西伯利亚鲟（Acipenser baeri）养殖群体遗传结构的AFLP分析

本研究构建了西伯利亚鲟（Acipenser baeri）扩增片段长度多态性（AFLP）分析体系,分析了DNA提取、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤。用7对引物对24尾西伯利亚鲟进行了AFLP分析,每对引物扩增的位点数在24～39之间,共扩增出了234个位点,多态性位点为116个,多态性比例为48.83%,平均Nei氏基因多样性指数H为0.4297,平均Shannon氏指数I为0.1233,个体间平均遗传距离为0.1872,表明西伯利亚鲟养殖群体的遗传多样性程度比较低,同时还构建了西伯利亚鲟的聚类图。     

魁蚶AFLP反应体系的建立

以魁蚶为研究材料,建立了魁蚶的AFLP分子标记反应体系。对其分析过程中的酶切链接、预扩增、选择性扩增等试验条件进行了检测与筛选,得到了清晰的AFLP指纹图谱,为探讨魁蚶种群之间的遗传关系和遗传图谱的构建等方面提供了新的技术手段。     

鲮原种群体的AFLP分析

运用AFLP技术,对广东鲮鱼原种场保存的鲮原种2个子群体(子群体h、q)各29个个体进行遗传多样性分析。25对AFLP引物组合中的6对引物扩增共产生173条条带,其中多态位点数为72,多态位点比例为41.5%。在6对引物产生的173条扩增条带中,有一条扩增条带在子群体h中的出现频率远远高于其在子群体q中的出现频率(72.4%>20.6%),编号为E2M4-1。遗传多样性分析结果表明,子群体h的遗传多样性指数高于子群体q(0.1367>0.0998)。该研究为筛选鲮原种群体2个子群体间的特异性分子标记做了初步尝试。     

光棘球海胆3个地理亚群的遗传多样性AFLP分析

应用AFLP技术对光棘球海胆的大连长海县群体、大连旅顺群体及日本青森县群体进行遗传多样性分析。试验结果表明,4对引物组合扩增得到231个扩增位点,其中168个多态位点,多态位点总比例为72.73%;3个群体的遗传多样性指数分别为0.3020±0.1925、0.2995±0.1977和0.2945±0.1935;Shannon多样性指数分别为0.4390±0.2767、0.4344±0.2820和0.4298±0.2766;群体内遗传变异系数为0.2987±0.0366,群体间遗传分化系数为0.0230,说明群体内的遗传多样性比较丰富。3个群体之间的遗传距离无显著差异,遗传相似度基本一致,用UPGMA方法对3个群体的143个个体的聚类分析显示,3个群体的个体交互混杂在一起,3个群体间没有因为地理隔离而产生明显的遗传分化。     

吉富罗非鱼不同选育群体的遗传多样性

用微卫星和扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记对海南、广州和青岛吉富品系尼罗罗非鱼的遗传多样性和遗传分化进行了研究。9对微卫星引物和4对AFLP引物的分析结果一致。微卫星分析表明,青岛吉富鱼的平均等位基因数(4.8)、平均观测杂合度(0.528)、平均多态信息含量(0.605)最高,海南吉富鱼的平均等位基因数(4.4)、平均观测杂合度(0.479)、平均多态信息含量(0.549)最低。AFLP分析显示,青岛吉富鱼的多态位点比例(48.4%)和基因多样性(0.245)最高,海南吉富鱼的多态位点比例(36.3%)和基因多样性(0.147)最低。这些表明,青岛吉富鱼的遗传多样性最高,海南吉富鱼最低。遗传分化分析表明,两两群体间遗传分化显著(微卫星FST为0.07~0.11,P<0.01;AFLPFST为0.24~0.29,P<0.01)。AMOVA分析显示,大部分遗传变异(微卫星的分析结果为91.26%;AFLP的为67.6%)来源于群体内个体间,表明吉富鱼选育品系尚具有进一步选育的潜力。     

福建东山和广东阳江褐毛鲿养殖群体遗传多样性的AFLP分析

应用AFLP分子标记技术对福建东山和广东阳江褐毛鲿养殖群体进行了遗传多样性分析。采用6对选择性引物组合对2个群体60个个体进行扩增,共扩增出313个位点,多态位点85个。东山和阳江褐毛鲿养殖群体的多态位点比例、Nei’s基因多样性指数、Shannon遗传多样性指数分别为24.60%和25.56%、0.0795和0.0768、0.1210和0.1176,2个群体的遗传多样性均处于较低水平。遗传分化系数Gst及AMO-VA分析表明,2个群体的遗传变异主要来源于群体内个体间。UPGMA聚类图及PCA分析显示,群体间具有典型的地理特征,且出现了一定程度的遗传分化。     

AFLP技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种新型分子标记。该技术原理简单,引物设计巧妙,既具有RFLP技术的可靠性又具有RAPD技术的高效性,被称为"最有力的分子标记"。近年来其在遗传多样性的研究中得到了广泛的应用。动物遗传多样性的研究对揭示生物群体的进化历史或对生物资源的有效利用与保护有着十分重要的现实意义。本文主要论述AFLP的技术原理、要求及特点,并介绍其在动物遗传多样性研究中的应用。     

长臀AFLP分析体系的建立

以珠江长臀(Cranoglanis bouderius)为试验材料,建立了长臀扩增酶切片段长度多态性(AFLP)分析体系,对其分析过程中的DNA提取、双酶切、单酶切、连接、预扩增、选择性扩增、银染等步骤进行了检测,并对反应条件进行了优化。用PstI/TaqI、EcoRI/MseI等2组双酶切组合构建长臀的AFLP指纹图谱,条带清晰可辨,扩增信号强,得到清晰的长臀AFLP指纹图谱。     

福建东山和广东阳江褐毛鳞养殖群体遗传多样性的AFLP分析

应用AFLP分子标记技术对福建东山和广东阳江褐毛鳞养殖群体进行了遗传多样性分析。采用6对选择性引物组合对2个群体60个个体进行扩增,共扩增出313个位点,多态位点85个。东山和阳江褐毛鳞养殖群体的多态位点比例、Nei’s基因多样性指数、Shannon遗传多样性指数分别为24．60％和25．56％、0．0795和0．0768、0．1210和0．1176,2个群体的遗传多样性均处于较低水平。遗传分化系数Gst及AMO．VA分析表明,2个群体的遗传变异主要来源于群体内个体间。UPGMA聚类图及PCA分析显示,群体间具有典型的地理特征,且出现了一定程度的遗传分化。     

基于AFLP的海萝野生群体遗传多样性分析

应用AFLP分子标记技术对广东深圳、汕头和山东长岛的海萝(Gloiopeltis furcata)群体进行遗传多样性分析。应用筛选出的10对多态性丰富的AFLP引物,对这3个地理群体的90个个体进行扩增,共得到427个位点,多态性位点数为392(91.75%),各引物扩增位点数为30~59。长岛群体扩增位点最多,多态性也最高。群体内的等位基因数(Na)、有效等位基因数(Ne)、遗传多样性指数(H)和Shannon信息指数(I)变化趋势一致,均由高到低依次为长岛群体、汕头群体、深圳群体,表明长岛群体遗传多样性最高。3个海萝群体遗传变异主要来自群体间。群体间的基因流为0.368 9,群体分化系数(Gst)为0.575 4,表明群体间有高度分化。深圳群体和汕头群体的遗传距离最小(0.110 2),与长岛群体的遗传距离最大(0.357 7)。UPGMA聚类分析显示广东深圳和汕头群体聚为一支,山东长岛群体为另一支,表明群体间的遗传差异与地理距离有关。本研究结果为海萝资源的保护与利用提供了基础数据。     

Application of amplified fragment length polymorphism markers to assess molecular polymorphisms in gynogenetic haploid embryos of turbot (Scophthalmus maximus)

Among the variety of cultured marine species, the turbot Scophthalmus maximus is a fish of growing importance in European aquaculture. In this paper, an advanced application of AFLPs to estimate the genetic diversity of haploid gynogenetic families with the aim of obtaining a preliminary genetic map is presented. Ten EcoRI/TaqI primer combinations were tested in four families comprising diploid mothers and their haploid progenies. The amplified fragment length polymorphism (AFLP) analysis revealed an average of 6.8 polymorphic bands per primer combination and a total number of 88 polymorphisms out of 579 fragments. Among various primer pairs, seven combinations were selected in relation to the quality of profiles and number of polymorphic fragments, to be used in the determination of genetic linkage relationship between AFLP markers within the largest haploid family. Co‐migration of non‐homologous fragments was also investigated in one primer combination adding a fourth selective nucleotide to the three used in the classic TaqI AFLP protocol. Surprisingly, a rate of 38.7% of non‐homologous fragments co‐migrating with monomorphic bands was identified, due to the combined effect of homoplasy and the protocol used. Additional polymorphic markers discovered by this protocol were included in the linkage map. The turbot AFLP linkage map comprises 52 AFLP markers distributed in 12 linkage groups. On the basis of this map, turbot expected total genome length sums up to 1225.6 cM. The results confirm the usefulness of AFLPs in revealing genome segregation in haploid turbot progeny.     

黄姑鱼群体遗传多样性的AFLP分析

对青岛和厦门黄姑鱼群体的遗传多样性进行了AFLP分析，5对选择性引物在两个群体47个个体中，共扩增出461个位点，多态位点265个。青岛和厦门群体的多态位点比例、Nei遗传多样性指数和Shannon遗传多样性指数分别为51.70%、51.99%，0.1022、0.0996，0.1643、0.1622；两个群体遗传多样性在同一水平上。基因分化系数G_st、Shannon遗传多样性指数和AMOVA分析均显示黄姑鱼的遗传变异主要来源于群体内个体间，而群体间无明显的遗传分化。群体的显性基因型频率分布和位点差异数分布显示两个群体有基本相同的群体遗传结构。结果表明，黄姑鱼青岛和厦门群体间无明显的遗传差异，群体间有明显的基因交流。     

大口黑鲈选育群体遗传多样性的AFLP分析

采用AFLP技术对大口黑鲈(Micropterus salmoides)F3、F4代选育群体的遗传结构进行分析,并计算2个选育群体和一个对照养殖群体的遗传多态度、遗传距离及分化系数。结果显示,8对AFLP引物共扩增到262条带,其中多态性条带有80条,每对引物检测到的多态性条带在5～13之间。F3、F4代选育群体和对照养殖群体的多态性位点比例分别为29.36%、29.20%、30.29%。Shannon多样性指数分别为0.2017,0.1955,0.2042。结果表明,选育群体相较于对照养殖群体多态位点比例和遗传多态度均有所下降。群体间的遗传变异平均为0.0752,由此可见,92.48%的遗传变异来自于群体内,而只有7.52%的遗传变异来自于群体间,这初步显示了大口黑鲈在遗传上的稳定性,群体尚具有一定的选育潜力,可继续进行人工选育。     

黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况,利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带,表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况,雌核发育家系7个SSR位点的平均纯合度为0.382,是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个,纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带,其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度,正常交配家系同父本和母本的遗传距离大致相同。研究结果表明,黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系,人工诱导雌核发育是促进基因纯合的一个有效途径,它不仅可以加速有利基因的纯合固定,还可以加速有害基因的淘汰,从而有效提高育种效率。     

Molecular identification of interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin using amplified fragment‐length polymorphism and microsatellite markers

Interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin were produced in this study. The hybridity of the interspecific hybrids was confirmed by using the methods of amplified fragment‐length polymorphism (AFLP) and microsatellite [simple sequence repeats (SSR)] markers. Five AFLP primer combinations were used to develop the AFLP profiles of H. discus hannai, H. gigantea and their reciprocal hybrids. AFLP analysis revealed that genetic variations of H. discus hannai and H. gigantea were relatively diverse and each species holds species‐specific bands. The AFLP profiles of reciprocal hybrids showed that all of the hybrids inherited bands specific to H. discus hannai and H. gigantea. Of a total of 20 microsatellite loci, which were selected from H. discus hannai microsatellite markers evaluated, eight loci were polymorphic in H. gigantea samples, with an average of 3.375 alleles per locus. Preliminary screening showed that, two of these eight microsatellite loci (Awb002 and Awb022) could be used as species‐specific markers to distinguish the hybrids and their parental species. Simple sequence repeats analysis showed that the reciprocal hybrids inherited one allele from each parent for both of the two SSR loci investigated. These data strongly suggest that the induced interspecific hybrid is a true hybrid between H. discus hannai and H. gigantea.     

基于遗传连锁图谱筛选虾夷扇贝性别相关AFLP分子标记的方法

基于近年来他人构建的遗传连锁图谱,对2011–2013年2–5月期间采集的雌性、雄性及雌雄同体虾夷扇贝进行性别相关AFLP分子标记筛选。从雄性和雌性遗传连锁图谱上选取合计18对引物组合,经3批次试验,有8对引物扩增出性别相关的条带,其中Eb Mc、Ej Mf和Ei Mk重复性好,有4对引物扩增出雌雄同体特异性条带,5对引物扩增出雌性特异性条带。结果表明,基于高密度遗传连锁图谱筛选性别相关分子标记的方法简单可行,雌雄同体的基因组DNA与雌性和雄性存在显著差异,可能是一个单独的群体。     

First Evidence for Hybrid Breakdown in the Backcross of Olive Flounder,Paralichthys olivaceus,and Summer Flounder,Paralichthys dentatus

Hybrid and parental backcross experiments were performed using olive flounder, Paralichthys olivaceus, summer flounder, Paralichthys dentatus, and their female F₁ hybrids to examine hybrid fitness in the backcross. Fertilization rate, hatching rate, and combined fitness measure (CFM, product of fertilization rate and hatching rate) were detected and the results showed that the CFM of hybrid and backcross generations was reduced significantly compared to pure olive flounder crosses. Genomic inheritance from parents to backcross progeny was also detected by amplified fragment length polymorphism (AFLP). Almost all AFLP bands observed in parents were presented in backcross progeny (backcross1, 218/227; backcross2, 265/282), although 9 and 17 parental bands were missing in backcross1 and backcross2, respectively. Novel bands (absent in parents) also occurred in progeny at the frequencies of 5.63% (13/231) in backcross1 and 3.28% (9/274) in backcross2. Additionally, 27.40 and 31.18% of AFLP markers deviated from expected Mendelian ratio in backcross1 and backcross2, respectively. The present study suggests that genetic incompatibilities may exist between olive flounder and summer flounder. Furthermore, possible implications of segregation distortion for reduced hybrid fitness in backcross generations are discussed.     

罗非鱼3个养殖群体的遗传多样性及特异性AFLP标记研究

对不同来源的养殖群体进行种质鉴定，探索在种苗阶段即区分新吉富罗非鱼（New GIFT strain Oreochromis niloticus）、吉诺玛罗非鱼（GenoMar strain O．niloticus）和奥杂罗非鱼（0．niloticus♀×O．aureus♂）3个养殖群体具有重要的意义。笔者选取了5对AFLP（amplified fragment length polymorphism）引物组合对3个罗非鱼养殖群体共60尾个体进行比较分析，引物组合E—ACA／M—CAT表现出较好的区分能力，有7条标记的分布在群体间表现出“有和无”的差别，引物组合E—ACA／M—CAG的扩增图谱中有2条带表现出“有和无”的差别。遗传多样性方面，平均基因多样性在新吉富罗非鱼和吉诺玛罗非鱼分别为0．1605和0．1595，而在奥尼罗非鱼为0．2214，多态位点百分数和Shannnon多样性指数也表现出相近的变化趋势。分析表明新吉富罗非鱼群体在遗传上已较为稳定，吉诺玛罗非鱼遗传多样性偏低。     

辽宁沿海海蜇与沙海蜇遗传多样性的AFLP分析

海蜇和沙海蛰均为腔肠动物门的大型食用水母,采用AFLP分子标记技术对辽宁沿海的海蜇野生群体、养殖群体和沙海蜇野生群体共90个个体进行了遗传多样性分析.10对引物共得到560个稳定扩增位点.3个群体的多态性位点比例为海蜇野生群体82.05%.海蜇养殖群体78.46%,沙海蜇野生群体74.10%;平均杂合度分别为0.2072,0.1850和0.2116,Shannon多样性指数分别为0.3248、0.2954和0.3262,海蜇野生群体与海蜇养殖群体和沙海蜇野生群体的遗传距离分别为0.0300和0.2702.分析结果表明,3个群体的遗传多样性均保持了相对较高的水平.