Potential role of the Macquaire perch 146-bp sexing region in regulation of the candidate sex-determining gene SOX1b
The 146-bp Macquarie perch sexing region was not annotated to any of the eight genes on scaffold 633 (Table D3), but was located ~38 Kb upstream of the candidate Macquarie perch sex-determining gene SOX1b (g10286). This intergenic region had both genomic characteristics of a cis-regulatory region: evolutionary conservation, and presence of putative transcription factor binding sites (Hardison & Taylor, 2012). Despite being a non-coding DNA, the 600-bp fragment containing the sexing region aligned to unannotated genomic regions of over a dozen other bony fishes from five taxonomic orders (Appendix E). In contrast, a 200-bp section of scaffold 633 containing the other XY gametologous SNP (94017) did not align to fish other than two percichthyids- golden perch and Murray cod. Transcription factor binding motifs for 145 transcription factors were predicted within 246 base pairs containing the 146-bp sexing region (Appendix F). Of these, 14 transcription factors had binding motifs present on Y-haplotype, but not on X-haplotype nor golden perch nor Murray cod sequences, implying potential gain of the binding sites due to mutations on Y-haplotypes (Table F1). In addition, binding motifs for three transcription factors were absent on Y-haplotype, but present on X-haplotype and the two outgroups, implying potential loss of the binding sites on Y-haplotype. The latter included SOX14 - a negative regulator of transcription, involved in the regulation of embryonic development and in the determination of cell fate.
The 146-bp sexing region was also ~41 Kb downstream of gene g10285 that encodes for a protein of the Dbl homology (DH) domain superfamily, associated with guanyl-nucleotide exchange factor activity (GO:0005085) and protein-binding function (interacting selectively with any protein or protein complex; GO:0005515; Appendix D, Table D3). The scaffold 633 sex-linked DArT SNP was located within intron 5 of this gene.