【作者】 赵燕；

【导师】 曾勇庆； 王慧；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 日本沼虾隶属十足目、长臂虾科、沼虾属，自然分布于中国、日本、越南及俄罗斯远东地区，在我国各地淡水水域中都有分布。日本沼虾的养殖始于二十世纪五十年代，从2004年起，我国日本沼虾的年养殖产量就超过了20万吨。多年来，日本沼虾在市场上一直供不应求，畅销不衰，是一种养殖效益非常可观的淡水虾类。但长期以来，繁殖日本沼虾用的亲本多为野生个体，未经过系统选育，日本沼虾的养殖单产较低，且多是套养在各种鱼类的养殖池塘中，主养日本沼虾的池塘较少。因此开展日本沼虾的种质资源保护、良种选育工作是当前日本沼虾养殖业中亟待解决的问题。由于当前已知的日本沼虾微卫星标记只有二十多个，这严重地限制了日本沼虾的群体遗传结构分析、种质资源评价等工作；此外，日本沼虾雄性生长速度显著大于雌性，开展单性苗种培育意义重大，但当前没有鉴别日本沼虾遗传性别的分子标记，使得单性苗种培育工作一直无法得以开展。本论文的研究内容主要包含如下3个方面：1.日本沼虾多态性微卫星标记的开发：（1）FIASCO法开发微卫星标记。通过生物素标记的探针（GT）13和（GA）13富集微卫星序列，共获得含微卫星的序列334个，成功设计引物110对，设计引物的序列含有的微卫星重复多为2碱基重复，重复数多数在10次以上。设计的引物扩增的目的条带大小在95-356bp之间。多态性分析显示，24对引物扩增出的条带为清晰稳定的多态，开发效率（多态性引物占总设计引物的百分比）为21.8%。各位点等位基因数介于2-9之间，各等位基因的大小范围在111-334bp之间；各位点期望杂合度（He）在0.1905-1.0000之间，各位点的观测杂合度（Ho）在0.1765-0.8090之间，除个别位点外（X781、X63、Z339）期望杂合度（He）与观测杂合度（Ho）均在0.3以上；各等位基因的多态信息含量（PIC）在0.1574-0.8272之间，其中X781与Z339两个位点的PIC值在0.25以下，属于低度多态性位点；X195等5个位点的PIC值在0.25与0.5之间，属于中度多态性位点；其余的17个位点的PIC值皆大于0.5，属于高度多态性位点。经Bonferroni法校正（P <0.003）后，24个多态性微卫星位点中有9个微卫星位点偏离了Hardy–Weinberg平衡，其中有2个位点（X1214、Z167）表现出显著的杂合子缺失（Hardy–Weinberg平衡偏离指数D<0，D=(Ho-He)/He），其他7个位点表现出显著的杂合子过剩（Hardy–Weinberg平衡偏离指数D>0）。（2）ISSR-PCR法开发微卫星标记。设计的8对简并引物中用于扩增CA/GT重复单元的引物PCA6、PGT6、PTG6、PAC6能够扩增出亮度较大的弥散带，将这四对简并引物克隆后测序。将前人已经开发出的微卫星同源序列去除后，共获得含微卫星的序列114个，成功设计引物45对。多态性分析显示，23对引物扩增条带为多态，开发效率为51.1%。各位点等位基因数介于2-12之间；各等位基因的大小范围在141-324bp之间；各位点期望杂合度（He）在0.2357-1.0000之间，各位点的观测杂合度（Ho）在0.3203-0.9169之间；各等位基因的多态信息含量（PIC）在0.2894-0.8877之间，其中4个位点（B78、B96、B75、D30）PIC值在0.25-0.5之间，属于中度多态位点，其他位点的PIC值都大于0.5，属于高度多态位点。经Bonferroni法校正(P <0.003)后，23个多态性位点中6个微卫星位点偏离了Hardy–Weinberg平衡，其中有三个位点（B96、B73、B92）表现出显著的杂合子缺失（Hardy–Weinberg平衡偏离指数D<0，D=(Ho-He)/He），另外三个位点（A89、 B75、 C2）表现出显著的杂合子过剩（Hardy–Weinberg平衡偏离指数D>0）。（3）微卫星序列特征分析对391条含微卫星重复的序列进行统计分析（图3-7），发现获得的微卫星序列长度范围在60-852bp之间，100bp以下的最少（16个）。大部分克隆片段大小在200bp以上，占总序列的83.1%。日本沼虾的微卫星序列中以二核苷酸重复最为丰富，占所有微卫星序列类型数目的92.4%，其次是三核苷酸（1.8%）或四核苷酸重复(1.6%)。二核苷酸重复中以（CA/GT）n类型所占比例最高，占总微卫星序列数的68.3%，其次是（GA/CT）n型，占25.1%，此外还有少量的（TA/AT）n型和（GC/CG）n型，分别占2.8%和0.6%。2.4个日本沼虾群体遗传结构分析利用8个微卫星标记（A28、A73、B13、B29、B44、B93、D7、Z337）对太湖、东平湖、微山湖、长江下游（崇明岛附近水域）4个群体的遗传结构进行分析，4个群体中长江下游（崇明岛附近水域）群体的平均等位基因数最多，为13.5000，其次是微山湖群体为12.2500，太湖群体最低为11.87。微山湖群体的平均期望杂合度和观测杂合度最高，分别为0.8403和0.8350；长江下游（崇明岛附近水域）群体的最低，分别为0.7145和0.7909。各群体间遗传距离分析显示，微山湖和东平湖群体的遗传距离最小，为0.0158；长江下游（崇明岛附近水域）和微山湖的遗传距离最大为0.3844。各群体间的遗传相似度分析也得出了类似的结果，微山湖和东平湖的遗传相似度最大（0.9162）；长江下游（崇明岛附近水域）和微山湖的遗传相似度最小（0.6312）。基于遗传距离的NJ和UPGMA聚类分析得到了相同的结果，东平湖群体和微山湖群体遗传距离最小首先聚类为一支，再与太湖群体聚为一支，最后和长江下游（崇明岛附近水域）群体聚类为一支。聚类分析的情况和实际的地理距离相符。4个日本沼虾群体的分子方差分析（AMOVA）显示，群体中89.29%的变异来源于个体内，仅有4.18%的变异来源于群体间，6.52%来源于群体内个体间，显著性检验分析显示这4个群体遗传分化极显著（P <0.01）。4个群体两两间遗传分化指数Fst从0.0031到0.0739，其中东平湖群体与长江下游（崇明岛附近水域）群体、微山湖群体与长江下游（崇明岛附近水域）群体的0.05<Fst <0.15，属于中等分化程度；其他群体间的Fst值均<0.05，分化程度较弱。日本沼虾群体的遗传结构分析对其种质资源保护和合理挖掘利用具有重要意义。（3）性别相关标记的筛选：在利用15雌和15雄日本沼虾对开发的47个微卫星标记进行多态性分析时，我们发现有8个SSR标记出现了雌性或雄性特异的等位基因。为此，我们合成了这8个微卫星标记的上游荧光引物，利用荧光SSR法，对另外的15雌和15雄日本沼虾进一步进行分析、验证。结果显示，有7个微卫星标记检测到了雌性或雄性特异的等位基因（等位基因出现次数在2次以上）。首先利用2个雌性个体和2个雄性个体对64对AFLP引物的扩增情况进行了筛选，对其中的9对引物进一步利用15个雌性个体和15个雄性个体进行大量分析。有8对检测到分布频率雌雄差异显著的等位基因，其中雄性占绝大多数（90.9%）的等位基因有1个，雌性占绝大多数（84.6%）的等位基因有2个，可为日本沼虾性别决定机制研究提供参考。更多还原

【Abstract】 Oriental river prawn Macrobrachium nipponense, a member of the Palaemonidae familyof decapod crustaceans, is widely distributed in freshwater and low-salinity regions ofestuaries in China, Japan, Vietnam and Russia. M. nipponense is commercially important andis cultured extensively throughout China and other Asian countries. The basic productiontechniques for M. nipponense were developed in China approximately40years ago. In2004,the culture yields of this prawn exceeded200,000tons, which accounts for the majority of itsworldwide production. Owing to its high benefit and excellent adaptability, its cultureproduction has gradually increased. The price of M. nipponense is relatively higher for longtime. However, the parents for reproduction are almost wild M. nipponense and the systematicselective breeding has not ever been done until now. The M. nipponense is almost cultured insome fish ponds such as grass carp and the yield per unit is much lower. Therefore, thegermplasm conservation and selective breeding of M. nipponense is very important.Nowadays, there are only reported polymorphic20microsatellite loci. Due to the precocity ofthe farmed prawns, especially the females, the growth performance has decreased in recentyears. On the other hand, there is a significant difference in growth performance betweenmales and females in this species. Males grow much faster than females and reach a largersize at harvest, similarly to many other Macrobrachium species. Monosex culture is acommon practice in aquaculture, and many attempts have been made to apply this technologyto crustacean aquaculture. However, there is not sex specific molecular marker foridentification of genetic sex of M. nipponense, which inhibited the development of monosexculture of M. nipponense. My thesis contained three contents:(1) Isolation of polymorphic M. nipponense microsatellite markers:The biotin labeling probe （GT）13and（GA）13are synthesized and334sequencescontaining microsatellites are obtained using FIASCO method.110Pairs of primers weredesigned successfully and the targeted amplified fragments ranged from95-356bp.24microsatellite loci were proved to be polymorphic. The allele number ranged from2to9,observed and expected heterozygosities ranged from0.1765to0.8090and from0.1905to1.0000, respectively. The polymorphic information contents (PIC) ranged from0.1574to0.8272. Among24microsatellite loci, the PIC of17loci are higher than0.5, belonging tohigh polymorphic loci. After Bonferroni correction,9among24microsatellite loci deviatedfrom Hardy-Weinberg equilibration.114containing microsatellite sequences were obtainedusing ISSR-PCR method.45Pairs of primers were designed successfully, and23 microsatellite loci were proved to be polymorphic. The observed and expectedheterozygosities ranged from0.3203to0.9169and from0.2357to1.0000, respectively. Thepolymorphic information contents (PIC) ranged from0.2894to0.8877. The PIC of four loci(B78、B96、B75、D30) ranged from0.25-0.5, belonging to middle polymorphic loci, andother microsatellite loci are high polymorphic. After Bonferroni correction,6among23microsatellite loci deviated from Hardy-Weinberg equilibration. These markers will bebenefit for the evaluation of genetic resource and the construction of genetic linkage map inM. nipponense.(2) Characteristics of isolated microsatellite markersAfter analyzing the391containing microsatellite sequences, it showed that themicrosatellite sequence fragment ranged from60-582bp, and83.1%sequences are longerthan200bp.92.4%sequences contained2base repeated microsatellites. Additionally,3basetype and4base type are also isolated, with the proportion of3.4%. The proportion of(GT/CA)nreached68.3%, and (GA/CT)n,(TA/AT)nand (GC/CG)nare relatively25.1%、2.8%、0.6%.(3) The genetic diversity of four populations:To assess the genetic status of this species, the genetic diversity of wild Macrobrachiumnipponense from four locations in China were investigated using8polymorphic microsatelliteDNA loci (A28、A73、B13、B29、B44、B93、D7、Z337). The mean number of allelesfor Changjiang population was13.5000, and only11.87in Taihu population. The observedand expected heterozygosities are0.8350and0.8403in Weishan lake, and only0.7909and0.7145in Changjiang population. Both the FST and AMOVA analyses showed that there issignificant difference on population differentiation among populations. The NJ and UPGMAclustering tree demonstrated that Dongping lake and Weishan lake population were clusteredtogether, and then clustered with Taihu population, with Dongping lake population in the end.AMOVA analysis for the four populations based on8microsatellite loci showed89.29%of the total genetic variation within the individuals. The genetic variation amongpopulations and between individuals within population was4.18and6.52%, respectively.Statistical analysis showed a significant genetic differentiation for the four populations andFST value ranged from0.0031to0.0739.(4) Development of sex specific molecular marker:In previous study, we found female or male specific alleles during charactering theisolated47polymorphic loci using15female and15male M. nipponense in8SSR markers. Therefore, these8fluorescence SSR markers are synthesized and further studied the sexspecific molecular markers in another15females and15males. The results showed thatfemale or male specific alleles are found in7SSR marker (the frequencies of allele are higherthan2).The differences of genomic DNA between females and males of M. nipponense werestudied using AFLP technique. Firstly,2females and2males were used for AFLP analysisand9pairs of AFLP primers were selected. During these9pairs of AFLP primers, manydifferent allele frequencies between females and males were found, such as a male majorallele (90.9%) and female major allele (84.6%). However, no sex specific AFLP was found.These markers will be benefit for the evaluation of genetic resource, selection ofbreeding base population, the development of monosex culture of M. nipponense, and theconstruction of genetic linkage map in M. nipponense.更多还原