Identifying sex-linked DArT SNPs for Macquarie perch and golden
perch
We used DArTseq (Kilian et al., 2012), a reduced-representation
sequencing method, to genotype 93 female and 78 male Macquarie perch
from the Dartmouth and Yarra populations, and 41 female and 25 male
golden perch from Macquarie, Murray and Murrumbidgee populations
(samples in Supplementary Material S1). DArTseq approach is similar to
double-digest restriction-associated sequencing, except it ensures high
SNP quality by including ~25% of technical replicates
from independent libraries in each sequencing lane and rejects SNPs with
low reproducibility between technical replicates. Sequencing libraries
were prepared at Diversity Arrays Technology Pty Ltd (Canberra,
Australia) following Kilian et al. (2012). DNA samples were digested
using a combination of restriction enzymes Pst I and Sph I
that target low-copy genomic regions. Pooled libraries were sequenced
using single-read technology on an Illumina HiSeq2500 (94 samples per
lane; details in Appendix A). SNP discovery and genotyping were
performed using DArT P/L’s proprietary analytical pipeline (detailed in
Nguyen, Premachandra, Kilian, & Knibb, 2018). Briefly, the primary
pipeline removed poor-quality sequences, applying more stringent
criteria to the barcode region than the rest of the sequence and
corrected low-quality bases from singleton tags using collapsed tags
with multiple members as a template. Then the secondary pipeline
(DArTsoft14) parsed clusters, comprising 69-bp sequenced tags differing
by no more than 3 bases, into separate SNP loci, while ensuring the
balance of read counts for the allelic pairs: loci with a 5-fold or
higher difference in read counts for each allele were rejected. SNPs
with a reproducibility <95% were removed, but no other
filtering was performed. DArT loci for each perch species were aligned
to their respective newly-assembled reference genomes (Table 1) using
BLAST, with e-value ≤5e-5 and sequence identity ≥90%.
We tested each DArT SNP locus for belonging to one of four types:
(i ) Y-linked :
homozygous in males, and absent in females; (ii )XY-gametologs: homozygous in females, heterozygous in all males;
(iii ) loci with male-specific allele : homozygous in
females and heterozygous in >10% of males; and (iv )loci with female-specific allele : heterozygous in
>10% of females and homozygous in males. The tests were
performed using the gl.sexlinkage function of the dartR package
(Gruber, Unmack, Berry, & Georges, 2018) in R (R Core Team, 2020)
(details in Appendix B). To reduce the number of false positives due to
small sample size, only loci successfully scored in >75%
(>58) of male Macquarie perch and >95%
(>23) male golden perch were considered, male sample sizes
being smaller than for females in both species.