scholarly journals A DNA-Based Procedure for In Planta Detection of Fusarium oxysporum f. sp. phaseoli

2002 ◽  
Vol 92 (3) ◽  
pp. 237-244 ◽  
Author(s):  
Fernando M. Alves-Santos ◽  
Brisa Ramos ◽  
M. Asunción García-Sánchez ◽  
Arturo P. Eslava ◽  
José María Díaz-Mínguez
Keyword(s):  
Common Bean ◽  
Fusarium Oxysporum ◽  
Scar Marker ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Rapd Marker ◽  
In Planta ◽  
Sequence Characterized Amplified Region ◽  
Polymerase Chain ◽  
Highly Virulent

We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of ≥4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.

scholarly journals New Virulence Groups in Fusarium oxysporum f. sp. phaseoli: The Expression of the Gene Coding for the Transcription Factor ftf1 Correlates with Virulence

2011 ◽  
Vol 101 (4) ◽  
pp. 470-479 ◽  
Author(s):  
José J. de Vega-Bartol ◽  
Raúl Martín-Dominguez ◽  
Brisa Ramos ◽  
María-Asunción García-Sánchez ◽  
José María Díaz-Mínguez
Keyword(s):  
Transcription Factor ◽  
Common Bean ◽  
Fusarium Oxysporum ◽  
Quantitative Polymerase Chain Reaction ◽  
In Planta ◽  
Gene Encoding ◽  
Runner Bean ◽  
Bean Plants ◽  
Highly Virulent ◽  
Common Bean Plants

Fusarium oxysporum f. sp. phaseoli strains isolated from runner bean plants showing Fusarium wilt symptoms were characterized. The analysis of the genetic diversity of these strains and the comparison with strains formerly isolated from diseased common bean plants grown in the same region of Spain indicated a close genetic similarity among them. Pathogenicity assays carried out on runner bean plants showed virulence differences that allowed the classification of these strains into three groups: super virulent, highly virulent, and weakly virulent. However, all the analyzed strains behaved as highly virulent when inoculated on common bean plants, indicating that virulence is specific of the host–pathogen interaction. We also analyzed the number of copies and expression of the gene encoding the transcription factor ftf1, which has been shown to be specific of virulent F. oxysporum strains and highly up-regulated during plant infection. In planta real-time quantitative polymerase chain reaction expression analysis showed that expression of ftf1 was correlated with the degree of virulence. The comparative analysis of the polymorphic copies of ftf1 detected in the strains here characterized and those detected in the genome sequence of F. oxysporum f. sp. lycopersici strain 4287 indicates that some of the copies are likely nonfunctional.

scholarly journals Random Amplified Polymorphic DNA (RAPD) Marker Variability between and within Gene Pools of Common Bean

1994 ◽  
Vol 119 (1) ◽  
pp. 122-125 ◽  
Author(s):  
Scott D. Haley ◽  
Phillip N. Miklas ◽  
Lucia Afanador ◽  
James D. Kelly
Keyword(s):  
Common Bean ◽  
Gene Pool ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
Major Gene ◽  
Rapd Marker ◽  
Gene Pools ◽  
Navy Bean ◽  
Breeding Programs ◽  
Polymerase Chain

The objective of this study was to evaluate the degree of RAPD marker variability between and within commercially productive market classes representative of the Andean and Middle American gene pools of common bean (Phaseolus vulgaris L.). Six sets of near-isogenic lines were screened with oligonucleotide primers in the polymerase chain reaction-based RAPD assay. Simultaneous analyses with at least three sets of lines enabled us to score RAPD markers between the two major gene pools, races within the same gene pool, and different genotypes of the same race (within race). A “three-tiered” pattern of polymorphism was observed: between gene pools> between races> within races. The overall level of polymorphism between the Andean and Middle American gene pools was 83.4%. The overall level of polymorphism between races within the same gene pool was similar for Andean races (60.4%) and Middle American races (61.7%). The level of polymorphism between related commercial navy bean lines was 39.2% and between related commercial snap bean lines was 53.6 %. The inherent simplicity and efficiency of RAPD analyses, coupled with the number of polymorphisms detectable between related commercial genotypes, should facilitate the construction of RAPD-based genetic linkage maps in the context of populations representative of most bean breeding programs.

scholarly journals Identification of Fusarium oxysporum f. sp. basilici Isolated from Soil, Basil Seed, and Plants by RAPD Analysis

Plant Disease ◽  
1999 ◽  
Vol 83 (6) ◽  
pp. 576-581 ◽  
Author(s):  
Annalisa Chiocchetti ◽  
Stefano Ghignone ◽  
Andrea Minuto ◽  
M. Lodovica Gullino ◽  
Angelo Garibaldi ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Dna Polymerase ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Selective Medium ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Polymerase Chain ◽  
Clear Differentiation ◽  
Pathogenicity Assay

Fifty-two isolates of Fusarium oxysporum, obtained from infected basil plants, seed, flower residues, and soil from different growing areas in Italy and Israel, were analyzed by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), coupled to a DNA extraction protocol from colonies grown on Fusarium-selective medium. In a pathogenicity assay, 35 isolates caused 32 to 92% disease on seedlings of the highly susceptible basil cultivar Fine verde, while 17 isolates were nonpathogenic on basil. Thirty of the F. oxysporum f. sp. basilici isolates obtained from soil or wilted plants gave identical amplification patterns using 31 different random primers. All tested primers allowed clear differentiation of F. oxysporum f. sp. basilici from representatives of other formae speciales and from nonpathogenic strains of F. oxysporum. RAPD profiles obtained from DNA of isolates extracted directly from cultures grown on Fusarium selective medium were identical to those obtained from DNA extracted from lyophilized mycelia.

scholarly journals Development of RAPD-SCAR markers for Lonicera japonica Thunb. (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning

2014 ◽  
Vol 62 (4) ◽  
pp. 1649 ◽  
Author(s):  
Luquan Yang ◽  
Md. Asaduzzaman Khan ◽  
Zhiqiang Mei ◽  
Manman Yang ◽  
Tiandan Zhang ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Molecular Marker ◽  
Scar Marker ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Vital Role ◽  
Lonicera Japonica ◽  
Scar Markers ◽  
Sequence Characterized Amplified Region ◽  
Young Leaves

<p>Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. <em>Lonicera japonica</em> is a traditionally used medicinal plant, which have been recently genetically characterized by an improved random amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific <em>L. japonica</em> variety. The DNAs were extracted from fresh young leaves of different samples of <em>L. japonica</em> collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with different species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the <em>L. japonica</em> samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other <em>L. japonica</em> samples). The marker JYH3-3 was found as specific molecular marker for the identification of <em>L. japonica</em>, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for <em>L. japonica</em> variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in <em>L. japonica</em>.</p><p>de cualquier organismo, que hemos aplicado con éxito en <em>L. japonica</em>.<strong></strong></p>

scholarly journals Isolates of Uromyces appendiculatus with Specific Virulence to Landraces of Phaseolus vulgaris of Andean Origin

Plant Disease ◽  
1999 ◽  
Vol 83 (2) ◽  
pp. 108-113 ◽  
Author(s):  
Craig M. Sandlin ◽  
James R. Steadman ◽  
Carlos M. Araya ◽  
Dermot P. Coyne
Keyword(s):  
Phaseolus Vulgaris ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Rust Fungus ◽  
Distinct Group ◽  
Uromyces Appendiculatus ◽  
Bean Rust ◽  
Banding Patterns ◽  
Highly Virulent ◽  
Specific Virulence

Five isolates of the bean rust fungus Uromyces appendiculatus were shown to be specifically virulent on bean genotypes of Andean origin. This specificity was demonstrated by the virulence of five pairs of isolates on a differential set of 30 Phaseolus vulgaris landraces. Each isolate pair was from a different country in the Americas and consisted of one Andean-specific isolate and one nonspecific isolate. Of the differential P. vulgaris landraces, 15 were of Middle American origin and 15 were of Andean origin. The Andean-specific rust isolates were highly virulent on Andean landraces but not on landraces of Middle American origin. Rust isolates with virulence to Middle American landraces were also generally virulent on Andean material; no truly Middle American-specific isolates were found. Random amplified polymorphic DNA (RAPD) analysis of the rust isolates also distinguished the two groups. Four of the Andean-specific rust isolates formed a distinct group compared to four of the nonspecific isolates. Two of the isolates, one from each of the two virulence groups, had intermediate RAPD banding patterns, suggesting that plasmagomy but not karyogamy occurred between isolates of the two groups.

scholarly journals Random amplified polymorphic DNA (RAPD) finger prints evidencing high genetic variability among marine angiosperms of India

2016 ◽  
Vol 97 (6) ◽  
pp. 1307-1315 ◽  
Author(s):  
Elangovan Dilipan ◽  
Jutta Papenbrock ◽  
Thirunavakkarasu Thangaradjou
Keyword(s):  
Genetic Variability ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Rapd Marker ◽  
Seagrass Species ◽  
Complex Species ◽  
Marine Angiosperms ◽  
Different Populations ◽  
High Degree ◽  
Identification Tool

In India 14 seagrass species can be found with monospecific genera (Enhalus, ThalassiaandSyringodium),Cymodoceawith two species andHalophilaandHalodulerepresented by more than two taxonomically complex species. Considering this, the present study was made to understand the level and pattern of genetic variability among these species collected from Tamilnadu coast, India. Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the level of polymorphism existing between the species. Out of the 12 primers tested, 10 primers amplified 415 DNA fragments with an average of 41.5 fragments per primer. Of the total 415 amplified fragments only 123 (29.7%) were monomorphic and the remaining 292 (70.3%) were polymorphic for Indian seagrass species. Among the 10 primers used four are identified as the key primers capable of distinguishing all the Indian seagrasses with a high degree of polymorphism and bringing representative polymorphic alleles in all the tested seagrasses. From the present investigation, this study shows that the RAPD marker technique can be used not only as a tool to analyse genetic diversity but also to resolve the taxonomic uncertainties existing in the Indian seagrasses. The efficiency of these primers in bringing out the genetic polymorphism or homogeneity among different populations of theHalophilaandHalodulecomplex still has to be tested before recommending these primers as an identification tool for Indian seagrasses.

Comparison of repetitive-sequence-based polymerase chain reaction with random amplified polymorphic DNA analysis for rapid genotyping of nontuberculosis mycobacteria

2012 ◽  
Vol 58 (8) ◽  
pp. 953-964 ◽  
Author(s):  
Sara Christianson ◽  
Joyce Wolfe ◽  
Hafid Soualhine ◽  
Meenu K. Sharma
Keyword(s):  
Repetitive Sequence ◽  
Dna Analysis ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Gene Sequencing ◽  
Variable Number ◽  
Outbreak Investigations ◽  
Hsp65 Gene ◽  
Polymerase Chain ◽  
Clinically Significant

Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit – variable number of tandem repeat (MIRU–VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU–VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.

Development of DNA fingerprinting keys for the identification of radish cultivars

2004 ◽  
Vol 44 (1) ◽  
pp. 95 ◽  
Author(s):  
A. Pradhan ◽  
G. Yan ◽  
J. A. Plummer
Keyword(s):  
Dna Fingerprinting ◽  
Random Amplified Polymorphic Dna ◽  
Rapd Marker ◽  
Morphological Characteristics ◽  
Group Method ◽  
Base Pairs ◽  
Difference Matrix ◽  
Pair Group ◽  
Young Leaves ◽  
Polymerase Chain

Identification of cultivars is extremely important both for cultivation and breeding of crop plants. Cultivar identification based on morphological characteristics can be difficult and complicated. Polymerase chain reaction technologies, such as random amplified polymorphic DNA (RAPD) analysis, can readily and quickly identify cultivars using seeds and young leaves. Sixty individuals representing 7 radish cultivars were examined for RAPD marker polymorphism. Based on the polymorphism generated, 5 primers were selected, out of the 14��examined, to fingerprint the cultivars. The 5 primers produced a total of 52 fragments, 6 monomorphic and 46�polymorphic fragments, ranging in size from 206 to 2258 base pairs. A total and mean character difference matrix was calculated based on the RAPD data and a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA). Three DNA fingerprinting keys were developed for the 7 cultivars and 5 markers derived from 3 primers was the minimum required to distinguish cultivars. Results demonstrated that RAPD markers could be effectively used for the identification of radish cultivars.

scholarly journals In Planta and Soil Quantification of Fusarium oxysporum f. sp. ciceris and Evaluation of Fusarium Wilt Resistance in Chickpea with a Newly Developed Quantitative Polymerase Chain Reaction Assay

2011 ◽  
Vol 101 (2) ◽  
pp. 250-262 ◽  
Author(s):  
Daniel Jiménez-Fernández ◽  
Miguel Montes-Borrego ◽  
Rafael M. Jiménez-Díaz ◽  
Juan A. Navas-Cortés ◽  
Blanca B. Landa
Keyword(s):  
Polymerase Chain Reaction ◽  
Fusarium Oxysporum ◽  
Fusarium Wilt ◽  
Plant Root ◽  
Quantitative Polymerase Chain Reaction ◽  
Infested Soil ◽  
In Planta ◽  
Field Plot ◽  
Chain Reaction ◽  
Polymerase Chain

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.

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