Amplified Fragment Length Polymorphism Patterns Research Articles

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae x P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.

Silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus Trametes

Silver stained denaturing polyacrylamide gels (PAGEs) and fluorescent denaturing automated capillary electrophoresis (CE) were used to detect amplified fragment length polymorphism (AFLP) patterns obtained from white-rot fungi belonging to the genus Trametes. AFLP fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of Trametes ochracea, nine strains of Trametes hirsuta and ten isolates of Trametes versicolor. UPGMA dendrograms derived from fluorescently labelled and silver stained AFLP patterns were similar, but a few differences were detected especially in the clustering of T. ochracea and T. hirsuta strains. Compared to silver-stained AFLP, detection of fluorescent AFLP was fast, reliable and easy to perform and it facilitated surveying with a computerized analysis system. Fluorescent CE seems to be well suited for studying similarity between Trametes species.

Analysis of genetic heterogeneity in Chlamydia trachomatis clinical isolates of serovars D, E, and F by amplified fragment length polymorphism.

Amplified fragment length polymorphism (AFLP) fingerprinting of clinical isolates of Chlamydia trachomatis serovars D, E, and F showed a low percentage of genetic heterogeneity, but clear differences were found. Isolates from index patients and partners had identical AFLP patterns and AFLP markers. Characterization of these AFLP markers could give more insight into the differences in virulence and clinical course of C. trachomatis infections.

Amplified fragment length polymorphism fingerprinting of Pseudomonas strains from a poultry processing plant.

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.

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.

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii (Plumbaginaceae) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii. Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.

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.

Development of Amplified Fragment Length Polymorphism (AFLP) Markers Suitable for Genetic Linkage Mapping of Catfish

Genomic research requires many molecular markers for construction of the genetic linkage map and for marker-assisted selection (MAS). Amplified fragment length polymorphism (AFLP) markers are inherited with Mendelian expectations in catfish and, thus, are suitable for use in gene mapping and MAS. To identify large numbers of AFLP markers, 64 primer combinations were tested to generate AFLP patterns between channel catfish Ictalurus punctatus and blue catfish I. furcatus. The 64 primer combinations amplified 7,871 bands, of which 3,081 were polymorphic between the two mating parents of channel catfish and blue catfish used to produce the reference families for mapping analysis. The primer combinations that produced fewer bands showed higher rates of polymorphism. Of the 64 primers, 53 primer combinations produced clean and reproducible AFLP profiles. Nine primer combinations produced fair AFLP profiles. The remaining two primers produced poor AFLP profiles with intensive background bands. The 53 excellent primer combinations produced 2,572 AFLP markers. These markers are suitable for use in gene and quantitative trait loci mapping of catfish by using the interspecific hybrid system. Analysis of genetic variations among strains of channel catfish indicated that AFLP should also be a valuable tool for genetic resource and population analyses in fish.

Amplified fragment length polymorphism analysis of vandaceous orchids

We investigated the application of the PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), in orchids. The optimal AFLP patterns have been determined using primer combinations of EcoRI +4 and MseI +3 selective nucleotides. The same reproducible AFLP patterns were demonstrated in genomic DNAs isolated both from: (1) different orchid tissues, e.g. leaves and flowers; and (2) orchid flowers collected at different times. Genomic variations among different cultivars of vandaceous orchid hybrids were successfully determined by AFLP analysis. More than 10% of the AFLP bands were polymorphic DNA when siblings, derived from the same original crosses (two cultivars of Aranda Christine, five cultivars of Mokara Willie How), were used. Only 0.3–0.7% of the AFLP patterns were shown to be polymorphic when different cultivars, originating from somatic mutations during meristem culture for massive propagation, were used (two cultivars of Ar. Christine, four cultivars of M. Chark Kuan).

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.

AFLP markers for DNA fingerprinting in cattle.

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with EcoRI and MseI is suggested, in mammals the enzyme combination EcoRI/TaqI produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 EcoRI/TaqI primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0.31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.

Development of AFLP markers in barley.

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley.