小麦品种Triticum spelta album中抗条锈病基因Yr5的RAPD标记

共用520个10碱基随机引物对小麦抗条锈基因Yr5的近等基因系进行了RAPD分析,发现了3个特异性DNA片段S1496、S14181950与Yr5基因连锁,其中S1496761与Yr5基因紧密连锁,遗传距离为2．7cM。经对特异性DNA片段S1496 761进行克隆,测序,设计了PCR扩增用专化引物SC－S1496 761a和SC－S149676b,用该引物可扩增出与原RAPD引物扩增出的相似的特异DNA片段,由于该引物还可扩增出迁移率极为相近的另1条非特异带,在琼脂糖凝胶上难以分辨,需用聚丙烯酰胺凝胶电泳结合银染进行检测,经用F2分离群体及部分相关品种材料检测,已证明该标记的可靠性。     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

甘蓝品种'争春'和'寒光2号'的DNA指纹图谱构建

用SDS法提取甘蓝（Brassfca oleraceavat．capitata）品种‘争春’、‘寒光2号’及其各自亲本的基因组DNA,通过SRAP、RAPD两种分子标记方法,构建其DNA指纹图谱,用于种子纯度鉴定。利用30对SRAP引物组合和200个RAPD随机引物,以各品种及其亲本的基因组DNA为模板组进行筛选,结果显示：多数SRAP引物组合对模板组的扩增带型一致,少数组合扩增出差异,但未能找到具有互补差异的引物组合;通过RAPD标记方法筛选出能鉴定2个品种纯度的引物分别为S42、S103、S193和S42、S89、S151,其中引物S42对2组材料均能扩增出特异的RAPD指纹图谱,并将RAPD指纹图谱转变为相应的数字指纹。     

利用RAPD分析鉴定花椰菜杂种纯度

应用随机扩增多态性DNA(RAPD)分析方法,鉴定花椰菜F1代杂种纯度。筛选出20个10bp随机引物对杂种F1代和父母本基因组DNA进行RAPD分析,共获得扩增片段124条,分子量在0.3-3kb之间,其中2个引物S120和S174可用来鉴定杂种纯度。RAPD分析结果与田间形态鉴定结果基本一致,表明RAPD分析方法适用于花椰菜杂种的纯度鉴定。     

外源DNA导入小麦的RAPD验证及遗传分析

将外源DNA特别是牛胸腺DNA转移给普通小麦,对在其后代中选育出的矮杆变异体进行了RAPD分子验证及其遗传学分析研究,获得了3个主要结果：（1）对变异体D111,受体814527,供体矮孟牛I型进行的RAPD验证中,通过137个引物由5个引物检测出了DNA的多态性,表明矮秆变异体D111的出现可能是供体的片段DNA进入了受体并得到稳定遗传。（2）对变异体D011,D1453,受体814527,供体牛胸腺DNA进行的RAPD验证中,在供试的180个引物中,变异体,受体扩增产物的带型绝大多数一致而与供体则完全不一致,其中有很少引物对变异体和受体扩增产物的带型表现不同,还有个别引物对变异体扩增产物的带型与供体的个别带一致,由此说明亲缘关系极远的牛胸腺DNA导入受体引起的变异更为广泛和复杂;（3）3个矮秆变异体的遗传分析表明,控制其株高的基因位于核基因组,在杂种后代中表现了明显降低株高的作用,其杂种后代的某些农艺性状也表现良好,因此,利用它们进行杂交育种或在杂种优势的利用上,都将是有较好应用价值前途的新矮源。     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

两个地区东方田鼠基因组RAPD分析比较研究

目的　从DNA的水平分析比较两个地区东方田鼠的分子遗传特征,探讨以RAPD标记鉴别两个地区的东方田鼠。方法　筛选6条10bp的随机引物对洞庭湖和青铜峡地区的东方田鼠基因组进行了随机扩增多态DNA(RAPD)分析,并对这两个地区的东方田鼠的基因组DNA进行了比较。结果　①两个地区东方田鼠的所有受试个体中共有的片段数为20条,这是两个地区东方田鼠的共性所在;②两个地区东方田鼠各有其特异性扩增片段;③引物S17和S80可作为鉴别两个地区东方田鼠的特异性引物;④不同地区的东方田鼠其不同个体之间的共享度较低,且存在较大差异;两个地区东方田鼠的遗传背景均呈非均一性。结论　运用RAPD方法可以作为鉴别不同地区东方田鼠的基因多态性的标记。     

沙梨8个栽培品种的DNA指纹鉴定

利用RAPD标记技术,对8个沙梨(Pyrus pyrifolia)主栽品种进行RAPD分析。结果显示,从33个10碱基随机引物中筛选出了2个多态性高的引物S2075和S1296,扩增位点分别为11个和10个,以此为基础建立了8个品种的DNA指纹图谱;根据这2个引物中任何一个的扩增图谱均可以将这8个品种区分。研究结果为沙梨品种的区别与鉴定提供了一种有效方法。     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

随机扩增多态性DNA技术对结核分支杆菌菌株鉴别及其耐药相关性分析

用引物G1和L1扩增的16S-23SrDNA间隔区作为模板DNA,再用引物AP50进行随机扩增多态性DNA（Random Amplified PolymorphicDNA,RAPD)分棉线AP50扩增出的各菌株DNA片段,用1.2%的琼脂糖凝胶电泳EB染色,可获得1或2个大小不同的DNA片段（390-1031bp)。各株间差异明显,结果表明,不同菌株有不同的DNA多态性,药物敏感株与耐药株之间多态性差异明显,耐药株中耐药谱相似或相同的菌株间其多态性也相似,RAPD有助于简便,快速,有效地了解菌株间的克隆相关性及耐药性传播。     

应用DNA指纹图谱技术鉴定整肠生菌株BL63516的研究

目的建立应用DNA指纹图谱技术鉴定微生态制剂——整肠生菌株BL63516的方法,提高菌种鉴别水平。方法应用RAPD（随机扩增多态性）方法,采用50条随机引物对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,选择多态性好、重复性好、稳定性强的随机引物,对BL63516与其他地衣芽胞杆菌进行区分。结果发现选用引物$87或$88分别对7株地衣芽胞杆菌进行基因组DNA指纹图谱分析,BL63516菌株扩增的DNA片段的大小、数量均与其他地衣芽胞杆菌有明显差异。结论此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂整肠生菌株的鉴别。     

Clonal relations among Salmonella enteritidis phage type 3 outbreak isolates traced by DNA fingerprinting.

Isolates of Salmonella enteritidis PT3, a rare phage type, were recovered from patients and strains were isolated from an outbreak of gastroenteritis that occurred during the summer of 1997 in North-East Sardinia, Italy. To investigate possible clonal involvement in the outbreak and to evaluate the capacity to discriminate among S. enteritidis PT3 strains, a number of molecular typing methods including ribotyping with a mixture of PstI and SphI (PS-ribotyping), PFGE with endonuclease XbaI and RAPD typing with four arbitrary primers was used. The typical XbaI endonuclease generated PFGE pattern also explained the prevalence of highly clonal S. enteritidis PT3 strains in the outbreak and adjacent areas. RAPD fingerprinting with primers OPA 4, OPB 15, OPB17 and P1254 exhibited a single but unique RAPD profile among the outbreak strains from various sources that differed significantly from control strains. The results of this study showed that when an appropriately chosen set of primers is employed, RAPD fingerprinting can be used as an alternative, rapid, highly reproducible technique for tracing the clonal relations of S. enteritidis PT3, and can be more discriminatory than PFGE. Furthermore, this study revealed the possibility of PT3 causing outbreak.     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

Application of randomly amplified polymorphic DNA (RAPD) analysis for typing avian Salmonella enterica subsp. enterica

Randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular genetic typing of 30 Salmonella enterica subsp. enterica strains isolated from chickens and ducks in Thailand. Six different primers were tested for their discriminatory ability. While some of the primers could only differentiate between the different serovars, the use of multiple primers showed that the RAPD method could also subdivide within a given serovar. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, we suggest that it may be a valuable new tool for studying the molecular genetic epidemiology of S. enterica ssp. enterica, both inter- and intra-serovars.     

应用RAPD技术鉴定地霉的实验研究

目的探讨RAPD技术在快速鉴定地霉中应用。方法用E.Z.N.A.yeastDNAkit提取地霉菌基因组DNA,采用随机引物AP3(5'-TCGTAGCCAA-3')、ATG(5'-ATGGATCGGC-3')、RP2(5'-AAGGATCAGA-3')、OPA-10(5'-GTGATCGCAG-3')对临床上致病性白地霉、林生地霉皮损株和血液株的基因组DNA进行扩增,对各病原菌的DNA指纹的特征进行分析。结果成功提取了地霉的基因组DNA,其纯度和浓度均能满足PCR反应的要求。利用4种引物对基因组DNA进行扩增,不同种真菌的DNA显示不同的DNA带型,分离自不同感染部位的同种不同株真菌的DNA显示的主要DNA带型基本一致。结论采用E.Z.N.A.yeastDNAkit提取的地霉基因组DNA可以用于PCR反应。RAPD法鉴定地霉菌简单、快速、特异,可用于临床诊断。     

Identification, mapping, and application of polymorphic DNA associated with resistance gene Pm21 of wheat.

A new powdery mildew resistance gene designated Pm21, from Haynaldia villosa, a relative of wheat, has been identified and incorporated into wheat through an alien translocation line. Cytogenetic and biochemical analyses showed that chromosome arms 6VS and 6AL were involved in this translocation. Random amplified polymorphic DNA (RAPD) analysis was performed on recipient wheat cultivar Yangmai 5, the translocation line, and H. villosa with 180 random primers. Eight of the 180 primers amplified polymorphic DNA in the translocation line, and the same results were obtained in four replications. Furthermore, RAPD analysis was reported for substitution line 6V, seven addition lines (1V-7V), and the F1, as well as F2 plants of (translocation line x 'Yangmai 5'), using two of the eight random primers. One RAPD marker, specific to chromosome arm 6VS, OPH17-1900, could be used as a molecular marker for the detection of gene Pm21 in breeding materials with powdery mildew resistance introduced from H. villosa. Key words : RAPD analysis, 6VS-specific marker, Pm21, Erysiphe graminis f.sp. tritici, Triticum aestivum - Haynaldia villosa translocation.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.