【作者】 何玉英；

【导师】 王清印； 李健；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 中国对虾（Fenneropenaeus chinensis）主要分布于我国的黄、渤海及朝鲜半岛沿海，是我国北方重要的渔业对象及海水养殖虾类。在养虾业发展盛期（1990年前后）曾占到我国对虾养殖产量的70％左右。但1993年以后，由于品种、病害和环境等因素的影响，养殖产量急剧下降，不到全国对虾养殖产量的1／3。缺乏经过人工选育的优良品种、培育的虾苗质量差是对虾养殖业存在的关键问题。我国目前养殖的中国对虾只有“黄海1号”1个新品种，难以满足日益增长的生产需求。另外，“野捕家养”的苗种供应系统也不能满足对虾养殖业可持续发展的需要。本研究在中国对虾“黄海1号”选育研究的基础上，进行了生长和抗逆性状选育的初步研究。获得的研究结果如下：1．中国对虾“黄海1号”与野生群体F₁代生长发育规律比较采用4种生长曲线模型对中国对虾“黄海1号”和野生群体F₁代15项形态性状的生长规律进行了拟合，以三次函数模型的拟合优度（R2）最高；采用三次函数模型拟合的2个群体各形态性状的生长曲线、拐点月龄以及拐点体重（各形态性状长度）结果表明，中国对虾“黄海1号"的拐点月龄为2．87（拐点体重14．98g），野生群体F₁代的拐点月龄为4．05（拐点体重26．26g）；中国对虾“黄海1号”各形态性状的拐点月龄分布在0．51～3．07之间，达到最大生长速度的顺序分别为：头胸甲长＞第1腹节宽＞头胸甲高＞腹1高＞头胸甲宽＞体长＞全长＞腹节5长＞腹节3和4长＞尾节长＞腹节2长＞腹节1长＞腹节6长；野生群体F₁代除第2腹节长的拐点月龄为0．45之外，其它性状的拐点月龄分布在2．38～3．08之间，达到最大生长速度的顺序分别为：腹节2长＞腹节1长＞腹节3长＞腹节4长＞头胸甲高＞腹节5长＞舞头胸甲宽＞全长＞腹1高＞腹1宽＞尾节长=头胸甲长=体长＞腹节6长，除第1和第2腹节长2个性状外，野生群体F₁代的其它性状均比中国对虾“黄海1号”发育迟缓了1个月左右。2．中国对虾生长性状的遗传力和遗传相关估计采用人工授精技术建立了51个全同胞家系（包括35个半同胞家系），分别测定了中国对虾150日龄时各家系的体长、头胸甲长、腹节长和体重。应用数量遗传学原理，采用方差、协方差分析的方法，研究了中国对虾150日龄时生长性状的遗传力及性状间的遗传相关和表型相关。研究结果表明，中国对虾体长遗传力的估计值在0．36～0．51之间，头胸甲长的遗传力估计值在0．14～0．24之间，腹节长的遗传力估计值在0．25～0．50之间，而体重的遗传力估计值在0．04～0．29之间。中国对虾各生长性状之间表现出高的正相关，其中体重和腹节长的遗传相关最大为0．920，其次为体长和腹节长（0．915）、体长和体重（0．880）、体重和头胸甲长（0．870）、腹节长和头胸甲长（0．861）之间的遗传相关，以体长和头胸甲长之间的遗传相关为最小（0．832）。各性状表型相关在0．795～0．905之间，t检验均达到极显著水平（P＜0．01）。3．中国对虾生长性状遗传标记的筛选采用RAPD技术对“黄海1号”中国对虾快速生长选育群体第6代大个体群体（CP-a）和小个体群体（CP-b）以及野生群体（WP）为对照组的各50尾个体进行扩增，获得可能与生长性状相关的9个RAPD遗传标记。对获得的标记进行克隆、测序并根据序列设计特异性引物对3个群体进行SCAR标记分析。其中6对引物（SCAR1、SCAR2、SCAR3、SCAR4、SCAR5和SCAR6）在3个群体中有扩增产物，前4对引物在3个群体共150尾个体中的扩增产物无多态性。SCAR5和SCAR6在3个群体中的扩增产物具有多态性。依据扩增产物在群体中出现的频率和变化规律进行分析表明，SCAR5扩增的多态片段在3个群体中的组成比例分别为78％、52％和54％，差异显著（P＜0．05）；SCAR6扩增的多态片段经电泳后产生3个等位基因，6种基因型，只有CP-b含有等位基因A。这2个标记可以作为与中国对虾生长性状相关的候选标记，为在生产实践中实行分子标记辅助育种奠定理论基础。4．中国对虾家系仔虾幼苗对氨氮和pH的耐受性比较采用人工授精技术建立中国对虾家系，对建立的20个家系幼体通过急性毒性试验进行抗氨氮和pH性状的比较。结果表明：不同试验时间中国对虾家系仔虾幼苗对氨氮和pH的耐受性差异极显著（p＜0．01）和显著（p＜0．05），24h、48h和72h对氨氮耐受性的平均半数致死值分别为62．15、30．31和15．60 mg／L，对pH耐受性的平均半数致死值分别为9．99、9．41和9．12。以平均LD50值为评价指标，综合24h、48h和72h各家系对氨氮和pH的耐受性，最终筛选出对氨氮耐受性最强的家系8个，对pH耐受性最强的家系10个，对氨氮和pH耐受性均较强的家系7个，为构建中国对虾抗逆基础群体，开展中国对虾抗逆新品系的选育工作提供了基础。5．中国对虾养殖群体生长和抗逆性状杂交优势与遗传相关分析对中国对虾3个养殖群体：中国对虾“黄海1号”昌邑群体（CY）、中国对虾“黄海1号”河北群体（HB）以及日照近海野生群体养殖F₁代（WP）及其6个杂交组合子一代150日龄的生长性状和对高氨氮和高pH的抗性进行了测定，计算了各项指标的杂种优势率及遗传相关。研究结果表明，在生长性状上，CY×WP组合的子一代无论在形态性状（体长、头胸甲长、腹节长）还是体重均较其它组合表现出最大的杂种优势（2．28％～18．20％），而CY×HB和WP×CY组合子一代的各生长性状表现出杂交劣势。在抗性方面，6种杂交组合的子一代均表现出一定程度的杂种优势（12．67％～69．33％），其中，以HB×WP组合的杂种优势最明显（69．33％）。而在对高pH的抗性方面，CY×HB、HB×WP和WP×HB组合的子一代表现出杂种劣势，而其它组合表现出杂种优势，其中以WP×CY组合子一代的杂种优势最明显（16．03％）。遗传相关分析表明，中国对虾各生长性状与对高氨氮和高pH抗性方面存在负的遗传相关和表型相关，各生长性状与高氨氮抗性之间的遗传相关在-0．528～0之间，表型相关在-0．103～0之间，各生长性状与高pH抗性之间的遗传相关在-0．221～0．027之间，表型相关在-0．042～0．005之间。因此，在中国对虾选育过程中，可以采用综合选择指数的方法对生长性状和抗逆性状进行聚合性状的选育。更多还原

【Abstract】 Chinese shrimp Fenneropenaeus chinensis is a commercially important speciesin China and the natural distribution is confined only in the Bohai Sea, Yellow Seaand the coast of Korean Peninsula. During the 1990s, the yield of Fenneropenaeuschinensis from farming ponds counted for 70% of the total marine shrimp farmingproduction. However, because of the factors of strains, diseases and environment, theyield decreased sharply, which counted for a quarter of the annual harvest in theprosperous stage and the economic losses reached to tens of billion RMB. Mostshrimp farmers rely on the capture of wild shrimp to stock their ponds, and there areconcern that harvesting wild shrimp for aquaculture may cause poor diseasesresistance, gene losses and degradation of good traits, which can not satisfy with thesustainable development of shrimp farming, so Studies on selective breeding of newshrimp strains must be strengthened so as to establish the genetic breeding system.Particularly, the selective breeding should be focused on those traits like growth,stress and disease resistance. Here, Genetic analysis linked to growth and stress-resistancetraits in Fenneropenaeus chinensis was studied on the basis of the selectivebreeding of Fenneropenaeus chinensis New Variety "Huanghai No. 1". The mainresults are as follows.1. The Comparison of Growth Pattern in "Huanghai NO. 1" and the First Generationof Wild Population in Chinese Shrimp Fenneropenaeus chinensisFour growth models were used to fit the growth pattern of morphologicalcharacters in the new variety "Huanghai NO.1" and the first generation of wild population in Fenneropenaeus chinensis based on fifteen morphological characters.The squared multiple correlation coefficient （R2） of the Cubic growth model was thehigh most than Line model, Power model and Exponential model, so the Cubicgrowth model was selected to investigate the growth pattern and age in month atinflexion. The results showed that ages in month at inflexion of body weight in"Huanghai NO. 1" and the first generation of wild population were 2.87 （body weightat inflexion was 14.98g） and 4.05（body weight at inflexion was 26.26g）, respectively.The ages in month at inflexion of other morphological characters in "Huanghai NO. 1"were from 0.51 to 3.07. CL had the most rapid growth rate, followed by AW, CH, AH,CW, BL, FL, AL5, AL3, AL4, TL, AL2, AL1, and AL6 had the slowest growth rate.The ages in month at inflexion of other morphological characters in the firstgeneration of wild population were from 2.38 to 3.08, except that of AL2, and theorder of achieving the most rapid growth rate was AL2, AL1, AL3, AL4, CH, AL5,CW, FL, AH, AW, TL, CL, BL, and AL6, that delayed one month than that of"Huanghai NO. 1" except AL2 and AL1.2. Studies on heritability and correlation for growth traits in Chinese shrimpFenneropenaeus chinensisFifty-one full-sib families （including thirty-five half-sib families） of the Chineseshrimp Fenneropenaeus chinensis were obtained by artificial insemination. Fourgrowth traits of these families were measured, including body length, cephalothoraxlength, abdorminal segment length and body weight. According to quantitativegenetics theory, the heritability of each growth trait, genetic and phenotypiccorrelation among the traits was statistically studied. The results show that theheritabilities were 0.36～0.51 for body length, 0.14～0.24 for cephalothorax length,0.25～0.50 for abdominal segment length, and 0.04～0.29 for body weight. Geneticcorrelations were high between growth traits. The genetic correlation between bodyweight and abdominal segment length was the highest （0.920）, followed by bodylength and abdominal segment length（0.915）, body length and body weight（0.880）,body weight and cephalothorax length（0.870）, abdominal segment length andeephalothorax length（0.861）, The genetic correlation between body length and cephalothorax length was the lowest（0.832）. The estimates of phenotypic correlationwere from 0.795 to 0.905 with a high significant difference （P＜0.01） among the fourgrowth traits by t-test.3. Candidate markers linked to growth traits in Fenneropenaeus chinensisRandom Amplified Polymorphic DNA （RAPD） technique was applied to analyze150 samples of three populations from the large body population （CP-a） and the smallbody population （CP-b） which were from the sixth generation of cultured populationsof Fenneropenaeus chinensis and from the wild populations （WP）. 240 randomprimers were screened for mixed templates and 65 differential fragments correlatedwith growth traits were obtained including 55 positive fragments and 10 negativefragments. Population analysis used primers screened from 240 RAPD primersshowed 9 RADP markers were correlated with growth traits including 7 positivemarkers and 2 negative markers. After cloning and sequencing we got 8 sequencesvarying in length from 500bp to 1000bp except marker amplified by S159.PrimerPremier 5 software was used to design specific primers on the basis of the sequenceand Sequence-Characterized Amplified Regions （SCAR） technique was used toinvestigate growth traits markers of Fenneropenaeus chinensis. There were 6 pairs ofprimers （SCAR1, SCAR2, SCAR3, SCAR4, SCAR5 and SCAR6） can obtainamplified products, but the fragments from the former four pairs of primers did notdifferentiate in three populations. The latter two pairs of primers amplifiedpolymorphic fragments according to the band frequency and variety rule appeared inthe samples. The numbers of polymorphic fragments amplified by primer SCAR5 inCP-a, CP-b and WP were 39,26 and 27, respectively, and the band frequencies were78%, 52% and 54%, respectively.χ2 test of polymorphic fragments in CP-a and WPpopulations showed significantly different （P＜0.05）, that indicated the polymorphicfragment from SCAR can be used as positive marker candidate.3 alleles and 6genotypes were found in three populations amplified by primer SCAR6, while onlyCP-b had the allele A, that revealed that locus A maybe was correlative or linkagewith some gene which can restrain growth speed of Fenneropenaeus chinensis. Theresults demonstrate that the two SCAR markers we had identified can he used as markers candidate correlated with growth traits of Fenneropenaeus chinensis andsupplied theory basis for marker-assisted selection （MAS） in the practice.4. Comparison for the Resistance to pH and Ammonia in Chinese ShrimpFenneropenaeus chinensis familiesTwenty families obtained using artificial insemination were used to compare theresistance to pH and ammonia by acute toxicity. The results showed that there wereextremely significant （p＜0.01） for the resistance to pH and ammonia betweendifferent test times, and the average LD₅₀ （median lethal dose） values of 24h, 48h and72h for resistance to ammonia were 62.15、30.31 and 15.60 mg/L, respectively, andfor resistance to pH were 9.99、9.41 and 9.12, respectively. Eight families wereselected for the high resistance to ammonia, and ten families were selected for thehigh resistance to pH, while seven families were selected for the high resistance toammonia and pH According to the average LD₅₀, which provide the basic conditionfor the production of stress-resistance populations and the selective breeding ofstress-resistance lines in Chinese shrimp Fenneropenaeus chinensis.5. Analysis of heterosis and genetic correlation of growth traits and the resistance tostress in Fenneropenaeus chinensisThe heterosis and genetic correlation of growth traits and the resistance to stressof Fenneropenaeus chinensis were analyzed in two groups of "Huanghai No.1"（CYand HB）, and the first generation of wild population based on diallel cross at their ageof 150 days. The hybrids of CY×WP was found to show the highest heterosis ratewith a range of values from 2.28% to 18.20% on the four growth traits of body length,body weight, cephalothorax length and abdominal length, while the hybrids ofCY×HB and WP×CY showed the poor heterosis among the six hybridizationcombinations. High Heterosis for the resistance to high ammonia was found with arange from 12.67% to 69.33% among six hybridization combinations, the hybrids ofHB×WP showed the highest heterosis rate （69.33%）. There was no heterosis inhybrids of CY×HB, HB×Wp and WP×HB for the resistance to high pH, the hybridsof WP×CY showed the highest heterosis （16.03%）. The results of genetic correlationsshowed that there were negative genetic correlation between growth traits and the resistance to high ammonia and pH. The estimates of genetic and phenotypiccorrelation were 0.528～0 and -0.103～0 between growth traits and the resistance tohigh ammonia, and -0.221～0.027 and -0.042～0.005 between growth traits and theresistance to high pH, which showed the method of selection index can be used for thecomplex traits.更多还原