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 nonmodel 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.