Identifying candidate sex-determining gene
To identify candidate sex-determining genes for both perches, we tested
whether the scaffolds containing the sex-determining genes known from
teleost fish also harbour sex-linked DArT SNPs or the Macquarie perch
sexing region detected using WGS data. We searched our InterProScan5
output for amh , anti-Mullerian hormone, amhr,anti-Mullerian hormone receptor, bcar1 , breast cancer
anti-resistance, brca2 , breast cancer type 2 susceptibility
protein, dmrt1 , doublesex- and mab-3 -related
transcription factor 1, dmY, dm-W, dmW, gsdf, gonadal soma
derived growth factor, sdY and SOX (Bao et al., 2019;
Graves, 2013; RodrÃguez-Marà et al., 2011) and Gene Ontology term
GO:0007530 (biological process: sex determination) and its child terms
(https://www.ebi.ac.uk/QuickGO/term/GO:0007530). To confirm gene
identity of the candidate sex-determining genes, we searched for similar
sequences using Blastn (Zhang, Schwartz, Wagner, & Miller, 2000) in
NCBI nucleotide database, retaining hits with annotations, an e-value
<10-10, query coverage >99%
and percent identity >90%. Finally, we tested the
Macquarie perch sexing region for two genomic signatures of a
cis-regulatory element (i.e. a DNA sequence that regulates tissue- and
time-specific candidate gene expression): evolutionary conservation of
non-coding regions, and the presence of multiple transcription factor
binding sites (i.e. ~10bp-long degenerate sequences
recognized by transcription factors) (Hardison & Taylor, 2012). We
queried the 600-bp region containing the sexing region (GenBank
accession SEMN01000633, bases 93001-93600) against the NCBI nucleotide
database (using Blastn, retaining all hits with e-value
<10-10). We also used CiiiDER (Gearing et
al., 2019) and the JASPAR database of transcription factor binding
profiles (Fornes et al., 2020) to search for potential binding-site
locations in ~246-bp-long X- and Y- specific sequences
(haplotypes) encompassing the sexing region. We investigated whether
some transcription factor binding sites are specific to Macquarie perch
X- or Y- haplotypes and if so, whether they are present in golden perch
and Murray cod sequence (outgroups). A potential gain of a binding site
on Y-haplotype was inferred if the site was present on Y- but absent on
X-haplotype and both outgroups, whereas a potential loss of a binding
site was inferred if it was absent on Y-haplotype but present on
X-haplotype and in both outgroups.