Finding the Females: New Reference Genome Leads to Better Sex Determination Technique in Tuna
Tuesday, November 5, 2019
The cultivation and conservation of one of the most important commercial fishes in the world may come down to sex determination — how can you successfully breed a species without knowing the sex of your stock?
A Japanese research team has come up with a solution, thanks to a new Pacific bluefin tuna reference genome and the male-specific DNA markers they were able to identify as a result.
In a study published recently in the Nature journal Scientific Reports, first author Ayako Suda and lead author Atushi Fujiwara of the Japan Fisheries and Education Agency of Yokohama, described how they developed a PCR assay to accurately identify male tuna, based on a new high-quality PacBio and Illumina assembly.
Wild populations of Thunnus orientalis have been in drastic decline due to overfishing, leading Japan and other nations to develop full-life-cycle tuna aquaculture systems as early as the 1970s. They have identified optimum rearing conditions for the species, but these conditions are difficult to achieve. Spawning is strongly influenced by environmental factors such as water temperature, for example, and only some females spawn in cultivation conditions, reducing genetic diversity. Controlling the sex ratio in sea cages could help increase the production of fertilized eggs, but the Pacific bluefin tuna lacks morphological sexual dimorphism, making it difficult to identify and remove males. Furthermore, identifying males through gonad inspection can be lethal and inconclusive in young fish.
So the researchers set out to improve the T. orientalis genome, first assembled in 2013. Despite being used as a reference, that assembly is highly fragmented, with a large number of gaps in its scaffolding.
By combining sequence data from PacBio long reads and Illumina short reads, the Japanese team created a 787 Mb genome assembly in only 444 scaffolds with a contig N50 of 3 Mb. This represents a 376-fold increase in contiguity and a 148-fold reduction in the number of gaps compared to the existing reference.
Through analyzing re-sequence data of several males and females, 250 male-specific SNPs were identified from more than 30 million polymorphisms, with seven distinct regions being identified. The team then focused on one in particular: a 3,174 bp section of a single scaffold that contained 51 male-specific variants. They created a PCR-based sex identification assay targeting this stretch of DNA and achieved high accuracy in testing across 115 fish.
“Sex identification using our PCR assay is easy, requiring minimal handling of individuals. Moreover, sex of juveniles can be identified using our method, allowing the sex ratio in cages to be adjusted at an early stage, which could enhance breeding programs,” the authors state.
They also note that the approach might be less stressful to tuna, and requires less effort than sampling approaches based on fin clips or muscle tissues from live individuals.
“It could also be used to obtain data from wild populations, providing useful information for the management and conservation of these natural stocks,” they add.
The assay could be implemented in surveys that evaluate sex ratio analyses, rather than waiting for sexual maturity, for example. Incorporating the sex of fish while tracking their migration patterns — a strategy used by Barbara Block and colleagues in California — could also provide valuable information for the management of wild tuna fisheries.
“Our improved draft genome provides a solid foundation for future population and resource management studies of Pacific bluefin tuna,” the authors conclude.