利用RAPD和ISSR引物对三系杂交水稻及其父母本的鉴定

为了鉴定杂交种子与父母本的亲缘关系，用20条随机引物和21条ISSR引物在供试的一组三系杂交水稻F1、父母本及对照进行扩增。20条RAPD引物共扩增出78个条带，其中28条为多态性条带；21条ISSR引物共扩增出72个条带，其中多态性条带为20条。将这些多态性条带进行聚类分析，F1与父、母亲缘关系较近归为一类．随后它们才与对照归为一类。结果表明RAPD和ISSR引物的PCR扩增均能有效地鉴定三系杂交水稻的杂种F1与组合中父、母本的遗传关系。与RAPD相比ISSR引物在三系杂交水稻及其父母本的鉴定中具有稳定性更好的优点。     

建鲤与黄河鲤的RAPD分子标记及其杂交优势的遗传分析

利用RAPD技术对建鲤、黄河鲤及其杂交子代进行了遗传分析,筛选的33个引物共扩增出155条带,其中多态性片段为105条,片段S18-1600、S472-300具有种的特异性,可作为鉴别建鲤与黄河鲤的分子遗传标记。建鲤、黄河鲤及其正、反交子代群体内的遗传相似系数分别为0.8240、0.7921、0.7920、0.8569,黄河鲤与正交F1具有较高的遗传变异水平,反交F1变异最小。正交F1、反交F1与亲本(建鲤、黄河鲤)的遗传距离分别为0.2233、0.2436、0.1749、0.2026,说明子代均继承了较多的建鲤的遗传物质。分析了子代与亲本的RAPD标记类型以及各类型的条带数。应用PIT标记系统测定了子代的杂种优势并探讨了优势产生的遗传机理。     

广东水稻纹枯病菌RAPD基因型与致病型关系

分析了广东水稻纹枯病菌(Rhizoctonia solani AG-1IA)致病型多样性和RAPD多态性之间的相互关系。结果表明,供试水稻纹枯病菌表现出较为丰富的致病型多样性(H=3.485)和遗传多样性,55个菌株接种9个水稻品种表现出39种致病类型。11条RAPD随机引物共扩增出91条谱带,其中多态性条带86条,多态率为94.51%。扩增出的22个选择性RAPD位点中,每个菌株都存在特有的RAPD分子指纹图谱。55个菌株在毒力数据、RAPD数据以及两者结合的数据聚类分析UPGMA树形图上形成几个不同的聚类类群,其中一个类群包括了大部分的菌株。毒力数据与RAPD分子数据的Jacarrd相似性距阵间的相关性系数比较低(r=0.0137),说明致病型与RAPD基因型性之间为相互独立的关系。     

三种笛鲷属鱼类的随机扩增多态性DNA初步研究

RAPD技术是由 Williams[1]和 Welsh[2 ]领导的两个科研小组于 1 990年几乎同时独立创立的一种 DNA多态性分析技术 ,它的原理是利用一系列随机排列的寡核苷酸单链引物 ,以研究对象的基因组 DNA为模板进行 PCR扩增 ,通过扩增产物 DNA片段的多态性来检测基因组 DNA的多态性。该技术已被广泛地应用于种质鉴定、遗传多样性、亲缘关系及系统分类研究 [3～ 6]。但对海水鱼类RAPD研究报道较少。红鳍笛鲷 (L utjanus erythopterus Bloch)、紫红笛鲷 (L utjanus argentimaculatus Forskal)和勒氏笛鲷 (L utjanus russelli Bleeker)具有…     

RAPD分析中最适样本量和位点数的研究

用25个引物,对40个样本扩增,研究了勒氏笛鲷随机扩增多态性DNA(RAPD)分析中的最适样本量和位点数。结果显示:在样本量达到20且位点数达到70个以上时,遗传距离趋于一个稳定的数值,即在RAPD分析中,样本数应达到20且位点数应达到70才能保证结果的可靠性。     

RAPD分析中最适样本量和位点数的研究

用25个引物，对40个样本扩增，研究了勒氏笛鲷随机扩增多态性DNA（啪）分析中的最适样本量和位点数。结果显示：在样本量达到20且位点数达到70个以上时，遗传距离趋于一个稳定的数值，即在RAPD分析中，样本数应达到20且位点数应达到70才能保证结果的可靠性。     

马氏珠母贝近交与杂交家系的DNA甲基化多态性比较

采用甲基化敏感多态性扩增技术(MSAP)检测马氏珠母贝(Pinctada fucata martensii)近交(F1)与杂交(Z1)家系闭壳肌的DNA甲基化修饰水平,对部分甲基化修饰片段进行回收、测序和注释分析。结果表明:使用20对引物进行选扩增PCR,最终F1和Z1分别获得(632.20±22.58)和(588.60±25.09)条条带,差异显著(P0.05);F1和Z1之间的组织甲基化水平分别为(9.93±1.60)%和(8.04±1.25)%,差异显著(P0.05)。对回收片段进行注释,F1家系共获得9条甲基化修饰片段,其中8条为功能蛋白,分别为E3泛素连接酶、逆转录病毒多聚蛋白、神经肽受体、交叉结点核酸内切酶、磷酸糖器、苯丙氨酸解氨酶、隐花色素蛋白、脆性位点相关蛋白;Z1家系共获得5条甲基化修饰片段且均为功能蛋白,分别为脆性位点相关蛋白、神经元乙酰胆碱受体α亚基、无调性的同源蛋白、麦芽糖酶、甲基胞嘧啶双加氧酶。初步阐明马氏珠母贝近交家系F1与杂交家系Z1间的DNA甲基化修饰水平和部分具甲基化修饰位点。     

奥利亚罗非鱼微卫星位点的筛选

从GenBank上选取40对尼罗罗非鱼(Oreochromis niloticus)的微卫星引物,分别在奥利亚罗非鱼(Oreo-chromis aureus)(83系)、红色奥利亚罗非鱼、奥利亚罗非鱼(02系)和尼罗罗非鱼的基因组上进行扩增。40对引物中有37对(92.5%)可进行有效扩增,32对引物(80%)可检测到个体间等位基因的多态性。其中引物UNH168与奥利亚罗非鱼的性别相关,在雌性个体中可扩增出二条大小不同的特异带(分别为135和171 bp),在雄性个体中则只有一条(171 bp)。将特异条带回收、克隆并测序,结果显示雌雄个体中171 bp条带的序列完全相同,包括103bp的侧翼序列和34个CA重复,在雌性个体中获得的135 bp条带则只有16个CA重复。引物UNH846、UNH860和UNH995可鉴别奥利亚罗非鱼和尼罗罗非鱼,UNH890可鉴别出红色奥利亚罗非鱼。可见,大部分的尼罗罗非鱼微卫星位点存在于奥利亚罗非鱼中。     

全基因组重测序筛选弓背青鳉三亚群体性别遗传标记

【目的】开发弓背青鳉（Oryzias curvinotus）三亚群体（下称“三亚青鳉”）遗传性别鉴定分子标记,为进一步挖掘其性别决定基因奠定基础。【方法】用染色体商（Chromosome quotient,CQ）法比较雌雄两性个体的全基因组重测序覆盖度,筛选潜在的性别特异性序列区域（CQ <0.1）并设计引物,通过PCR扩增验证引物区分三亚青鳉群体及其子代性别的适用性。【结果】经CQ分析筛选,CQ <0.1的特异性区域279个,设计引物279对。随机挑选的60对引物中,29对标记引物在三亚青鳉群体雄鱼中扩增得一个DNA条带,雌鱼无条带,均可鉴定三亚青鳉遗传性别。从29对引物中随机挑选2对引物（命名为Marker1和Marker2）进一步验证,引物Marker1、Marker2在雄鱼中扩增产物分别为791、556 bp,两对引物对三亚青鳉子代群体的扩增结果与亲本一致。用两对性别特异性标记对三亚群体的F2代全同胞家系94个个体进行扩增,结果发现F2群体自然性别比约1:1。【结论】性别特异性引物Marker1和Marker2均可对三亚青鳉群体进行遗传性别鉴定,在三亚青鳉野生亲本及...     

运用RAPD技术对笛鲷属3种鱼的群体遗传学分析

应用RAPD技术,从9个系列180种随机引物中筛选出16种,对笛(鱼周)属的画眉笛(鱼周)(Lutja-nus vitta)、金焰笛(鱼周)(Lutjanus fulviflamma)、金带笛(鱼周)(Lutjanus vaigiensis)进行种群内及种群间遗传学分析,并利用UPGMA确定了它们之间的亲缘关系。结果表明,三种鱼的种内遗传多样性指数(H)分别为,画眉笛(鱼周)0.1949,金带笛(鱼周)0.1107,金焰笛(鱼周)0.1673;三者的多态位点比例(P)分别为69.4％、47.8％和59.0％;金带笛(鱼周)具有较低的遗传多样性,且全带笛(鱼周)与金焰笛(鱼周)有较近的亲缘关系。三种鱼共检出10个可作为种的特异性鉴定的条带,可用于种质鉴定。     

Assessing the Germplasm of Laminaria （Phaeophyceae） with Random Amplified Polymorphic DNA （RAPD） Method

Eighteen gametophytes including L.japonica ,L.ochotensis and L.longissima,were verified with random amplified polymorphic DNA (RAPD) technique.Eighteen ten-base primers were chosen from 100 primers selected for final amplification test.Among the total of 205 bands amplified ,181(88.3%) were polymorphic ,The genetic distance among different strains ranged from 0.072 to 0.391,The dendrogram constructed by unweighted pair -group method with arithmetic(UPGMA) method showed that the female and male gamtophytes of the same cell lines could be grouped in pairs respectively,It indicated that RAPD analysis could be used not only to distin-gusih different strains of Laminariz ,but also to distinguish male and female gametophyte within the same cell lines,There is ambiguous systematic relationship if judged mereely by the present data It seems that the use of RAPD marker is limited to elucidstion of the phylogenetic relationship among the species of Laminaria.     

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria. Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences. This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090).     

Genetic diversity analysis with ISSR PCR on green algae Chlorella vulgaris and Chlorella pyrenoidosa

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR (ISSR PCR). Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity (h *) and Shannon index of diversity (I *) in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones (0.190 3 and 0.274 8), which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Identification of Phase and Sex-related ISSR Markers of Red Alga Gracilaria lemaneiformis

Six ISSR primers are employed to display the polymorphism of different phases and sexes of red alga Gracilaria lemaneiformis, and two of them, P1 and P3, amplified distinct band patterns. The ISSR pattern amplified by primer P1 of the female gametophyte is identical to that of tetrasporophyte, but distinct from that of male gametophyte. Of the bands produced by primer P3, one is specific to female gametophyte. Three morphologically similar fronds can be easily identified using ISSR technique. Two specific markers, SM1 and SF3, related to male gametophyte and female gametophyte, are cloned and sequenced. The homologous sequences of SM1 are found to encode a hypothetical protein. There is no homologous sequence of SF3 that can be found in GenBank.     

Invasion and spreading of Cabomba caroliniana revealed by RAPD markers

Applying randomly amplified polymorphic DNA （RAPD）, the genetic variation of Cabomba caroliniana Gray （cabomba or fanwort）, a new alien plant in China, was analyzed in this paper. Total 143 bands, including 47 polymorphic bands, were amplified from 23 primers in 20 samples. The sampling distance was large, but its genetic diversity was low. The main results were that： （1） Cabomba, which grew and dispersed mainly in fragment, was an abundant and dominant species in freshwater, and its main dispersal mechanism was vegetative reproduction. （2） Cabomba was originally introduced into China as an aquarium submerged plant. Somehow, those discarded cabomba became invasive species in the areas of Hangzhou, Shanghai, and Meicheng, and other places. （3） Although the level of genetic diversity in cabomba was low, their rapid dispersion and propagation could seriously harm to local aquatic community. Therefore, specific measure should be used to control cabomba from uncontrolled spreading and damage to local vegetation communities.     

Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Invasion and spreading of Cabomba caroliniana revealed by RAPD markers

Applying randomly amplified polymorphic DNA (RAPD), the genetic variation ofCabomba caroliniana Gray (cabomba or fanwort), a new alien plant in China, was analyzed in this paper. Total 143 bands, including 47 polymorphic bands, were amplified from 23 primers in 20 samples. The sampling distance was large, but its genetic diversity was low. The main results were that: (1) Cabomba, which grew and dispersed mainly in fragment, was an abundant and dominant species in freshwater, and its main dispersal mechanism was vegetative reproduction (2) Cabomba was originally introduced into China as an aquarium submerged plant. Somehow, those discarded cabomba became invasive species in the areas of Hangzhou, Shanghai, and Meicheng, and other places. (3) Although the level of genetic diversity in cabomba was low, their rapid dispersion and propagation could seriously harm to local aquatic community. Therefore, specific measure should be used to control cabomba from uncontrolled spreading and damage to local vegetation communities. Supported by National Natural Science Foundation of China (No. 39970145) and Major State Basic Research Development Program of China (No. G2000046803).     

Application of RAPD in genetic diversity study onGracilaria lemaneiformis III. Phase and sex related markers

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 random primers (P-1 to P-10) inGracilaria lemaneiformis. Phase and sex specific bands were amplified by primers P-2, P-6, P-7 and P-8: for P-2 a 1.4 kb band was found in female gametophytes and tetrasporophytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.76 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in female gametophytes and tetrasporophytes; for P-8, a 0.73 kb band only appeared in male gametophytes.