Amplified Fragment Length Polymorphism Assay Research Articles

Evaluation of genetic diversity of Panicum turgidum Forssk from Saudi Arabia.

The genetic diversity of 177 accessions of Panicum turgidum Forssk, representing ten populations collected from four geographical regions in Saudi Arabia, was analyzed using amplified fragment length polymorphism (AFLP) markers. A set of four primer-pairs with two/three selective nucleotides scored 836 AFLP amplified fragments (putative loci/genome landmarks), all of which were polymorphic. Populations collected from the southern region of the country showed the highest genetic diversity parameters, whereas those collected from the central regions showed the lowest values. Analysis of molecular variance (AMOVA) revealed that 78% of the genetic variability was attributable to differences within populations. Pairwise values for population differentiation and genetic structure were statistically significant for all variances. The UPGMA dendrogram, validated by principal coordinate analysis-grouped accessions, corresponded to the geographical origin of the accessions. Mantel’s test showed that there was a significant correlation between the genetic and geographical distances (r = 0.35, P < 0.04). In summary, the AFLP assay demonstrated the existence of substantial genetic variation in P. turgidum. The relationship between the genetic diversity and geographical source of P. turgidum populations of Saudi Arabia, as revealed through this comprehensive study, will enable effective resource management and restoration of new areas without compromising adaptation and genetic diversity.

Analysis of Genetic Diversity of Indian Tea Accessions Using Two Modified Amplified Fragment Length Polymorphism Methods

The authors compared the utility of two variants of Amplified Fragment Length Polymorphism (AFLP) technique, viz. 4-selective AFLP and three Endonuclease Amplified Fragment Length Polymorphism (TE-AFLP), for accurate assessment of genetic diversity in tea. These assays were optimized to resolve the reported discrepancies in detected levels of genetic variation in tea through conventional AFLP assay. The 4-selective AFLP assay involved the use of four selective nucleotides in MseI primer instead of three, in combination with EcoRI primers with three selective nucleotides. This led to a significant reduction in the total number of bands and improved the overall band readability as compared to conventional AFLP. However, presence of a large number of “shadow bands” of varying intensities across lanes, still disallowed an error free scoring. This was effectively resolved by TE-AFLP assay where selective adapter ligation, in addition to selective amplification, contributes to reduction in the complexity of fingerprint pattern. The major advantage of TE-AFLP over 4-select AFLP was its clear and unambiguous banding profiles that simplified the scoring of bands. Both methodologies yielded comparable results with China types clearly separating from Assam types. In contrast to previous reports, the dendrogram revealed an overall tendency of the accessions to cluster based on their morphotypes and their geographic origin. Although, 4-select AFLP detected higher polymorphism information content values, the overall reliability of TE-AFLP bands was higher due to their unambiguous nature and easy scorability. A major fraction of variation (75 %) was found to be partitioned within the morphotypes indicating predominant out-crossing in this species.

Population genetic analysis and trichothecene profiling of Fusarium graminearum from wheat in Uruguay.

Fusarium graminearum sensu stricto (F. graminearum s.s.) is the major causal agent of Fusarium head blight of wheat worldwide, and contaminates grains with trichothecene mycotoxins that cause serious threats to food safety and animal health. An important aspect of managing this pathogen and reducing mycotoxin contamination of wheat is knowledge regarding its population genetics. Therefore, isolates of F. graminearum s.s. from the major wheat-growing region of Uruguay were analyzed by amplified fragment length polymorphism assays, PCR genotyping, and chemical analysis of trichothecene production. Of the 102 isolates identified as having the 15-ADON genotype via PCR genotyping, all were DON producers, but only 41 strains were also 15-ADON producers, as determined by chemical analysis. The populations were genotypically diverse but genetically similar, with significant genetic exchange occurring between them. Analysis of molecular variance indicated that most of the genetic variability resulted from differences between isolates within populations. Multilocus linkage disequilibrium analysis suggested that the isolates had a panmictic population genetic structure and that there is significant recombination occurs in F. graminearum s.s. In conclusion, tour findings provide the first detailed description of the genetic structure and trichothecene production of populations of F. graminearum s.s. from Uruguay, and expands our understanding of the agroecology of F. graminearum and of the correlation between genotypes and trichothecene chemotypes.

Phenotypic and Genotypic Analysis of Newly Obtained Interspecific Hybrids in the Campanula Genus.

Interspecific hybridisation creates new phenotypes within several ornamental plant species including the Campanula genus. We have employed phenotypic and genotypic methods to analyse and evaluate interspecific hybridisation among cultivars of four Campanula species, i.e. C. cochleariifolia, C. isophylla, C. medium and C. formanekiana. Hybrids were analysed using amplified fragment length polymorphism (AFLP), flow cytometry and biometrical measurements. Results of correlation matrices demonstrated heterogeneous phenotypes for the parental species, which confirmed our basic premise for new phenotypes of interspecific hybrids. AFLP assays confirmed the hybridity and identified self-pollinated plants. Limitation of flow cytometry analysis detection was observed while detecting the hybridity status of two closely related parents, e.g. C. cochleariiafolia × C. isophylla. Phenotypic characteristics such as shoot habitus and flower colour were strongly influenced by one of the parental species in most crosses. Rooting analysis revealed that inferior rooting quality occurred more often in interspecific hybrids than in the parental species. Only interspecific hybrid lines of C. formanekiana ‘White’ × C. medium ‘Pink’ showed a high rooting level. Phenotype analyses demonstrated a separation from the interspecific hybrid lines of C. formanekiana ‘White’ × C. medium ‘Pink’ to the other clustered hybrids of C. formanekiana and C. medium. In our study we demonstrated that the use of correlation matrices is a suitable tool for identifying suitable cross material. This study presents a comprehensive overview for analysing newly obtained interspecific hybrids. The chosen methods can be used as guidance for analyses for further interspecific hybrids in Campanula, as well as in other ornamental species.

The phenotypic and genomic diversity of Aspergillus strains producing glucose dehydrogenase

Twelve Aspergillus sp. strains producing glucose dehydrogenase were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was investigated. Moreover, partial gdh gene sequences were determined and aligned. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of twelve Aspergillus isolates. Using one PstI restriction endonuclease and five selective primers in an AFLP assay, 556 DNA fragments were generated, including 532 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished twelve Aspergilli fungi. The AFLP-based dendrogram generated by the UPGMA method grouped all the Aspergillus fungi studied into two major clusters. All the Aspergillus strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the analyzed strains up to three folds. All of the studied strains mainly decomposed carbohydrates.

Exploration of amplified fragment length polymorphism analysis as an effective tool for discriminating pathogenic strains of bacteria

Exploration of amplified fragment length polymorphism analysis as an effective tool for discriminating pathogenic strains of bacteria K Weinbrecht,1 A Taylor,2 C Beaumann,3 RW Allen11Department of Forensic Sciences, Center for Health Sciences, Oklahoma State University, Tulsa, OK; 2Centers for Disease Control, Atlanta, GA; 3PharmaNet-i3, Indianapolis, IN, USABackground: The detrimental effect pathogens used as bioweapons could have on the US is sufficient to warrant extensive efforts to establish preparedness to prevent, and, if necessary, respond to such an event. The objective of this research was to adapt and refine amplified fragment length polymorphism (AFLP) analysis for DNA profiling of microbial strains for use in forensic and clinical case work.Methods: Included in this study were multiple strains of Pseudomonas syringae that were restricted in their respective host ranges (ie, the pathovars maculicola and tomato), Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus. AFLP profiles were reduced to numeric haplotype codes that conveyed the size characteristics of the profiles.Results: The AFLP assay exhibited over 97% reproducibility and every analyzed strain produced a unique haplotype code. Conserved areas of similarity were identified in haplotype codes of closely related strains of the same species, indicating that AFLP may also be a useful species identification tool, a possibility supported by cluster analysis of AFLP codesConclusion: The results of this study demonstrate that AFLP technology is sufficiently reproducible, powerful, and reliable for use as a broadly effective molecular screening tool in microbial forensics.Keywords: amplified fragment length polymorphism, pathogenic strains, bacteria

Assessment and characterization of genetic diversity in Withania somnifera (L.) Dunal using RAPD and AFLP markers

Genetic diversity of 23 accessions of Withania somnifera collected from different geographical regions of India was estimated by employing Random Amplification of Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) markers. Eighteen RAPD primers and six AFLP primer combinations revealed 37.82 and 43.94% polymorphism, respectively, among 163 and 286 genetic loci amplified. The AFLP assay revealed higher levels of polymorphism among the tested W. somnifera accessions compared to the RAPD. Mean genetic diversity based on Shannon index ranged from 1.33 (RAPD) to 5.13 (AFLP). Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. The matrix reveals, two main clusters, wild accessions formed one cluster and the cultivated accessions formed the other. The cultivated accessions are well separated from the wild ones at a low similarity value of 0.3, indicating that cultivated and wild accessions are highly distinct. Morphologically cultivated accessions were also quite distinct from the wild ones and the cluster analysis of RAPD and AFLP fingerprints clearly discriminated the five cultivated accessions of W. somnifera . A strong correlation was observed between morphology and molecular marker systems. Identification of specific markers to wild as well as cultivated accessions is yet another important finding in the present study. Such genetic diversity is useful in facilitating the development of large number of new varieties through hybridization, transfer of useful genes, thus maximizing the use of such available germplasms as genetic resource materials for breeders. The present input, first of its kind in Ashwagandha, will thus assist the marker assisted crop improvement programme. Key words: Withania somnifera, genetic diversity, RAPD, AFLP, polymorphism, Shannon index.

Improved AFLP protocol using dual-suppression PCR and its application to species with large genomes

To improve the amplified fragment length polymorphism assay, dual-suppression PCR was introduced into the preamplification step of the assay. The dual-suppression PCR blocked completely the amplification of fragments with the same sequence (Bsp1407I-Bsp1407I or NlaIII-NlaIII) at both ends and amplified selectively fragments with different adaptor sequences (Bsp1407I-NlaIII) at each end. Two protocols, referred to as A and B, were established for species with medium- and large-sized genomes, respectively. Both protocols incorporated the dual-suppression PCR. Protocol A resulted in high-quality electrophoretic profiles for black cottonwood and rice, which have medium-sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.

Characterization and pharmacological properties of in vitro propagated clones of Echinacea tennesseensis (Beadle) Small

Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinacea tennesseensis (Beadle) Small (ETN 03 and ETN 11) were identified as high and low activity based on the activation of human monocytes. Phenotypic analyses of ETN 03 and ETN 11 clones were done using AFLP (Amplified Fragment Length Polymorphism) assay. Results of the AFLP assay revealed that no mutation has occurred during in vitro multiplication, storage, and acclimatization into soil. Plants of ETN 03, ETN 11 clones were cultivated for two growing seasons. Extracts of their dry leaves and roots exhibited immune enhancing activity; however, the variation in activity noticed between clones during micropropagation diminished and was no longer statistically relevant.

A New Type of Strain of Xanthomonas euvesicatoria Causing Bacterial Spot of Tomato and Pepper in Grenada.

Bacterial spot of tomato and pepper (BSTP) can be caused by several Xanthomonas genospecies (2). BSTP is a major disease in Grenada where A and B phenotypic groups (Xanthomonas euvesicatoria and X. vesicatoria, respectively, [2]) have been reported (3). There is no previous report of group A strains, which are strongly amylolytic and pectolytic, in Grenada. In March 2007, tomato and pepper leaves with lesions typical of BSTP were collected in Saint David and Saint Andrew parishes of Grenada. Bacterial isolations were performed on KC semiselective agar medium (4), resulting in isolation of five yellow-pigmented, Xanthomonas-like strains. Three strains isolated from tomato or pepper in Saint David were negative for starch hydrolysis and pectate degradation, two tests that were found useful for strain identification in the 1990s (2). Two strains isolated from pepper in Saint David were strongly amylolytic and degraded pectate. Amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) assays targeting atpD, dnaK, efp, and gyrB were performed on the five strains from Grenada together with a type strain of each of X. euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria as well as other reference strains of X. euvesicatoria and X. perforans as described previously (1). All strains from Grenada were identified as X. euvesicatoria regardless of the typing technique. On the basis of AFLP assays, the two strains with phenotypic features not reported in Grenada were closely related (distances of ≤0.002 nucleotide substitutions per site [1]) to a group of strains from India (ICMP 3381, LMG 907, LMG 908, and LMG 918). These two strains were also identical to the Indian strains based on MLSA, but differed from the X. euvesicatoria type strain by at least one nucleotide substitution in all loci examined. The three strains from Grenada that were negative for starch hydrolysis and pectate degradation had sequences identical to that of the type strain. Young leaves of tomato plants of cv. Marmande and pepper plants of cvs. Yolo Wonder and Aiguille were infiltrated (six inoculation sites per leaf, three replicate plants per cultivar per experiment, and the experiment was replicated once) using inoculum of each of the five strains from Grenada made from suspensions in Tris buffer containing approximately 1 × 105 CFU/ml. Two reference strains of X. euvesicatoria (NCPPB 2968 and LMG 922) were also inoculated as positive control treatments. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical water-soaked lesions that developed into necrotic spots were observed 3 to 8 days after inoculation (dai) for all strains on all cultivars, except NCPPB 2968, which was not pathogenic on pepper cv. Aiguille. Xanthomonas population sizes from lesions plated onto KC agar medium (4) 25 dai ranged from 3 × 106 to 5 × 107, 8 × 107 to 2 × 108, and 9 × 106 to 2 × 108 CFU/lesion on tomato cv. Marmande and pepper cvs. Yolo Wonder and Aiguille, respectively. The epidemiological importance of this previously unreported group of X. euvesicatoria strains in Grenada needs to be assessed.

The effect of boat propeller scarring intensity on genetic variation in a subtropical seagrass species

Abstract We report here the effect of one form of disturbance, boat propeller scarring, on genetic variation in the subtropical seagrass Halodule wrightii. We developed an amplified fragment length polymorphism assay to measure genetic variation in plots representing four levels of scarring intensity: reference (0% scarring), low (1–5%), moderate (5–15%) and severe (&gt;15%). Although we found severely scarred plots to have the lowest, and moderately scarred plots to have the highest, mean genetic diversity estimates (H e, P), differences among scarring levels were found to be non-significant (α=0.05). Analysis of molecular variance also showed no significant effect of scarring intensity. While propeller scarring can cause significant habitat loss, scarring intensities of up to 20% may not yet have seriously affected those factors (population size, flowering density, recruitment, gene flow) that strongly influence population genetic variation. The relatively recent occurrence of this type of disturbance, however, could mean that any long-term effects have yet to be detected.

Methodological comparison of DNA extraction from Holcocerrus hippophaecolus (Lepidoptera: Cossidae) for AFLP analysis

Amplified fragment length polymorphism (AFLP) is a powerful DNA fingerprinting technique for studying genetic relationships and genetic diversity in insects. However, the crucial prerequisite for AFLP analysis is to extract DNA of high quality. In this study, we evaluate four different protocols (SDS method, improved SDS method, CTAB method and a complex method with SDS and CTAB) for isolating DNA from the seabuckthorn carpenter moth (Holcocerrus hippophaecolus (Lepidoptera: Cossidae)). The results indicate that the CTAB method does not produce DNA suitable for AFLP analysis. The SDS method and the complex method with SDS and CTAB are comparatively time-consuming and resulted in low yields of DNA and were therefore not used for AFLP assay. The improved SDS method is recommended for preparing DNA templates from H. hippophaecolus for AFLP analysis.

Glycosyl linkage characteristics and classifications of exo-polysaccharides of some regionally different strains of Lentinula edodes by amplified fragment length polymorphism assay and cluster analysis

We report here the first combined amplified fragment length polymorphism (AFLP) analysis of genomic DNA fingerprinting data and cluster analysis of the exo-polysaccharide glycosyl linkage data of 10 regionally different strains of Lentinula edodes to compare their genetic and structural similarities and differences. In addition, the monosaccharide compositions, molecular weights, glycosyl structural linkages were investigated for the exo-polysaccharides extracted from these different phylogenetic groups of regionally different L. edodes. All exo-polysaccharides had similar molecular weight distribution between 1 × 10 4 and 3 × 10 6 Da and the monosaccharide composition analysis revealed the presence of heterogeneous materials containing glucose, mannose, xylose, galactose, fucose, rhamnose and arabinose in different ratios. Among these monosaccharides, the glucose contents are the highest for all but one strain, indicating that glucose probably is the building block of the backbones of these exo-polysaccharides. The AFLP assay data helped to classify the 10 L. edodes strains into three distinct genetic groups. Gas chromatographic and mass spectrometric (GC–MS) data revealed five different glycosyl linkage types for these exo-polysaccharides. Most of the exo-polysaccharide backbone structures contain (1 → 4)-linked- d-glucopyranosyl and (1 → 6)-linked- d-glucopyranosyl moieties. Arabinose 1 → 4 linkages and mannose 1 → 2 linkages also exist in all strains. The only differences among these linkages are their monosaccharide compositions leading to different degree of backbone and branch formations. Cluster analyses of the GC–MS data of the exo-polysaccharides of the 10 strains resulted in 10 dendrograms. However, four of the 10 dendrograms were identical and were obtained using the average, Ward and weighted linkage type method of Manhattan distance and using the Ward method of Euclidean distance. The results of cluster analyses were not very much different from that of the AFLP assay and allowed the comparison of genetic and structural similarities and differences.

Detecting DNA Polymorphism and Genetic Diversity in a Wide Pistachio Germplasm: Comparison of AFLP, ISSR, and RAPD Markers

There are limited numbers of pistachio (Pistacia vera L.) cultivars in the world and their phenotypic appearance and productivity are variable. Understanding such variation would facilitate their use in cultivar breeding programs. Therefore, in this study, 69 pistachio cultivars and genotypes originating from seven countries were characterized by randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and amplified fragment-length polymorphism (AFLP) markers. The results showed that all three marker systems were able to reveal variability between pistachio cultivars and genotypes. The correlation coefficients for genetic distances were statistically significant among all three molecular marker types. The correlation between RAPD and AFLP data was the highest (r = 0.73) and the value between RAPD and ISSR data was the lowest (r = 0.58). AFLP proved to be the best technique among them. ISSR and AFLP assays were reliable and produced reproducible bands. ISSR was preferred over RAPD, especially when financial investment and technical knowledge are limited. The constructed unweighted pair group method with arithmetic averages (UPGMA) dendrogram obtained from combined data separated the genotypes into two main clusters: one cluster (“Iranian”) included genotypes originating from Iran and the second cluster (“Mediterranean”) contained most other genotypes. The “Mediterranean” cluster further divided into three subclusters: one (“Siirt”) consisted of the cultivars Siirt and Hacireso with a few other selections; the second subcluster (“Turkish”) included Turkish cultivars; and the third subcluster contained Syrian, Italian, and the remaining cultivars. The closeness of the clusters was “Iranian” - “Siirt” - “Turkish”/“Syrian.” These findings reveal a new understanding in the diffusion of pistachio cultivation from its center of origin, the Iranian-Caspian region, via southeastern Turkey to Syria, the Mediterranean region of Europe, and northern Africa.

Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of Black

The R10 and R11 late blight differentials of Black (tetraploid clones 3681ad1 and 5008ab6) were crossed with the susceptible potato (Solanum tuberosum) cultivar Maris Piper and the progeny were assessed for blight resistance in a whole plant glasshouse test using race 1,2,3,4,6,7 of Phytophthora infestans. The disease scores for the R10 population displayed a continuous distribution whereas the progeny in the R11 population could be categorised as resistant or susceptible. A bulk segregant analysis using amplified fragment length polymorphism assays was done on the ten most resistant and ten most susceptible progeny in each population and two closely linked markers were found to be associated with resistance. R11 mapped to 8.5 cM from marker PAG/MAAG_172.3 and R10 mapped as a quantitative trait locus in which marker PAC/MATC_264.1 explained 56.9% of the variation in disease scores. The results were consistent with R10 and R11 being allelic versions of genes at the R3 locus on chromosome 11. The implications are discussed for mapping R-genes which fail to give complete immunity to a pathogen.

Analysis of genetic diversity among different geographical populations and determination of biotypes of Bemisia tabaci in China

Abstract: Analysis of the genetic diversity among 27 different geographical populations of Bemisia tabaci and determination of biotypes of B. tabaci in China based on amplified fragment‐length polymorphism (AFLP) and the mitochondrial cytochrome oxidase I (mtDNA COI) gene sequences were conducted. In AFLP assay, the use of five primer combinations selected from 64 primer combinations allowed the identification of 229 polymorphic bands (97.03%) from 60 to 500 bp, suggesting abundant genetic diversity among different geographical populations of B. tabaci. To further identify biotypes of B. tabaci in China, the mtDNA COI gene sequences of nine representative populations from China, Israel and Spain were obtained. Molecular phylogenetic tree based on AFLP and mtDNA COI gene analyses revealed the presence, in China, of at least four different genetic groups of B. tabaci. B biotype, Q biotype and two non‐B/Q biotype. B biotype was distributed nationwide. Q biotype was present only in the local region of China including the YunNan province and BeiJing city. This was also the first report about the invasion of Q biotype into China. Of the other two non‐B/Q biotype groups, one was found in ShanDong and HeBei provinces, and another in ZheJiang province. The non‐B/Q biotype ZheJiang population showed very high similarity with another Asian population India‐IW (AF110704) in mtDNA COI sequences and was possibly a Chinese indigenous population. The close monitoring of the Q biotype in locales of China where commercial plants were exported or imported, is now essential to avoid the further accidental distribution of the Q biotype.

A comparison of AFLP and ERIC-PCR analyses for discriminating Escherichia coli from cattle, pig and human sources

Amplified fragment length polymorphism (AFLP) and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genomic fingerprinting assays were compared for their ability to differentiate Escherichia coli isolates obtained from various host sources, and with respect to their pathogenicity. One hundred and ten verotoxigenic, enterotoxigenic and non-pathogenic E. coli isolates obtained from cattle, humans and pigs were used in this study. The AFLP assay was shown to be highly effective in predicting both the host source and pathogenicity of the E. coli isolates. A stepwise discriminant function analysis showed that 91.4, 90.6 and 97.7% of the human, bovine and pig isolates were classified into the correct host types, respectively. The analysis also distinguished the non-pathogenic E. coli from the verocytotoxigenic and enterotoxigenic virulence phenotypes at 100, 100 and 90.9% accuracy, respectively. Sixty-two E. coli strains from the collection were subjected to the ERIC-PCR fingerprinting analysis. Using this method, only 28.6, 0 and 75.0% of the human, bovine and pig isolates were classified into the correct host types, respectively. Overall, the AFLP method was able to ascribe host source with a high level of confidence and readily discriminate pathogenic from non-clinical isolates of E. coli.

Genetic relationships among biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae) based on AFLP analysis.

Genetic similarities between 13 samples belonging to nine reference biotypes and two field populations of Bemisia tabaci (Gennadius), one field population of B. medinae Gómez-Menor and another of B. afer Priesner & Hosny, were evaluated using amplified fragment length polymorphism (AFLP) markers. The results indicate that B. tabaci biotypes can be grouped together with a minimum similarity coefficient of 0.32 and separated from the two other species with a similarity coefficient of 0.07. Bemisia tabaci biotypes were grouped in four clusters which comprised: (i) Near East and Indian subcontinent biotypes; (ii) B and Q biotypes plus a Nigerian population from cowpea; (iii) New World A biotype; and (iv) S biotype and a Nigerian population from cassava. These results were consistent with a previous grouping of biotypes based on RAPD-PCR analysis. The AFLP assay allowed the scoring of a total of 354 polymorphic bands in two reaction events with the use of two primer combinations.

A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates.

We have performed amplified-fragment length polymorphism (AFLP) fingerprinting on a collection of Salmonella enterica subsp. enterica serovar typhimurium strains with a restriction endonuclease combination (BglII and MfeI) that has previously been used successfully for typing Campylobacter jejuni isolates with high resolution. Additionally, a variation of the AFLP assay in which two rare cutting restriction enzymes (XbaI and BsrGI) in combination with the frequent cutter (HinP1I) was examined. The BglII and MfeI enzyme combination offered low resolution for genotyping Salmonella typhimurium isolates and is not recommended for this common serovar. The three-enzyme combination gave a higher discrimination, and is thus a new alternate way of performing AFLP fingerprinting of S. typhimurium.

A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII– MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates

We have performed amplified-fragment length polymorphism (AFLP) fingerprinting on a collection of Salmonella enterica subsp. enterica serovar typhimurium strains with a restriction endonuclease combination ( BglII and MfeI) that has previously been used successfully for typing Campylobacter jejuni isolates with high resolution. Additionally, a variation of the AFLP assay in which two rare cutting restriction enzymes ( XbaI and BsrGI) in combination with the frequent cutter ( HinP1I) was examined. The BglII and MfeI enzyme combination offered low resolution for genotyping Salmonella typhimurium isolates and is not recommended for this common serovar. The three-enzyme combination gave a higher discrimination, and is thus a new alternate way of performing AFLP fingerprinting of S. typhimurium.