AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

A PCR-based DNA fingerprinting technique: AFLP for molecular typing of bacteria.

Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique. In AFLP analysis, bacterial genomic DNA is digested with restriction enzymes, ligated to adapters, and a subset of DNA fragments are amplified using primers containing 16 adapter defined sequences with one additional arbitrary nucleotide. Polymorphisms of different Escherichia coli strains or Agrobacterium tumefaciens strains were demonstrated as distinct, unique bands in a denaturing sequencing gel using AFLP. The polymorphisms detected between BL21 and BL21F'IQ and between DH5 alpha and DH5 alpha F'IQ strains indicated that AFLP is able to resolve differences in F' episomal DNA (approximately 100 kb).     

AFLP fingerprinting of the human genome

Elucidation of the genetic basis of complex traits and diseases in humans includes the use of genome-wide association studies that depend on the analysis of a large number of diallelic markers. We describe the application of the amplified fragment length polymorphism (AFLP) technique as an efficient approach for rapidly identifying and scoring multiple variants in the human genome. Using a commercially available kit, we found that AFLP yields reproducible DNA fingerprints consisting of 42-132 fragments, 8% of which show variability between individuals. These variant markers appear to be from different chromosomes, and the majority of them is diallelic. Based on the information obtained in this study, it is possible to approximate the minimum number of selective AFLP primer combinations needed to approach a desired coverage density of all chromosomes. To our knowledge, this is the first study showing the general applicability of AFLP in humans and providing a constructive guide for the design of genomic studies in Homo sapiens with this robust methodology.     

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-sevenPorphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27Porphyra lines has its unique AFLP fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germ-plasm identification-AFLP) was designed for identification of the 27Porphyra lines. In addition, 21 specific AFLP markers from 15Porphyra lines were identified; 6 AFLP markers from 4Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of thePorphyra lines.     

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.     

Differentiation of European cattle by AFLP fingerprinting

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.     

AFLP fingerprinting reveals pattern differences between template DNA extracted from different plant organs.

AFLP (amplified fragment length polymorphism) fingerprinting of cultivars of bread wheat (Triticum aestivum) and some of its wild relatives has allowed the efficient detection of large numbers of polymorphic amplified fragments. While the reproducibility of fingerprints in repeated experiments is high, pattern differences were observed between fingerprints obtained from seed and leaf DNA template from the same wheat accession. These distinct organ specific amplified DNA fragments were shown to be due neither to genotypic mixtures nor to pathogen contamination. They are likely a result of differences in DNA methylation between organs. Even greater numbers of organ specific amplified fragments were observed when fingerprints obtained from the root and shoot of individual seedlings of the wheat relatives Aegilops mutica and Aegilops speltoides were compared. This phenomenon underlines the importance of ensuring that DNA is extracted from physiologically uniform tissue in phylogenetic studies based on AFLP fingerprints. For this purpose, mature seed is a convenient source.     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

DNA fingerprinting techniques for microorganisms

A whole array of DNA-fingerprinting techniques, which provide indirect access to DNA sequence polymorphism in order to assess species or clonal identity of bacterial organisms or in order to study bacterial genome composition, have been described during past decades. Nomenclature has been sometimes erroneous and/or confusing, also because of hybrid techniques that combine different approaches. It can be shown that most techniques study the sequence polymorphism of only the chromosome, or only the plasmid(s) or only a gene or gene fragment and that the sequence polymorphism is revealed by AFLP (amplified fragment length polymorphism) or by RFLP (restriction fragment length polymorphism) or by special electrophoresis techniques. Starting from these considerations, some taxonomy of techniques, which enables more appropriate nomenclature, can be developed.     

Towards rice genome scanning by map-based AFLP fingerprinting

Map-based DNA fingerprinting with AFLP markers provides a fast method for scanning the rice genome. Three hundred AFLP markers identified with ten primer combinations were mapped in two rice populations. The genetic maps were aligned and almost full coverage of the rice genome was obtained. The transferability of AFLP markers between indica × japonica and indica × indica crosses was tested. The chromosomes were divided into DNA Fingerprint Linkage Blocks (DFLBs) defined by specific AFLP markers. Using these blocks, the degree of similarity or divergence within specific chromosome regions was calculated for nine varieties. Applications of map-based fingerprinting for biodiversity studies and maker-assisted selection are discussed. Received: 6 June 1998 / Accepted: 11 November 1998     

Phylogeny of bovine species based on AFLP fingerprinting

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.     

Genomic diversity of Astragalus cicer microsymbionts revealed by AFLP fingerprinting

DNA polymorphism among 36 Astragalus cicer nodule isolates and 9 reference mesorhizobia was evaluated by a simplified PstI based AFLP procedure with three selective primers: Pst-A, Pst-G, and Pst-GC. The DNA profiles were found to be highly specific for nearly each strain, although DNA bands characteristic for most A. cicer microsymbionts were also noted. The overall topologies of dendrograms, generated by AFLP patterns in PCR reaction with three primers, were very similar to one another and to that constructed by phenotyping. Also the strain compositions in the particular clusters on pheno- and genomograms were in good agreement. The obtained results indicate that AFLP technique can be a useful tool for typing of A. cicer rhizobia as well as for studying their diversity.