Production and verification of a 2(nd) generation clonal group of Japanese flounder, Paralichthys olivaceus

Clonal fishes are useful tools in biology and aquaculture studies due to their isogenicity. In Japanese flounder (Paralichthys olivaceus), a group of homozygous clones was created by inducing meiogynogenesis in eggs from a mitogynogenetic homozygous diploid. As the clones reached sexual maturity, meiogynogenesis was again induced in order to produce a 2(nd) generation clonal group of Japanese flounder. After 3 months, there were 611 healthy, surviving individuals. Twenty-four microsatellite markers, that covered all the linkage groups of Japanese flounder, were used to identify the homozygosity of the 2(nd) generation clones; no heterozygous locus was detected. This indicates that the production of a 2(nd) generation clonal group of Japanese flounder was successful. Restriction-site DNA associated sequencing at the genomic level also confirmed the homozygosity and clonality of the 2(nd) generation clonal group. Furthermore, these 2(nd) generation clones had a small coefficient of variation for body shape indices at 210 days of age and showed a high degree of similarity in body characteristics among individuals. The successful production of 2(nd) generation clones has laid the foundation for the large-scale production of clonal Japanese flounder.