Genetic verification of doubled haploid Japanese flounder, Paralichthys olivaceus by genotyping telomeric microsatellite loci

Mitotic gynogenetic diploid or doubled haploid Japanese flounder Paralichthys olivaceus were produced by activating eggs with UV irradiated sperm of red sea bream (Pagrus major), followed by hydrostatic pressure treatment to block the first mitotic division. To verify the doubled haploid, we measured the homozygosity of 21 maternally heterozygous microsatellite loci that are located in the telomeric region of chromosomes. These 21 loci have large marker-centromere distances of 43 to 50 cM estimated from four meiotic gynogenetic diploid lines, suggesting the location at telomeric regions of chromosomes. These 21 loci cover 16 out of the total 24 linkage groups of Japanese flounder. Microsatellite genotyping of these 21 markers in four mitotic gynogentic diploid lines shows that 75% (58 out of 77) individuals are homozygous at all 21 loci, indicating doubled haploids. The other 25% are heterozygous at 1 to 13 loci, probably representing spontaneously occurring meiotic gynogenetic diploids. These results demonstrate the high efficiency of generating doubled haploids in just one generation by mitotic gynogenesis. We have used these doubled haploids to successfully generate clonal lines via a second round of gynogenesis.

► Doubled haploids Japanese flounder were produced.
► Selection of microsatellites which show high recombination rates.
► These loci were used to analyze doubled haploid progeny.
► Genetic status of doubled haploids and clones was confirmed.