Screening Of Gender-related Molecular Markers Of Takifugu Rubripes And Differences In Morphology Of Male And Female

Takifugu rubripes is one of the excellent sea fish which is economic value in thenorthern of China. In recent years,there are more and more studies in T.rubripes,such asgenetic polymorphisms analyzing of different populations and breeding familyestablishment and cultivation.Howevre, gender-related research is extremely rare.Firstly,this paper summarized research status of fish sex-related genes and molecularmarkers in order to provide feasible program for screening of gender-related molecularmarkers in T.rubripes.Then, summarized the research progress in selective breeding inthreshold traits in the fish so as to expansion ideas about selective breeding in stressresistance family. Secondly, AFLP and SSR were used to screen sex-related molecularmarkers in T.rubripes. Modern molecular biology techniques were used to distinguishbetween male and female fish and screened male fish for farming in the early growthstages which not only had important practical significance in improving economicefficiency and market value but also provided a theoretical basis for sex-determiningmechanism and sex-related genes.Finally, based on morphological indicators,multivariate statistical analysis was used to analyze differences in morphology of maleand female and build discriminant equations of male and female which was aimed atproviding a kind of new method and theoretical basis for sex identification of T.rubripes during cultivation and breeding.The main results of this study are as follows:1.AFLP was used to screen gender-related molecular markers of T. rubripes.Fiveindividuals of male and female were used to screen16primers combination. At about800bp, primers combination（E+2-3，M+2-4）amplified bands in all of the five maleindividuals,but none of the female individuals. Other15selective amplification primercombinations didn’t amplify gender specific bands in5individuals of male and female.The results of screening primers showed that only one primers combination could beused as candidate. Significantly different bands amplified by primers combination （E+2-3，M+2-4）in30individuals of male and female population showed that therewas no differences of male and female at the level of individual DNA. Thus, thecandidate band was not gender-related AFLP markers. Other DNA markers wereselected to screen gender-related molecular markers in T. rubripes.2.Soon afterwards, microsatellites based on bulked segregation analysis (BSA)were used to screen the gender differences of male and female population of Takifugurubripes. Two gene pools were constructed using30individuals of male and femalepopulation separately, and were scanned by66pairs of microsatellite primers.8pairs ofmicrosatellite primers amplified different bands between two gene pools. Differentbands amplified by the8pairs of primers were verified for the first round in the60individuals constructing the gene pools. The results showed that bands amplified byprimers f1050and f383in male and female individuals differed extremely significantly（P<0.01）.And in the second round verification, bands amplified by primers f1050inmale and female individuals did not differ significantly, while bands amplified byprimers f383in male and female individuals differed extremely significantly（P<0.01）.The results showed that different bands amplified by primers f383were twicesignificantly correlated with gender in male and female population, and the correlationcoefficient were0.710and0.673separately. Therefore, primers f383and male havepositive correlation in T. rubripes. After recovering、cloning and sequencing themale-related bands, the analysis of BLAST alignment showed that this sequence is apart of chromosome19of T. rubripes, homology is98%and fraction of coverage is100%.3.Not only molecular biology methods were used in sex discrimination ofT. rubripes, but also multivariate statistical analysis methods were used to analyzedifferences in morphology of male and female.17normalized traits of male and femalepopulation in T.rubripes were researched showing that Principal Component Analysisobtained three principal components, the contribution rate of which was46.776%、27.668and7.122%, respectively. The cumulative contribution rate was81.566%. Mostof the female and the male could be told apart in the three-dimensional space composedof three principal components. The result of Stepwise Discriminant Analysis showedthat two normalized traits body width/body length and body girth(2)/body length werepicked out. The discriminant equations of female and male T. rubripes were built andthe accuracy rate of72samples was81.9%. The other38T. rubripes were used forverification and the accuracy rate was84.2%. Analysis of variance showed that width/body length differed extremely significantly（P<0.01）in male and femalepopulation in T. rubripes, body girth(2)/body length differed significantly（P<0.05）inmale and female population in T. rubripes. The result showed that the male has widerbody width and longer body girth (2) than female. The discriminant equations of femaleand male T. rubripes provide a kind of new method and theoretical basis for sexidentification of T. rubripes during cultivation and breeding.