Mapping the sex determination locus in the hāpuku (Polyprion oxygeneios) using ddRAD sequencing

Jeremy K. Brown, John B. Taggart, Michaël Bekaert, Stefanie Wehner, Christos Palaiokostas, Alvin N. Setiawan, Jane E. Symonds, David J. Penman

Hāpuku (Polyprion oxygeneios) is a member of the wreckfish family (Polyprionidae) and is highly regarded as a food fish. Although adults grow relatively slowly, juveniles exhibit low feed conversion ratios and can reach market size in 1-2 years, making P. oxygeneios a strong candidate for aquaculture. However, they can take over 5 years to reach sexual maturity in captivity and are not externally sexually dimorphic, complicating many aspects of broodstock management. Understanding the sex determination system of P. oxygeneios and developing accurate assays to assign genetic sex will contribute significantly towards its full-scale commercialisation. DNA from parents and sexed offspring (n = 57) from a single family of captive bred P. oxygeneios was used as a template for double digestion Restriction-site Associated DNA (ddRAD) sequencing. Two libraries were constructed using SbfI - SphI and SbfI - NcoI restriction enzyme combinations, respectively. Two runs on an Illumina MiSeq platform generated 70,266,464 raw reads, identifying 19,669 RAD loci. A combined sex linkage map (1367 cM) was constructed based on 1575 Single Nucleotide Polymorphism (SNP) markers that resolved into 35 linkage groups. Sex-specific linkage maps were of similar size (1132 and 1168 cM for male and female maps respectively). A single major sex-determining locus, found to be heterogametic in males, was mapped to linkage group 14. Several markers were found to be in strong linkage disequilibrium with the sex-determining locus. Allele-specific PCR assays were developed for two of these markers, SphI6331 and SphI8298, and demonstrated to accurately differentiate sex in progeny within the same pedigree. Comparative genomic analyses indicated that many of the linkage groups within the P. oxygeneios map share a relatively high degree of homology with those published for the European seabass (Dicentrarchus labrax). P. oxygeneios has an XX/XY sex determination system. Evaluation of allele-specific PCR assays, based on the two SNP markers most closely associated with phenotypic sex, indicates that a simple molecular assay for sexing P. oxygeneios should be readily attainable. The high degree of synteny observed with D. labrax should aid further molecular genetic study and exploitation of hāpuku as a food fish.