Analysis Of Genetic Traits, Development Of Molecular Markers, And Isolation Of Sex-related DNA Markers In Two Kinds Of Flounder Fish

Half-smooth tongue sole（Cynoglossus semilaevis） and starry flounder（Platichthys stellatus） are the two important cultured marine flounder-fish species in the northern China. Half-smooth tongue sole mainly distributed in the Yellow Sea and the Bohai Sea,which is the native fish species of China, starry flounder mainly distributed in North Pacific Ocean and its traces could be found in the deep water of the northern Yellow Sea,which is of very small amounts for its wild-resources. In aquaculture, they have been the most popular fish species together with turbot（Scophthalmus maximus） and olive flounder（Parelichthys olivaceus） in the northern China. In order to benefit the molecular genetics and breeding of fish species, the three items were studied in this paper including:（1） The phenotypic and genetic traits analysis of ZZ males and ZW males（sex-inverted males） of Cynoglossus semilacvis;（2）Identification, Analysis of microsatellite sequences in starry flounder Platichthys stellatus;（3）Isolation of sex-related DNA markers in the starry flounder Platichthys stellatus. The mainly research results are as follows:1. The phenotypic and genetic traits analysis of ZZ males and ZW males（sex-inverted males） of Cynoglossus semilacvisThere are two kinds of male in breeding populations of the half-smooth tongue sole Cynoglossus semilacvis, natural males（ZZ） and neomales（ZW）. It is of great significance to study their phenotypic and genetic characteristics for breeding and excellent-seed cultivating of the half-smooth tongue sole. In this study, we established several maternal half-sib families with both ZZ and ZW males. Comparative analysis of population genetic structure were processed in maternal half-sib families with ZZ and ZW males by using 50 pairs of specific microsatellite markers. Results showed that the pcr products amplified from 22 of 50 pairs of markers have polymorphism. There were232 alleles identified in 4 half-sib families, with higher mean allelic number（Na） andIV mean observed heterozygosity（He）. The average polymorphism information content（PIC） were 0.4566、0.4254、0.5069、0.4795. There were 3, 3, 4 and 5 loci with significant deviations from Hardy-Weinberg expected genotypic frequencies. The Nei’s genetic distances among the 4 families were 0.2101⁰.5840, and the genetic similarities were0.5577⁰.8105. In this study, it was indicated that there were significant differences in growth traits between ZZ and ZW males of C. semilacvis. A higher genetic similarity, a lower genetic distance and similar genetic diversity index appeared among the mother`s half-sib families.2. Identification, Analysis of microsatellite sequences in starry flounder Platichthys stellatusWe found 48,185 microsatellite sequences in the male and femal gene pool of the starry flounder Platichthys stellatus by using RAD-seq technology, the length of the whole sequencing sequence is 89,810,018 bp.The number of the complete-type microsatellite sequences is 36,804, the percentage of microsatellite sequences at the total length sequencing sequence is 0.82%. We discovered six types of microsatellite sequence, respectively: mononucleotide, dinucleotide, trinucleotide,tetranucleotide,pentanucleotide and hexanucleotide. Among all types of microsatellite sequence, dinucleotide repeat sequence have the largest number, occupied 72.79% of the total microsatellite sequence, trinucleotide motif arraies second place,accounting for10.80%. Among the dinucleotide repeat type, AC motif accounts for dinucleotide type for 76.56%. Changes in the number of repetitions of different repeat units were analyzed, the results showed that the average of the copy number of repeating units showed a negative correlation（r=-0.789）. Along with the increasation in the length of the repeating unit, the copy number in reducing. To statistic the variation coefficient of different types of microsatellite, dinucleotide microsatellite has the lagest variation coefficient, 0.51. During this study, we counted the content of AT base group, positive correlation was found among the content of AT base group, the total number of the total length of different microsatelltie repeat unit and the average of the copy number.3. Isolation of sex-related DNA markers in the starry flounder Platichthys stellatusThe genomic DNA of each 48 individuals of male and female of starry flounder were scanned by 26 markers that repeated more than 10 times were differently expressed between males and females, which was verified for the first round. 1 pair of microsatellite primers amplified different bands between 48 individuals of male andfemale. Different bands amplified by the 1 pair of primers was verified for the second round in the 96 individuals of male and female of starry flounder. The results showed that bands amplified by primer RAD_PE_through₂37042 in male and female individuals differed extremely significantly（p ﹤ 0.01）. And in the second round verification, the different band amplified by primer RAD_PE_through₂37042 with29 times in 96 female individuals, a higher frequency of 30.21%, and with 11 times in96 male individuals, a lower frequency of 11.46. After recovering, cloning and sequencing the male-related bands, the analysis of DNASTAR alignment showed that difference fragments were less than indifference fragments of a AT repeat. Therefore,primers RAD_PE_through₂37042 and male of starry flounder have positive correlation in T.rubripes.