Genetic variation, pathogenicity and mycelial growth rate differentiation between Gaeumannomyces graminis var. tritici isolates derived from winter and spring wheat

The variability of Gaeumannomyces graminis var. tritici ( Ggt ) isolates was evaluated at an intravarietal level using non-molecular and molecular methods. Pathogenicity and linear growth rate of the pathogen were estimated. Very high pathogenicity was found in 44% of the isolates, medium in 20% and low only in 8%. Significant differences in mycelial growth rate were observed. The quickest linear growth rate of Ggt mycelium was observed at 25°C. Isolates derived from winter wheat grew faster than those obtained from spring wheat. The correlation between growth rates and pathogenicity was not significant. DNA polymorphism determined by random amplified polymorphic DNA (RAPD)–PCR was used to assess genetic variation among isolates. Thirty-two RAPD markers revealed DNA polymorphism suitable for assessing variability among isolates examined. Cluster analysis of RAPD data identified a few groups of isolates. RAPD markers associated with pathogenicity as well as mycelium growth rate were found.     

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Application of different molecular techniques for deciphering genetic diversity among yeast isolates of traditional fermented food products of Western Himalayas

Forty-three yeast isolates derived from various fermented foods, alcoholic beverages and traditional inocula of Western Himalayas were characterized by using traditional and molecular techniques. Traditional characterization identified these isolates as belonging to seven genera and eight species. Twenty-three yeast isolates were identified as Saccharomyces cerevisiae, six as Debaromyces hansenii, five as Issatchenkia orientalis, four as Saccharomyces fermentati, two as Schizosaccharomyces pombe and one each as Endomyces fibuliger, Brettanomyces bruxellensis and Candida tropicalis. The molecular characterization using four marker systems i.e. universal rice primers (URP), randomly amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and delta typing was carried out, which revealed strainal level differences along with geographical origin clustering of various yeast isolates which otherwise could not be revealed through conventional characterization. URP markers were found to be best for revealing the genetic polymorphism hidden among forty-three yeast isolates followed by delta typing, RAPD and ISSR. In the above study, URP 6R and URP 9F were found to be species specific thereby producing specific banding pattern for a specific species.     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

Use of RAPD and ISSR Markers in Detection of Genetic Variation and Population Structure among Fusarium oxysporum f. sp. ciceris Isolates on Chickpea in Turkey

Genetic variation among the isolates of Fusarium oxysporum f. sp. ciceris, the causal agent of chickpea wilt worldwide, was analysed using pathogenicity tests and molecular markers – random amplified polymorphic DNA (RAPD) and inter‐simple sequence repeat (ISSR) polymorphism. Hundred and eight isolates were obtained from diseased chickpea plants in 13 different provinces of Turkey, out of which 74 isolates were assessed using 30 arbitrary decamer primers and 20 ISSR primers. Unweighted pair‐grouped method by arithmetic average cluster analysis of RAPD, ISSR and RAPD + ISSR datasets provided a substantially similar discrimination among Turkish isolates and divided into three major groups. Group 1, 2 and 3 consisted of 41, 18 and 15 isolates, respectively. These methods revealed a considerable genetic variation among Turkish isolates, but no correlation with regard to the clustering of isolates from different geographic regions. Analysis of molecular variance confirmed that most genetic variability resulted from the differences among isolates within regions. Our results also indicated that the low‐genetic differentiation (F_ST) and high gene flow (Nm) among populations had a significant effect on the emergence and evolutionary development of F. oxysporum f. sp. ciceris. This is the first report on genetic diversity and population structure of F. oxysporum isolates on chickpea in Turkey.     

AFLP and RAPD Characterization of Phaeoacremonium aleophilum Associated with Vitis vinifera Decline in Spain

The genetic diversity among Spanish isolates of the fungus Phaeoacremonium aleophilum, one of the major causes of grapevine decline, was determined using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) techniques. Using RAPD, a large genetic variation was observed among 36 Pm. aleophilum single‐spore cultures, with 76 (82.6%) polymorphic bands generated by 12 RAPD primers. A neighbour‐joining dendrogram showing the RAPD patterns of diversity revealed four groups of haplotypes. The Bayesian and principal components clustering analysis revealed three groups of haplotypes. When more than one isolate of Pm. aleophilum was obtained from a single vine, different haplotypes were found. Seventeen single‐spore isolates were used for AFLP analysis. Five primer combinations produced 358 scorable markers, of which 309 (86.3%) were polymorphic. The analysis based on genetic distance as well as clustering analysis confirmed three main groups largely in agreement with those returned by the RAPD results. The Mantel correlation between the RAPD and AFLP distance matrices ranged from R = 0.5931 to R = 0.6294. The high level of haplotype diversity among the RAPD and AFLP markers suggests that sexual reproduction and genetic recombination may occur between Pm. aleophilum haplotypes in Spain. The AFLP approach revealed a greater number of polymorphic markers. A relationship between the genetic profile of the infecting isolate of Pm. aleophilum and the age or decline symptoms of the grapevines may exist.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Molecular diversity analysis of Rhizoctonia solani isolates infecting various pulse crops in different agro-ecological regions of India

Genetic diversity of 89 isolates of Rhizoctonia solani isolated from different pulse crops representing 21 states from 16 agro-ecological regions of India, 49 morphological, and 7 anastomosis groups (AGs) was analyzed using 12 universal rice primers (URPs), 22 random amplified polymorphic DNA (RAPD), and 23 inter-simple sequence repeats (ISSR) markers. Both URPs and RAPD markers provided 100?% polymorphism with the bands ranging from 0.1 to 5?kb in size, whereas ISSR markers gave 99.7?% polymorphism with the bands sizes ranging from 0.1 to 3?kb. The marker URP 38F followed by URP13R, URP25F, and URP30F, RAPD marker R1 followed by OPM6, A3 and OPA12 and ISSR3 followed by ISSR1, ISSR4, and ISSR20 produced the highest number of amplicons. R. solani isolates showed a high level of genetic diversity. Unweighted pair group method with an arithmetic average (UPGMA) analysis grouped the isolates into 7 major clusters at 35?% genetic similarity using the three sets of markers evaluated. In spite of using three different types of markers, about 95?% isolates shared common grouping patterns. The majority of the isolates representing various AGs were grouped together into different sub-clusters using all three types of markers. Molecular groups of the isolates did not correspond to agro-ecological regions or states and crops of the origin. An attempt was made for the first time in the present study to determine the genetic diversity of R. solani populations isolated from different pulse crops representing various AGs and agro-ecological regions.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Virulence and molecular diversity in <Emphasis Type="Italic">Colletotrichum graminicola</Emphasis> from Brazil

Genetic diversity among 37 isolates of the sorghum anthracnose pathogen Colletotrichum graminicola, from four geographically distinct regions of Brazil, was evaluated by RAPD and RFLP-PCR markers and virulence characters on a set of 10 differential sorghum genotypes. Twenty-two races were identified and race 13B was the most frequent, but present in only two regions. RAPD analysis revealed 143 polymorphic bands that grouped the isolates according to their geographic origin, but not by their virulence phenotypes. RFLP with HaeIII, MspI, HinfI, HhaI, HpaII, EcoRI, HindIII, PstI, RsaI, Taq I, and AluI enzymes over ITS domains and 5.8 rDNA genes of C. graminicola did not show differences among the isolates, indicating high conservation of these restriction sites. Molecular polymorphism was observed among isolates belonging to the same race. No association between virulence phenotypes and molecular profiles was observed.     

The Detection of Genome Polymorphism in Stachys Species Using RAPD

Molecular genome analysis was for the first time carried out in the genus Stachys. RAPD analysis proved to be suitable for identifying the species-specific markers, studying the interspecific DNA polymorphism, and detecting the genetic changes that arise during in vitro culturing of Stachys sieboldii. RAPD was also used for screening genetic variation in S. sieboldii regenerants obtained at various phytohormone concentrations. High cytokinin concentrations and multiple regeneration were shown to induce genetic changes detectable in RAPD patterns. High DNA polymorphism was detected for two types of S. sieboldii callus cultures and for plants regenerated from a callus culture.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Population Structure of Puccinia recondita in Western Europe During 1995, as Assessed by Variability in Pathogenicity and Molecular Markers

The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty‐five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.