2.2 NextRAD sequencing
Genomic DNA was converted into nextRAD genotyping-by-sequencing
libraries (SNPsaurus, LLC, Eugene, OR, USA) as in Russello, Waterhouse,
Etter, and Johnson, (2015). Genomic DNAs were first fragmented with
Nextera DNA Flex reagent (Illumina, Inc., San Diego, CA, USA), which
also ligates short adapter sequences to the ends of the fragments. The
Nextera reaction was scaled at a 1/5th reaction, and 5 ng of genomic DNA
was used due to the presence of inhibitors in the samples. Fragmented
DNAs were then PCR amplified for 26 cycles at 73ÂșC, with one of the
primers matching the adapter and extending 9 nucleotides into the
genomic DNA with the selective sequence GTGTAGAGC. Thus, only fragments
starting with a sequence that matched the selective sequence of the
primer were efficiently amplified. NextRAD libraries were sequenced in a
150 bp single-end reads mode in a lane of a HiSeq 4000 (SNPSaurus at
University of Oregon). Raw sequence data are available at the National
Centre for Biotechnology Information (NCBI), under the BioProject
accession number PRJNA593002.