Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

Tissue Expression and Stock Variation of Isozymes of Stone Flounder(Kareius bicoloratus)

Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD＊, GDH＊, G3PDH-2＊ and ADH-2＊ were detected only in liver, SDH-1＊, MDH-1＊ and ADH-1＊ only in muscle, and LDH-B＊ and LDH-C＊ only in eyes. In comparison, MDH-2＊, GPI-3＊ and SDH-2＊ were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci （P0.99） were 29.17% and 25.00%, the observed heterozygosities （H0） were 0.028 ±0.014 and 0.040 ± 0.019, and the expected heterozygosities （He） were 0.039±0.017 and 0.052±0.022 in Qingdao and Wethai stock, respectively. The coefficient of gene differentiation （Fst） and genetic distance （D） between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity ofK. Bicoloratus were at a middle level.

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype,allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.