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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.

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