应用SSR和SRAP标记构建青虾遗传连锁图谱

采用SSR和SRAP标记结合拟测交策略构建青虾(Macrobrachium nipponense)遗传连锁图谱。共有175个标记(含27个SSR、148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6.7~91.2 cM之间,相邻标记间最大间隔为49.0 cM,最小为1.4 cM,平均间隔为13.1 cM。青虾框架图谱长度为997.2 cM,图谱观察总长度为2 270.5cM,根据估算,青虾遗传连锁图谱预期长度为4 380.6 cM,图谱的覆盖率为51.83%。本研究构建了青虾遗传连锁图谱,该图谱也是淡水虾蟹类第一张遗传连锁图谱,可为青虾QTL定位、基因克隆、遗传选育等提供指导,并为进一步构建高密度的青虾遗传连锁图谱奠定了基础。     

坛紫菜遗传连锁图谱的构建

以野生型坛紫菜纯系(♀)和红色型坛紫菜纯系(♂)作为杂交亲本,结合四分子分析法及单个体细胞克隆的丝状体途径,创建了由157个株系组成的坛紫菜DH作图群体,并用经过筛选的24对SRAP引物和16对SSR引物对父母本及作图群体各株系进行双标记分析,获得了224个多态性标记,其中157个标记符合孟德尔分离规律。根据标记间的连锁规律,首次构建了坛紫菜的分子遗传连锁图谱,所构建的遗传图谱由包含124个标记(含SRAP标记104个,SSR标记20个)的5个连锁群组成,总长度为879.2cM,平均标记间隔为7.09cM,各个连锁群长度为134.2～213.6cM,包含标记18～31个。最后采用3种不同方法计算得到坛紫菜的估计基因组长度平均为955.3cM,由此得到坛紫菜遗传连锁图谱的基因组覆盖率为92.0%。     

牙鲆遗传连锁图谱的构建

利用基因组测序得到的大量微卫星序列,以681383B为父本、6812E36为母本杂交获得的F1为作图群体,构建了牙鲆(Paralichthys olivaceus)微卫星标记(SSR)遗传连锁图谱。雌雄图谱共定位SSR标记529个,其中雄性连锁图谱包括418个标记,分布在24个连锁群上,总长度1 418.1 cM,标记平均间隔3.62 cM,图谱覆盖率为88.7%;雌性连锁图谱包括437个标记,分布在24个连锁群上,总长度1 298.1 cM,标记平均间隔为3.16 cM,图谱覆盖率为89.1%。牙鲆中密度遗传图谱的构建为QTL分析以及分子标记辅助育种进一步奠定基础,并可以有效推动牙鲆的遗传改良工作,推动牙鲆养殖业的可持续发展。     

中国对虾遗传连锁图谱的构建

利用RAPD、SSR和AFLP三种标记技术结合"拟测交"策略,以中国对虾"黄海1号"雌虾与野生雄虾作为亲本进行单对杂交产生的F1家系为作图群体,初步构建了中国对虾雌、雄性遗传连锁图谱.对460个RAPD引物和44对SSR引物进行筛选,共选出61个.RAPD和20对SSR引物,结合88对AFLP引物组合对父母本和82个F1个体进行了遗传分析.共得到783个分离标记(RAPD标记237个,微卫星标记45个,AFLP标记501个),761个标记用于连锁分析.雌性图谱包括40个连锁群和15个三联体,20个连锁对,标记间平均间隔为12.5 cM,图谱共覆盖2835.5 cM,覆盖率为73.5%;雄性图谱包括41个连锁群和6个三联体,12个连锁对,标记间平均间隔为11.9 cM,图谱共覆盖2776.7 cM,覆盖率为73.3%.中国对虾遗传图谱的构建为其分子标记辅助育种、比较基因组作图及数量性状位点(QTL)的定位与克隆奠定了基础.     

牙鲆遗传作图及生长性状QTL定位

采用牙鲆日本群体和韩国群体杂交的92个F₁个体作为分离群体, 利用微卫星标记和Joinmap 4.0作图软件构建了牙鲆遗传连锁图谱。共有221个SSR标记用于连锁图谱构建, 雌性图谱中, 共178个微卫星标记定位到22个连锁群上, 观测总长度为(G _oa )599.0 cM, 覆盖率(C _oa )达76.27%。雄性图谱中, 共194个微卫星标记定位到23个连锁群上, G _oa 为693.4 cM, C _oa 为78.82%。对全长、体质量、体高3组数据进行主成分分析处理, 得到可解释3个性状的89.6%特征的一组数据, 命名为牙鲆生长性状GT。用WinQTLCart 2.5软件的复合区间作图,在已构建的遗传连锁图谱上对牙鲆生长性状GT进行QTL定位, 取LOD经验值2.5为QTL存在的阈值; 对微卫星标记进行性状—标记之间的回归分析。本研究共定位3个与牙鲆生长性状GT相关的QTLs, qGT-f4 qGT-m20 qGT-f20,可解释表型变异率分别为27.60%, 13.74%, 10.27%。在性状—标记之间的回归分析中, 得到22个与生长性状GT相关(P<0.05)的微卫星标记, 单个标记可解释表型变异率介于3.70%~10.42%, 其中6个微卫星标记scaffold558_51720、scaffold558_26183、scaffold903_69232、scaffold485_47120 、scaffold1262_77386、scaffold809_65154与生长性状GT之间呈极显著相关(P><0.01), 可解释表型变异率分别为10.42%、7.31%、10.07%、10.07%、8.39%和11.26%。     

三疣梭子蟹遗传连锁图谱的初步构建

利用AFLP和SSR标记技术结合"拟测交"策略,以三疣梭子蟹莱州湾、舟山野生群体杂交(1♂×3♀)产生的F2代家系为作图群体,初步构建了三疣梭子蟹雌、雄性遗传连锁图谱。用经过筛选的60对AFLP引物和3对SSR引物对亲本及108个F2代个体进行遗传分析,共得到母本分离标记214个,其中155个标记(AFLP标记153个,SSR标记2个)符合1∶1孟德尔分离规律;父本分离标记195个,139个标记(AFLP标记138个,SSR标记1个)符合1∶1孟德尔分离规律。雌性图谱包括100个遗传标记,分布在9个连锁群,6个三联体,15个连锁对,图谱总长度为1544cM,标记平均间隔22.0cM,总覆盖率为52.9%。雄性图谱包括71个遗传标记,分布在6个连锁群,6个三联体,11个连锁对,图谱总长度1174.2cM,标记平均间隔24.0cM,总覆盖率为49.5%,图谱中遗传标记分布比较均匀。     

一种杂交鲤四种经济性状的QTL定位分析

以柏氏鲤和荷包红鲤抗寒品系杂交F21个家系的92尾个体为材料,用300个微卫星(SSR)标记构建了遗传连锁图谱,其长度为2 471.7 cM,标记间平均间隔为10.75 cM,在此基础上对体长(SL)、体厚(BT)、体高(H)和体质量(W)进行QTL定位分析.共检测到17个QTL区间分布于6个连锁群.4个体长的QTL中,位于LG5、LG7和LG19的QTL为显著水平(P＜0.05),位于LG32的QTL为极显著水平(P＜0.01),可解释表型变异率为5.14％～10.2％;4个体厚的QTL中,位于LG11(两个QTL)和LG32上的QTL为显著水平(P＜0.05),位于LG5的QTL达极显著水平(P＜0.01),可解释表型变异率为6.42％～ 8.43％;6个体高的QTL中,LG5,LG10,LG11,LG19,LG32上的QTL为显著水平(P＜0.05),LG7上达极显著水平(P＜0.01),可解释表型变异率为5.92％ ～ 8.95％;3个体质量的QTL中,位于LG5和LG7上的为显著水平(P＜0.05),可解释表型变异率为5.55％和8.36％,LG32上达极显著水平(P＜0.01),可解释表型变异率为6.44％.本研究采用了两个不同品系的杂交子代为作图群体,丰富了QTL研究群体的多样化,为今后不同鲤品系或品种间的QTL在全基因组水平上的比较和共性QTL的发掘等奠定基础,进而指导鲤分子育种.     

合浦珠母贝遗传连锁图谱的构建

用AFLP标记构建了印度合浦珠母贝(Pinctada fucata)全同胞家系的遗传图谱。用经过筛选的36对引物组合对父母本和62个子代个体进行遗传分析,共得到1 547个标记,包括581个1∶1分离标记。母本分离标记294个,其中178个符合1∶1孟德尔分离规律;父本分离标记287个,其中182个符合1∶1孟德尔分离规律。雌性框架图包括33个遗传标记,分布在14个连锁群中,标记间平均间隔24.3 cM,有2个3联体,11个连锁对,图谱总长度为488.5 cM。雄性框架图包括53个遗传标记,分布在19个连锁群中,标记间平均间隔30.5 cM,有4个3联体,10个连锁对,图谱总长度为1 035.5 cM。雌雄两个框架图中AFLP标记的分布都较均匀。雌雄基因组估算长度分别为1 168.4 cM和2 037.1 cM,图谱覆盖率分别为41.8%和50.8%。本研究为进一步构建高密度遗传连锁图谱及QTL定位分析奠定了基础。     

镜鲤酸性磷酸酶的QTL分析

利用992个微卫星标记检测了德国镜鲤(Cyprinus carpio)F1代190个个体基因组DNA进行基因型检测,共组成51个连锁群,覆盖基因组总长度为5138.2cM,标记间平均距离为5.19cM;利用软件MapQTL 6.0采用区间作图法对酸性磷酸酶性状进行数量性状基因座(QTL)定位分析。研究结果共检测到9个与酸性磷酸酶有关的QTLs,分布于8个连锁群。其中LG24连锁群LOD值最大(5.37),位于LG24连锁群,最大的可解释表型变异为13.8%。通过BLAST与斑马鱼进行序列比对,找到了与斑马鱼富含亮氨酸重复蛋白、横跨膜蛋白50a、ADP核糖化因子和细胞因子受体家族b8同源的分子标记。本研究结果对鲤鱼分子标记辅助育种和疾病调控具有重要应用价值。     

半滑舌鳎微卫星标记遗传连锁图谱的构建

利用全基因组测序方法筛选出微卫星标记,以渤海近海野生个体和人工养殖的半滑舌鳎(Cynoglossus semi-laevis)为亲本交配产生的F1全同胞家系为作图群体,构建了半滑舌鳎雌、雄微卫星标记遗传连锁图谱。用320对引物对父母本和92个F1个体进行遗传分析,共得到288个分离标记,其中包含112个偏分离标记(P<0.05)。其中雌性框架图包含242个标记,分布在21个连锁群上,总长度1 311.9 cM,标记间平均距离为4.9 cM,图谱覆盖率为83.3%;雄性框架图定位标记218个,21个连锁群,总长度1 316.2 cM,标记间平均距离为5.5 cM,覆盖率为82%。半滑舌鳎遗传连锁图谱的构建为半滑舌鳎重要经济性状QTL定位、分子标记辅助育种和性别控制奠定了重要基础。     

Genetic linkage map of bay scallop, Argopecten irradians irradians (Lamarck 1819)

The bay scallop (Argopecten irradians irradians Lamarck 1819) has become one of the most important aquaculture species in China. Genetic improvement of cultured bay scallop can benefit greatly from a better understanding of its genome. In this study, we developed amplified fragment length polymorphisms (AFLPs) and simple sequence repeat markers from expressed sequence tags (EST‐SSRs) for linkage analysis in bay scallop. Segregation of 390 AFLP and eight SSR markers was analysed in a mapping population of 97 progeny. Of the AFLP markers analysed, 326 segregated in the expected 1:1 Mendelian ratio, while the remaining 74 (or 19.0%) showed significant deviation, with 33 (44.6%) being deficient in heterozygotes (A/a). Among the eight polymorphic EST‐SSR loci, one marker (12.5%) was found skewing from its expected Mendelian ratios. Eighteen per cent of the markers segregating from female parent were distorted compared with 21% of the markers segregating from male parent. The female map included 147 markers in 17 linkage groups (LGs) and covered 1892.4 cM of the genome. In the male map, totally 146 AFLP and SSR markers were grouped in 18 LGs spanning 1937.1 cM. The average inter‐marker spacing in female and male map was 12.9 and 13.3 cM respectively. The AFLP and SSR markers were distributed evenly throughout the genome except for a few large gaps over 20 cM. Although preliminary, the genetic maps presented here provide a starting point for the mapping of the bay scallop genome.     

基于SSR和RSAP的龙须菜作图群体多态性标记 开发与亲本间遗传距离的估计与比较

遗传连锁图谱的构建是实现分子标记辅助育种与数量性状定位的必要手段,为构建龙须菜(Gracilariopsis lemaneiformis)遗传连锁图谱提供多态性分子标记,本实验共采用400对SSR引物和91对RSAP引物对组合,对龙须菜两个F1代配子体作图群体的亲本进行了多态性标记筛选,并对4个亲本间的遗传距离进行了评估.得到的SSR多态性标记在GL-F3/GL-M8间为15个,GL-F6/GL-M9间为8个;多态性RSAP标记在GL-F3/GL-M8间为88个;GL-F6/GL-M9间为93个.分析了基于SSR、RSAP技术的两对亲本间的遗传距离,SSR结果显示GL-F3/GL-M8间遗传距离为0.071 2,GL-F6/GL-M9间的遗传距离为0.043 6;RSAP结果显示GL-F3/GL-M8间遗传距离为0.228 4,GL-F6/GL-M9间的遗传距离为0.253 7.结果表明,在引物组合平均覆盖的位点和引物组合平均产生的多态性方面,RSAP均远高于SSR,在GL-F3/GL-M8间RSAP分别是SSR的4.48与12.13倍,在GL-F6/GL-M9间RSAP分别是SSR的5.81与25.5倍,RSAP标记更适合应用于龙须菜的遗传距离估计和资源的遗传多样性研究.本研究揭示出作图群体亲本间遗传多样性特征,SSR与RSAP分子标记的开发为龙须菜遗传连锁图谱的构建奠定了基础.     

A first‐generation genetic map of the Japanese scallop Patinopecten yessoensis‐based AFLP and microsatellite markers

We constructed genetic linkage maps of the Japanese scallop Patinopecten yessoensis using AFLP and microsatellite markers. With 32 AFLP primer combinations, a total of 413 markers (209 from the female parent and 204 from the male parent) segregated in a 1:1 ratio, corresponding to DNA polymorphisms which were heterozygous in one parent and null in the other. Among the six microsatellite markers we used, there were four polymorphic loci. Two segregated in the female parent, and the other two segregated in both parents. In the maternal parent, 161 framework markers were mapped in 20 linkage groups, with a total coverage of 2198.8 cM. In the paternal parent, 166 framework markers established a map with 21 linkage groups, spanning a genome length of 2137.6 cM. The AFLP markers on the maps were randomly distributed with an average spacing between markers of 14.7–15.6 cM. The estimated coverage for the framework maps are 77.9% both for the female and the male. These are the first linkage maps for P. yessoensis, which constitute a basis for further genome studies and provide a useful framework for consensus map construction by adding orthologous anchor markers developed in P. yessoensis.     

鲤头长、体厚、体高性状的QTL定位及遗传效应分析

用174个SSR、41个EST、345个SNP标记对以镜鲤良种后代为祖父母本所培育的杂交F2群体的68个个体进行基因型检测,运用JoinMap4.0软件包构建遗传连锁图。利用MapQTL5.0区间作图法(interval mapping,IM)和多QTL区间定位法(MQM mapping,MQM)进行QTL检测,通过置换实验(1000次重复)确定连锁群显著性水平阈值。在对体高、头长、体厚的区间定位中,共检测到6个与体高性状相关的QTLs区间,分布在LG1(SNP1339-SNP1490)、LG10(HLJE469-SNP1491)、LG12(SNP0922-HLJ1316)、LG13(SNP0937-HLJ328)、LG25(SNP1041-HLJ594)、LG35(SNP1425-SNP0389)等6个连锁群上,解释表型变异范围为20.0%～43.3%。其中,SNP1339-SNP1490区间LOD值最大为3.64,解释表型变异35.4%。6个与头长相关的QTLs,分布在LG1(SNP1339-SNP1490)、LG12(HLJ071-HLJ336)、LG13(SNP0937-HLJ328)、LG24(SN...     

双须骨舌鱼转录组EST-SSR标记开发与引物筛选

利用MISA软件对双须骨舌鱼(Osteoglossum bicirrhosum)性腺转录组测序获得的188 461条Unigene进行SSR检测,结果在11 917个Unigene中共检测到SSR位点12 578个,其中包含2个及以上SSR位点的Unigene有633个。SSR的发生频率为6.32%,平均分布距离为9 949 bp。在筛选到的SSR位点中,优势重复基序为二核苷酸、三核苷酸和四核苷酸,其中二核苷酸重复基序数量最多,占总数的50.52%。重复基元类型共216种,其中数量最多的类型为二核苷酸重复AC/GT,占总数的20.66%。从所有筛选出的SSR位点中随机选取50个位点并运用Primer Premier 5.0设计引物,共有25对引物成功扩增出特异性产物,并且均在同科的美丽硬仆骨舌鱼(Scleropages formosus)的三个亚种中通用。结果表明,转录组测序产生的EST序列是开发SSR标记的有效来源,开发所得EST-SSR引物可用于双须骨舌鱼及其近缘物种遗传分析、遗传图谱构建以及分子标记辅助育种等工作中。     

A genetic linkage map of the Japanese flounder, Paralichthys olivaceus

We report the first genetic linkage map of the Japanese flounder (Paralichthys olivaceus) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.     

基于微卫星标记整合长牡蛎遗传图谱

为了提高长牡蛎遗传图谱上的微卫星标记密度,实验采用6个家系图谱间的共有微卫星标记作为锚定标记,构建了长牡蛎的整合图谱.该整合图谱共有161个微卫星标记,覆盖10个连锁群,图谱长度和平均间距分别为615.4和3.8 cM.各连锁群的标记数介于10 ～ 24个之间,连锁群长度为47.3～73.3 cM,是目前密度最高的长牡蛎微卫星图谱.不同作图家系连锁群上的标记分组保持一致,但标记顺序出现差异,可能与长牡蛎自然群体中存在大量的染色体重排现象有关.结果表明,该图谱可以为今后长牡蛎的遗传育种研究提供新的遗传工具.     

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.