界定；NewHybrids软件、杂交指数（hybrid index）以及HIest工具均确认，来自假定杂交种群的48只个体存在遗传混合，证明该套标记具备识别种间杂交的潜力。我们预期本研究设计的这套标记可为肋突螈属的杂交分析提供高分辨能力。