| Oriental river prawn, Macrobrachium nipponense, subordinated to Decapoda, Palaemonidae, Macrobrachium, extensively distributed everywhere in China. It is an important speciese for aquaculture in China with an annual cultured production of about205,000tons and an annual cultured output value of near10billions RMB(?). Oriental river prawn farmed in China with the characteristics of large-scale, fast development and great potential. However, its economic characters seriously declined due to genetic retrogression in recent years. Because of the late beginning and very less work in genetic breeding, the relevant research cannot match the demand of production. As a result, it’s very important to strengthen the investigation of genetic background, establish the feasible techniques of genetic breeding and develop excellent varieties in Oriental river prawn. In this paper, three aspects of researches were carried out as follows:1. Isolation and characterization of polymorphic microsatellite markersMicrosatellite markers, as a highly polymorphic and codominant marker, were very popular in research of genetics and genetic breeding. However, reported microsatellite loci of oriental river prawn were limited in number. In this paper, a repeat-echriched genomic library ((GT) n (AG) n) was constructed by FIASCO (fast isolation by AFLP of sequences containing repeats) in oriental river prawn. One hundred and forty sequences were obtained, of which123contained microsatellite repeats (n>5), the positive ratio was87.8%; the microsatellite sequences could be categorized structurally as follows:perfect(66.7%), imperfect(20.3%), andcompound(13.0%).Senventy-five pairs of primers were designed successfully by using Primer Premier5.0, and54amplified specific products of the expected size. Finally28microsatellite loci were proved to be polymorphic.These28microsatellite sequences along with other12developed by our lab before have been deposited in GenBank (Accession no GU189600-GU189639).Microsatellite polymorphism of forty loci was evaluated using two populations from China with32samples each. One wild population was collected from Taihu Lake (THW) and the other cultured population was the3rd generation captive breeding progenies from Taihu Lake (TH3) from Yixing city in Jiangsu province. Thirty-one loci showed high polymorphism (PIC>0.5), and8had intermediate polymorphism (0.25<PIC<0.5),only one had low polymorphism (PIC<0.25). In wild population (THW), the mean observed and expected heterozygosities (Ho and HE) were0.536and0.650respectively, the average number of allele was5.5. Three loci (WXM08, WXM10and WXM19) displayed significant deviations from the expectations of Hardy-Weinberg equalifiation (P<0.05). In cultured population (TH3), the mean Ho and HE were0.417and0.571respectively, the average number of allele was4.8. Nine loci (WXM01, WXM08, WXM10, WXM16, WXM18, WXM19, WXM20, WXM37and WXM38) displayed significant deviations from the expectations of Hardy-Weinberg equalifiation (P<0.05).Cross-species amplification was performed using a closely related species, Macrobrachium hainanense, with ten individuals. Twenty-four out of40(60%) markers amplified specific products, of which19(47.5%) were polymorphic with the number of alleles ranging from two to seven.2. Studies on genetic diversity of wild populations of oriental river prawn from different segment of the Yangtze RiverThe Yangtze River is an excellent gene pool of oriental river prawn as well as an important parent pool for its farming. The investigation on the genetic resources of oriental river prawn in the Yangtze River is essential for its conservation and scientific utilization.Twenty polymorphic microsatellite markers were applied to investigate the genetic diversity of6oriental river prawn populations in the Yangtze River. The sampling sites included Chongqing, Wanzhou, Yichang, Wuhan, Jiujiang and Jiangyin. The average number of alleles (A) and effective numbers of alleles (Ne) in6populations were5.25and3.4622respectively. The mean PIC value of20microsatellite loci was0.5894. Expected heterozygosity (He) of6populations was as follows:Jiangyin (0.6308), Jiujiang (0.6096), Yichang (0.5945), Wuhan (0.5934), Wanzhou (0.5844) and Chongqing (0.5821), and their mean value were0.6296. The analysis of molecular variance (AMOVA) indicated that almost majority of the variance in the M. nipponense was within populations (93.08%), and6.92%was among populations. The Fst values between populations was0.0253-0.0838(P<0.05), Which showed the genetic divergence between populations in the Yangtze River was intermedieate but lower than that in the lakes. That was probably because running water promoted the exchanges between populations. Hardy-Weinberg equilibrium analysis indicated that deficiency of heterozygote existed in all6populations, which was probably because of rare allele deficiency or null alleles. The genetic distances among populations ranged from0.0620to0.1809. The UPGMA tree showed that:Jiangyin stock formed an independent clade and resident5populations formed another one in which Jiujiang and Wuhan populations clustered at first.3. Construction of genetic linkage map of oriental river prawn using microsatellite and SRAP markersGenetic linkage map is a very important data of genetic background with great value. However, oriental river prawn, as one of primary species for freshwater aquaculture in China, no work on genetic linkage map construction was reported so far. In this paper, a first genereation genetic linkage map of oriental river prawn was constructed using microsatellite and SRAP markers and pseudo-testcross mapping strategy. Of136microsatellite markers,52segregated in the parents.Thirty-eight out of52makers,11in the female and10in the male and17in both parents, segregated at1:1or1:1:1:1ratios. A hundred SRAP primer combinations produced2724bands with an average27.2per primer combination, including493polymorphic bands in the parent with an average4.9per primer combination. A total of409SRAP markders segregated according to the expected1:1Mendelian ratio, of which132segregated in the female parent and177segregated in the male parent.All these segregating makers were used to construct a genetic linkage map of oriental river prawn. A total of175makers including25microsatellite and150SRAP makers were mapped in53linkage groups with the number of markers per group ranged from2to8. Thirty-five groups included at least three markers.The average number of markers per group was3.3. The length of groups ranged from6.7cM to91.2cM (Kosambi). The maximum average distance of linkage group was30.4cM in LG15and the minimun was6.7cM in LG36. The framework map contained LG1to LG16with the length of997.2cM. The total length of the map was2270.5cM with an average distance of13.1cM. An average estimated genome size for oriental river prawn was4380.6cM. On the basis of the estimated genome lengths, genome coverage of map was51.83%.In this paper, microsatellite loci were isolated and characterizated and it provided new molecular tools for genetic research in oriental river prawn. Studies on genetic diversity of wild populations in the Yangtze River accumulated the data of genetic background of oriental river prawn and are helpful for the conservation and scientific utilization of wild resources. The first genetic linkage map of oriental river prawn was constructed and it should be useful for quantitative trait loci (QTL) mapping, molecular marker-assisted breeding (MAS) and construction of a higher density of genetic linkage map. |
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