鹅膏菌属真菌RAPD分析及亲缘关系的研究

本文对采自湖南莽山的２６种鹅膏菌属（Ａｍａｎｉｔａ）真菌进行了随机扩增多态性ＤＮＡ（ＲＡＰＤ）分析,４０个随机引物中筛选出扩增效果较好的６个引物,每个引物能产生１～１０条ＤＮＡ条带,获得的ＲＡＰＤ谱带清晰并呈现多态性ＯＰＧ１５、ＯＰＨ０４两个引物扩增的ＲＡＰＤ谱带能将２６种鹅膏菌完全区分开来,通过６个引物的ＲＡＰＤ分析获得的平均相似性系数表明种与种之间的相关系数在２０－６０％之间,平均链锁聚类分析     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

RAPD应用于蕈菌研究中的条件优化探讨

在进行鹅膏菌属蕈菌遗传多样性的ＲＡＰＤ分析时,对ＲＡＰＤ分析过程中的ＤＮＡ提取方法、ＤＮＡ纯度、模板ＤＮＡ和ｄＮＴＰ浓度、循环次数、ＤＮＡ不同来源等影响因素进行了大量的实验探索,结果表明：ＤＮＡ不同提取方法具有相同的扩增产物,ＤＮＡ模板中的ＲＮＡ对扩增产物无影响,模板浓度在一个相当大的范围内（５０～４００μｇ）不影响扩增结果．ｄＮＴＰ浓度达０．７５ｍｍｏｌ／Ｌ时无带谱出现,引物浓度达１μｍｏｌ／Ｌ时出现非特异性带谱,从菌丝体和子实体中提取的ＤＮＡ可获得一致的扩增产物．扩增循环４０～４５周期条件下扩增效果较好,本实验证明了ＲＡＰＤ产物具有很好的重复性,建立了适合蕈菌ＲＡＰＤ分析的ＰＣＲ程序及条件,为ＲＡＰＤ应用于蕈菌的遗传研究打下了良好的基础．     

白鲢和镛鱼的扩增多态DNA分析

根据鱼类外周血细胞都有核的特点,采用从冷冻和低渗双重处理分离的细胞核的取基因组ＤＮＡ。以此法获得的白鲢和镛鱼的基因组ＤＮＡ为模板,和Ｏｐｅｒｏｎ公司生产的ＯＰＮ和ＯＰＭ两个组共４０个随机引物,对这两种鱼进行了随机扩增多态ＤＮＡ（ＲＡＰＤ）分析;确定了对这两种鱼基因组相关区域可进行随机ＰＣＲ扩增的有效引物,特别是哪些可产生种群内或群体的ＲＡＰＤ遗传标记即可产生个体特异性和群体特异性ＲＡＰＤ带谱的引物     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究(英文)

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

多孢节丛孢DNA随机扩增多态性及变种分析

本文对少孢节丛孢的１０个菌株进行了胡机扩增多态性ＤＮＡ（ＲＡＰＤ）分析。４个随机引物获得的ＲＡＰＤ谱带并呈多态性,单个引物获得的ＲＡＰＤ片段数在２～１３个之间。供试的１０个菌株来自不同的地方,形态差异较大,但四个随机引物的扩增结果基本上都体现了少孢节丛孢种的特征“指纹”,基于遗传距离分析和应用ＵＰＧＭＡ方法构建的分子系统树显示,少孢节丛孢不同地理群体间不存在遗传分化,提示在少孢节丛孢群体中没有显著     

冬虫夏草的随机扩增多态DNA及其遗传分化

本文对来自青藏高原３个区域５个具有代表性地方的１３个冬虫夏草样本进行随机扩增多态ＤＮＡ（ＲＡＰＤ）分析。１９个随机引物获得的ＲＡＰＤ谱带清晰并呈现多态,单个引物获得的ＲＡＰＤ片段数在３￣１０个之间。该１９个引物在每个样本中扩增的ＲＡＰＤ片段总数平均约为６５个。基于遗传距离分析,受试的１３个冬虫夏草样本中,来自同一地方的样本间遗传差异甚微,同一区域不同地方的样本间遗传差异较大,不同区域的样本间遗传差     

随机扩增多态性对白色念珠菌分型的估价

利用随机扩增多态性（ＲＡＰＤ－ＰＣＲ）的方法对４８株白念珠菌Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ Ｂｅｒｋｈ进行了分析。由初步试验中,随机选用了１８种引物?筛选出ＯＰＡ－１４引物,该引物扩增的带型清晰可辨,不同菌株之间扩增的带数６－１２条不等,共有６条主带。扩增片段的长度粗略估计在３００－２０００ｂｐ左右。除个别菌株的带型相同以外,大多数菌株之间呈多态性分布,其带型数目和扩增的片段存在差异。对简单的Ｄ     

烟草黑胫病菌株亲缘关系的RAPD分析

从２２０个ＲＡＰＤ（Ｒａｎｄｏｍ Ａｍｐｌｉｆｉｅｄ Ｐｏｌｙｍｏｒｐｈｉｃ ＤＮＡｓ）随机引物中所选出的多态扩增性强的２１个引物对来源不同的３３个烟草黑胫病菌株进行全基因组ＤＮＡ遗传多样性分析和指纹构建。选用引物对受试菌株进行ＲＡＰＤ－ＰＣＲ扩增,共产生２４３条ＤＮＡ标记图带,其中１９１条为多态性图带,多态检测率为７８．６％。利用ＵＰＧＭＡ（Ｕｎｗｅｉｇｔｈｔｅｄ Ｐａｉｒ－ｇｒｏｕｐ Ｍｅｔｈ     

利用RAPD技术对木耳属菌株进行分类鉴定的研究

利用ＲＡＰＤ技术对木耳属不同种和种内不同菌株进行了分子鉴定。结果表明,在供试的２６个随机引物中,有１０个引物可扩增出清晰、稳定的ＤＮＡ带型,其中引物Ｓ４和Ｓ６与供试木耳属菌株的基因组ＤＮＡ结合位点少而保守,扩增图谱具有种的特异性,可用于种的快速准确鉴定;其综随机引物的扩增ＤＮＡ多态性较强,可用于种内不同菌株的鉴定;聚类分析表明,在７５％相似水平下,可将供试的木耳属菌株分成四大类,其中有三大类各代表一个形态鉴定的种。研究结果表明了ＲＡＰＤ技术可有效地用于木耳属种或菌株的快速准确鉴定。     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

AN EVALUATION OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) FOR THE IDENTIFICATION AND PHYLOGENY OF FRESHWATER SNAILS OF THE GENUS BULINUS (GASTROPODA: PLANORBIDAE)

The Random Amplified Polymorphic DNA (RAPD) assay was used tostudy genetic variation within and between 9 species of thegenus Bulinus and to determine whether RAPD profiles could beused as markers for identification purposes. RAPDs were generatedwith 8 primers of two different sizes (l0mers & 15mers)and were visualised using both polyacrylamide gel electrophoresis(PAGE) with silver staining and agarose gel electrophoresiswith ethidium bromide staining. The species groups of Bulinushad few similarities in their RAPD profiles and there was interspecificvariation within groups. Intrapopulation variation was observed,with all primers, for B globosus collected from a single sitein Zimbabwe PAGE/silver staining methods visualised a greaternumber of RAPDs in comparison with agarose/ethidium bromidemethods. Phenetic analysis indicated that distance estimatesbetween taxa were sometimes non-additive and the phylo-geneticanalysis of such non-metnc data is discussed. The resultantphenograms, constructed using a least squares method, were constrainedalmost into a polytomy with topologies often differing betweendata sets. It was concluded that this phenomenon was most likelyattributable to large nucleotide divergences between the speciesgroups which go beyond the phylogenetic scope of RAPD analysis.RAPD profiles, when used in conjunction with other taxonomicmethods, may contribute to the identification of species ofBulnus on a regional basis, but the observed variability ina natural population suggests that a diagnostic RAPD profilefor each species throughout its geographic range is unlikely. (Received 19 April 1995; accepted 1 September 1995)     

Biochemical and Molecular Characterization of Some Centaurea Species Growing in the Eastern Anatolia Region of Turkey

Random amplified polymorphic DNA (RAPD) and fatty acid (FAME) profiles were used to examine phenotypic and genetic relationships among 16 Centaurea species growing wild in the eastern Anatolia region of Turkey. Thirteen decamer primers were used to examine polymorphism. According to the RAPD results, 99 amplicons in the size range of 50–1000 bp were produced from 13 primers in 16 Centaurea species. Genetically four distinct groups were determined among the species of Centaurea, which represents high genetic variation. In the 16 species, 14 fatty acids were determined according to FAME results. Both FAME and RAPD results showed that C. virgata is genetically different from other species. The differences in the composition of fatty acids among Centaurea species suggest that fatty acid profiles could be used to differentiate among some of these species. Results of this study show that RAPD and FAME analyses are consistent.     

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.     

Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve Salmonella Serotypes

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

稻恶苗菌异核体三种核型核DNA的RAPD分析

利用RAPD技术,对稻恶苗菌TJ47-26异核体的紫、红、白三种核型(同核体)核DNA进行比较,用80种10bp长的随机引物进行DNA扩增,共检测了基因组中近400个位点,未发现三种核型的基因组间有明显差异,表明紫、红、白三种核型的DNA碱基序列高度相似。由此推测TJ47-26异核体的三种不同核型是来自同一个核的变异体。     

26种冬青属植物遗传多样性分析

以26种冬青属植物种质资源为研究材料,利用RAPD和AFLP技术对基因组DNA进行扩增,以研究其物种间遗传多样性以及亲缘关系.结果表明:在RAPD分析中,从400条10个碱基的寡核苷酸引物中筛选出反应稳定、扩增性强、重复性好的引物20个,共扩增出312条多态性条带,多态率为95.41%;聚类分析显示26种冬青属植物间,布利奥特夫人枸骨叶冬青和黄果在AFLP分析中,10对选择性引物组合均扩增出了丰富的多态性片段,共扩增出350条谱带,其中336条具有多态性,占95.96%.综合RAPD和AFLP聚类结果,枸骨、无刺枸骨和日拉斯纳尔逊枸骨的亲缘关系较近,钝齿冬青、金宝石钝齿冬青和龟甲冬青三者的亲缘关系较近,可为冬青属植物的杂交育种与种质创新提供理论依据.