为研究不同诱导方法对香鱼(Plecoglossus altivelis)人工雌核发育的影响,本研究采用紫外线灭活同源精子刺激卵子发育、冷刺激法、热刺激法和静水压法抑制受精卵第二极体排出诱导香鱼雌核发育,并采用微卫星(SSR)标记和相关序列扩增多态性(SRAP)标记分析了雌核发育子代样本的遗传特征.结果表明,香鱼雌核发育冷刺激组受精率、发眼率和孵化率分别为(39.6±2.5)%、(25.3±3.5)%和(6.7±1.2)%;热刺激组受精率、发眼率和孵化率分别为(61.0±2.0)%、(34.7±2.1)%和(11.0±2.0)%;静水压组受精卵、发眼率和孵化率分别为(80.3±2.1)%、(66.3±1.5)%和(57.0±2.0)%,诱导方法以静水压法最佳,孵化率显著高于冷刺激组和热刺激组(P<0.01),15日龄子代存活率为(15.2±8.2)%,而冷刺激组存活率低于1%,热刺激组未发现存活个体.SSR标记分析表明,静水压法可以通过抑制第二极体排出诱导香鱼雌核发育,受检测的16尾子代标本有14尾未出现父本条带,15日龄鱼苗雌核发育比例达87.5%.雌核发育子代在Pag-003位点均为纯合子,但着丝点与Pag-051和Pag-076位点之间的重组率分别为28.6%和93.3%.SRAP标记K-group引物组合在母本中扩增出5条带,其中母本特有条带4条,父本中扩增出4条清晰带,其中父本特有条带3条.母本特有条带a4在14尾雌核发育子代标本中出现的比例(显隐性比)为l∶1(P=l.000),条带al、a3和a5在后代中的显隐性比例基本符合孟德尔定律(1.33∶1)(P=0.705); SRAP标记P-group引物组合在母本中仅能扩增出1条带,无母本特异条带,父本中扩增出8条带,父本特有条带7条.No.7和No.14标本检测到父本特有条带,属于精子灭活不完全鱼苗(12.5%).与微卫星标记相比,SRAP标记可通过一次反应可同时实现对香鱼基因组多个位点检测,获得更多的遗传信息.本研究数据显示?
To investigate the effectiveness of ayu (Plecoglossus altivelis) artificial gynogenesis by different inducing methods, in this paper, the development of ovum was triggered by inactivated ayu sperm, then cold shock, heat shock and hydrostatic pressure treatment were carried out to retain the second polar body, respectively. The genetic feature of the offspring was analyzed by simple sequence repeats (SSR) and sequence-related amplified polymorphism (SRAP). Our data suggested that the fertility rate, eyespot rate and hatching rate of clod shock group were (39.6±2.5)%, (25.3±3.5)% and (6.7±1.2)%, respectively. The fertility rate, eyespot rate and hatching rate of heat shock group were (61.0±2.0)%, (34.7±2.1)% and (11.0±2.0)%, respectively. And the fertility rate, eyespot rate and hatching rate of hydrostatic pressure group were (80.3 ± 2.1)%, (66.3±1.5)% and (57.0±2.0)%, respectively. These data showed that hydrostatic pressure treatment was the optimum approach to induce ayu gynogenesis. And the hatching rate was significantly higher than that of cold shock and heat shock with P〈0.01. The survival rate of 15-day-old offspring of hydrostatic pressure group was (15.2±8.2)%, while that of clod shock group was less than 1% and no survival was found in heat shock group. The results of SSR suggested ayu gynogenesis could be induced by hydrostatic pressure treatment through retaining the second polar body. In tested gynogenesis offspring samples, 14 samples in 16 were detected without male-specific bands. The gynogenesis rate of offspring was 87.5%. In the locus of Pag- 003, the pattern showed that the gynogenesis offspring was all homozygote. While, in the locus of Pag-051 and Pag-076, the pattem showed that the recombination rates were 28.6% and 93.3% respectively between centromere and SSR locus. The result of SRAP showed that 5 bands in the female parent and 4 bands in the male parent were amplified respectively including 4 female-specific bands and 3 mal