Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H (O)) and expected (H (E)) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.

Microsatellites assessment of Chinese sturgeon (Acipenser sinensis Gray) genetic variability

Four microsatellites were used to examine the genetic variability of the spawning stocks of Chinese sturgeon, Acipenser sinensis, from the Yangtze River sampled over a 3-year period (1999-2001). Within 60 individuals, a total of 28 alleles were detected over four polymorphic microsatellite loci. The number of alleles per locus ranged from 4 to 15, with an average allele number of 7. The number of genotypes per locus ranged from 6 to 41. The genetic diversity of four microsatellite loci varied from 0.34 to 0.67, with an average value of 0.54. For the four microsatellite loci, the deviation from the Hardy-Weinberg equilibrium was mainly due to null alleles. The mean number of alleles per locus and the mean heterozygosity were lower than the average values known for anadromous fishes. Fish were clustered according to their microsatellite characteristics using an unsupervised 'Artificial Neural Networks' method entitled 'Self-organizing Map'. The results revealed no significant genetic differentiation considering genetic distance among samples collected during different years. Lack of heterogeneity among different annual groups of spawning stocks was explained by the complex age structure (from 8 to 27 years for males and 12 to 35 years for females) of Chinese sturgeon, leading to formulate an hypothesis about the maintenance of genetic diversity and stability in long-lived animals.

青藏高原东缘青杨（Populus cathayana Rehd.）遗传多样性研究

青杨（Populus cathayana Rehd.）是青杨派杨树的主要树种之一，为我国特有乡土树种，其主要分布区之一是我国的青藏高原，集中分布地带在甘肃省中部及青海省东部，四川省西北部岷江上游和松潘等地区。本研究以青藏高原东缘青杨天然分布区的6个群体143个个体为材料，用AFLP、SSR和叶绿体SSR分子标记分析青杨天然群体的遗传多样性，分析其遗传结构和分化，比较6个群体间遗传多样性的高低和群体间的遗传关系。旨在为青杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据。通过以上研究，得出如下主要研究结果： 1 AFLP分子标记研究结果 采用4对选择性引物对6个青杨天然群体143个个体进行分析，扩增谱带分析共检测到175个位点，其中173个位点表现为多态，多态位点百分率高达98.9%。从整体上表现出较高的遗传多样性，Nei’s基因多样度(h)水平为0.306。从青杨天然群体位点分布来看，有高达20%的位点（32位点）为群体所特有，仅有9.14%的位点（16位点）在所有群体中存在。群体间的遗传分化极大，所有遗传变异中，有48.9%的遗传变异存在于群体间。在个体群丛（Individuals cluster）和主坐标（PCO analysis）分析中，青杨各群体未呈现任何地理模式，Mantel检测也显示各群体间遗传距离与地理距离无明显相关。研究认为，由于地理和空间上大尺度的隔离和地形地貌复杂使得群体间无法进行基因交流，导致群体间遗传分化极大，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 2 SSR分子标记研究结果 在SSR分析中，7个位点在6个青杨天然群体143个个体中共检测到79个等位基因，每位点检测到的等位基因数在5-16之间，平均11.3个，总体上多态位点百分率达100%。平均观察杂合度和期望杂合度分别为0.792和0.802。Hardy-Weinberg平衡检验表明青杨大部分群体都处于非平衡状态，群体大部分位点都是偏离哈迪-温伯格平衡（76.3%），只有23.7%的测验满足哈迪-温伯格平衡。分析青杨天然群体内和群体间的遗传变异，基因分化系数（GST）为0.373，即有62.7%的遗传变异存在群体内，37.3%的遗传变异存在群体间。群体内的遗传变异高于群体间水平。根据各群体遗传距离UPGMA聚类分析，有来自相临分布区、近似气候类型的群体聚在一起的趋势，但Mantel检测反映遗传距离与地理距离间并无明显相关性。 3 cpSSR分子标记研究结果 分析来自青藏高原东缘6个青杨天然群体，所用cpSSR引物中有5对cpSSR引物（CCMP2、CCMP5、SCUO01、SCU03、SCU07）都表现较高的多态性，单个引物检测的片段数都在4以上。5对cpSSR引物共检测片段数26个，组成了12种叶绿体DNA单倍型。各群体的单倍型分布和频率有较大差异，群体单倍型多样性范围为0-0.4926，TS、JZ、PW和SHY群体单倍型多样性高于QHY和LED群体水平。本研究发现，分布在青藏高原东缘的青杨天然群体，群体间不存在共享的单倍型，各群体间存在极大的遗传分化（GST=0.9223）。从青藏高原东缘地区经历的地质历史事件来看，第四纪的冰期气候变迁可能是造成青杨现今遗传结构模式的主要因素之一。根据单倍型在各群体的分布情况，进行青杨群体聚类分析结果，各群体无明显的分组现象，青杨各群体也未呈现任何清晰地理模式。 由于不同分子标记在对群体遗传多样性检测能力与效率上存在差异，所以三种标记检测的青藏高原东缘青杨天然群体遗传多性水平也不尽一致，但在与用同种方法检测其它物种或同一物种不同种源群体比较，三种分子标记方法都揭示了青藏高原东缘青杨天然群体具有中等偏上的遗传多样性水平。结果分析表明，群体间遗传分化极大，这是由于青杨天然群体分布于青藏高原东缘，既有高原又有高山峡谷，由于地理和空间上大尺度的隔离和地形地貌复杂导致了基因流物理上的阻隔。三种分子标记研究结果经Mantel分析检测，遗传距离与地理距离之间都无明显相关性。较为一致的解释是，青杨分布区域地理和空间上大尺度的隔离和和地形地貌复杂导致群体之间不存在均匀扩散现象，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 The wide geographical and climatic distribution of P. cathayana Rehd. indicates that there is a large amount of genetic diversity available, which can be exploited for conservation, breeding programs and afforestation schemes. The results are as follows: 1 Research results of AFLP genetic diversity In present study, genetic diversity was evaluated in the natural populations of P. cathayana originating from southern and eastern edge of the Qinghai-Tibetan Plateau of China by means of AFLP markers. For four primer combinations, a total of 175 bands were obtained, of which 173 (98.9%) were polymorphic. Six natural populations of P. cathayana possessed different levels of genetic diversity, high level of genetic differentiation existed among populations (GST=0.489) of P. cathayana. Individuals cluster and PCO analysis based on Jaccard’s similarity coefficient also showed evident population genetic structure with high level population genetic differentiation. The long evolutionary process coupled with genetic drift within populations, rather than contemporary gene flow, are the major forces shaping genetic structure of P. cathayana populations. Moreover, there is no correspondence between geographical and genetic distances in the populations of P. cathayana, seldom gene exchange among populations and different selection pressures may be the causes. Our finding of different levels of genetic diversity within population and high level of genetic differentiation among populations provided promising condition for further breeding or conservation programs. 2 Research results of SSR genetic diversity In this study, the genetic diversity of P. cathayana was investigated using microsatellite markers. In a total of 150 individuals collected from six natural populations in the southeastern part of the Qinghai-Tibetan Plateau in China, a high level of microsatellite polymorphism was detected. At the seven investigated microsatellite loci, the number of alleles per locus ranged from 5 to 16, with a mean of 11.3, the observed heterozygosities across populations ranged from 0.408 to 0.986, with a mean of 0.792, and the expected heterozygosities across populations ranged from 0.511 to 0.891, with a mean of 0.802. The proportion of genetic differentiation among populations accounted for 37.3% of the whole genetic diversity. The presence of such a high level of genetic diversity could be attributed to the features of the species and the habitats where the sampled populations occur: The southeastern part of the Qinghai-Tibetan Plateau is regarded as the natural distribution and variation center of the genus Populus in China. Variation in environmental conditions and selection pressures in different populations, and topographic dispersal barriers could be factors associated with the high level of genetic differentiation found among populations. The populations possessed significant heterozygosity excesses, which may be due to extensive population mixing at the local scale. The cluster analysis showed that the populations are not strictly grouped according to their geographic distances but the habitat characteristics also influence the divergence pattern. In addition, we suggest that population SHY should be regarded as an ecologically divergent species of P. cathayana. 3 Research results of cpSSR genetic diversity Genetic diversity of six natural populations of P. cathayana originating from the southeastern part of the Qinghai-Tibetan Plateau in China was studied by use of cpSSR markers. Based on 5 pairs of polymorphic primers screened from 12 pairs of primers, twenty-six different length fragments and twelve different kinds of haplotypes were reduced in 143 samples. There were significant variant haplotypes among the populations．There were no shared haplotypes found among populations, analysis of molecular variance indicated that a high proportion of the total genetic variance was attributable to variations among populations (92.23%). The pattern of genetic structure which is associated with spatial separation, variation in environmental conditions and selection pressures in different populations, is also the result of geological historical factor. A molecular phylogenetic tree based on the 12 haplotypes showed that the populations are not strictly grouped according to their geographic distances.

西藏特有植物砂生槐天然居群遗传多样性研究

采用等位酶淀粉凝胶电泳技术对西藏雅鲁藏布江中游砂生槐 (Sophoramoorcroftiana) 10个天然居群的遗传多样性进行了研究。 13个酶系统 2 4个酶位点 ( 46个等位基因 )的检测结果表明 ,砂生槐具较低的遗传变异水平。居群水平上的遗传多样性指标分别为 :多态位点百分率Pp =2 5 .0 %～ 37.5 % ,等位基因平均数Ap=1.3～ 1.7,平均期望杂合度Hep=0 .112～ 0 .16 9;种水平上的遗传多样性 (Ps=37.5 % ,As=1.9,Hes=0 .171)低于长寿命木本被子植物的平均值 (Ps=5 9.5 % ,As=2 .10 ,Hes=0 .183)。居群遗传结构的分析显示 ,10个居群中随机交配的偏差为FIS=- 0 .0 0 71,表明砂生槐在居群水平上存在轻微的杂合子过量现象 ,偏离了Hardy Weinberg平衡 ;FST=0 .1748,表明砂生槐是居群间分化较大的一类多年生木本植物 ,主要原因是环境恶化和人类活动干扰 (过度砍伐、放牧等 )导致其生境片断化 ,从而影响了居群间基因交流而造成基因流水平较低 (Nm =1.180 2 )。砂生槐高海拔居群H2 (谢通门 )、H31(江当 1)、H32 (江当 2 )、H5 (朗塞岭 )包含着绝大部分等位基因 ,显示了相对较高的遗传多样性水平 ,应加以保护和管理 ,作为砂生槐种质资源就地保存的基地。

牙鲆群体遗传多样性及鲽形目鱼类分子系统学初步研究

本文以山东近海野生和养殖牙鲆Paralichthys olivaceus(T．& S.)为研究对象，采用同工酶电泳和随机扩增多态性DNA(RAPD)两种方法，进行了群体遗传学研究；另外，用PCR扩增了牙鲆、桂皮斑鲆Pseudorhombus cinnamomeus(T．& S.)、石鲽Kareius bicoloratus，Basilewsky和大菱鲆Psetta maxima 4种鲽形目鱼类mtDNA 16s rRNA基因区的部分片段，采用生物信息、学方法构建了鲽形目分子系统树。主要结果如下：1．首先建立了适于牙鲆同工酶分析的水平淀粉凝胶和垂直聚丙烯酰胺凝胶电泳系统；对获得的牙鲆15种同工酶基本酶谱进行了生化遗传分析，进而对自然和养殖群体的生化遗传结构进行了分析，共记录了29个基因座位，发现了9个多态座位。2．野生群体的生化遗传参数多态基因座位比例(31．O％)、等位基因平均数(1．38)和群体平均杂合度(0．0802)都明显高于养殖群体(24．1％，1．28，O．0788)；在野生群体中有9个多态基因座位，而养殖群体仅7个多态基因座位；其中，除了Cat和Idhp-1(仅养殖群体)(P < 0．05)有显著差异、Ldh-C(P < O．01)完全偏离Hardy-Weinberg定律外，其余多态座位基因频率均符合Hardy-Weinberg遗传平衡定律。野生和养殖群体的遗传相似性系数(I)为0．9877，它们的遗传距离(D)是0．0124；两群体间的遗传分化系数G_(st)为0．0681，D_m为0．01，表明总变异中的6．8％的遗传变异产生于群体间的基因差异。3．采用11个随机引物对20个野生个体和24个养殖个体进行了RAPD群体遗传多样性分析，分别扩增出88条和86条DNA带，片段大小在200-2500bp之间，平均每个引物扩增的带数是7．8-8．0。两个群体的多态座位比例分别是43．2％和34．9％，平均杂合度是0．2739和0．2255，而Shannon遗传多样性指数表明两群体的遗传变异中有88．12％的遗传变异来自种群内，只有11．88％的变异来自群体间。遗传分化指数G_(st)的结果也验证了Shannon遗传多样性指数的结果：总群体的遗传变异中约有12％是由两群体间的基因差异产生的。4．本文对牙鲆两个群体的同一批样品分别采用经典的同工酶方法和RAPD方法进行了较系统的比较分析。发现，RAPD所显示的多态性要比同工酶的高得多，因为大部分RAPD的变异是源于非编码区和重复DNA，可以遍布整个基因组，而同工酶仅是功能基因的产物，只表现编码区的变异。因此，自然选择在同工酶编码区的作用要多于RAPD标记。在遗传相似性系数(I)和遗传距离(D)上，RAPD的分析结果与同工酶的分析结果也是有差异的，用同工酶分析两个群体遗传距离只有0．0124，而用RAPD研究可达0．0508。遗传分化指数的差异也很大，同工酶为0．0681，RAPD为0．1237。5．RAPD和同工酶的分析结果是类似的，即自然群体的多态座位比例和平均杂合度要比养殖群体高，降低幅度在同工酶中界于1．7～22．3％之间，在RAPD中则界于15．9～19．2％之间。这充分证明了养殖群体的遗传多样性水平已有明显的丧失，值得我们注意。6．构建了鲽形目鱼类mtDNA 16S rRNA基因的分子系统树。通过分子克隆法将牙鲆、桂皮斑鲆、大菱鲆和石鲽mtDNA 16S rRNA目的基因片段连接到质粒载体上，经MegaBACE测序仪测序，分别获得了590、595、582和590bp序列，通过生物信息学方法对其进行了序列分析和核酸变异比较，结合NCBI上6种鲽形目鱼类的同源序列探讨了这4种鱼类在鲽形目中的遗传分化和分子系统进化，构建了系统树，其中，桂皮斑鲆的16S rRNA基因在系统树中的位置与物种形态资料的系统演化不相符，而其它三种很好地呈现了它们在鲽形目中的系统位置。同时，可以看出mtDNA 16S rRNA基因片段可以构建一个相对准确的树，特别是NJ树和ML树比较接近，更为客观一些。由比对序列获得的物种之间的遗传距离也基本可以反映种、属、科间的不同变异水平。

牙鲆和大黄鱼经济性状候选基因的DNA分子标记研究

本文利用不同的分子标记方法，分别对牙鲆及大黄鱼不同养殖群体的生长、抗病等经济性状的候选基因进行了序列多态性研究，检测到了几个SNP位点和微卫星的多态性位点，并分析了它们与经济性状之间的相关性；同时，利用微卫星的多态性位点对牙鲆2个养殖群体的遗传变异进行了分析，这些均为海水鱼类遗传育种及标记辅助选育工作提供了基础数据。 在牙鲆胶南养殖群体中，以100个个体为实验材料，根据其生长激素（GH）基因的6个外显子序列设计引物，通过SSCP分析技术显示该群体GH基因的第4外显子存在多态性，检测到2种基因型，AA型和AB型。DNA测序结果表明，AB型在第1763位发生碱基突变，c→t，与AA型同源性达到99%。连锁分析结果表明：这2种基因型的个体在体重和头长上表现出显著的差异，AB型个体的体重和头长都明显大于AA型个体(P<0.05)，由此推测等位基因B是一个对牙鲆体重和头长都有利的等位基因；这2种基因型个体之间在其体型性状上也存在显著差异（P<0.05）；同时，该多态位点的Hardy-Weinberg平衡性检验结果表明，该群体处于Hardy-Weinberg平衡状态。在牙鲆GH基因第1外显子区域还发现了一个微卫星位点，对该位点进行多态性分析，检测到5种基因型、3种等位基因，one-way ANOVA统计结果显示，基因型AC个体的体重、头长和体高明显大于其它基因型个体（P<0.05），C是一个对体重、头长和体高有利的等位基因。 对2个大黄鱼养殖群体的GH基因进行SSCP分析后发现，浙江群体大黄鱼GH基因在第196位存在1个SNP（g→a）位点，检测到2种基因型，AA和AB。t检验结果表明，AA型个体的体高比AB型个体的高(P≤0.05)，但AB型个体在体长/体高上占优势(P≤0.05)，提示该突变位点可以作为大黄鱼体型性状的候选标记。福建群体大黄鱼GH基因在第692位有1个SNP位点(t→c)，共检测到2种基因型，CC型和CD型，其中，CD基因型个体的体重和全长显著大于CC基因型个体(P≤0.01)，提示该位点可以作为大黄鱼体重和头长性状的候选标记。 在牙鲆胶南和日照2个养殖群体中，采用牙鲆GHR基因5’端Promoter区的一个微卫星标记，进行了群体遗传变异的研究，并探索了该基因多态性位点与牙鲆生长性状之间的相关性。结果表明，2个群体在该位点检测到的等位基因数为12和9个，有效等位基因数为6.26和5.04个。两个群体该位点的Hardy-Weinberg遗传偏离指数均为正值，并没有显示出杂合子缺失，但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析发现，在胶南群体中，IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的，但仅在体重上极其显著的大于全部其它基因型个体；在日照群体中，BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的；而CJ基因型对应的个体在体长和头长这两组数据中是最大的。由此认为，该位点IM基因型可以作为牙鲆体重性状的潜在标记。 在进行牙鲆抗病性状标记的筛选时，利用迟缓爱德华氏菌（Edwardsiella tarda）LSE40对牙鲆鱼进行攻毒感染实验，得到死亡群体和未死亡群体。选择Toll样受体基因中的TLR2、TLR3和TLR9基因作为候选基因，分别对这3个基因中的部分序列共设计7对引物进行扩增，同时对扩增产物进行RFLP多态性分析，目前只在TLR3基因内检测到一个EcoRI的酶切多态性位点，测序后发现，这是由于在TLR3基因第3806位的EcoRI酶切位点在某些个体中缺失所致。酶切产物共呈现出3种基因型，分别定义为AA，AB和BB。χ2检验证明该多态性位点与牙鲆抗迟缓爱德华氏菌LSE40的能力有一定关系。利用多因素非条件Logistic回归分析对死亡组和存活组牙鲆的各种形态学数据以及不同基因型之间进行了分析，发现体长、头长和体高均具有显著的相关性（P<0.05），而这几个因素与体重的相关性不显著（P>0.05）。多因素非条件Logistic分析后发现：AA基因型对死亡率具有显著的影响（P<0.05），是主要的危险因素，而AB基因型的作用不显著（P>0.05）；头长是主要的保护因素（P<0.05），体重对死亡率的影响很小。χ2检验证明，等位基因A是对死亡的主要危险等位基因，B是对存活有利的主要等位基因。推测该位点可以作为牙鲆抗迟缓爱德华氏菌的潜在标记。

栉孔扇贝抗鳗弧菌感染性状候选基因的多态性研究

栉孔扇贝（Chlamys farreri）是我国北方地区主要的养殖贝类之一，曾为沿海各省带来巨大的经济效益。但自1997年以来，陆续爆发的病害问题给扇贝养殖业造成了巨大的经济损失，严重影响了该产业的健康发展。目前认为培育抗病性强的扇贝优良品种是解决病害问题的根本途径。由于传统的育种方法费时费力，无法满足对良种的迫切需求，因此有必要通过分子手段加快抗病品种的培育步伐。标记辅助育种（marker assisted selection，MAS）是成功应用于动物育种中的分子手段之一，但由于缺乏与抗病性状相关的标记，MAS目前还无法在软体动物中得到应用。因此，寻找与抗病性状相关的分子标记是在软体动物中发展MAS的关键。 本研究利用鳗弧菌（Listonella anguillarum）对栉孔扇贝进行攻毒感染实验，初步得到敏感群体和抗病群体后采用PCR、PCR-RFLP、Bi-PASA PCR等方法研究了CfLysG、CfC1qDC和CfLITAF基因多态性及其与栉孔扇贝对鳗弧菌抗性的关系。 研究发现，栉孔扇贝CfLysG的基因序列中共有104个单核苷酸多态性（SNP）位点和29个插入/缺失（I/D）多态性位点。有17个多态性位点位于启动子区域，选择其中的-753 I/D、-391A/G和-284I/D多态性进行检测，发现这三个位点的基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。其中-753 ID基因型和-284 ID基因在抗病群体中的频率高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。-391 AG基因型在抗病群体中的频率显著高于敏感群体（P=0.007），表明-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。为验证这一相关性，对-391位点不同基因型的扇贝进行攻毒感染实验。统计发现，具有-391 AA基因型的扇贝累计死亡率显著高于具有-391 AG基因型的扇贝（P=0.001），进一步证实了CfLysG基因-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因的外显子共有3处SNP，其中仅第三外显子上的+3473 A/C为非同义突变。统计分析表明，+3473位点不同基因型在敏感群体中的分布频率符合Hardy-Weinberg平衡（P>0.05），而在抗病群体中则偏离Hardy-Weinberg平衡（P<0.01）。+3473 AA基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.022），表明+3473 AA基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因第1内含子存在+96 I/D和+487 I/D两处大片段的I/D多态性。统计发现，这两个位点的基因型在敏感群体和抗病群体中的分布频率均符合Hardy-Weinberg 平衡（P>0.05）。其中+96 DD基因型和+487 ID基因型在抗病群体中的频率均略高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。表明这两个位点的多态性与栉孔扇贝对鳗弧菌的抗性无显著相关性。对CfLysG基因各多态性位点的统计分析表明，各位点之间存在不同程度的连锁不平衡，提示有单体型的存在。对19种频率>1%的单体型在敏感群体及抗病群体中的频率进行分析，发现-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型在抗病群体中的频率显著高于敏感群体（P=0.044），表明该单体型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfC1qDC基因cDNA序列上共发现14处SNP。对+423 T/C多态性与栉孔扇贝对鳗弧菌抗性的关系进行了分析。统计发现，+423位点各基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。+423 TT基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.005），表明+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfLITAF基因cDNA序列中共发现3处SNP及1处I/D多态性。对+145 I/D多态性进行研究，发现所有敏感个体及抗病个体中均同时存在+145 位点所有等位基因，表明+145位点多态性与栉孔扇贝对鳗弧菌的抗性不相关。 以上研究表明，栉孔扇贝CfLysG基因-391 AG基因型、+3473 AA基因型、-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型以及CfC1qDC基因+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关，提示它们可作为与栉孔扇贝抗病相关的候选分子标记应用于贝类抗病育种中，为贝类的标记辅助育种提供参考。此外，抗病相关分子标记的发现还有利于加深对扇贝发病机理的理解，并有助于发掘预防及治疗贝类疾病的新方法。

中国近海栉孔扇贝群体遗传结构及遗传变异与生长的关系

本文以凝胶电泳方法，以等位基因酶和可溶性蛋白为探针研究了中国近海栉孔扇贝(Chlamys farreri)5个不同自然分布群体的遗传结构及遗传变异与生长的关系。在所研究的23个位点中，只有Mdh-1在5个群体中为纯合状态，其余位点都不同程度地处于多态或杂合态。大多数位点的等位基因及基因型分布频率都处在不平衡状态。对各位点的D值检验表明，大多数位点都杂合子缺失。杂合子过剩或处于Hardy-Weinberg平衡的位点为19.64-22.32%。杂合度以海洋岛和长岛群体最高，都是0.304，荣成和大连群体次之，分别为0.290和0.287，青岛群体为0.219。位点有效等位基因数及密码子差数在群体间的变化与杂合度基本一致。用这三个参数构建的遗传变异综合评价指数在这5个群体从北至南依次为0.726，0.542，0.658，0.547和0.133。多态位点比例在P ≤ 0.99水平下，海洋、大连、长岛及荣成4个群体都在0.800以上，仅青岛群体为0.565。在Est7个位点从高纬度向低纬度在等位基因数目上存在梯度递减渐变性。在某些等位基因的分布频率或从高纬度或从低纬度向相对方向递变。除了单向渐变外，也存在双向渐变现象。栉孔扇贝这5个群体在遗传相似性方面基本上是群体间的差异（I > 0.900）。相比之下，大连、长岛两群体间的相似性最大。由于辽东沿岸流和黄海冷水团的作用，海洋岛群体与邻近群体的基因交流较差，使之在遗传上相对较为独立。从遗传变异不平、杂合子缺失特征及等位基因的渐变性也证明，该群体已具相当程度的分化，这主要是由于环境隔离所造成的“孤岛效应”的结果。通过对5个群体形态数量性状生与群体遗传变异的比较表明，遗传变异不平较高的群体，其生长速度也较快，二者基本呈平行关系，唯海洋岛与长岛的结果相悖，其原因可能与两海区间初级生产力水平有关。从个体杂合度与第一年生长壳高关系的研究可以发现，海洋、大连和青岛群体在杂合度与生长间基本呈正相关关系，而在长岛与荣成群体内存在杂合度与生长具不同相关性的子群体，并证明个体杂合度超过0.300以上，其与生长的关系基本上处于不定向状态。在环境和遗传双重压力下，青岛群体的变异水平较低。

Yellow perch strain evaluation I: Genetic variance of six broodstock populations

As a prelude to strain selection for domestication and future marker assisted selection, genetic variation revealed by microsatellite DNA was evaluated in yellow perch, Perca flavescens, from four wild North American populations collected in 2003-2004 (Maine, New York, North Carolina, and Pennsylvania,), and two captive populations (Michigan and Ohio). For the loci examined, levels of heterozygosity ranged from H-e=0.04 to 0.88, genetic differentiation was highly significant among all population pairs, and effective migration ranged from low (N(e)m=0.3) to high (N(e)m=4.5). Deviation from Hardy-Weinberg equilibrium was regularly observed indicating significant departures from random mating. Instantaneous measures of inbreeding within these populations ranged from near zero to moderate (median F=0.16) and overall inbreeding levels averaged F-IS=0.18. Estimates of genetic diversity, Phi(ST), and genetic distance were highest between Michigan and all other broodstock groups and lowest between New York and Ohio. Genetic differentiation among groups did not correlate with geographic distance. Overall, the patterns of variation exhibited by the captive (Michigan and Ohio) populations were similar to patterns exhibited by the other wild populations, indicating that spawning and management practices to date have not significantly reduced levels of genetic variation. (C) 2007 Elsevier B.V. All rights reserved.

Quantitative Analysis of Single Nucleotide Polymorphisms within Copy Number Variation

Background Single nucleotide polymorphisms (SNPs) have been used extensively in genetics and epidemiology studies. Traditionally, SNPs that did not pass the Hardy-Weinberg equilibrium (HWE) test were excluded from these analyses. Many investigators have addressed possible causes for departure from HWE, including genotyping errors, population admixture and segmental duplication. Recent large-scale surveys have revealed abundant structural variations in the human genome, including copy number variations (CNVs). This suggests that a significant number of SNPs must be within these regions, which may cause deviation from HWE. Results We performed a Bayesian analysis on the potential effect of copy number variation, segmental duplication and genotyping errors on the behavior of SNPs. Our results suggest that copy number variation is a major factor of HWE violation for SNPs with a small minor allele frequency, when the sample size is large and the genotyping error rate is 0~1%. Conclusions Our study provides the posterior probability that a SNP falls in a CNV or a segmental duplication, given the observed allele frequency of the SNP, sample size and the significance level of HWE testing.

Framingham Heart Study 100K project: genome-wide associations for cardiovascular disease outcomes

BACKGROUND:Cardiovascular disease (CVD) and its most common manifestations - including coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF) - are major causes of morbidity and mortality. In many industrialized countries, cardiovascular disease (CVD) claims more lives each year than any other disease. Heart disease and stroke are the first and third leading causes of death in the United States. Prior investigations have reported several single gene variants associated with CHD, stroke, HF, and AF. We report a community-based genome-wide association study of major CVD outcomes.METHODS:In 1345 Framingham Heart Study participants from the largest 310 pedigrees (54% women, mean age 33 years at entry), we analyzed associations of 70,987 qualifying SNPs (Affymetrix 100K GeneChip) to four major CVD outcomes: major atherosclerotic CVD (n = 142; myocardial infarction, stroke, CHD death), major CHD (n = 118; myocardial infarction, CHD death), AF (n = 151), and HF (n = 73). Participants free of the condition at entry were included in proportional hazards models. We analyzed model-based deviance residuals using generalized estimating equations to test associations between SNP genotypes and traits in additive genetic models restricted to autosomal SNPs with minor allele frequency [greater than or equal to]0.10, genotype call rate [greater than or equal to]0.80, and Hardy-Weinberg equilibrium p-value [greater than or equal to] 0.001.RESULTS:Six associations yielded p <10-5. The lowest p-values for each CVD trait were as follows: major CVD, rs499818, p = 6.6 x 10-6; major CHD, rs2549513, p = 9.7 x 10-6; AF, rs958546, p = 4.8 x 10-6; HF: rs740363, p = 8.8 x 10-6. Of note, we found associations of a 13 Kb region on chromosome 9p21 with major CVD (p 1.7 - 1.9 x 10-5) and major CHD (p 2.5 - 3.5 x 10-4) that confirm associations with CHD in two recently reported genome-wide association studies. Also, rs10501920 in CNTN5 was associated with AF (p = 9.4 x 10-6) and HF (p = 1.2 x 10-4). Complete results for these phenotypes can be found at the dbgap website http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:No association attained genome-wide significance, but several intriguing findings emerged. Notably, we replicated associations of chromosome 9p21 with major CVD. Additional studies are needed to validate these results. Finding genetic variants associated with CVD may point to novel disease pathways and identify potential targeted preventive therapies.

Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study

INTRODUCTION:Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study.METHODS:Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency [greater than or equal to] 0.10, call rate [greater than or equal to] 80%, and Hardy-Weinberg p-value [greater than or equal to] 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated.RESULTS:There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10-5 by GEE and five SNPs with p < 10-5 by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

The Framingham Heart Study 100K SNP genome-wide association study resource: overview of 17 phenotype working group reports

BACKGROUND:The Framingham Heart Study (FHS), founded in 1948 to examine the epidemiology of cardiovascular disease, is among the most comprehensively characterized multi-generational studies in the world. Many collected phenotypes have substantial genetic contributors; yet most genetic determinants remain to be identified. Using single nucleotide polymorphisms (SNPs) from a 100K genome-wide scan, we examine the associations of common polymorphisms with phenotypic variation in this community-based cohort and provide a full-disclosure, web-based resource of results for future replication studies.METHODS:Adult participants (n = 1345) of the largest 310 pedigrees in the FHS, many biologically related, were genotyped with the 100K Affymetrix GeneChip. These genotypes were used to assess their contribution to 987 phenotypes collected in FHS over 56 years of follow up, including: cardiovascular risk factors and biomarkers; subclinical and clinical cardiovascular disease; cancer and longevity traits; and traits in pulmonary, sleep, neurology, renal, and bone domains. We conducted genome-wide variance components linkage and population-based and family-based association tests.RESULTS:The participants were white of European descent and from the FHS Original and Offspring Cohorts (examination 1 Offspring mean age 32 +/- 9 years, 54% women). This overview summarizes the methods, selected findings and limitations of the results presented in the accompanying series of 17 manuscripts. The presented association results are based on 70,897 autosomal SNPs meeting the following criteria: minor allele frequency [greater than or equal to] 10%, genotype call rate [greater than or equal to] 80%, Hardy-Weinberg equilibrium p-value [greater than or equal to] 0.001, and satisfying Mendelian consistency. Linkage analyses are based on 11,200 SNPs and short-tandem repeats. Results of phenotype-genotype linkages and associations for all autosomal SNPs are posted on the NCBI dbGaP website at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:We have created a full-disclosure resource of results, posted on the dbGaP website, from a genome-wide association study in the FHS. Because we used three analytical approaches to examine the association and linkage of 987 phenotypes with thousands of SNPs, our results must be considered hypothesis-generating and need to be replicated. Results from the FHS 100K project with NCBI web posting provides a resource for investigators to identify high priority findings for replication.

A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study

BACKGROUND:Blood lipid levels including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are highly heritable. Genome-wide association is a promising approach to map genetic loci related to these heritable phenotypes.METHODS:In 1087 Framingham Heart Study Offspring cohort participants (mean age 47 years, 52% women), we conducted genome-wide analyses (Affymetrix 100K GeneChip) for fasting blood lipid traits. Total cholesterol, HDL-C, and TG were measured by standard enzymatic methods and LDL-C was calculated using the Friedewald formula. The long-term averages of up to seven measurements of LDL-C, HDL-C, and TG over a ~30 year span were the primary phenotypes. We used generalized estimating equations (GEE), family-based association tests (FBAT) and variance components linkage to investigate the relationships between SNPs (on autosomes, with minor allele frequency [greater than or equal to]10%, genotypic call rate [greater than or equal to]80%, and Hardy-Weinberg equilibrium p [greater than or equal to] 0.001) and multivariable-adjusted residuals. We pursued a three-stage replication strategy of the GEE association results with 287 SNPs (P < 0.001 in Stage I) tested in Stage II (n ~1450 individuals) and 40 SNPs (P < 0.001 in joint analysis of Stages I and II) tested in Stage III (n~6650 individuals).RESULTS:Long-term averages of LDL-C, HDL-C, and TG were highly heritable (h2 = 0.66, 0.69, 0.58, respectively; each P < 0.0001). Of 70,987 tests for each of the phenotypes, two SNPs had p < 10-5 in GEE results for LDL-C, four for HDL-C, and one for TG. For each multivariable-adjusted phenotype, the number of SNPs with association p < 10-4 ranged from 13 to 18 and with p < 10-3, from 94 to 149. Some results confirmed previously reported associations with candidate genes including variation in the lipoprotein lipase gene (LPL) and HDL-C and TG (rs7007797; P = 0.0005 for HDL-C and 0.002 for TG). The full set of GEE, FBAT and linkage results are posted at the database of Genotype and Phenotype (dbGaP). After three stages of replication, there was no convincing statistical evidence for association (i.e., combined P < 10-5 across all three stages) between any of the tested SNPs and lipid phenotypes.CONCLUSION:Using a 100K genome-wide scan, we have generated a set of putative associations for common sequence variants and lipid phenotypes. Validation of selected hypotheses in additional samples did not identify any new loci underlying variability in blood lipids. Lack of replication may be due to inadequate statistical power to detect modest quantitative trait locus effects (i.e., < 1% of trait variance explained) or reduced genomic coverage of the 100K array. GWAS in FHS using a denser genome-wide genotyping platform and a better-powered replication strategy may identify novel loci underlying blood lipids.

Genetic correlates of longevity and selected age-related phenotypes: a genome-wide association study in the Framingham Study

BACKGROUND: Family studies and heritability estimates provide evidence for a genetic contribution to variation in the human life span. METHODS:We conducted a genome wide association study (Affymetrix 100K SNP GeneChip) for longevity-related traits in a community-based sample. We report on 5 longevity and aging traits in up to 1345 Framingham Study participants from 330 families. Multivariable-adjusted residuals were computed using appropriate models (Cox proportional hazards, logistic, or linear regression) and the residuals from these models were used to test for association with qualifying SNPs (70, 987 autosomal SNPs with genotypic call rate [greater than or equal to]80%, minor allele frequency [greater than or equal to]10%, Hardy-Weinberg test p [greater than or equal to] 0.001).RESULTS:In family-based association test (FBAT) models, 8 SNPs in two regions approximately 500 kb apart on chromosome 1 (physical positions 73,091,610 and 73, 527,652) were associated with age at death (p-value < 10-5). The two sets of SNPs were in high linkage disequilibrium (minimum r2 = 0.58). The top 30 SNPs for generalized estimating equation (GEE) tests of association with age at death included rs10507486 (p = 0.0001) and rs4943794 (p = 0.0002), SNPs intronic to FOXO1A, a gene implicated in lifespan extension in animal models. FBAT models identified 7 SNPs and GEE models identified 9 SNPs associated with both age at death and morbidity-free survival at age 65 including rs2374983 near PON1. In the analysis of selected candidate genes, SNP associations (FBAT or GEE p-value < 0.01) were identified for age at death in or near the following genes: FOXO1A, GAPDH, KL, LEPR, PON1, PSEN1, SOD2, and WRN. Top ranked SNP associations in the GEE model for age at natural menopause included rs6910534 (p = 0.00003) near FOXO3a and rs3751591 (p = 0.00006) in CYP19A1. Results of all longevity phenotype-genotype associations for all autosomal SNPs are web posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. CONCLUSION: Longevity and aging traits are associated with SNPs on the Affymetrix 100K GeneChip. None of the associations achieved genome-wide significance. These data generate hypotheses and serve as a resource for replication as more genes and biologic pathways are proposed as contributing to longevity and healthy aging.