曲霉菌的RAPD分析及其在酿造工业中的应用

以米曲霉沪酿3.042(AS3.951)、黄曲霉GIM3.18、酱油曲霉AS3.495为参照,利用RAPD分子标记技术对16株曲霉菌进行系统发育分析。通过改进提取方法,获得了质量较好的模板DNA,凝胶电泳结果和分光光度法检测结果表明其适合用于进一步的RAPD-PCR试验。从9个待选引物中筛选到3个扩增产物谱带多、特征好、覆盖面广的引物:Primer1、Primer2、Primer5,重复实验证明其RAPD-PCR扩增图谱具有较好的稳定性,扩增产物谱带一般8~14条,各试验菌株主带4~9条,次带丰富。由此构建的系统进化树较好地吻合了传统的形态分类学,证实了RAPD分子标记技术在此类微生物系统发育分析中应用的可行性,也为酿造工业中检出产黄曲霉毒素的污染菌株提供了理论基础。     

利用RAPD标记分析东亚地区桔梗的亲缘关系

对取自中国、日本、韩国和朝鲜等东亚地区的24个桔梗种质资源,利用RAPD-PCR标记方法,分析了其遗传变异和遗传关系,旨在为桔梗的品种鉴定和杂交育种的亲本选择提供理论依据。结果表明： （1）用11个引物扩增出131条谱带,平均每个引物扩增出11.9条谱带,并扩增出61个多态性谱带,平均每个引物扩增出5.5条多态性谱带,显示出了相对高的多态率（46.6%）。（2）得到的RAPD数据的遗传相似性范围为0.668～0.994,并以遗传相似系数0.78为标准,将24个桔梗种质资源分为7个类群。     

Identification and phylogenetic analysis of Lactobacillus using multiplex RAPD-PCR

Multiplex RAPD-PCR was used to generate unique and identifying DNA profiles for isolates of the genus Lactobacillus. The method that was used was based on the combination of two 10-mer oligonucleotides in a single PCR. The generated RAPD profiles enabled discrimination of all lactobacillus strains that were used in this study. A dendrogram was generated from the RAPD profiles. The results of genetic relatedness obtained from the dendrogram were compared with the results obtained using carbohydrate fermentation profiles. Most of the gastrointestinal isolates studied could not be grouped using carbohydrate fermentation profiles. The RAPD profiles provided sufficient information to prepare a dendrogram of genetic relatedness. The gastrointestinal isolates were clustered together on the dendrogram. Furthermore an isolate originating from the stomach (strain ML004) was closely related to Lactobacillus fermentum. It was concluded that multiplex RAPD-PCR was useful for characterisation and inference of relatedness of Lactobacillus isolates.     

Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Genetic variation in an introduced aphid pest (Metopolophium dirhodum) in New Zealand and relation to individuals from europe

RAPD-PCR was used to determine the genetic variation of Metopolophium dirhodum collected in a winter wheat field and in a nearby 2.5-m-high suction trap at Lincoln, New Zealand. Over three collection dates, five distinct genotypes were identified, using two primers (OPK16 and OPC09) independently. There was a significant temporal effect on the ratio of genotypes in populations collected in the field. There was no significant spatial aggregation or association of these genotypes in the field. Two of the genotypes present in the field were also detected in the suction trap sample. Using a higher resolution method of RAPD-PCR (with the Stoffel fragment of Taq polymerase), a total of 124 genotypes were distinguished from 142 individuals collected from Scotland and New Zealand. The Jaccard similarity index ( S ) was used to measure similarity between individual aphids within and between populations from both hemispheres. All populations were very diverse ( S < 0.33). However, at similar crop growth stages, M. dirhodum was significantly more diverse in Scotland than in New Zealand. The results are discussed in relation to the value of monitoring aphid flights for pest forecasting, and in terms of the most appropriate RAPD-PCR techniques.     

Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data

Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD). Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands. Phenetic analysis based on a similarity matrix corresponded well with previous reports on X. fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X. fastidiosa. Cladistic analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.     

Differentiation of Aeromonas genomospecies using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to investigate the differentiation of the genus Aeromonas at the genomospecies level. Of 20 primers evaluated, six produced profiles which contained multiple bands capable of differentiating the genomospecies. These six primers were also used in RAPD-PCR analyses of clinical and environmental isolates of the different genomospecies. In most cases. each strain gave a unique fingerprint, illustrating genetic heterogeneity at the genomospecies level. However, some homogeneity in fragment sizes was seen among strains within a genomospecies which was not apparent in strains from different genomospecies. This study therefore complements and supports the current classification of Aeromonas into genomospecies. These results also show that RAPD-PCR has the potential to differentiate between the genomospecies of Aeromonas.     

RAPD-PCR analysis of genetic diversity in the Manchurian pheasant

The genetic diversity of a local population of the Manchurian pheasant Phasianus colchicus pallasi was studied using RAPD-PCR. Based on the DNA patterns obtained in PCR with five arbitrary decanucleotide primers, we assessed genetic polymorphism of this population, estimated genetic distances between individuals, and constructed an NJ phylogenetic tree, and an UPGMA dendrogram of genetic similarity. The population was shown to exhibit high average genetic polymorphism (P = 79.4%) and genetic distances (D = 0.267). Possible reasons for the high genetic diversity of this local population are discussed.     

Genetic diversity of the natural strains of Streptococcus thermophilus

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.     

利用RAPD-PCR技术分析东北地区16种小卷蛾的亲缘关系

利用RAPD-PCR技术对东北地区黄卷蛾族的16种小卷蛾的基因组进行随机扩增,构建出系统发育树,对其亲缘关系进行分析.系统树显示褐卷蛾属的3个种、条卷蛾属的两个种和黄卷蛾属的4个种的聚类方式和传统形态分类法基本一致.也发现了一些与传统分类结论不完全一致的现象,如同属的忍冬双斜卷蛾和棉花双斜卷蛾在系统树上没有聚在一起,而和不同属的松褐卷蛾、樱桃铅卷蛾聚在一起等.分析同属和不同属种间的遗传距离,初步得出以下结论,遗传距离0.7可以作为属的分类依据,同一属的种间遗传距离小于0.7,不同属的种间遗传距离大于0.7.     

Application of random amplified polymorphic DNA and inter-simple sequence repeat markers in the genus Crataegus

Hawthorn ( Crataegus spp.) has a long history as an ornamental and a source of medicine. We report the use of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers to determine genetic relationships in the genus Crataegus . Twenty-eight accessions, including eight species ( Crataegus pinnatifida , Crataegus bretschneideri , Crataegus maximowiczii , Crataegus kansuensis , Crataegus altaica , Crataegus songarica , Crataegus dahurica and Crataegus sanguinea ) and two botanical varieties ( C. pinnatifida var. major and C. maximowiczii var. ninganensis ) were analysed. Twelve RAPD primers reproducibly and strongly amplified 128 fragments of which 116 were polymorphic; similarly, 13 ISSR primers generated 127 products of which 119 were polymorphic. Dendrograms based on unweighted pair group method with arithmetic average analysis were constructed from both the RAPD and the ISSR data. Similarity coefficient based on RAPD and ISSR markers ranged from 0.22 to 0.98 and 0.23 to 0.98, respectively. The range in similarity coefficient indicated that the genus has a high level of genetic diversity. The Mantel test on the similarity matrices produced by RAPD and ISSR markers gave r  = 0.86, showing high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions. RAPD and ISSR appear to be reliable methods for the analysis of genetic relationships among hawthorns.     

Discrimination of three Typhlodromalus species (Acari: Phytoseiidae) using random amplified polymorphic DNA markers

We compared the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) banding patterns obtained from four laboratory cultures representing three phytoseiid mite species (Typhlodromalus limonicus (Garman and McGregor), two cultures of Typhlodromalus manihoti (de Moraes) and Typhlodromalus tenuiscutus (McMurtry and de Moraes). The RAPD-PCR was conducted on the pooled DNA from five adult female mites. For each culture, three samples of five females were analysed with each of eight RAPD-PCR primers. Five of the eight primers could be used individually to distinguish the species. To quantify the within- and between-species variation, genetic distances were calculated based on the proportion of shared scorable bands. The within-species genetic distances (0.072-0.186) were much lower than the between-species genetic distances (0.407-0.656). We believe that this technique could be used effectively to identify other cryptic mite species.     

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.     

Identification of Spanish barbel species using the RAPD technique

The RAPD-PCR technique was employed to identify three endemic Spanish species of Barbus: Barbus bocagei, B. graellsii and B. sclateri , that present very similar morphologies. Using seven primers, six diagnostic bands were found in B. bocagei , 11 in B. graellsii and nine in B. sclateri . Cluster analysis of the genetic similarity values obtained from RAPD data indicated that the species B. bocagei and B. graellsii are more related to each other than to B. sclateri .     

Comparative study of the population structure and population assignment of sockeye salmon Oncorhynchus nerka from West Kamchatka based on RAPD-PCR and microsatellite polymorphism

Using two types of molecular markers, a comparative analysis of the population structure of sockeye salmon from West Kamchatka as well as population assignment of each individual fish were carried out. The values of a RAPD-PCR-based population assignment test (94–100%) were somewhat higher than those based on microsatellite data (74–84%). However, these results seem quite satisfactory because of high polymorphism of the microsatellite loci examined. The UPGMA dendrograms of genetic similarity of three largest spawning populations, constructed using each of the methods, were highly reliable, which was demonstrated by high bootstrap indices (100% in the case of RAPD-PCR; 84 and 100%, in the case of microsatellite analysis), though the resultant trees differed from one another. The different topology of the trees, in our view, is explained by the fact that the employed methods explored different parts of the genome; hence, the obtained results, albeit valid, may not correlate. Thus, to enhance reliability of the results, several methods of analysis should be used concurrently.     

RAPD variation in Mediterranean turtle Testudo graeca L. (Testudinidae)

The polymerase chain reaction with arbitrary primers (RAPD-PCR) was used to study intraspecific variation in Mediterranean turtle Testudo graeca, which is represented by the Dagestan (T. g. pallasi) and Nikolskii (T. g. nikolskii) subspecies in Russia. To study the phylogenetic relationships, the RAPD variation was also compared in two other T. graeca subspecies (T. g. ibera and T. g. terrestris), two closely related Testudo species (T. kleinmanni and T. marginata), and Central Asian turtle Agrionemys horsfieldii. Parameters of RAPD variation showed that the sample from different geographical regions of Dagestan was more polymorphic and heterogeneous than that from Central Asia. The two samples differed in the mean number of RAPD fragments N (48.761 vs. 40.400), number of polymorphic fragments P (78.7 vs. 32.3), and within-group similarity index APS (0.607 vs. 0.784). In T. g. pallasi, no significant difference in N, P, or APS was observed between samples from different localities of Dagestan or between groups of turtles with four- or five-clawed forelegs. A dendrogram of genetic similarity between the species and subspecies under study contained two clusters, one comprising all A. horsfieldii individuals and the other, all turtles of the genus Testudo. In the latter, T. marginata and T. kleinmanni showed higher similarity to each other than to T. graeca. The four T. graeca subspecies clustered separately from each other with a high reliability, T. g. nikolskii and T. g. ibera (Turkey) being more similar to each other than to T. g. terrestris or T. g. pallasi. The possible causes of the presence of four claws on a foreleg and the relationships among members of the genus Testudo were discussed.     

Genetic Diversity within an Italian Population of Forest Armillaria gallica Isolates as Assessed by RAPD-PCR Analysis

The genus Armillaria includes harmful fungal pathogens that cause root rot and wood decay in a broad range of host plants throughout the world. The aim of this study was to detect, by means of Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers, the level of intraspecific variability within isolates of an Armillaria gallica population sampled from a Quercus spp. stand located in Gravina in Puglia, southern Italy. UPGMA cluster analysis of RAPD profiles generated by decamer primers grouped the isolates in subclusters demonstrating relatively low intraspecific genetic variability. Moreover, RAPD pattern analysis yielded clusters which did not correspond to the groups discriminated by vegetative compatibility tests performed by a previous investigation on the same population. The findings of this research pose the question of whether somatic incompatibility, which involves an undefined number of genes and alleles per gene, might still be considered an effective tool for the epidemiological studies of A. gallica , whereas molecular analyses are more useful for assessing genomic variation within the species.     

Comparative study of the population structure and population assignment of sockeye salmon Oncorhynchus nerka from West Kamchatka based on RAPD-PCR and microsatellite polymorphism

Using two types of molecular markers, a comparative analysis of the population structure of sockeye salmon from West Kamchatka as well as population assignment of each individual fish were carried out. The values of a RAPD-PCR-based population assignment test (94-100%) were somewhat higher than those based on microsatellite data (74-84%). However, these results seem quite satisfactory because of high polymorphism of the microsatellite loci examined. The UPGMA dendrograms of genetic similarity of three largest spawning populations, constructed using each of the methods, were highly reliable, which was demonstrated by high bootstrap indices (100% in the case of RAPD-PCR; 84 and 100%, in the case of microsatellite analysis), though the resultant trees differed from one another. The different topology of the trees, in our view, is explained by the fact that the employed methods explored different parts of the genome; hence, the obtained results, albeit valid, may not correlate. Thus, to enhance reliability of the results, several methods of analysis should be used concurrently.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

Genetic differentiation of subspecies of the house mouse Mus musculus and their taxonomic relationships inferred from RAPD-PCR data

Genetic differentiation of six subspecies of the house mouse Mus musculus (Mus musculus musculus. M. m. domesticus, M. m. castaneus, M. m. gansuensis, M. m. wagneri, and M. m. ssp. (bactrianus?) was examined using RAPD-PCR analysis. In all, 373 loci of total length of about 530 kb were identified. Taxon-specific molecular markers were detected and the levels of genetic differences among the subspecies were estimated. Different degree of subspecific genetic differentiation was shown. The most similar subspecies pairs were M. m. castaneus--M. m. domesticus and M. m. musculus--M. m. gansuensis. In our phylogenetic reconstruction, M. m. wagnery proved to be most different from all the other subspecies. Genetic distances between it and other subspecies were two- to threefold higher than those between the "good"' species of the subgenus Mus (e.g., between M. m. musculus and M. spicilegus, M. musculus and M. abbottii). The estimates of genetic similarity and the taxonomic relationships between six house mouse subspecies inferred from RAPD partially conformed to the results based on cytogenetic and allozyme data. However, they were considerably different from phylogenetic reconstructions based on sequencing of the control mtDNA region, which reflects mutual inconsistency of different systems of inheritance.