The mulloway (Argyrosomus japonicus) is an iconic Australian gamefish species, known as the ‘silver ghost’, prized for being one of the largest fish that you can land from the beach. They grow to 75kg and almost 2m in length. In NSW, mulloway stocks have collapsed. As a result, this species is the focus of restocking efforts. Mulloways are sexually monomorphic externally; lacking distinguishing features present in other fish species. Sex can typically only be determined by lethal sampling or gonadal biopsy via gonopore cannulation. Gonadal biopsy is only possible on fish in the advanced stages of gametogenesis shortly before or during the reproductive season and can injure the fish. Sexual maturation takes 2–3 years in captivity, so non-lethal sexual identification can only be performed after this period. In this study we report the development of molecular markers for the purpose of determining sex in mulloway non-invasively. This marker is based on a 95bp indel in the dmrt1 gene only present in males. This marker was validated on known sex mulloway caught for restocking programs in NSW.
Fish sampled were NSWDPI broodfish of known sex, currently used for restocking. DNA was extracted from finclips of 6 females and 6 males and sent for Illumina sequencing. The resulting dataset had an average read depth of 40x for each fish. Each sequence was aligned to a published mulloway genome reference (ASM1571009v1). Both the aligned male and female sequences were next searched for known sex-associated genes; these gene sequences were inspected for differences between sexes. The most prominent differences were used to create oligonucleotide primers. These primers were tested on known sex individuals and the products of the most robust pair were sequenced (Figure 1).
After sequencing, areas of heterogeneity identified in the products were used to develop a second round of primers. This second round of primers were tested in all possible combinations and the most effective ‘sex marker’ pair were determined.
These markers were validated on 31 mature, known sex, mulloway. In all instances (100%), sex markers matched observed sex. These markers are based on the promotor region of the dmrt1 gene and target a 95bp deletion in the male sequence. This deletion is most likely located on the y sex chromosome as males are heterozygous.
These markers enable sex to be determined for sub-adult or non-spawning mulloway. They have applications for managing the sex ratio of mulloway broodstock and can be utilised to answer biological questions such as natural sex ratio, habitat usage and dispersal of wild mulloway. Additionally, the techniques demonstrated here present a pipeline for the development of an effective tool for developing sex markers for other sexually monomorphic species.