Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.     

Application of AFLP markers to genome mapping in poultry

The amplified fragment length polymorphism (AFLP) technique has been used to enhance marker density in the East Lansing reference chicken genome map, using a backcross family derived from a Red Jungle Fowl by White Leghorn mating with White Leghorn as the recurrent parent. To date, 204 AFLP markers have been added, expanding overall map coverage by about 25%. To the limits of our resolution, AFLP markers are distributed relatively evenly across the EL reference map. AFLP are about 60% as frequent in a cross within White Leghorns (line 7(2) x 6(3)) in comparison to the more divergent reference map population. Based on apparent identity of size, about 40% of the 7(2) x 6(3) cross AFLP fragments were also polymorphic in the reference map cross. Primer pairs in which one primer contains 3' extensions of three selective nucleotides and the other has two selective nucleotides successfully generated AFLP from chicken DNA, but such pairs appeared to amplify only a subset of those fragments to which they have an exact sequence match. Three different restriction enzymes with 4 bp recognition sites (TaqI, HinP1I and MspI) were found to work well with EcoRI as the rarer of the two AFLP restriction enzymes used, with HinP1I being the most effective of the three. AFLP markers are likely to provide an economical method with which to enhance framework linkage maps of chicken and probably other avian genomes.     

The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints

BACKGROUND AND AIMS: Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used. METHODS: AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used. KEY RESULTS: Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid. CONCLUSIONS: We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.     

Genetic variation in Oryza species detected by MITE-AFLP

MITE-AFLP markers were successfully used to study the genetic variation and species relationship in Oryza species. Analysis of 53 accessions of Oryza species with seven MITE-AFLP primer combinations detected a total of 250 polymorphic fragments. High polymorphism was detected within and between Oryza species. Species relationships were analyzed by the pattern of presence or absence of homologous fragments, because nucleotide sequences of the detected MITE-AFLP fragments revealed that the same fragments in different species shared very high sequence homology. The genetic distances (GDs) between species were higher than those within species and the GDs in O. sativa complex were higher than those in O. officinalis complex. The phylogenetic tree recognized two major groups at 62% genetic similarity; group I consists of all AA genome species of the O. sativa complex, and group II consists of BB-, CC-, EE- and BBCC genome species of the O. officinalis complex. Therefore, this study demonstrated that the MITE-AFLP technique provide a tool for studying the genetic variation and species relationship in Oryza species.     

Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, recently derived from the Amplified Fragment Length Polymorphism (AFLP) technique, produces amplified fragments containing a retrotransposon LTR sequence at one end and a host restriction site at the other. We report the application of this procedure to the LTR of the Tms1 element from Medicago sativa L. Genomic dot-blot analysis indicated that Tms1 LTRs represent about 0.056% of the M. sativa genome, corresponding to 16 x 10(3) copies per haploid genome. An average of 66 markers were amplified for each primer combination. Overall 49 polymorphic fragments were reliably scored and mapped in a F(1) population obtained by crossing diploid M. falcata with M. coerulea. The utility of the LTR S-SAP markers was higher than that of AFLP or SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) markers. The efficiency index of the LTR S-SAP assay was 28.3, whereas the corresponding values for AFLP and SAMPL markers were 21.1 and 16.7, respectively. The marker index for S-SAP was 13.1, compared to 8.8 for AFLP and 9.5 for SAMPL. Application of the Tms1 LTR-based S-SAP to double-stranded cDNA resulted in a complex banding pattern, demonstrating the presence of Tms1 LTRs within exons. As the technique was successfully applied to other species of the genus Medicago, it should prove suitable for studying genetic diversity within, and relatedness between, alfalfa species.     

Nuclear markers reveal that inter-lake cichlids' similar morphologies do not reflect similar genealogy

The apparent inter-lake morphological similarity among East African Great Lakes' cichlid species/genera has left evolutionary biologists asking whether such similarity is due to sharing of common ancestor or mere convergent evolution. In order to answer such question, we first used Geometric Morphometrics, GM, to quantify morphological similarity and then subsequently used Amplified Fragment Length Polymorphism, AFLP, to determine if similar morphologies imply shared ancestry or convergent evolution. GM revealed that not all presumed morphological similar pairs were indeed similar, and the dendrogram generated from AFLP data indicated distinct clusters corresponding to each lake and not inter-lake morphological similar pairs. Such results imply that the morphological similarity is due to convergent evolution and not shared ancestry. The congruency of GM and AFLP generated dendrograms imply that GM is capable of picking up phylogenetic signal, and thus GM can be potential tool in phylogenetic systematics.     

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

High density whole genome fingerprinting of methicillin-resistant and -susceptible strains of Staphylococcus aureus in search of phenotype-specific molecular determinants

AFLP is a selective restriction fragment amplification method generating DNA fingerprints for microbial isolates. We present high-throughput AFLP (htAFLP) to characterize molecular markers associated with bacterial phenotypes. Methicillin-resistant and -susceptible isolates of Staphylococcus aureus have been used for this model study in conjunction with the available S. aureus genome sequences. This facilitates the calculation of theoretical AFLP fingerprints, comparison of these fingerprints with genuine experimental fingerprints, and the subsequent identification of polymorphic AFLP markers without sequence analysis. Analysis of 46 MRSA and 46 MSSA strains by 39 different AFLP reactions generated more than 2500 fragments per strain and an overall number of 6180 scorable markers within all strains. We successfully identify MRSA specific markers and elaborate on the general applicability of the htAFLP approach. This method can be applied to any microbial species for which at least one full-genome sequence is available.     

Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.

Bacillus anthracis causes anthrax and represents one of the most molecularly monomorphic bacteria known. We have used AFLP (amplified fragment length polymorphism) DNA markers to analyze 78 B. anthracis isolates and six related Bacillus species for molecular variation. AFLP markers are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca. 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations. Extensive variation was observed among taxa, and both cladistic and phenetic analyses were used to construct a phylogeny of B. anthracis and its closest relatives. This genome-wide analysis of 357 AFLP characters (polymorphic fragments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subtilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers with B. anthracis and were used as outgroups to root the analysis. In contrast to the variation among taxa, only rare AFLP marker variation was observed within B. anthracis, which may be the most genetically uniform bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pXO2 plasmids and detected 31 polymorphic chromosomal regions among the 79 B. anthracis isolates. Cluster analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribution are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing anthrax epidemic in Canada and the northern United States is due to a single strain introduction that has remained stable over at least 30 years and a 1,000-mile distribution.     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

Identification of three distinct Polytomella lineages based on mitochondrial DNA features

Polytomella is composed of colorless green algae closely related to Chlamydomonas reinhardtii. Species in the genus have been used in diverse fields of biological research, most recently to study mitochondrial function and mitochondrial genome evolution in the Chlorophyceae, but the phylogenetic relationship between the various available taxa has not yet been clarified and it is not known whether they also possess fragmented mitochondrial genomes, as reported for Polytomella parva. We therefore examined cox1 sequence from seven Polytomella taxa with the goal of establishing their phylogenetic relationships and relating this information to their mitochondrial DNA (mtDNA) fragmentation pattern. We found that the Polytomella isolates examined fall into three distinct lineages, two of which possess fragmented mitochondrial genomes. The third and earliest branching lineage, represented by Polytomella capuana, appears to possess an intact mtDNA. In addition, there is evidence for variation in both size and number of mtDNA fragments between various Polytomella isolates, even within the same lineage. The considerable amount of sequence divergence between lineages seems to correlate with the geographic origin of the strains, leading us to believe that greater amounts of sequence divergence could be uncovered by a broader sampling of Polytomella.     

Optimization of AFLP for extremely large genomes over 70 Gb

Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8‐base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost‐effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo‐cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).     

AFLP variation in 25 Avena species

Current molecular characterization of ex situ plant germplasm has placed more emphasis on cultivated gene pools and less on exotic gene pools representing wild relative species. This study attempted to characterize a selected set of germplasm accessions representing various Avena species with the hope to establish a reference set of exotic oat germplasm for oat breeding and research. The amplified fragment length polymorphism (AFLP) technique was applied to screen 163 accessions of 25 Avena species with diverse geographic origins. For each accession, 413 AFLP polymorphic bands detected by five AFLP primer pairs were scored. The frequencies of polymorphic bands ranged from 0.006 to 0.994 and averaged 0.468. Analysis of molecular variance revealed 59.5% of the total AFLP variation resided among 25 oat species, 45.9% among six assessed sections of the genus, 36.1% among three existing ploidy levels, and 50.8% among eight defined genome types. All the species were clustered together according to their ploidy levels. The C genome diploids appeared to be the most distinct, followed by the Ac genome diploid A. canariensis. The Ac genome seemed to be the oldest in all the A genomes, followed by the As, Al and Ad genomes. The AC genome tetraploids were more related to the ACD genome hexaploids than the AB genome tetraploids. Analysis of AFLP similarity suggested that the AC genome tetraploid A. maroccana was likely derived from the Cp genome diploid A. eriantha and the As genome diploid A. wiestii, and might be the progenitor of the ACD genome hexaploids. These AFLP patterns are significant for our understanding of the evolutionary pathways of Avena species and genomes, for establishing reference sets of exotic oat germplasm, and for exploring new exotic sources of genes for oat improvement.     

AFLP-based analysis to study genetic variability and relationships in the Spanish species of the genus Aegilops

Amplified fragment length polymorphism (AFLP) DNA markers were used to characterize the genetic diversity and relationships in wild species of the genus Aegilops. Fifty populations, which included the species Aegilops biuncialis (UUMM), Ae. neglecta (UUMMNN), Ae. ovata (UUMM), Ae. ventricosa (DDNN) and Ae. triuncialis (UUCC) were selected. These populations are distributed in the Iberian peninsula and Balearic islands. Five AFLP selective primer combinations generated a total of 527 amplification products of which 517 (98.10%) detected polymorphisms. Aegilops neglecta showed the least variation in contrast with Ae. biuncialis that presented the highest degree of polymorphism. Genetic relationships within the populations were evaluated by generating a similarity matrix based on the Jaccard index. In the resulting phenogram Ae. ventricosa appears segregated from the other species, probably owing to the influence of the D genome. The species sharing the U genome are located in the main cluster. The branching pattern of the U genome group reflects the proximity of the species sharing the M genome. Ae. biuncialis and Ae. ovata are clearly separated suggesting that the super index system should be used to differentiate the M genomes of both species. The variation among populations within species in relation to their geographical origin and results previously obtained by the authors using biochemical and molecular markers are discussed.     

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.     

Genetic structure of Eurasian and North American Leymus (Triticeae) wildryes assessed by chloroplast DNA sequences and AFLP profiles

Leymus is a genomically defined allopolyploid of genus Triticeae with two distinct subgenomes. Chloroplast DNA sequences of Eurasian and North American species are distinct and polyphyletic. However, phylogenies derived from chloroplast and nuclear DNA sequences are confounded by polyploidy and lack of polymorphism among many taxa. The AFLP technique can resolve phylogenetic relationships between closely related species, with a curvilinear relationship expected between the proportion of shared bands and nucleotide substitution rate (D), up to about 0.100 D. The objective of this study was to compare D and phylogenetic relationships among 16 Leymus taxa, based on chloroplast DNA sequences and multi-locus AFLP genotypes. Estimates of chloroplast D between taxa were 0.002 and 0.013 within and among continental regions, respectively. Estimates of AFLP D between taxa were 0.076 and 0.093 compared within and between continental regions, respectively, versus 0.024 within taxa. Bayesian and neighbor-joining cluster analyses effectively separated all AFLP genotypes by species, but showed that North American L. ambiguus is a hybrid species with nearly equal contributions from sympatric L. cinereus and L. salinus taxa. Two hierarchical AFLP clades, containing six North American taxa and four Eurasian taxa, had more than 98% bootstrap confidence with 0.071 and 0.055 D among taxa. Three other Eurasian taxa clustered with 79% and 89% confidence, with up to 0.79 D between taxa. These estimates provide benchmarks for phylogenetic comparisons of AFLP profiles, but three taxa could not be reliably grouped, which may reflect concurrent radiation of multiple lineages or lack of homologous AFLP characters caused by a high D.     

The status of AFLP in the genomics era and a pipeline for converting AFLPs into single-locus markers

Even though next-generation sequencing (NGS) has now become the predominant state-of-the-art technique for genotyping populations, amplified fragment length polymorphism (AFLP) DNA fingerprinting is still a relevant method, thanks to its versatility, cost-effectiveness, independence of prior sequence information and broad applicability. Even though the number of AFLP studies reached its peak in 2012, it is still applied extensively for phylogenetic analysis, genotyping or identifying non-model species, which often feature complex and large genomes. For these purposes, tools continue to be developed for designing AFLP studies, scoring AFLPs or handling AFLP data. Moreover, AFLP studies embrace the NGS technology; for example, the whole-genome sequence of model species is used to design more efficient AFLP studies for non-model species. Conversely, in complexity reduction of polymorphic sequences and restriction site-associated DNA sequencing studies, polymorphisms are often found to be present in many restriction sites, which can still be studied as AFLPs. We discuss the latest advances in AFLP-based studies in the era of NGS and anticipate that AFLP will remain a relevant method in the near future, even for species with a known genome, owing to its many promising new features such as methylation-sensitive-AFLP. Here, we also present an optimized pipeline for converting AFLP markers into single-locus markers, which can be applied in both traditional AFLP and NGS studies.     

Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size

We investigate the distribution of sizes of fragments obtained from the amplified fragment length polymorphism (AFLP) marker technique. We find that empirical distributions obtained in two plant species, Phaseolus lunatus and Lolium perenne, are consistent with the expected distributions obtained from analytical theory and from numerical simulations. Our results indicate that the size distribution is strongly asymmetrical, with a much higher proportion of small than large fragments, that it is not influenced by the number of selective nucleotides nor by genome size but that it may vary with genome-wide GC-content, with a higher proportion of small fragments in cases of lower GC-content when considering the standard AFLP protocol with the enzyme MseI. Results from population samples of the two plant species show that there is a negative relationship between AFLP fragment size and fragment population frequency. Monte Carlo simulations reveal that size homoplasy, arising from pulling together nonhomologous fragments of the same size, generates patterns similar to those observed in P. lunatus and L. perenne because of the asymmetry of the size distribution. We discuss the implications of these results in the context of estimating genetic diversity with AFLP markers.     

Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.     

Phylogeny of ten species of the genus Hordeum L. as revealed by AFLP markers and seed storage protein electrophoresis

The phylogenetic relationships of 60 accessions representing ten species of the genus Hordeum were investigated based on AFLP markers and seed storage protein SDS-PAGE electrophoresis. A total of 339 AFLP polymorphic markers were scored as a result of fingerprinting the studied taxa using seven AFLP primer combinations, whereas 46 polymorphic protein bands resulted from the water soluble and water non-soluble seed storage protein electrophoresis. The phylogenetic tree deduced from AFLP analysis is concordant in a large extent with that deduced from seed storage protein electrophoresis. The studied taxa were clustered according to their genome type into two main groups representing the Old and New World’s species. Inside each group the species were clustered according to their genome type. Highly significant cophenetic correlation coefficient was obtained between both AFLP (0.96) and seed storage protein (0.89) indicating the reliability of the results.