| 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. |
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