Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology

Summary We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter-and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single expreiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations, depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter-and intraspecific contaminations.     

DNA fingerprinting of Cryptosporidium parvum isolates using amplified fragment length polymorphism (AFLP)

The genetic variability of 10 Cryptosporidium parvum isolates of human and animal origin was investigated using amplified fragment length polymorphism (AFLP). Analysis of fluorescent dye-labeled amplified products was carried out using an ABI PRISMS 377 DNA sequencer and ABI PRISMS GeneScan software. One-hundred and twelve primer combinations were evaluated using a single C. parvum isolate. The patterns generated were highly reproducible. For subsequent study, a subset of 9 primer pairs that yielded 30-90 DNA fragments after the polymerase chain reaction, within the size range of 50-500 bp, was used to screen the 10 C. parvum isolates, including 7 bovine, 1 equine, and 2 of human origin. The animal isolates produced identical fingerprint patterns with every primer combination tested. Of the 2 human isolates tested, 1 of the isolates, passaged in calves, generated the same AFLP DNA banding patterns as the animal isolates, whereas the other isolate, obtained directly from human feces, produced unique patterns. Polymorphism, detected by comparison of the fingerprint patterns of the latter human isolate with the common pattern shared by all other isolates, ranged from 17 to 35% for the 9 primer pairs. The results show that AFLP is a useful method for differentiating C. parvum isolates into 2 distinct genotypes.     

High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

Molecular characterization of Fusarium head blight resistance in Wangshuibai with simple sequence repeat and amplified fragment length polymorphism markers.

Molecular mapping of Fusarium head blight (FHB) resistance quantitative trait loci (QTL) and marker-assisted selection of these QTL will aid in the development of resistant cultivars. Most reported FHB resistance QTL are from 'Sumai 3' and its derivatives. 'Wangshuibai' is a FHB-resistant landrace that originated from China and is not known to be related to 'Sumai 3'. A mapping population of 139 F(5:6) recombinant inbred lines was developed from a cross of 'Wangshuibai' and 'Wheaton'. This population was developed to map the FHB-resistant QTL in 'Wangshuibai' and was evaluated twice for Type II FHB resistance. A total of 1196 simple sequence repeat and amplified fragment length polymorphism markers were screened on this population, and four FHB resistance QTL were detected. A major QTL near the end of 3BS explained 37.3% of the phenotypic variation. Another QTL on 3BS, located close to the centromere, explained 7.4% of the phenotypic variation. Two additional QTL on 7AL and 1BL explained 9.8% and 11.9% of the phenotypic variation, respectively. The simple sequence repeat and amplified fragment length polymorphism markers closely linked to these QTL may be useful for stacking QTL from 'Wangshuibai' and other sources to develop cultivars with transgressive FHB resistance.     

Amplification of reproducible allele markers for amplified fragment length polymorphism analysis.

A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipoprotein B gene (apoB) by re-amplifying a mixture of PCR products from individuals with known alleles. These allele markers allow precise and discrete determination of the VNTR alleles at these loci using the Amp-FLP technique that should prove suitable in forensic analyses, paternity testing and population studies.     

Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

Genetic diversity among Japanese indigenous common buckwheat (Fagopyrum esculentum) cultivars as determined from amplified fragment length polymorphism and simple sequence repeat markers and quantitative agronomic traits.

We assessed the genetic diversity in Japanese indigenous common buckwheat (Fagopyrum esculentum) cultivars using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers and investigated the relationships between the genetic diversity and agronomic traits. The average expected intracultivar hetero zygosity was 0.303 for AFLP and 0.819 for SSR. The differentiations among agroecotypes, among cultivars within an agroecotype, and among cultivars were small (0.002, 0.024, and 0.026 for SSR and 0.013, 0.013, and 0.026 for AFLP, respectively) but statistically significant from zero except for the SSR differentiation among agroecotypes. In principal coordinates analysis, cultivars within the same agroecotype tended to cluster, indicating that agroecotypes well reflected the genetic relationships among cultivars. In AFLP, the differentiation among the agroecotypes was more distinct than in SSR, and genetic distance showed a moderate correlation with the difference in quantitative traits, indicating that AFLP can resolve the relationships among cultivars with better resolution than SSR. By contrast, SSR may be more sensitive to demographic changes. Four of the five SSR markers showed a significant positive correlation (Kendall's tau = 0.382-0.607) between allelic richness and variation in flowering timing, indicating that cumulative bottleneck events have occurred during the population history, with a decline in the variation of photosensitivity of flowering.     

optiFLP: software for automated optimization of amplified fragment length polymorphism scoring parameters

Amplified fragment length polymorphism (AFLP), a widely used method for DNA fingerprinting, has shifted from polyacrylamide gel to capillary electrophoresis over the last years. Currently, most AFLP data are generated in a computer-readable format, and several programs are available that automatically score raw data into binary profiles. Good scoring parameters are the key to good AFLP profiles. optiFLP is the first open source program for automatic optimization of AFLP scoring parameters. It searches parameter space to maximize the contrast among groups of AFLP profiles, with the allocation of profiles to groups in either a supervised or an unsupervised mode. The software produces output files ready for use in a range of downstream applications.     

Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

Genotyping of Brazilian Erysipelothrix spp. strains by amplified fragment length polymorphism

One hundred and fifty-one Erysipelothrix spp. isolates from diseased and carrier swine from Brazil were identified by PCR, submitted to serotyping and analyzed by amplified fragment length polymorphism with a single enzyme (AFLP).Reference strains from Australia and the United Kingdom were also examined. The 151 strains were classified into 18 different serotypes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 and 25), being serotype 2b the most frequent (39.7%). By associating serotyping and PCR results, it was possible to identify 146 strains as E. rhusiopathiae and five strains as E. tonsillarum. Despite the fact that for this genus AFLP did not cluster all isolates according to serotype, origin, disease or isolation data, the execution of the technique was easy and fast, demonstrating high discriminatory power. The results produced by the AFLP analysis of Erysipelothrix spp. could also support its use as a discriminatory tool for E. rhusiopathiae and E. tonsillarum species.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

Molecular typing is an important tool in surveillance and outbreak investigations of human Salmonella infections. In this study, three molecular typing methods were used to investigate the discriminatory ability, reproducibility and the genetic relationship between 110 Salmonella enterica subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately the seven housekeeping genes used in this MLST scheme lacked diversity and the ability to discriminate between isolates were higher with both PFGE and AFLP. The discriminatory power of AFLP and PFGE were similar but PFGE fingerprints were both easier to reproduce, interpret and less time-consuming to analyze when compared to AFLP. PFGE is the therefore the preferred molecular typing method for surveillance and outbreak investigations, whereas AFLP is most useful for local outbreak investigations.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Genetic diversity of Echinacea species based upon amplified fragment length polymorphism markers.

The taxonomy of Echinacea is based on morphological characters and has varied depending on the monographer. The genus consists of either nine species and four varieties or four species and eight varieties. We have used amplified fragment length polymorphisms (AFLP) to assess genetic diversity and phenetic relationships among nine species and three varieties of Echinacea (sensu McGregor). A total of 1086 fragments, of which approximately 90% were polymorphic among Echinacea taxa, were generated from six primer combinations. Nei and Li's genetic distance coefficient and the neighbor-joining algorithm were employed to construct a phenetic tree. Genetic distance results indicate that all Echinacea species are closely related, and the average pairwise distance between populations was approximately three times the intrapopulation distances. The topology of the neighbor-joining tree strongly supports two major clades, one containing Echinacea purpurea, Echinacea sanguinea, and Echinacea simulata and the other containing the remainder of the Echinacea taxa (sensu McGregor). The species composition within the clades differs between our AFLP data and the morphometric treatment offered by Binns and colleagues. We also discuss the suitability of AFLP in determining phylogenetic relationships.     

Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists

Recently, the amplified fragment length polymorphism (AFLP) technique has gained a lot of popularity, and is now frequently applied to a wide variety of organisms. Technical specificities of the AFLP procedure have been well documented over the years, but there is on the contrary little or scattered information about the statistical analysis of AFLPs. In this review, we describe the various methods available to handle AFLP data, focusing on four research topics at the population or individual level of analysis: (i) assessment of genetic diversity; (ii) identification of population structure; (iii) identification of hybrid individuals; and (iv) detection of markers associated with phenotypes. Two kinds of analysis methods can be distinguished, depending on whether they are based on the direct study of band presences or absences in AFLP profiles ('band-based' methods), or on allelic frequencies estimated at each locus from these profiles ('allele frequency-based' methods). We investigate the characteristics and limitations of these statistical tools; finally, we appeal for a wider adoption of methodologies borrowed from other research fields, like for example those especially designed to deal with binary data.     

Construction of a high-density linkage map of Italian ryegrass (Lolium multiflorum Lam) using restriction fragment length polymorphism, amplified fragment length polymorphism, and telomeric repeat associated sequence markers.

To construct a high-density molecular linkage map of Italian ryegrass (Lolium multiflorum Lam), we used a two-way pseudo-testcross F1 population consisting of 82 individuals to analyze three types of markers: restriction fragment length polymorphism markers, which we detected by using genomic probes from Italian ryegrass as well as heterologous anchor probes from other species belonging to the Poaceae family, amplified fragment length polymorphism markers, which we detected by using PstI/MseI primer combinations, and telomeric repeat associated sequence markers. Of the restriction fragment length polymorphism probes that we generated from a PstI genomic library, 74% (239 of 323) of randomly selected probes detected hybridization patterns consistent with single-copy or low-copy genetic locus status in the screening. The 385 (mostly restriction fragment length polymorphism) markers that we selected from the 1226 original markers were grouped into seven linkage groups. The maps cover 1244.4 cM, with an average of 3.7 cM between markers. This information will prove useful for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.     

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats.

Genetic diversity can be measured by several criteria, including phenotype, pedigree, allelic diversity at marker loci, and allelic diversity at loci controlling phenotypes of interest. Abundance, high level of polymorphism, and ease of genotyping make simple sequence repeats (SSRs) an excellent molecular marker system for genetics diversity analyses. In this study, we used a set of mapped SSRs to survey three representative groups of barley germplasm: a sample of crop progenitor (Hordeum vulgare subsp. spontaneum) accessions, a group of mapping population parents, and a group of varieties and elite breeding lines. The objectives were to determine (i) how informative SSRs are in these three sets of barley germplasm resources and (ii) the utility of SSRs in classifying barley germplasm. A total of 687 alleles were identified at 42 SSR loci in 147 genotypes. The number of alleles per locus ranged from 4 to 31, with an average of 16.3. Crop progenitors averaged 10.3 alleles per SSR locus, mapping population parents 8.3 alleles per SSR locus, and elite breeding lines 5.8 alleles per SSR locus. There were many exclusive (unique) alleles. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94. The cluster analysis indicates a high level of diversity within the crop progenitors accessions and within the mapping population parents. It also shows a lower level of diversity within the elite breeding germplasm. Our results demonstrate that this set of SSRs was highly informative and was useful in generating a meaningful classification of the germplasm that we sampled. Our long-term goal is to determine the utility of molecular marker diversity as a tool for gene discovery and efficient use of germplasm.     

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.