合浦珠母贝印度家系F↓（1）代的AFLP分析

用AFLP标记对合浦珠母贝(Pinctada fucata)印度家系的亲本及其子一代(F1)进行了遗传分析。27对引物共扩增出1 013个位点,其中835个位点在F1代分离,178个位点不分离,多态位点比例82.4%。分离位点中,符合孟德尔分离规律的位点458个,占分离位点数的54.9%。偏孟德尔分离规律的位点377个,占分离位点数的45.1%。分离比为3∶1的位点共有482个,占分离位点数的57.7%,符合孟德尔分离规律的位点251个,占52.1%(占分离位点数的30.1%);分离比为1∶1的位点共有353个,占分离位点数的42.3%,符合孟德尔规律的位点207个,占58.6%(占分离位点数的24.8%)。半数以上的标记符合孟德尔遗传规律。表明AFLP标记适合于合浦珠母贝的遗传图谱构建。[中国水产科学,2007,14(1):52-58]     

半滑舌鳎微卫星标记的开发及其在F1家系中分离方式分析

利用富集文库-菌落原位杂交的方法筛选半滑舌鳎( Cynoglossus semilaevis)微卫星标记,构建富含(AC)和(AG)序列重复的半滑舌鳎微卫星富集文库,随机从文库中挑选1060个克隆,筛选得到883个(83.3％)阳性信号,菌落PCR检测742个阳性克隆片段的长度在500～1 200 bp范围内,随机挑选其中50个克隆进行测序,共设计引物33对,进行退火温度优化、多态性检测后,共得到20个多态的半滑舌鳎微卫星标记.对其进行分离方式分析,其中有17个位点符合孟德尔分离定律,共表现5种分离方式,可用于半滑舌鳎遗传连锁图谱的构建；另外有3个位点偏离孟德尔分离定律(P＜0.05).结果同时证明富集文库-菌落原位杂交是半滑舌鳎微卫星标记大量筛选和高密度遗传连锁图谱构建的一种行之有效的方法.     

RAPD标记在合浦珠母贝家系F1代的分离方式

对RAPD标记在合浦珠母贝F1代全同胞家系共27个样品中的分离方式进行了研究。从21条RAPD随机引物中筛选了8条引物用于扩增分析,结果共扩增出49个位点,其中正常位点47个(95.9%),异常位点2个(4.1%),即子代有扩增带而双亲无。47个正常位点中3个位点在F1中不分离(6.4%),44个位点发生分离(93.6%,即多态位点)。卡方检验表明,44个分离位点中32个符合孟德尔分离规律(72.7%),12个位点发生偏分离(27.3%)。符合孟德尔分离位点中按11分离位点占43.8%,31分离位点占56.2%,其中,父本特有标记、母本特有标记和双亲共有标记分别占28.2%,15.6%和56.2%,父本特有标记略多于母本特有标记。根据“双假测交”理论,11分离位点是构建F1代家系的作图位点。较高的11分离位点表明,利用RAPD标记构建合浦珠母贝遗传连锁图谱是可行的。     

中华绒螯蟹微卫星多重PCR体系的建立及其亲子鉴定应用

利用自主开发的微卫星标记对中华绒螯蟹(Eriocheir sinensis)进行微卫星多重PCR体系构建,最终成功建立4组多重体系,每组体系包含4个微卫星位点,并成功应用于3个家系的亲子鉴定中。结果显示:(1)本研究筛选的16个微卫星标记,平均观测杂合度为0.8206,平均期望杂合度为0.8164,平均多态信息含量为0.7927,具有丰富的多态性;(2)运用Cervus 3.0软件对已知系谱信息的3个中华绒螯蟹家系共95个子代个体进行亲子鉴定分析,结果显示,选用任意两组微卫星多重PCR体系时,累积实际正确鉴定率均超过94.74%;使用任意三组微卫星多重PCR体系时,累积实际正确鉴定率均大于98.95%;使用四组微卫星多重PCR体系时,累积实际正确鉴定率达到100%。并且当选用组合1、2和组合3,或者选用组合1、3和组合4时,对3个家系的累积正确鉴定率达到了100%,因此选用这些组合不但可以获得准确的系谱信息,还能减少工作量,降低成本。本研究构建的微卫星多重PCR体系能为中华绒螯蟹的种群选育和家系管理提供便捷、高效的途径。     

尼罗罗非鱼♀×萨罗罗非鱼♂杂交后代的遗传分离分析

应用微卫星标记对尼罗罗非鱼(Oreochromis niloticus)♀×萨罗罗非鱼(Sarotherodon melanotheron)♂杂交后代(F1、F2)的遗传分离情况进行了初步研究.在86对微卫星引物中,筛选出21对在尼罗罗非鱼和萨罗罗非鱼中存在差异等位基因的特异性引物,共检测出59个等位基因,包含32个尼罗罗非鱼和24个萨罗罗非鱼特异等位基因.F1观测杂合度(Ho=0.998)大于 F2(Ho=0.739), F2有效等位基因数(Ne=2.48)、平均多态信息含量(PIC=0.497)与 F1的(Ne=2.45、PIC=0.489)相似.F1中所有位点均表现为尼罗罗非鱼与萨罗罗非鱼杂合基因型.F2中位点等位基因分离情况各异,通过卡方检验,有18个位点符合孟德尔遗传(P>0.05),2个位点GM145、UNH003表现为偏离孟德尔遗传(P<0.05),1个位点GM276不分离.在18个孟德尔遗传位点中, F2中尼罗罗非鱼特异等位基因频率为48.3%,萨罗罗非鱼特异等位基因频率为47.7%;基因型出现F1型、尼罗罗非鱼型、萨罗罗非鱼型的比例分别为58.2%、19.9%、21.9%.结果表明, F2较好地继承了F1的遗传杂合性,但也存在一定程度的分离.     

利用中国明对虾单对杂交亲本及其F↓（2）群体构建RAPD遗传连锁图谱

由中国明对虾(Fenneropenaeus chinensis)单对杂交亲本(G♀和G♂)及其F2为作图群体,构建了中国明对虾RAPD分子标记的遗传连锁图谱。109个引物共产生284个符合孟德尔分离规律的位点,符合1∶1孟德尔分离类型的位点共234个,符合3∶1孟德尔分离类型的位点50个。利用拟测交理论分别构建中国明对虾雌虾、雄虾的遗传连锁图谱。107个1∶1分离标记分布于雌虾连锁图谱中,包括31个连锁群,图谱总长度为1 406.1 cM,所有标记间的平均间隔为18.5 cM;91个1∶1分离标记分布于雄虾连锁图谱中,包括26个连锁群,图谱总长度为1 187.7 cM,所有标记间的平均间隔为18.27 cM;利用F2自交模型构建了雌虾和雄虾共同的分子标记,35个3∶1分离标记分布于雌雄共有的连锁图谱中,包括10个连锁群,图谱总长度为432.9 cM,所有标记间的平均间隔为17.3 cM。     

三疣梭子蟹莱州湾、舟山野生个体定向交配产生F2代家系的AFLP分析

以三疣梭子蟹莱州湾、舟山野生群体杂交（莱州湾♀×舟山♂）产生F2代家系63个个体及F1代亲本为实验群体，采用AFLP分子标记对其进行遗传分析。35对选择性引物共扩增出1742个位点，其中分离位点为417个，不分离位点为1325个，多态位点比例为23．9％。在分离位点中，符合1：1孟德尔分离比例的位点为226个，占总分离位点数的54．2％；偏离1：1孟德尔分离比例的位点为31个，占总分离位点数7．4％。符合3：1孟德尔分离比例的位点为128个，占总分离位点数的30．7％；偏离3：1孟德尔分离比例的位点为32个，占总分离位点数的7．7％。家系内基因多样性指数为0．130，Shannon指数为0．193。遗传距离分析显示，子代个体与母本Nei’s遗传距离介于0．064～0．111之间，与父本Nei’s遗传距离介于0．065～0．107之间。其中，63号个体与48号个体分别与父、母本亲缘关系最远。本实验中，偏分离位点比例属正常范围，杂交F2代家系遗传多样性偏低。本研究结果为三疣梭子蟹野生群体及家系遗传多样性分析，遗传连锁图谱构建等方面提供一定的技术支持。     

中国对虾近交两代的DNA随机扩增多态性及其遗传规律分析

为评估DNA随机扩增多态性标记在中国对虾遗传连锁图谱构建中的应用前景，利用中国对虾单对交配亲本及其子二代材料，对RAPD标记及其遗传规律进行了研究。22条RAPD随机引物扩增结果的统计分析表明，标记在中国对虾F2的遗传规律可归为不分离标记和分离标记：不分离标记，指在亲本和后代中均不分离的标记，占总位点的54.1%；分离标记占总位点的45.9%。其中，分离标记又包括符合孟德尔遗传分离的标记、偏离孟德尔遗传分离标记和异常分离标记。符合孟德尔分离的标记中，分离比例为3:1的标记占分离标记的14.7%;总的1:1标记占分离标记的64.7%；偏离孟德尔分离和异常分离的标记分别占分离标记的11.7%和8.9%。在这些分离的标记中，有76.5%的位点在“双假测交理论”的策略中适合构建中国对虾的遗传连锁图谱，这为以中国对虾F2为作图群体，并利用RAPD标记构建中国对虾遗传连锁图谱提供了理论支持。     

中国对虾连续两代的DNA随机扩增多态性及其遗传规律分析

为评估DNA随机扩增多态性标记在中国对虾遗传连锁图谱构建中的应用前景,利用中国对虾单对交配亲本及其子二代材料,对RAPD标记及其遗传规律进行了研究。22条RAPD随机引物扩增结果的统计分析表明,标记在中国对虾F2的遗传规律可归为不分离标记和分离标记:不分离标记,指在亲本和后代中均不分离的标记,占总位点的54.1%;分离标记占总位点的45.9%。其中,分离标记又包括符合孟德尔遗传分离的标记、偏离孟德尔遗传分离标记和异常分离标记。符合孟德尔分离的标记中,分离比例为3∶1的标记占分离标记的14.7%;总的1∶1标记占分离标记的64.7%;偏离孟德尔分离和异常分离的标记分别占分离标记的11.7%和8.9%。在这些分离的标记中,有76.5%的位点在"双假测交理论"的策略中适合构建中国对虾的遗传连锁图谱,这为以中国对虾F2为作图群体,并利用RAPD标记构建中国对虾遗传连锁图谱提供了理论支持。     

七里海中华绒螯蟹遗传多样性的RAPD和ISSR分析

利用RAPD-PCR及ISSR-PCR两种分子标记技术,分析了七里海中华绒螯蟹第6代繁育群体的遗传多样性。用10个多态性高的RAPD引物对24个七里海中华绒螯蟹样本个体进行扩增,共检测出107个不同的扩增位点,扩增片段大小为300~2300bp。其中多态位点总数为66个,平均多态位点比例为61.32%,群体内香农-维纳多样性值为0.1714。用8个多态性高的ISSR引物对24个个体共检测到106个位点,扩增片段为200~1500bp。其中多态性位点79个,平均多态位点百分率为74.53%,群体内香农-维纳多样性值为0.1778。结果显示,该群体的多态位点比例和香农-维纳多样性值均较大,说明其有较丰富的遗传多样性,同时也说明RAPD和ISSR技术用于七里海中华绒螯蟹核基因组的遗传多样性分析,具有较高的检出率和灵敏度。     

Isolation of microsatellite markers by in silico screening implicated for genetic linkage mapping in Japanese pufferfish Takifugu rubripes

ABSTRACT:   The search for dinucleotide repeat microsatellites within scaffolds 1–25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.     

半滑舌鳎抗哈维氏弧菌病相关微卫星标记筛选及QTL定位

为了筛选出与抗哈维氏弧菌病相关的微卫星标记并进行QTL定位,以2014年感染实验中存活率为52.22%的半滑舌鳎(Cynoglossus semilaevis)家系F1412(Family+年份+家系号)为材料,采用混合分离子分析法(bulked segregant analysis,BSA)对169个微卫星标记(SSR)进行筛选,得到1个可能与哈维氏弧菌病相关的微卫星标记scaffold479_23523。用scaffold479_23523所在的连锁群LG18上的37个SSR对94尾抗病、感病个体进行基因分型,得到3个图谱:整合图谱(LG18)、雌性图谱(LG18F)和雄性图谱(LG18M),并用两种不同的模式进行单标记分析和QTL定位。模式一得到3个显著性相关标记(P0.05)和1个极显著性相关标记(P0.01),图谱LG18F定位出一个区间qE-F1;模式二得到4个显著相关标记(P0.05)和1个极显著相关标记(P0.01),图谱LG18M定位出两个区间qE-M1、qE-M2。在全基因组序列的分布范围上,区间qE-F1包含了qE-M1及qE-M2,因此qE-F1更可能与抗病性状相关。本研究开发出了抗哈维氏弧菌病性状相关微卫星标记及QTL区间,为抗病新品种的培育奠定了基础。     

欧、美中华绒螯蟹源于中国长江水系中华绒螯蟹的遗传证据

用包括Z2和Opp17两个10碱基随机引物在内的10个引物,对来自荷兰斯科克莱（Skodely)、美国加州圣何塞（San Jose)的中华绒螯蟹群体与中国长江水系中华绒螯蟹群体进行RAPD遗传比较分析。结果发现：（1）中华绒螯蟹群体特有的Z2引物扩增的700bp标记带（Z2^700bp）,在荷兰与美国2个中华绒螯蟹群体中同时出现,而不出现日本绒螯蟹南流江种群中特有的880bp标记带（Z2^880bp）,表明欧洲、美国中华绒螯蟹与中国中华绒螯蟹为同种Eriocheir sinensis,而非日本绒螯蟹Eriocheir japonicus;（2）Opp17引物扩增的947bp片段在中国长江、荷兰及美国3个中华绒螯蟹群体内的出现频率均达100%。结合Z2引物扩增结果,欧洲与美国中华绒螯蟹群体极可能是从中国长江水系中华绒螯蟹引入繁衍的。     

草鱼7个插入/缺失型突变多态性及与幼鱼生长性状关联分析

对草鱼生长性状进行数量性状基因座(quantitative trait locus,QTL)定位过程中,在15号连锁群中定位到一个与体质量相关的QTL,本研究根据已有的草鱼遗传连锁图谱和基因组序列,拟用短片段重复序列(short tandem repeat,STR)分型技术对该连锁群的3个scaffolds中插入/缺失型突变位点进行筛选,以降低分型成本,同时将筛选出的7个多态性位点与草鱼幼鱼生长性状进行关联分析。结果显示,(1)草鱼3个scaffolds中44个插入/缺失型突变位点中有17个简单重复序列(又称微卫星,simple sequence repeats,SSRs),2个位点引物设计不成功,设计的25对引物中仅22对扩增和分型成功;(2) 22个位点中仅有7对引物在4个亲本中存在多态性,直接测序结果发现,STR分型技术不仅可准确对插入/缺失型突变位点进行分型,同时可降低分型成本;(3)将7个插入/缺失型突变位点与323尾选育F2草鱼的生长性状进行关联分析发现,除了位点ID-10H和ID-41F以外,其余5个位点都与草鱼幼鱼的一个或多个生长性状显著相关,其中ID-6H与幼鱼肥满度性状显著相关,位点ID-11F、ID-15F、ID-32F和ID-39F分别与草鱼幼鱼体质量、体长、体宽和体高性状显著相关,可将以上5个与草鱼幼鱼生长性状相关的突变位点用于草鱼生长性状QTL加密和分子标记辅助育种。     

Genetic linkage map of the pearl oyster,Pinctada martensii (Dunker)

Genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) and microsatellite markers for the pearl oyster, Pinctada martensii (Dunker), the main bivalve used for marine pearl production in Asia. Twenty‐four AFLP and 84 microsatellite primer pairs were used for linkage analysis in a full‐sib family with two parents and 78 offspring. Of the 2357 AFLP fragments generated, 394 (16.7%) were polymorphic and segregating. Most (340 or 86.2%) of the markers segregated according to expected Mendelian ratios. Female and male linkage maps were constructed using 230 and 189 markers, including 15 and 10 microsatellites respectively. The female map consisted of 110 markers in 15 linkage groups, covering 1415.9 cM, with an average interval of 14.9 cM. The male map consisted of 98 markers in 16 linkage groups, with a total length of 1323.2 cM and an average interval of 16.1 cM. When unlinked doublets were considered, genome coverages were 78.5% for the female and 73.5% for the male map. Although preliminary, the genetic maps constructed here should be useful for future linkage and quantitative trait loci mapping efforts.     

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.     

A preliminary genetic map of Zhikong scallop (Chlamys farreri Jones et Preston 1904)

Zhikong scallop (Chlamys farreri Jones et Preston 1904) is one of the most important aquaculture species in China. The development of a genetic linkage map would provide a powerful tool for the genetic improvement of this species. Amplified fragment length polymorphism (AFLP) is a PCR‐based technique that has proven to be powerful in genome fingerprinting and mapping, and population analysis. Genetic maps of C. farreri were constructed using AFLP markers and a full‐sib family with 60 progeny. A total of 503 segregating AFLP markers were obtained, with 472 following the Mendelian segregation ratio of 1:1 and 31 markers showing significant (P<0.05) segregation distortion. The male map contained 166 informative AFLP markers in 23 linkage groups covering 2468 cM. The average distance between markers was 14.9 cM. The female genetic map consisted of 198 markers in 25 linkage groups spanning 3130 cM with an average inter‐marker spacing of 15.8 cM. DNA polymorphisms that segregated in a 3:1 ratio as well as the AFLP markers that were heterozygous in both parents were included to construct combined linkage genetic map. Five shared linkage groups, ranging from 61.1 to 162.5 cM, were identified between the male and female maps, covering 431 cM. Amplified fragment length polymorphism markers appeared to be evenly distributed within the linkage groups. Although preliminary, these maps provide a starting point for the mapping of the functional genes and quantitative trait loci in C. farreri.