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.

Triturus transcriptome-based gene models in FASTA formatTriturus transcriptome-based gene models in FASTA formatTriturus_transcriptome_contigs_sorted_to_length.fasExcel sheet and macro to compose multiplexesExcel sheet and macro to compose multiplexesExcel_multiplex_check.zipRaw Ion Torrent reads in FASTQ formatRaw Ion Torrent reads in FASTQ formatFASTQ_IonTorrent.zipBWA alignments in SAM formatBWA alignments in SAM formatSAM_alignments.zipRaw SNP reports in VCF formatRaw SNP reports in VCF formatVCF_raw_SNP_reports.zipFASTA files of reconstructed sequencesFASTA files of reconstructed sequencesReconstructed_sequences_FASTA.zipInput files for the BAPS analysis (in GENEPOP format), NewHybrids and HIest.Input files for the BAPS analysis (in GENEPOP format), NewHybrids and HIest.input_files.zipScript associated with the bioinformatics pipelineScript associated with the bioinformatics pipeline. The first script strings the programs together (used in Linux environment). The second script determines the SNPs and allelic variants. The third script creates the consensus sequences.Scripts.zip