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 ta...下一代测序（Next-generation sequencing）是一种快速且经济高效的技术，可针对非模式生物的众多标记及多个个体获取序列数据。本研究描述了一套可获取冠螈（两栖纲：蝾螈科：真螈属（Triturus））直系同源标记的实验方案，适用于种间杂交分析。我们利用单一真螈属物种的转录组数据，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，我们针对覆盖真螈属全部分类类群的多个物种，通过单重PCR（uniplex PCR）对这些标记进行验证。最终筛选出52个标记，在所有受试冠螈物种的凝胶电泳中均能稳定呈现单一条带且片段长度符合预期。随后我们针对132只蝾螈个体，通过5重多重PCR（每重反应包含10或11个引物对）对这些标记进行扩增：其中84个个体代表7个（候选）物种，剩余48个个体来自疑似杂交种群。在将每个个体的多重PCR产物混合后，独特的ta...