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在牙鲆家系选择育种中,为了快速的获得具有优良性状的纯系,本研究利用冷冻保存的鲈鱼(Lateolabrax japonicus)精子和2007年构建的牙鲆(Paralichthys olivaceus)优良家系为实验材料,进行了牙鲆卵裂雌核纯系的诱导建立、遗传倍性分析及遗传标记检测等相关研究。卵裂雌核发育诱导结果表明,在水温为17℃条件下,利用灭活鲈鱼精子受精后58 min,以静水压力为590 kg/cm2处理6 min,获得了(32.30±3.34)%的诱导成功率(P<0.05)。利用流式细胞仪对正常胚胎、雌核发育胚胎和单倍体胚胎进行检测,结果表明,单倍体的相对DNA含量为17.07,卵裂雌核发育二倍体相对DNA含量为27.75,正常二倍体相对DNA含量为25.63。雌核家系和近交家系的生长对比结果表明,两者差异极为显著(P<0.05),生长至6个月,近交家系平均体重可达(48.89±17.01)g,而雌核发育纯系体重为(22.09±6.94)g。利用30对微卫星引物对近交家系和卵裂雌核发育纯系进行鉴定与分析,30个微卫星位点在近交系中均表现为杂合多态基因,24个位点在雌核群体中具有多态性,并对这些位点的等位基因数进行了卡方检验,结果表明,等位基因均符合等位基因的1∶1的遗传分离定律。最后计算得到这两个群体的遗传相似性为0.813 9,遗传距离为0.205 9。研究结果提示,利用冷冻保存的异源精子可成功诱导和建立牙鲆有丝分裂雌核发育系,为构建牙鲆优良纯系提供了理论技术依据。
In order to obtain the pure lines with excellent traits rapidly in the selection breeding of Japanese flounder, the study was conducted using the frozen-preserved sperm of Lateolabrax japonicus and the excellent families of Paralichthys olivaceus constructed in 2007 as experimental materials Induced establishment of cleavage line of pure virginity of Paralichthys olivaceus, genetic analysis of ploidy and detection of genetic markers. The results of gynogenetic induction of cleavage showed that the induction of (32.30 ± 3.34)% induction at 58 ℃ with hydrostatic pressure of 590 kg / cm2 for 58 min after inoculation of inactivated seabass sperm at water temperature of 17 ℃ Rate (P <0.05). The normal embryos, gynogenetic embryos and haploid embryos were detected by flow cytometry. The results showed that the relative DNA content of haploid was 17.07, the relative DNA content of diploid of cleavage gynogenetic chromosome was 27.75, The ploidy relative DNA content was 25.63. The comparison of the growth of gynogenetic line and inbred line showed that the difference between them was extremely significant (P <0.05). The average body weight of inbred lines reached 48.89 ± 17.01 g when grown to 6 months, Body weight was (22.09 ± 6.94) g. Thirty pairs of microsatellite primers were used to identify and analyze the gynogenetic lines of inbreeding and cleavage. All 30 microsatellite loci showed heterozygous polymorphisms in inbred lines, with 24 loci in the gynogenetic nucleus Polymorphism was observed in the population and chi-square test was performed on the number of alleles at these loci. The results showed that all the alleles were in accordance with the rule of 1: 1 genetic segregation of alleles. Finally, the genetic similarity between the two populations was 0.813 9 and the genetic distance was 0.205 9. The results suggest that cryopreserved allogeneic sperm can successfully induce and establish the mitotic gynogenetic line of Paralichthys olivaceus, which provides a theoretical and technical basis for the construction of excellent pure lines of Paralichthys olivaceus.