【作者】 张天时；

【导师】 王清印； 孔杰；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2010， 博士

【摘要】 本研究采集了中国沿海不同地理群体、朝鲜半岛群体以及黄海所培育的“黄海1号”和抗病品系“即抗98”等不同中国对虾群体,构建了基础群体。采用人工定向交尾和人工授精技术,共构建了中国对虾家系。以构建的家系为材料,以杂交育种和选择育种为目标,进行了系统的中国对虾杂交育种试验、不同性状遗传参数分析工作。结果表明以不同地理群体杂交作为基础群体,然后采用系统的多性状选择育种方法可以获得较好的选择效果。本文研究结果也表明,利用多性状复合育种技术,培育综合性状优良的中国对虾新品种是一个正确而必要的育种策略。这些试验结果为中国对虾合理系统的育种工作提供了理论基础和数据支持。其具体结果如下：1.对虾选择育种操作规程中国对虾选择育种操作规程主要包括：1)基础群体的建立：采集了中国沿海不同地理群体、朝鲜半岛群体以及黄海所培育的“黄海1号”和抗病品系“即抗98”等不同中国对虾群体,构建了基础群体。2)亲虾的越冬和促熟：通过摘除眼柄的方式增加产卵亲虾的比例。3)定向交尾和人工授精技术：在中国对虾家系建立过程中,我们采用定向交配对子代进行选育的方法；采用4种中国对虾人工授精方法,即整个精荚移植,精荚切块移植,输精管移植和精液移植。4)苗种培育与中间暂养：建立中国对虾苗种培育的技术。5)幼体标记：所有家系均在控制条件下育苗、养殖和抗wssv能力测试。6)养成：养成后对遗传上表现优良的家系所有备选个体分别用独特的眼柄进行标记。7)成体标记：采用个体眼柄标记的方法为养成个体提供身份标记。8)生长、存活率与wssv抗性性状的检测：不同家系标记的中国对虾在共同的环境中进行生长、存活率和wssv人工攻毒实验,并且记录所有与这些性状检测相关的信息。2.中国对虾家系的建立及其生长发育分析采用4种中国对虾人工授精方法建立中国对虾家系,共进行了155例雌虾的人工授精实验,有108尾雌虾成功产卵并孵化出仔虾,成功率为69.7%,在此基础上目前已经建立了101个全同胞家系(包括29个半同胞家系组)。在受精卵孵化、育苗的基础上,同时进行了不同家系幼体生长发育情况的研究,结果表明受精率、孵化率、存活率以及初期生长在4个实验组间都没有显著性差异(P>0.05)。另外对其他家系之间的比较也得出同样的结果,说明不同家系在早期的生长发育中,没有表现出显著性差异(P>0.05)。而在养成阶段,方差分析结果表明家系间在生长上表现出差异极其显著(P<0.01)。2.饵料和养殖密度对暂养期家系生长和存活的影响在实验用虾遗传背景一致的情况下,分析了3种饵料(配合饲料、冰冻鲜鱼肉和活卤虫)和4个不同的养殖密度对小水体中国对虾幼虾生长和存活率的影响。结果表明,饵料和饲养密度对中国对虾幼虾生长及存活率有显著影响。幼虾的生长在饵料、养殖密度单因子实验及饵料和养殖密度相结合的双因子实验,均表现出极其显著差异(P<0.01),活卤虫对幼虾的生长的效应尤为突出。而养殖密度对中国对虾的行为生物学、个体间体重增量均有影响。随着养殖密度的提高,中国对虾增重变慢；同时,个体间体重增量差异变大。随着养殖密度的增加,中国对虾幼虾的存活率呈下降趋势,但不同饵料对存活率影响变化幅度较大,波动在58.1%～85.2%之间,其中投喂活卤虫养殖密度为50尾/桶的存活率最高(85.2%)；投喂配合饵料4个养殖密度梯度的存活率变化不明显；而投喂冰冻鱼肉4个养殖密度梯度的存活率变化较大。3.中国对虾不同群体间的杂交效果分析对中国对虾两个不同地理位置的选育群体—中国乳山湾选育群体(RS)和朝鲜半岛南海选育群体(KN)及其2个杂交组合后代、3个复式杂交组合后代的4月龄时生长发育情况和存活率进行了研究。结果表明,2个杂交后代在体长、体重、存活率3个指标上均表现不同程度的杂种优势(3.586%～13.892%),其中RS♀×KN♂杂交效果较好；ANOVA分析结果表明,中国乳山湾选育群体(RS)与朝鲜半岛南海选育群体(KN)及其杂交子一代4月龄时在体重和体长指标上在群体间均达到显著水平(P<0.05),而在在存活率指标上在群体间未达到显著水平(P>0.05)；LSD多重比较结果显示,2个杂交后代在体长指标上与双亲差异显著(P<0.05)。在3个复式杂交组合后代中,RS♀×(KN’×RS’)♂杂交在3个指标上均大于同龄(RS’×KN’)♀×RS♂,比同龄(KN’×RS’)♀×RS♂稍高；ANOVA分析结果表明,中国乳山湾选育群体(RS)与朝鲜半岛南海选育群体(KN)及其杂交子一代以及复式杂交后代的4月龄时在体长、体重指标上在群体间均达到显著水平(P<0.05); LSD多重比较结果显示,在体长和存活率指标上RS♀×(KN’×RS’)♂与(RS’×KN’)♀×RS♂差异显著。在所有两群体的纯繁后代、杂交组合后代和复式杂交组合后代中,RS♀×(KN’×RS’)♂杂交后代生长发育最快,成活率最高。5.对虾早期生长的遗传参数估计的动物模型分析利用中国对虾21个父系半同胞组共1387个个体体重数据,将家系标记时的平均体重、全同胞组效应等因子组合,建立了4种不同的动物模型,应用BLUP法估计体重育种值。四种模型的估计结果分析表明,家系标记时的平均体重和全同胞家系效应是2个重要影响因子,建立的AFB动物模型比其它模型所估计的结果更准确。AFB动物模型估计中国对虾145d的体重遗传力为0.14～0.076,据此计算育种值并进行模拟留种分析,结果显示,在根据表型值或育种值选种的情况下,育种值选种的留种家系或个体的育种值比表型值选种分别提高50%和80.59%,育种值选择的效率更高。6.中国对虾体重、存活率和WSSV抗性的遗传参数评估利用单性状动物模型估计中国对虾170d的体重、抗WSSV存活时间和存活率三个性状的育种值,在家系水平上进行性状间表型值和育种值相关分析。估计的三个性状的遗传力分别为：0.22±0.16、0.14±0.12和0.03±0.021,并据此计算各性状个体育种值。家系性状表型值的相关分析表明,家系170d体重和抗WSSV存活时间之间存在一定程度相关(r=0.35,P<0.05),其余性状间相关系数很小,且差异不显著。三个性状家系育种值间的相关系数均较小,170d体重与抗WSSV存活时间的相关系数最高(0.038),170d体重与存活率,抗WSSV存活时间与存活率之间为负相关(r=-0.24,r=-0.027),并且统计检验未达到显著性水平。另用所有家系和个体估计得到的生长、存活率和抗WSSV的育种值计算了家系和个体的选择指数值,并且依据选择指数值进行了选种以生产下一代家系。7.应用BLUP法分析中国对虾选择的遗传进展为了评估中国对虾最佳线性无偏预测(BLUP)的育种效果,利用亲本评估的个体育种值,组建3个高育种值家系为实验组,3个平均值育种值家系为对照组,进行了生长对比测试。6个家系的苗种经过独立培育、中间暂养,体重达到1g左右时用荧光标记进行了标记,标记后的幼虾同时放养于对虾养殖池中。在生产养殖池中养殖63天后进行生长性状的测定。BLUP法预测结果显示：中国对虾一代选育后的预期遗传进展为0.78g,相对于选育前全群的平均值6.68g,提高了约11.68%。实际对比测试结果表明,高育种值家系后代平均体重为21.55g,平均育种值家系的平均体重为19.03g,选育一代的体重提高13.28%。结果表明：BLUP育种技术对中国对虾生长性状的选育效果显著。更多还原

【Abstract】 Brood stock was collected from both domesticated breeds ("Huang Hai 1" and "Jikang-98") and wild populations in China and South Korea to establish a base population of Fenneropenaeus chinensis with a broad genetic variation. The families were successfully produced using oriented mating and artificial insemination. The crossbreeding of F. chinensis and genetic parameters estimates were carried out. The results indicated that obvious genetic improvement was gained in F. chinensis. Therefore, the multi-traits selection was necessary for shrimp genetic breeding program. The results would provide theoretic basis for indirect and early breeding for F. chinensis. The following is the results in detail.1.Technical operation of selective breeding for F. chinensisTechnical operation of Selective breeding for F. chinensis is established.1) Composite of base population:base population was collected from both domesticated breeds ("Huang Hai 1" and "Jikang-98") and wild populations in China and South Korea to establish a base population of F. chinensis with a broad genetic variation.2) Over-wintered and ripened of spawners:the percentage of spawning females are increased by Eyestalk ablation.3) Oriented mating and Artificial insemination: F. chinensis families were produced by carrying out oriented mating so as to select the offsprings in selective breeding of F. chinensis. We used 4 different methods of artificial insemination (AI), namely the entire spermatophores transplantation, the fractional spermatophores transplantation, the sperm duct transplantation and the seminal fluid transplantation.4) Larvae rearing and post-larval nursing:larvae rearing of standardized technical operation for F. chinensis.5) Post-larvae tagging:family coding using elastomers:all families, which are cultured in the controlled environment, are challenged with White Spot Syndrome Virus (WSSV).6) Post-larval rearing.7) Individual tagging:at harvest, all breeding candidates from the genetically superior families are individually tagged using unique eye-tags.8) Testing of growth, survival and WSSV-challenge:tagged test animals from different families were tested with growth, survival and WSSV-challenge in a common test environment. The growth, pond survival and WSSV-resistance and pond survival were measured including all recorded data.2.Establishment of families and their growth and development for F. chinensisF. chinensis families were produced by carrying out 4 different methods of artificial insemination (AI).155 cases of artificial insemination experiment were totally applied,108 spawners succeeded in spawning and hatching the larvae, with 69.7% of the ratio. And then on basis of the experiment,101 full-sib families that included 29 half-sib families of F. chinensis were established. The larval growth and development were studied, such as fertilization rate, hatching rate, survival rate and growth were compared. There were no significant differences among four different experimental groups (RS (♀)×RS (♂),QD(♀)×RS(♂),KC (♀)×RS (♂) and KC (♀)×QD (♂)). In addition, there were no significant differences between other families that were studied in the experiment at early life history stage of F. chinensis. During growout, analyses of variance for growth indicated there was most significant difference (P<0.01) among families.3. The effects of different diets and stocking density on the growth and survival rate of post-larval nursing of families of F. chinensisUnder the identical genetic ground, we studied the effect of different diets and stocking density on the growth and survival rate of juvenile shrimp F. chinensis in small water bodies. Groups of juvenile shrimps F. chinensis were cultured using three diets of artificial pellets, frozen fresh fish and living Artemia sp. at four stocking densities (50,100,150,200 shrimps per tank, respectively). The results showed that the diets and the stocking density had great effects on growth and survival rate of juvenile shrimps F. chinensis. Analyses of variance for growth of juvenile shrimps F. chinensis indicated there were significant differences among different diets, densities, diets and densities (P<0.01). The growth of juvenile shrimps F. chinensis fed on Artemia sp. was the fastest in growth. The behavior and the weight gain of shrimps were affected by stocking density. The results showed that shrimps held at the higher density showed lower weight gain than those at lower densities. The variation in shrimp weight gain was greatest in the highest density group. The results showed that the four different diets and stocking density had a significant effect on survival rates over the experimental period. Shrimps held at different stocking density showed with increase of stocking density the survival rates of fish was suppressed. However, different diets had different effect on survival rates from 58.1% up to 85.2%. The group of juvenile shrimps F. chinensis fed on Artemia sp. with stocking density (50 shrimps per tank) had the highest survival rate of 85.2%. The group of juvenile shrimps F. chinensis fed on artificial pellets had not significant effect on survival rates at four different stocking densities. The group of juvenile shrimps F. chinensis fed on frozen fresh fish had significant effect on survival rates.4. Studies on hybridization among the different populations of shrimp F. chinensisF. chinensis were collected from two different geographic areas in Rushan Bay of China and the South Coast of the Korean Peninsula. Intra-and intercross populations were produced between Rushan (RS) and Korean (KN) selected populations. Descendants of 3 groups were produced using hybrid duplex. All descendants of the above group were selected for the research on the growth trait and viability at the end of fourth month. The results indicated that growth trait and viability of two intercross populations showed a range of heterosis, ranging from 3.586% to 13.892%, and that KN♀×RS♂has a stronger heterosis than KN♀×RS♂. The result of ANOVA indicated that there was significant difference (P<0.05) on BL, BW of hybrids of Rushan (RS) and Korean (KN) selected populations with their parental populations while Sur was not significantly different (P>0.05). The multiple comparisons of LSD test indicated that the two intercross populations were significantly different (P<0.05) from their parents in BL. The results of hybrid duplex indicated that RS♀×(KN’×RS’)♂had a better growth and viability than (KN’×RS’)♀×RS♂and (RS’×KN’)♀×RS♂, (KN’×RS’)♀×RS♂had a better growth and viability than (RS’×KN’)♀×RS♂. The result of ANOVA indicated that the descendants of all populations and cross combinations were significantly different in BL and Sur. The multiple comparisons of LSD test indicated that there was significantly difference between RS♀×(KN×RS)♂and (RS×KN)♀×RS♂(P<0.05) in BL and Sur. In addition, among descendants of intra-, intercross populations and hybrids duplex RS♀×(KN×RS)♂had a best growth and viability.5. Analysis of Animal Models and Estimation of Genetic Parameters of growth in juvenile F. chinensisBased on the data of 1387 animals from 21 half-sib groups of Fenneropenaeus chinensis, breeding value of body weight of all individuals were estimated using four kinds of animal (A、AB、AF、AFB) models of BLUP (Best linear unbiased prediction). These models were composing of different combinations which included average body weight when tagging family (covariate) or full-sib group effect (uncorrelated random effect) or these two factors. Results implied that these two factors were very import for estimating breeding value of body weight. The accuracy of breeding value estimated by ABF model was higher than other models. The heritability estimated of 145d body weight by ABF model was 0.14±0.076. For body weight selection, the efficency of breeding value selection is increased by 50% and 80.59% in family selection and individual selection, compared to phenotypic value selection.6. Estimation of genetic parameters of body weight, survival rate and resistance to WSSV in shrimp F. chinensisThe breeding values of three traits about the body weight of 170 days, the survival time for resistance to White Spot Syndrome Virus (WSSV) and the survival rate were estimated using single trait animal model, and the correlation analyses between the phenotypic value and the breeding value among traits was performed at family level. The results indicated that the estimated heritability of three traits were 0.22±0.16,0.14±0.12 and 0.03±0.021, respectively, and the breeding value of each trait was calculated basing on these values. At family level, the correlation analyses of trait phenotypic value showed that the correlation coefficient between the average body weight of 170 days and the resistance to WSSV is positive and significant (r=0.35, P<0.05), and the correlation coefficients among other traits were very small, and the differences were not distinct. The correlation coefficients of family breeding value between three traits were all small, among which the correlation coefficient between body weight of 170 days and resistance to WSSV was highest (0.038), and the correlations between body weight of 170 days and tank survival, between the survival time of resistance to WSSV and pond survival were negative (r=-0.24, r=-0.027), respectively, and their differences were not distinct. Estimated breeding values for the above traits were combined in a multi-trait selection index to estimate a total breeding value of the breeding candidates. The next generation of families were Selected to produced based on Selection index values.7.Genetic gain for selection of F. chinensis applying BLUP methodIn order to evaluate genetic gain of BLUP (best linear unbiased prediction) for Fennropenaeus chinensis, the experiment on growth comparison of breeding generation of F. chinensis was carried out. Groups of the experiment on growth comparison consisted of three breeding sires of families of the high breeding value as the experimental group and three middling breeding value as the control group. The offspring of six families were stocked into separate larvae-culture tanks for rearing until tagging. The six families produced were tagged with a unique family code by injecting different colors of "Visible Implant Fluorescent Elastomers" (VIE). The animals had an average body weight of 1.0 grams at tagging. The breeding candidates were stocked in the tanks and harvested after 18 weeks. The expecting genetic gain of the growth trait was estimated to be 0.78g,11.68% increased to average value 6.68g. The test results show that the average body weight of the offspring of high breeding value families was 21.56g, the average body weight of the offspring of the middling breeding value families was 19.03g, the phenotypic gain of the body weight was 13.28%. The results indicated that obvious genetic improvement was gained by BLUP breeding value estimation.更多还原