【作者】 乔慧；

【导师】 傅洪拓；

【作者基本信息】 南京农业大学 ， 水产养殖， 2010， 博士

【摘要】 青虾,学名日本沼虾(Macrobrachium nipponense),隶属十足目(Decapoda),长臂虾科(Palaemonidae)、沼虾属(Macrobrachium),广泛分布于全国各地。青虾养殖年产量约20.5万吨,养殖年产值近100亿元,是我国重要的淡水养殖虾类。我国青虾养殖规模大、发展快、潜力大,但近年来普遍出现了品种退化现象,而相应的遗传育种研究工作起步很晚且相对落后,离产业发展需求还有较大距离。因此,加强青虾遗传背景研究,建立起适合于青虾的育种技术,培育青虾优良品种显得尤为重要。为此,本文拟开展如下三个方面的研究：1.青虾微卫星标记的开发与筛选微卫星标记由于其具有多态性好、共显性遗传等特点,在遗传学和遗传育种研究中备受青睐。已报道的青虾微卫星标记数量很少,不能满足青虾遗传学研究的需要。本文采用FIASCO (fast isolation by AFLP of sequences containing repeats)方法构建了青虾基因组富集文库((GT)n+(AG)n),测序140个克隆,含有重复次数5次以上的微卫星序列克隆123个,阳性率达到87.8%；其中,完美型微卫星序列共有82个,占66.7%；非完美型的25个,占20.3%；复合型的16个,占13.0%。采用Primer Premier5.0共设计出75对微卫星引物,其中共有54对引物能够扩增出目的产物,具有多态性的共有28对引物。将此28个微卫星序列连同本实验室之前开发的12个具有多态性微卫星序列整理编号,共40个位点已登录GenBank (注册号：GU189600-GU189639)。采用野生太湖青虾群体(THW)和野生太湖青虾在池塘养殖的第三代(TH3)进行多态性的比较分析,40个位点在两个青虾群体中均呈多态性,等位基因数量为2-10个,多态信息含量PIC值从0.186到0.900不等,其中高度多态位点31个(PIC>0.5),中度多态位点8个(0.25<PIC<0.5),低度多态位点1个(PIC<0.25)。THW群体的观测杂合度和期望杂合度值分别为0.536和0.650,平均等位基因数为5.5,其中3个位点(WXM08. WXM10和WXM19)显著偏离了Hardy-Weinberg平衡(P<0.05)；TH3群体的观测杂合度和期望杂合度值分别为0.417和0.571,平均等位基因数为4.8,其中9个位点(WXM01、WXM08、WXM10、WXM16WXM18、WXM19、WXM20、 WXM37和WXM38)显著偏离了Hardy-Weinberg平衡(P<0.05)。采用上述40个青虾微卫星标记对同属不同种的海南沼虾(Macrobrachium hainanense)基因组DNA进行通用性检测,有24对(60%)能在海南沼虾中稳定扩增,其中19对(47.5%)呈现多态性,每个位点的等位基因数量从2到7个不等,说明在海南沼虾中具有较高的通用性。2.长江不同江段青虾遗传多样性研究长江是青虾的优良种质资源库,也是我国青虾养殖的重要亲本库,长江野生青虾种质资源状况的研究对于青虾种质资源保护和科学利用均具有重要意义。本文采用20个微卫星分子标记对长江不同江段共6个青虾群体进行了遗传多样性分析,采样点包括重庆、万州、宜昌、武汉、九江和江阴。结果表明：6个青虾群体平均等位基因数(A)为5.25,平均有效等位基因数(Ne)为3.4622；20个位点平均多态信息含量PIC为0.5894；6个群体期望杂合度平均值为0.6296,表明长江青虾遗传多样性丰富；期望杂合度由高到低依次为：江阴群体(0.6308)、九江群体(0.6096)、宜昌群体(0.5945)、武汉群体(0.5934)、万州群体(0.5844)、重庆群体(0.5821).分子方差分析(AMOVA)结果表明长江青虾6群体间遗传变异6.92%来自群体间,93.08%来自群体内部,两两群体间Fst值在0.0253-0.0838之间,表明群体间已明显出现分化(P<0.05),但分化程度中等。Hardy-Weinberg平衡检验表明,6群体均出现杂合子缺失现象,可能是由于稀有等位基因缺失或无效等位基因造成；6群体间遗传距离在0.0620-0.1809之间,UPGMA聚类分析表明,江阴群体单独聚为一类,其余5个群体聚为另一类,其中九江和武汉群体最先聚到一起,其次万州、宜昌和重庆群体聚为一类。3.利用微卫星和SRAP标记构建青虾遗传连锁图谱遗传连锁图谱是具有重要价值的遗传背景资料,而青虾作为我国重要的淡水养殖品种,尚未见遗传连锁图谱构建方面的报道。本文采用微卫星和SRAP标记构建青虾的遗传连锁图谱。共采用青虾微卫星位点136个,其中40个来自本文第一章,其余均由Genbank下载青虾微卫星序列设计获得。经筛选,有52个位点在亲本中表现分离。共有38个位点在亲本和作图群体中呈现孟德尔分离,其中母本标记有11个,占分离标记总数的28.9%；父本标记有10个,占分离标记总数的26.3%；剩余44.7%的标记为双亲共有标记。100对SRAP引物组合共扩增出2724条片段,平均每个引物组合扩增出27.2个片段。其中有493个条带在父母本中呈现多态,平均每个引物组合产生4.9个多态标记。344个多态标记符合孟德尔分离,其中309个标记符合1：1分离,母本标记132个占多态标记总数的38.4%；父本标记177个,占多态标记总数的51.4%。将符合孟德尔分离的38个微卫星标记和符合1：1分离的309个SRAP标记进行连锁分析,构建青虾的遗传连锁图谱。共有175个标记(含25个微卫星、150个SRAP标记)分布在53个连锁群上。每个连锁群含2-8个标记,其中不少于3个标记的连锁群有35个,连锁对18个,平均每个连锁群的标记数为3.3个；连锁群长度在6.7-91.2cM之间,15号连锁群的平均间隔最大,为30.4cM,36号连锁群的平均间隔最小,仅为6.7cM。青虾框架图谱长度为997.2cM；青虾连锁图谱观察总长度为2270.5cM,平均间隔为13.1cM；根据估算,青虾遗传连锁图谱预期长度为4380.6cM,图谱的覆盖率51.83%。本文开发了青虾微卫星位点,为青虾遗传学研究提供了新的分子标记；对长江不同江段野生青虾种质状况和遗传多样性进行了研究,积累了青虾遗传背景资料,可为长江青虾资源的保护和科学利用提供指导；构建了第一张青虾遗传连锁图谱,可为青虾QTL定位、基因克隆、分子标记辅助育种等提供参考,并为进一步构建高密度的遗传连锁图谱奠定了基础。更多还原

【Abstract】 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.更多还原