海湾扇贝微卫星标记的开发及遗传连锁图谱的构建

海湾扇贝（Argopecten irradians）不连续分布于美国大西洋沿岸和墨西哥湾沿岸，自1982年以来北部亚种（A. i. irradians）和南部亚种（A. i. concentricus）被先后引进到中国，由于其生长速度快、繁殖周期短和适宜温度范围广的特点，迅速成为我国海水养殖的重要品种。近年来飞速发展的分子标记技术为优良品种的选育注入了新的活力，相对于传统的表型选择来说，标记辅助选择不易受环境的影响，尤其是对于低遗传力性状和后期表达的性状，能增强选择效率，提高选择的准确度，缩短育种周期。本文通过构建海湾扇贝微卫星富集文库获得大量的微卫星DNA序列，筛选多态的微卫星标记构建了海湾扇贝的遗传连锁图谱，并应用复合区间作图法对生长相关性状进行了QTL定位。 本研究利用富集文库-菌落原位杂交法筛选海湾扇贝微卫星DNA，吸附(AC)15和(AG)15探针的尼龙膜捕捉并富集含有微卫星序列的片段，菌落原位杂交结果显示阳性克隆率达到40%，测序比对后获得521个独立的阳性克隆，其中微卫星506个，小卫星15个。微卫星中，完美型248个，占49.0%，非完美型216个，占42.7%；复合型42个，占8.3%；AG/TC重复占大多数（356个，70.4%），AC/TG重复有150个（29.6%）。设计合成了382对引物，利用38个海湾扇贝个体对其中15个微卫星位点进行了遗传多样性评价，不同位点扩增得到的等位基因数从3到7个不等，期望杂合度和观测杂合度的范围分别为0.198~0.813和0.083~0.833，实验结果表明富集文库-菌落原位杂交法适合大规模筛选微卫星标记。 利用8个微卫星标记对海湾扇贝1个野生种群和3个养殖群体的遗传多样性与分化进行了比较和分析。8个位点共扩增得到35个等位基因，平均每个位点4.38个等位基因，平均有效等位基因数为2.30，平均观测杂合度和期望杂合度分别为0.41和0.46。相比于野生群体（美国），养殖群体（北卡罗来那、浙江和胶南）的等位基因数和杂合度都有所降低，在封闭环境下养殖19代的浙江群体等位基因数丢失最严重，共有9个等位基因丢失（25.7%）。经过多代人工养殖后，海湾扇贝养殖与野生群体之间和养殖群体之间出现了明显的遗传分化，胶南群体与野生群体的遗传距离最大，而胶南群体与浙江群体的遗传距离已经超过了胶南群体（北部亚种）和北卡罗来那（南部亚种）群体的遗传距离，这种分化将有利于海湾扇贝的杂交选育。 利用167个微卫星标记和1个壳色标记，以海湾扇贝2个全同胞F1代为作图群体，构建了海湾扇贝的性别遗传连锁图谱。整合的雌性连锁图谱含有118个标记，覆盖了16个连锁群，每个连锁群含有的标记数目从4到16个不等，平均每个连锁群上有7.4个标记，图谱总长度为761.0 cM，标记间的平均间隔为8.55 cM，图谱的覆盖率为73.5%；整合雄性连锁图谱含有126个标记，覆盖了17个连锁群，每个连锁群含有的标记数目从2到11个不等，平均每个连锁群上有7.4个标记，图谱总长度为729.1 cM，标记间的平均间隔为6.75 cM，图谱的覆盖率为74.7%。雌性亲本的重组率高于雄性，雌雄亲本共享标记间的重组率比值为1.18：1。偏分离标记在性别间呈现不对称分布，雄性亲本的偏分离高于雌性亲本，可能与雄性亲本来源于亚种间杂交的遗传背景相关。 利用海湾扇贝微卫星遗传连锁图谱在两个作图家系中对5个生长性状的QTL进行了定位，5个生长性状的表型相关均达到极显著水平（P < 0.01），Pearson相关系数均超过0.781，总重、壳长、壳宽、壳高和壳重的QTL（LOD > 2.0）的数目分别为8、6、6、7和6个。这些QTL成簇分布于CC5家系的LG1、LG3、LG4、LG8和CC10家系的LG1、LG3、LG6、LG8、LG9连锁群，单个QTL可解释的表型方差为5.5%到29.2%，QTL成簇分布现象说明这些生长相关的性状可能具有共同的遗传基础，家系特异性QTL暗示在不同的遗传背景和环境下存在不同的主效QTL。本研究定位的QTL，尤其在两个家系中共享的QTL为下一步分子标记辅助选择提供了参考区间。

牙鲆Paralichthys olivaceus（♀）×夏鲆Paralichthys dentaus（♂）分子遗传学及数量遗传学研究

本论文采用扩增片段长度多态性（AFLP）技术对牙鲆Paralichthys olivaceus群体和夏鲆Paralichthys dentaus 群体进行了种群鉴定和遗传结构分析。共用8 对引物对两个群体（每个群体30 个个体）进行了分析。总共产生了379 个条带，条带大小在60－1000bp。牙鲆与夏鲆的遗传多态性分别为53.83%和22.22%。牙鲆群体的多态位点数显著低于夏鲆群体。牙鲆群体和夏鲆群体种的特异条带分别为27.3% 和 29.61%。两群体平均杂和度分别为0.0701 和 0.1556，香农氏多态指数分别为0.1044 和0.2387。群体内平均遗传距离分别为0.0705 （0.0214 到0.1377）和0.1656 （0.0629 到 0.2338），两群体间的平均遗传距离为0.6328。 AFLP 技术是进行种间群体结构分析的一个很好的分子标记方法，对牙鲆和夏鲆亲本群体进行遗传背景析为今后的杂交育种阐明种间杂交的遗传机理奠定基础。 在本论文中我们采用5×8 因子交配设计建立了牙鲆P. olivaceus（♀）× 夏鲆P. dentaus（♂）杂交家系，对生长相关性状的遗传力进行分析。所有家系都混合养殖在同一养殖池中，40 日龄，在养殖池中随机采集600 个个体，测量体长，体宽，体重等数量性状；然后提取杂交子代DNA 进行家系的鉴定。首先从牙鲆的微卫星中筛选了在牙鲆、夏鲆以及杂交子代扩增多态性较好的10 对微卫星引物（Po1, Po13, Po20, Po35, Po42, Po48, Po56, Po58, Po91, Poli 23TUF）。我们采用了三对引物对600 个个体进行了家系鉴定，共有400 多个体鉴定出自己的亲本，成功率达到80％以上。基于以上结果我们认为微卫星标记可以做为一个有效的标记来代替现实的物理标记，并且可以在子代的早期进行亲本的鉴定。 在养殖过程中，我们对自交和杂交家系生长状况进行了跟踪分析，在181刘清华 牙鲆（♀）×夏鲆（♂）分子遗传学及数量遗传学研究 博士学位论文II日龄之前杂交鲆体长和体重均为未表现出杂种优势，杂种优势率值始终为负值，但是绝对值在逐渐减小。从196 日龄之后杂交鲆杂种优势开始表现出来，并且在196 日龄之后杂种优势率显著的增加。256 日龄体长杂种优势率为14.29%，体重杂种优势率为59.78%, 271 日龄体长杂种优势率达到27.36%，体重杂种优势率为 102.32%。 本研究对杂交鲆在40 日龄的体长，体高、尾柄长、尾柄高，以及全长和体重的遗传力进行了估计，5 个性状的体长半同胞遗传力h2 S 为0.00146－0.719，5个性状的全同胞遗传力h2D 为0.00121－0.632，5 个性状的半同胞和全同胞的平均遗传力h2SD 为0.001335－0.6755。其中全长的遗传力最大。实验结果说明对早期幼鱼进行体长、体重等性状实施选育策略可能会显著影响后期杂交鲆的生长。

海带、裙带菜和羊栖菜的遗传分析

利用AFLP标记对海带在太平洋西北沿岸的6个主要栽培品系和6个野生地理隔离种群进行遗传多样性和亲缘关系分析。通过10对选择性引物总共检测到547个位点。在品系和种群水平，多态性位点比例（P），基因多样性（H）和香农指数（I）在大连野生种群中最高（P: 59.05%; H: 0.2057; I: 0.3062），而在连江栽培品系中最低（P: 9.87%; H: 0.0331; I: 0.0497）。在物种水平（即对所有品系和种群来说），P, H 和I 的值分别是85.01%，0.1948和0.3096。以个体间的相似系数（Dice）和种群间的遗传距离为基础，用非加权类平均法（UPGMA）分别构建个体和种群间的系统树图。AMOVA分析显示，大部分的遗传变异（60.21%）存在于品系和种群间，少部分（39.79%）存在于品系或种群内。遗传分化系数GST的值是0.6226，与FST的值（0.6021）非常接近，基因流（Nm）的值是0.1515，这三个值表明种群（品系）间存在高度的遗传分化。Mantel检验发现6个野生种群的遗传距离或遗传分化与地理距离呈正相关性（相关系数分别是r=0.8870, P=0.007 和 r=0.7962, P=0.011），符合“距离隔离（isolation by distance, IBD）”模型。总体来说，种群（品系）内的遗传多样性处于低到中等水平（大连种群除外），而它们之间的遗传分化程度非常高。 用AFLP和微卫星标记对裙带菜配子体克隆单一交配组合的孢子体后代的遗传一致性进行分析。在这项研究中，建立了2个配子体克隆单一交配系（M1和M2），2个自交系（S1和S2）并采集1个野生种群（W）。11组AFLP引物总共检测到318个位点。M1, M2, S1, S2 和W的多态性位点比例分别是4.7%，0.3%，17.9%，16.4%和36.5%。M1和M2个体间的遗传相似度（95.6-100%）要高于S1和S2（87.7-98.4%）以及W（81.5-92.1%）。在微卫星分析中，用了6个位点。M1在其中的5个位点基因型一致，而在Up-AC-2B2位点显示出不同的基因型。M2在所有6个微卫星位点的基因型都一致。而S1, S2和W都在2个以上的位点检测到不同的基因型。总之，AFLP与微卫星的分析结果一致，即配子体克隆单一交配组合的孢子体后代呈现高度遗传相似性，但也存在细微的差异。 对中国羊栖菜主产区浙江省洞头县的12个羊栖菜养殖品系的重要形态特征进行了比较研究，并利用AFLP技术对一个典型养殖种群的遗传多样性进行了分析。结果显示，这12个品系在全长、全湿质量、侧枝长、侧枝湿质量、侧枝密度等方面存在显著或极显著差异（P<0.05或P<0.01）。8组AFLP引物在这个典型养殖种群中扩增出198个片段，其中多态性片段为166个，多态性位点比例为83.8%。根据个体间的遗传距离，以UPGMA法构建了个体间的系统树图，27个羊栖菜个体聚为一枝，作为对照的铜藻为另一枝。本研究从形态和DNA分子水平说明了浙江洞头羊栖菜养殖种群具有高度遗传多样性。

中国对虾微卫星标记的筛选

该文运用PCR法在中国明对虾（Fenneropenaeuschinensis）的小片段基因组文库中快速筛选含有微卫星序列的重组阳性克隆.利用生物信息学的方法,使用Repeatmasker软件对中国对虾ESTs数据库中10446条EST序列进行了微卫星序列的筛选.在发现的微卫星序列中选取了19条微卫星序列设计了引物,在16对能够扩增出产物的引物中,经过PCR扩增、聚丙烯酰胺凝胶电泳和硝酸银染色,最终共筛选出有多态性PCR产物的微卫星引物11对,其中适合进行微卫星分析的有8对.在中国对虾20个左右样本的8个微卫星位点上,等位基因的数目从5到15不等,等位基因的大小分布在165bp到305bp之间,基本符合引物设计时的理论产物长度.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.

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.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

A preliminary genetic linkage map of the pacific abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F, family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (alpha = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.

Stress corrosion cracking of AISI 321 stainless steel in acidic chloride solution

The stress corrosion cracking (SCC) of LambdaISI 321 stainless steel in acidic chloride solution was studied by slow strain rate (SSR) technique and fracture mechanics method. The fractured surface was characterized by cleavage fracture. In order to clarify the SCC mechanism, the effects of inhibitor KI on SCC behaviour were also included in this paper. A study showed that the inhibition effects of KI on SCC were mainly attributed to the anodic reaction of the corrosion process. The results of strain distribution in front of the crack tip of the fatigue pre-cracked plate specimens in air, in the blank solution (acidic chloride solution without inhibitor KI) and in the solution added with KI measured by speckle interferometry (SPI) support the unified mechanism of SCC and corrosion fatigue cracking (CFC).

小麦抗条锈病分子标记辅助育种研究进展

小麦条锈病是小麦生产的主要病害之一。在中国，尤其是西北麦区，小麦条锈病是小麦生产面临的最严重问题。筛选和培育抗锈基因是防治小麦条锈病最为经济、安全、有效的方法。综述了分子标记辅助育种技术在小麦抗条锈病育种上的研究概况和发展运用。

波动方程偏移速度分析

On the subject of oil and gas exploration, migration is an efficacious technique for imagining structures underground. Wave-equation migration (WEM) dominates over other migration methods in accuracy, despite of higher computational cost. However, the advantages of WEM will emerge as the progress of computer technology. WEM is sensitive to velocity model more than others. Small velocity perturbations result in grate divergence in the image pad. Currently, Kirrchhoff method is still very popular in the exploration industry for the reason of difficult to provide precise velocity model. It is very urgent to figure out a way to migration velocity modeling. This dissertation is mainly devoted to migration velocity analysis method for WEM: 1. In this dissertation, we cataloged wave equation prestack depth migration. The concept of migration is introduced. Then, the analysis is applied to different kinds of extrapolate operator to demonstrate their accuracy and applicability. We derived the DSR and SSR migration method and apply both to 2D model. 2. The output of prestack WEM is in form of common image gathers (CIGs). Angle domain common image gathers (ADCIGs) gained by wave equation are proved to be free of artifacts. They are also the most potential candidates for migration velocity analysis. We discussed how to get ADCIGs by DSR and SSR, and obtained ADCIGs before and after imaging separately. The quality of post stack image is affected by CIGs, only the focused or flattened CIGs generate the correct image. Based on wave equation migration, image could be enhanced by special measures. In this dissertation we use both prestack depth residual migration and time shift imaging condition to improve the image quality. 3. Inaccurate velocities lead to errors of imaging depth and curvature of coherent events in CIGs. The ultimate goal of migration velocity analysis (MVA) is to focus scattered event to correct depth and flatten curving event by updating velocities. The kinematic figures are implicitly presented by focus depth aberration and kinetic figure by amplitude. The initial model of Wave-equation migration velocity analysis (WEMVA) is the output of RMO velocity analysis. For integrity of MVA, we review RMO method in this dissertation. The dissertation discusses the general ideal of RMO velocity analysis for flat and dipping events and the corresponding velocity update formula. Migration velocity analysis is a very time consuming work. Respect to computational convenience, we discus how RMO works for synthetic source record migration. In some extremely situation, RMO method fails. Especially in the areas of poorly illuminated or steep structure, it is very difficult to obtain enough angle information for RMO. WEMVA based on wave extrapolate theory, which successfully overcome the drawback of ray based methods. WEMVA inverses residual velocities with residual images. Based on migration regression, we studied the linearized scattering operator and linearized residual image. The key to WEMVA is the linearized residual image. Residual image obtained by Prestack residual migration, which based on DSR is very inefficient. In this dissertation, we proposed obtaining residual migration by time shift image condition, so that, WEMVA could be implemented by SSR. It evidently reduce the computational cost for this method.

Lg尾波衰减成像研究——北京及邻区衰减成像

With the development of the technology of earthquake observation, more and more researchers work at many fields' of seismicity using seismic kinetic property, as the result, the study of attenuation has also made great progress, especially in the mechanism of the attenuation and the physical process. Aki put forward single back scattering theory to explain the forming of the seismic coda wave in 1969. Then, researchers started to develop the study in seismic scattering and attenuation. My thesis is also based on that theory. We assume that the Lg wave is a superposition fo high-mode surface waves, the coda of Lg is caused by scattering. Sato proposed Single Isotropic Scattering model (SIS model) to interpreted the scatter property, and he also formulated the geometrical spreading term. Then Xie (1988) developed the single spectral-ratio (SSR) method to obtain the Lg coda Q and the frequency dependent factor n. Later, he get to lateral images in the area of scatter ellipse. SSR method is explored and used in the study of Lg coda waves of regional earthquakes in my thesis. Choosing the earthquakes records with high ratio of signal-noise ,which were recorded at the stations from 1989 to 1999, we obtain the single trace Lg coda Q and its frequency dependent factor n. The results proved that SIS model is the reasonable model to explain the Lg coda wave, and SSR method also can be used to process Lg coda of regional earthquakes to get to the satisfied Lg coda Q. Based on the Lg coda Q we obtained using the former method, we explore the programs to inverse the regional Lg coda Q independently, and then make use of them to inverse the Lg coda Q of Beijing and adjacent area. The inversion result is satisfied. We conclude that the distribution of Qo (Q in lHz) is marked by the inhomogeneity, which is related to the tectonic structure: The value of Qo in uplift area, for example, Yanshan uplift, Taihang uplift, Luxi Uplift, is higher than the depression area, for example, Jizhong depression, Huanghua depression, and Jiyang depression, and the border between the higher Q area and lower Q area is very clear; Lg coda Q is also related to the velocity structure, higher velocity area is also with higher Q, lower velocity area is with lower Q; and higher heat-flow area is companied with lower Q. All in all, the value of Q reflects the difference of characteristics of lithofaces, porosity, the liquid content between the pores and heat flow. So, the Q value difference between uplift area and depression area reveals the difference of tectonic structure, lithology and physical character of the rock. So, the study of Lg coda Q is help to understand the earthquakes propagation mechanism through the inhomogenous medium, the cause of the coda, attenuation mechanism of the coda. Making use of the lateral images of Q, with velocity images, heat flow results, and other experimental result, we will be promoted to understand the complex structure of the crust, its inhomogenous character, and so on.

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n = 11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.