IDENTIFication of male and female characteristic dna marker of PACIFIC BLUEFIN TUNA  

Pacific bluefin tuna (Thunnus orientalis, PBF) aquaculture is very important, because the fish wild stock is suffering continuous reduction. Fertilized eggs collection at ocean cage is the first step of PBF aquaculture production. Only small portion of the females participate in spawning event under aquaculture conditions, female rich broodstock organization is advantageous for stable and mass fertilized egg collection. However, it is impossible to distinguish the PBF sexes by morphological features, and the observation of the large-sized adult fish gonad with dissection is impractical. Even if sex distinction in adult fish is possible, large and active fish transportation is impractical. In the previous studies, we found a sex-linked DNA marker of the PBF males, which enabled to conduct sex distinction of PBF at any stages from a part of fin or a drop of mucus by DNA analysis.

A 400 bp male characteristic fragment was identified by AFLP analysis. Nucleotide sequence of the fragment was analyzed and found that the sequence was redundant in both sexes however, 6 bp continuous deletion was characteristic in males. The PCR genotyping primers for sex distinction was developed depending on the sequence difference between male and female fish. The concordance of the genotyping was 93 % in F3 cultured PBF (n=64). Then we conducted genome walking experiment, male linked DNA sequence was extended to 2 kbp. Then we determined the difference of the sequence in both sexes and found the female characteristic sequence polymorphism, which was 210 bp continuous deletion when compared to male ones. A female PBF genotyping primers were developed and the concordance of the female PCR test was 92% in F3 cultured PBF (n=64). These DNA markers will be useful for tuna sex distinction and female rich brood stock organization for stable fertilized egg collection.