【作者】 李益云；

【导师】 王志勇；

【作者基本信息】 集美大学 ， 水产养殖学， 2007， 硕士

【摘要】 本文研究大黄鱼人工雌核发育诱导条件的优化,对胚胎发育进行连续观察,并利用微卫星标记对雌核发育一代和雌核发育二代的基因纯合度进行分析,通过建立减数分裂型人工雌核发育家系,研究了22个微卫星标记与着丝粒之间的遗传距离。主要结果如下:1.大黄鱼人工雌核发育二倍体诱导条件的优化。用不同剂量紫外线辐射过的精子诱导雌核发育,原肠期存活率和孵化率都呈现出明显的Hertwig效应。综合受精率、原肠期存活率、孵化率和初孵仔鱼形态正常率的观测结果,253800μw.cm-2– 406080μw.cm-2是用紫外线进行大黄鱼精子遗传失活的适宜照射剂量。对辐射持续时间、冷休克处理起始时间和持续时间进行3因素3水平正交试验的结果表明,大黄鱼减数分裂型雌核发育二倍体诱导的最适宜条件为紫外线辐射剂量355320μw.cm-2,授精后3 min开始冷休克,休克持续时间为10 min,在此条件下诱导率(形态正常二倍仔鱼的孵化率)可达到20%。2.利用微卫星标记分析了大黄鱼雌核发育一代(meio-G1)和雌核发育二代(meio-G2)的杂合度以及减数分裂型雌核发育的近交效应。对普通养殖群体(C)、雌核发育一代群体和雌核发育二代群体的6个微卫星位点进行了分析,共检测到21个等位基因。三群体等位基因数从多到少依次为C(21)>G1(20)>G2(13);雌核发育一代(G1)和二代(G2)的平均观测杂合度(HO)分别为0.195和0.031,均明显低于普通养殖群体(0.494),且G2平均观测杂合度比G1减少了84.1%,G1的实际近交系数为0.605,G2的实际近交系数为0.937。可见人工诱导雌核发育可导致基因快速地纯合,是快速建立大黄鱼遗传纯系的有效途径。3.利用筛选出来的22对母本基因型为杂合型的微卫星引物分别对两个减数分裂型雌核发育(meio-gynogenetic)一代家系和正常二倍体对照家系进行遗传分析。结果表明,两个雌核发育家系的雌核发育个体的比例分别为100%和96.9%;在所观察的22个微卫星标记位点中,4个位点的遗传偏离了孟德尔分离比;假设在完全干涉的条件下,余下的18个位点与着丝粒间的距离介于0～50cM之间,平均重组值y为0.586,近交系数为0.414,相当于全同胞近交系数的1.67倍。其中有10个微卫星标记的重组率大于0.667,说明大黄鱼减数分裂型雌核发育一代在这些位点上的等位基因存在高度重组的现象。更多还原

【Abstract】 In this paper, the conditions of induction of meiotic gynogenetic diploid in large yellow croaker (Pseudosciaena crocea) were optimized and the development of genogenetic hapoid and diploid embryos was observed. The gene homozygosity and inbreeding coefficient of 1st and 2nd generations of meiotic gynogens (meio-G1 and meio-G2) were evaluated by using microsatellite markers. The recombination rates between 22 microsatellite markers and the centromere was analyzed in the induced meio-gynogenetic families. The main results were shown as follows:1. The conditions of induction of meiotic gynogenetic diploid in large yellow croaker, including UV radiation dosage for genetically inactivation of sperms, the starting time and duration of cold shock treatment for chrosome set duplication were optimized. Hertwig effects were observed in the survival rate of gastrula embryos and hatchability when eggs fertilized with UV irradiated semen. It was suggested that the appropriate dosage of UV to genetically inactivate sperm of large yellow croaker was 253800μw.cm-2 to 406080μw.cm-2. An orthogonal experiment in three factors and three levels was designed to investigate the effects of irradiation dose, the starting time of cold shock and the duration of cold shock on induced gynogenetic diploid in large yellow croaker. The results showed the hatching rate of genogenetic diploid with highest of 20% when the egg inseminated with semen irradiated with UV for 355320μw .cm-2, and cold shocked for 10 min started at 3 min post insemination.2. The variation of heterozygosity (H) and inbreeding effects in meio-G1 and meio-G2 were analyzed by using micromsatellites. Six micromsatellite loci were investigated in common cultured stock (C), meio-G1 and meio-G2. A total of 21 different alleles were found in all the 6 loci in the present study. Among the 3 population, the number of alleles were ranged from C (21) > meio-G1 (20) > meio-G2 (13). The average observed heterozygosity (Ho) of the meio-G1 and meio-G2 populations were 0.195 and 0.031, respectively, significantly less than that in C (0.494), and the Ho of meio-G2 was decreased 84.1% comparing to meio-G1. The observed inbreeding coefficient of the meio-G1 and meio-G2 population reached 0.605 and 0.937, respectively. This results suggest that gynogenesis is an effective method to pure the genome and useful for quickly establishing pure-lines in aquaculture.3. Twenty-two microsatellite (MS) loci which were demonstrated heterozygous in the female parents were used to evaluate the inheritance in two meio-G1 families and their control families of normal diploid. Four of the 22 microsatellite loci were deviated significantly from Mendelian expectations, and the other 18 loci were observed followed the Mendelian’s segregation. The recombination rates (y) were ranged from 0 to 1.0 with an average y value of 0.586 in the 18 loci, corresponding to a fixation index of 0.414, which showed 1.67 times comparing to full-sib inbreeding. The map distances between MS loci and centromere from 0 to 50cM under the assumption of complete interference for these 18 loci. Among which, 10 loci showed high M-C recombination with frequency more than 0.667. The results indicated that high recombination existed in the meio- G1.更多还原