Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Molecular Characterization of the Plant Pathogen Verticillium dahliae Kleb. Using RAPD-PCR and Sequencing of the 18SrRNA-Gene

Thirty-four isolates of Verticillium dahliae Kleb. from nine different genera of dicotyledonous host plants and a broad range of geographic regions were analysed genotypically, Random amplified polymorphic DNA (RAPD) markers were used for the estimation of the genetic variability within the species. Using four primers for the analysis, 79 distinct fragments were obtained. The derived phenogram clustered the isolates in two main groups; one consisted almost entirely of V. dahliae isolates from oilseed rape ( Brassica napus napus ), the other group comprised isolates from a wide range of host plants. No correlation between geographic location of the isolates and the RAPD-pattern was observed.
Sequencing of the gene for the 18SrRNA and calculation of the phylogenetic tree integrated the deuteromycetous fungus V. dahliae into the sexual system of the filamentous ascomycetes.     

Characterization of Moroccan Isolates of Verticillium dahliaeKleb Using RAPD Markers

Isolates of Verticillium dahliae were sampled from different olive tree orchards in Morocco. These olive trees were located in different commercial culture locations in southern, central and northern Morocco. The isolates were characterized using genetic markers obtained after their DNA PCR amplification with random amplified polymorphic DNA (RAPD) primers. Among the 40 primers tested, 10 generated a total of 66 polymorphic fragments. Among the 38 isolates of V. dahliae tested, RAPD markers were successful in the characterization of groups based on their geographic origin. With the exception of one specific isolate, no correlation could be established among the isolates, based on the morphological appearance of the colony in culture.     

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

海南和两广地区柱花草炭疽菌遗传多态性的RAPD分析

利用8个随机引物对来自海南、广东和广西的114个柱花草胶孢炭疽菌(Colletotrichum gloeosporioides)的菌株进行了随机扩增多态性DNA(RAPD)分析, 扩增谱带大小0.2-3.0 kb。8个随机引物中以CW38114扩增的谱带重现性和稳定性最好, 共扩增出786条谱带, 其中多态性谱带558条。供试菌株扩增图谱在260-650 bp处有3条明显的共同特征谱带, 在650-3 000 bp之间的谱带显示较大的多态性差异。聚类结果表明: 供试菌株主要分为四大遗传类型, 其中第I类和第III类有两个亚类; 三省区的菌株遗传多态性丰富程度依次为海南>广西>广东, 并表现出地区性分布差异。研究结果还表明柱花草炭疽菌株表现出一定程度的专化寄生性。     

RAPD标记在紫菜遗传多样性检测和种质鉴定中的应用

用ＲＡＰＤ技术对４类紫菜（Ｐｏｒｐｈｙｒａ ｙｅｚｏｅｎｓｉｓ,Ｐ．ｈａｉｔａｎｅｎｓｉｓ,Ｐ．ｋａｔａｄｎｉ ｖａｒ．ｈｅｍｉｐｈｙｌｌａ和Ｐ．ｏｌｉｇｏｓｐｅｒｍａｔａｎｇｉａ）的１５个无性系丝状体进行了遗传多样履分析,从５０个ＯＰＥＲＯＮ引物中经过初筛,其中６个引物可以扩增出稳定的可重复的图谱。这６个引物共扩增出了６０条带,多态性比例达９７．１％。根据ＲＡＰＤ结果将这１５个无性了紫菜的ＤＮＡ     

The Application of RAPD Markers in Diversity Detection and Variety Identification of Porphyra

RAPD (random amplified polymorphic DNA) markers were generated from filaments of 15 Porphyra lines representing four important groups, P. yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia. Among the total 69 fragments generated by 6 selected primers (among 50 primers), 67 appeared to be polymorphic (97.1%). Cluster analysis based on the RAPD results was performed. The 15 Porphyra lines were divided into 3 groups. This result was consistent with that from taxonomy analysis. A DNA fingerprinting based on 8 bands amplified with OPN-02 and OPJ-18 was constructed and might be used in Porphyra variety identification. Five specific RAPD fragments of 5 Porphyra lines were isolated and cloned into pGEM-T easy vector. These five RAPD fragments may be useful in germplasm identification and property protection of Porphyra.     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Use of conserved randomly amplified polymorphic DNA (RAPD) fragments and RAPD pattern for characterization of Lactobacillus fermentum in Ghanaian fermented maize dough.

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.     

中国大豆疫霉菌遗传多样性的RAPD分析

采用随机扩增多态性DNA(RAPD)技术,利用13个引物对75个中国大豆疫霉菌分离物和11个美国分离物进行PCR扩增。在78个RAPD标记中,多态性标记为68个,占87.2%。RAPD指纹聚类分析表明,当以相异距离0.3为阈值,86个分离物被划为12个RAPD遗传组,其中J组有54个分离物,占总数的62.8%,包括44个中国分离物和10个美国分离物。在中国大豆疫霉菌群体内,多数分离物之间遗传相似性较低,在DNA水平上存在显著的遗传变异,具有较丰富的遗传多样性。RAPD分组结果未表明大豆疫霉菌DNA多态性特征与病原菌毒力基因构成之间和分离物地理来源之间存在相关性,证明中国不同地区的大豆疫霉菌群体在与大豆品种的互作中发生了广泛的遗传变异,具有DNA遗传进化方向和毒力基因演变的多样性。美国大豆疫霉菌分离物间遗传距离较近,而中国分离物在总体上与美国分离物的遗传距离较远,表明中国大豆疫霉菌具有比较独特的遗传背景。     

中国大豆疫霉菌遗传多样性的RAPD分析*

采用随机扩增多态性DNA(RAPD)技术,利用13个引物对75个中国大豆疫霉菌分离物和11个美国分离物进行PCR扩增。在78个RAPD标记中,多态性标记为68个,占87.2%。RAPD指纹聚类分析表明,当以相异距离0.3为阈值,86个分离物被划为12个RAPD遗传组,其中J组有54个分离物,占总数的62.8%,包括44个中国分离物和10个美国分离物。在中国大豆疫霉菌群体内,多数分离物之间遗传相似性较低,在DNA水平上存在显著的遗传变异,具有较丰富的遗传多样性。RAPD分组结果未表明大豆疫霉菌DNA多态性特征与病原菌毒力基因构成之间和分离物地理来源之间存在相关性,证明中国不同地区的大豆疫霉菌群体在与大豆品种的互作中发生了广泛的遗传变异,具有DNA遗传进化方向和毒力基因演变的多样性。美国大豆疫霉菌分离物间遗传距离较近,而中国分离物在总体上与美国分离物的遗传距离较远,表明中国大豆疫霉菌具有比较独特的遗传背景。     

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.     

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.     

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.     

Host-based genetic differentiation in the aphid Aphis gossypii Glover, evidenced from RAPD fingerprints

Samples of the aphid Aphis gossypii (Glover) were collected from different host plants at 18 locations in southern France, La Réunion, Portugal and Laos. RAPD (random amplified polymorphic DNA) patterns of the 480 aphids were obtained using three random primers. A large number of RAPD bands were shared by all aphids of the 18 populations, but some RAPD bands appeared to be population specific. Over all aphids, a total of 37 polymorphic bands were identified and defined 142 RAPD phenotypes. A cluster analysis based on genetic distance revealed that the 18 aphid populations were divided into two groups, depending on whether they were collected on a cucurbit host plant. An analysis of molecular variance ( AMOVA ) was also performed and confirmed the differentiation into two groups. Several RAPD bands that were obtained using random primer A11 could be considered as diagnostic loci as they were fixed in populations collected on cucurbits and were always absent in those collected on noncucurbit host plants. These results represent the first evidence for genetic structuring within the species A. gossypii , according to host-plant type.     

RAPD markers associated with quercetin accumulation in Psidium guajava

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。