Studies On Early Sex Differentiation And Sex-associated SNPs Screening In Turbots (Scophthalmus Maximus L.)

The turbot （Scophthalmus maximus L.） is a flatfish of great commercial value,where a strong sexual dimorphism exists for growth rate. In this study, by usinghaematoxylin-eosin staining of histology section of the gonads and macroscopicexamination fistly, phenotypic sex of juvenile individuals from3to5months age wereidentified. Then sex ratio of4022individuals within37families at4months wereanalyzed both at family and population level. Meanwhile,2254individuals from20families among37families were randomly selected for analysis of sexual dimorphismin body weight.Candidate SNP loci come from large-scale high-throughput sex specialtranscriptome454sequencing system of turbot （Scophthalmus maximus L.）. Accordingto the candidate SNP loci, primers were designed to test and verify. And by use of MassArray （MALDI-TOF MS Array）, a high-throughput genotyping technology for SNPs,398sex associated candidate SNPs were screened and verified with20males andfemales at8months age. Then the other35sex-associated candidate SNPs of6contigswere verified and conducted haplotypes analysis. The main results are as follows:1. By dissecting individuals at4months age directly, significant shapedifference of gonads between male and females were observed.Females’ were shortrod-like while males a slender line-like. Then, sex of most4months age individualscould be identified by macroscopic examination. Few individuals that shape of gonadsshowed no significant difference between sexes, still should be identified by use ofhaematoxylin-eosin staining of histology section of their gonads. And a shape differenceof gonads was observed between4and6months age individuals, which suggested thatshape of turbot gonads was continuously changing during early stage.2. Phenotypic sex of4022individuals composed of37families at4months age wereidentified and counted. The statistical analysis showed that the number of female andmale were2426and1596, respectively, which reflected a significant difference （P<0.01） in sex ratio to the hypothesis of a balanced sex ratio （50%males/50%females）. And atfamily level, there were19families among37families exhibited significantmale-deviation from a balanced sex ratio （1:1）（P<0.05）.Although the other18familiesshowed no significant deviation （P>0.05）, among those13families were male-biased.In a short, the significant male-deviation in sex ratio has been confirmed in culture stockof turbot juvenile at4months age, and within families, most of them were alsomale-deviation in sex ratio. Meanwhile, there were20families among37families,80-209individuals in each family were randomly selected and weighted. The resultsdemonstrated that no significant deviation of body weight has been observed betweenfemale and male individuals within populations and families for4months juvenile fish,which indicated that the selection of weight traits in juvenile stage in selective breedingproject did not affect the sex ratio among selective breeding stock.3. By using Mass Array （MALDI-TOF MS Array） genotyping method,524SNPloci were genotyped,165of which were validated and used to investigate the geneticstructure of a breeding population of Scophthalmus maximus L. in2013. Transition（A G or C T） and transversion （C A, C G, A T） of165validate loci were58.18%,41.82%respectively. Besides, the genetic diversity estimation of165SNP locishowed that: the effective number of alleles ranged from1.0512to2.000, the averagevalues of observed and expected heterozygosity ranged from0.0000to0.9333and from0.0500to0.5000respectively, the minimum allee frequency （MAF） ranged from0.0250to0.5000,the average value of polymorphism information content （PIC） values was0.2739, the shannon information index ranged from0.1169to0.6931and frequencies ofmost SNP loci conformed to the hardy-weinberg equilibrium （HWE）. Meanwhile,21SNPs that might have a correltion with sex detemination were screened, however40more samples verified that they were all fake.4.35SNPs of7groups on6contigs（UN_xiong₁4522、UN_ci₆70、UN_ci₅009、UN_ci₇774、UN_ci₇877and UN_xiong₁4976, respectively） were choosen andverified by sequencing directly within70males and70females at8months age. And12haplotypes were found on3contigs（UN_xiong₁4522、UN_ci₅009and UN_ci₇774）.One of them （TTTAG） on contig UN_ci₇877（2543bp） was verified to be sexassociated with male individuals （P<0.05）. In this paper, Sex identification and differenciation, body weight deviation,screening of sex associated SNPs and haplotypes of juvenile turbots were studied inorder to do some effort for supplying accurate sex information of brood stock forefficient mating project, revealing sex differenciation mechanisim and other sexassociated research on turbot.