牙鲆F_1代近交系、雌核发育系的建立及遗传检测

Construction and genetic analysis of gynogenetic clone and inbreeding strain of F_1 generation families for Japanese flounder Paralichthys olivaceus

在牙鲆育种中基于构建的大量家系,已经筛选出生长快成活率高的F1代家系0719,0750和0751,为了使其优良性状进一步纯化,采集以上3个优良家系中成熟个体的精子和卵子,分别利用家系内兄妹近交和鲈鱼冷冻精子诱导雌核发育的方式,构建了近交系3个、雌核发育系7个,在其生长至127 d和190 d时,测定体长和体重两个性状,并利用方差分析、Student-Newman-Keuls多重比较及绝对增重比较等法对两个性状进行分析,发现0751的雌核发育系较其他近交系和雌核发育系生长更快(P<0.05)。利用倍性分析仪对0751近交系和雌核发育系的染色体倍性进行分析。结果显示近交系和雌核发育系分别在G0-G1期和G2-M期DNA相对含量相近,G0-G1期DNA指数均为1.000,都具有二倍体的特点。利用筛选的14对具有多态性的微卫星引物分别对近交系和雌核发育系的遗传特征进行检测,发现近交系和雌核发育的等位基因数(Na)分别为2.214 3和2.000 0,有效等位基因数(Ne)分别为2.082 9和1.936 9,观测杂合度(Ho)分别为0.564 2和0.413 8(P<0.05),期望杂合度(He)分别为0.515 4和0.490 1,遗传杂合度(Ave_Het)分别为0.506 6和0.482 8,多态信息含量(PIC)分别为0.403和0.366,雌核发育系的遗传指数均低于近交系。除EKOP-E1-In,EKOP17-Li,EKOP-E1-Ey 3个位点仅在近交系中为高度多态,其他位点在两个系中均为中度多态。近交系和雌核发育系的遗传偏离指数(d)分别为0.091 5和-0.157 3,反映了近交系杂合子的比例远高于理论上的数值,纯合子缺失;雌核发育系杂合子缺失,纯合子比例高。利用雌核发育较近交方式更有利于基因的纯合,在牙鲆育种中具有较大的应用价值。

Three F1 generation families of Japanese flounder Paralichthys olivaceus（0719,0750 and 0751） with fast growth rate and high survival rate were screened out by selective breeding.In order to further purify these families＇good traits,three inbreeding strains and seven gynogenetic clones induced by cryopreserved sperms of sea perch（Lateolabrax japoniaus） were constructed.The body lengths and masses of these two lines at 127 d and 190 d were analyzed by ANOVA,Student-Newman-Keuls and AGR.The results showed that 0751 gynogenetic clone grew fastest in all families（P 0.05）.The chromosome ploidy analysis of lines from 0751 family revealed that inbreeding line and gynogenetic clone had the diploid characters with the similar DNA content at G0-G1 and G2-M phases,as well the same DNA index at G0-G1 phase.Further,the genetic diversity detection of these strains by 14 pairs of microsatellite markers showed that the allelic number Na was 2.214 3 and 2.000 0,the effective allelic number Ne was 2.082 9 and 1.936 9,the observed heterozygosity（Ho） was 0.564 2 and 0.413 8（P0.05）,the expected heterozygosity（He） was 0.5154 and 0.490 1,the average genetic heterozygosity（Ave_Het） was 0.5066 and 0.4828,and the polymorphism information content（PIC） was 0.403 and 0.366,respectively.Expect for three loci EKOP-E1-In,EKOP17-Li,EKOP-E1-Ey being highly polymorphic in the inbreeding line,others detected moderate polymorphism in both lines.Finally,the genetic deviation index（d） of the two lines was 0.091 5 and-0.157 3,suggesting that heterozygous excess was present in the inbreeding line,and heterozygosity deficiency and homozygous excess was present in the gynogenetic clone.This study shows that gynogenesis is a more effective method to promote gene purification and has great value in Japanese flounder breeding.