牙鲆杂合克隆的鉴定及其生长性状的遗传分析

Identification of Heterozygous Clone and Genetic Analysis of Growth Traits in Japanese Flounder

采用经紫外线灭活的真鲷精子激活牙鲆卵子,利用静水压处理抑制卵子发生第一次有丝分裂,制备牙鲆有丝分裂雌核发育二倍体。培育二倍体至性成熟,选择性腺发育良好的雄性个体9尾和雌性个体9尾进行交配,制备杂合克隆9个(HC1～HC9)。同时,诱导2尾雌性个体进行减数分裂雌核发育,制备纯合克隆2个(C1～C2)。由牙鲆第二代遗传连锁图谱筛选22个高多态性的微卫星标记,鉴定牙鲆杂合克隆的遗传背景,分析等位基因杂合位点数量与其生长性状表型值之间的关联性。鉴定结果表明,所有杂合克隆子代基因型完全一致,为亲本等位基因的组合,证实杂合克隆制备成功。2个纯合克隆生长性状表型值最低,其杂合位点数为0;9个杂合克隆中,随着杂合位点数的逐渐增加,杂合克隆的生长性状表型值呈现先升高后降低的趋势。HC5的生长性状表型值最高,其杂合位点数为5;HC9杂合位点数最多,但其生长性状表现值并不是最优,此现象还需今后进一步研究。

In this study, mitotic gynogenetic diploid Japanese flounder （Paralichthys olivaceus） were produced by activation of ultraviolet irradiated sperm of red sea bream （Pagrosomus major）, and by hydrostatic pressure treatment to block the first mitotic division to identity the heterozygous clone. Nine heterozygous clones （HC1--HCP） of Japanese flounder were produced by crossing nine males with nine females after these diploid flounder were cultivated until sexual maturity. Simultaneously, two homozygous clones （C1--C2） were prepared by inducing two females to carry out meiotic gynogenesis. A set of 22 microsatellite markers with high potymorphism were selected from the second genetic linkage maps of Japanese flounder. Genetic status of these heterozygous clones was identified and the association between the number of heterozygous loci at alleles and phenotypic value for growth traits of different heterozygous clones were analyzed. The results showed that the genotypes of heterozygous clone offsprings were identical and the combination of parental alleles, indicating the successful development of heterozygous clones. The association analysis revealed that the minimal phenotypic values for growth traits were found in 2 homozygous clones including zero heterozygous loci. In 9 beterozygous clones, the phenotypic value for growth traits of corresponding heterozygous clone was first increased and then decreased with increase in the number of heterozygous loci. The maximal phenotypic value for growth traits were found in HC5 containing 5 heterozygous loci. The HC9 had the maximum number of heterozygous loci, but its phenotypic value for growth traits was not the optimum. Thus, there was to some extent relationship between the number of heterozygous loci at alleles and phenotypic value for growth traits, which would provide theoretical references for parent selection of heterozygous clone and genetic improvement of objective traits in Japanese flounder in the future.