DOI: 10.5061/dryad.bg79cnp9v
title: Mitochondrial DNA assay of 63 Chinook-Coho salmon hybrids

description: Mitochondrial DNA sequences can identify the maternal species involved in
 hybridization events and provide behavioural clues of the
 matings. In this study, Mitochondrial DNA
 indicated hybrids were the offspring of female Coho salmon
 spawning with Chinook salmon males (all but two individuals from a
 reciprocal cross). This finding suggested two possible scenarios:
 accidental fertilization in crowded spawning grounds or heterospecific
 choice of mate when conspecifics were not available (e.g.
 differential abundance).
description: Two amplicon types for the characterization of salmonid were
 applied to the extracted DNA samples. Amplicons used were a ~270 bp
 segment of the chordate (16SChord) (Deagle <i>et al.</i>
 2009), and a ~260 bp segment of the cytochrome oxidase I (COI) gene
 amplified with primers designed primarily for the amplification of
 salmonids (Thomas <i>et al.</i> 2017). The
 16SChord amplicons were multiplexed in one PCR reaction and the COI was
 performed in a separate reaction. All PCR amplifications were performed in
 20 uL volumes using the Multiplex PCR Kit (Qiagen). Reactions contained 10
 uL (2X) master mix, 0.25 uM of each primer, and 2 uL template DNA. Thermal
 cycling conditions were as follows: 95 °C for 15 min followed by 34 cycles
 of 94 °C for 30 s, 57 °C for 90 s and 72 °C for 60 s, and a final
 extension at 72 °C for 600 s. Amplified 16SChord and
 COI amplicon samples were barcoded with unique, matching 10 bp Forward and
 Reverse tags with an edit distance of 5. The 16SChord and COI amplified
 samples were pooled by amplicon into single libraries. These were cleaned
 and concentrated using DNA Clean and Concentrate-5 columns (Zymo) prior to
 library preparation and indexing using the KAPA Low Throughput Library
 Preparation kit (KAPA) for Illumina platforms as per manufacturer’s
 instructions. The quantity of the indexed library pools was assessed using
 dsDNA High Sensitivity kit (Qubit) and quality and size assessed using the
 DNA 1000 Bioanalyzer chip (Agilent). Finally, the pools were combined into
 a single library of 1:1 and processed on a 301bp single end MiSeq V2 chip
 (Illumina) with a 5% phiX spike in. The data was de-multiplexed by library
 pool using the TruSeq<sup>TM</sup> adapter sequences on the
 sequencer which produced fastq files. FASTQ sequence
 files for each indexed library pool were imported into QIIME for
 demultiplexing by sample and sequence assignment to species (Caporaso
 <i>et al. </i>2010). For a sequence to be assigned to sample
 it had to match the full forward and reverse primer sequences, and match
 the 10 bp primer tag for that sample (allowing for up to 2 mismatches in
 either primer or tag sequence). USEARCH was used to cluster OTUs with a
 similarity threshold = 0.99, a minimum cluster size = 3 and de novo
 chimera detection. For all DNA sequences successfully assigned to a
 sample, a Genbank BLAST search (Altschul <i>et al. </i>1990;
 Edgar 2010) was done against our custom 16S or COI reference databases
 whereby an assignment was made based on the best match in the database
 (threshold BLASTN e-value &lt; 1e-20 and a minimum identity of 0.9).
 Samples were excluded if they contained &lt; 20 identified prey DNA
 sequences. Deagle,
 B.E., Kirkwood, R., and
 Jarman, S.N. 2009. Analysis of Australian fur seal diet by pyrosequencing
 prey DNA in faeces. <i>Molecular Ecology</i>
 18:2022– 2038 Thomas, A.C.,
 Nelson, B.W., Lance, M.M., Deagle, B.E., and Trites, A.W. 2017. Harbour
 seals target juvenile salmon of conservation concern. <i>Canadian
 Journal of Fisheries and Aquatic Sciences</i> 74:907-921.
 Caporaso, J.G., J. Kuczynski, J.
 Stombaugh, K. Bittinger, F.D. Bushman, E.K. Costello, N. Fierer, A.G.
 Pena, J.K. Goodrich, and J.I. Gordon. 2010. QIIME allows analysis of
 high-throughput community sequencing data. Nature Methods
 7:335-336. Altschul, S.F., Gish, W., Miller,
 W., Myers, E.W. and Lipman, D.J., 1990. Basic local alignment search
 tool. <i>Journal of molecular
 biology</i>, <i>215</i>(3),
 pp.403-410. Robert C. Edgar, Search and
 clustering orders of magnitude faster than BLAST, <em>Bioinformatics</em>, Volume
 26, Issue 19, 1 October 2010, Pages 2460–2461, https://doi.org/10.1093/bioinformatics/btq461
description: Dataset in text format.