Amplified Fragment Length Polymorphism Fingerprinting Research Articles

Population structure of the Dory snapper, Lutjanus fulviflamma, in the western Indian Ocean revealed by means of AFLP fingerprinting

The genetic structure of spatially separated populations of the Dory snapper, Lutjanus fulviflamma, was investigated in seven areas along the East African coast and one area in the Comoros archipelago in the western Indian Ocean, using amplified fragment length polymorphism (AFLP). Phylogenetic and multidimensional scaling analyses did not show any clear clustering of individuals into the spatially separated populations. The analysis of molecular variance clearly showed that the variation was partitioned within populations and not between populations, leading to low genetic differentiation among populations. No clear relationship between genetic distance and geographic distance between populations was observed. These observations suggest that populations of Lutjanus fulviflamma have an open structure and are possibly genetically connected on a large geographic scale in the western Indian Ocean.

Circumpolar phylogeography of Juncus biglumis (Juncaceae) inferred from AFLP fingerprints, cpDNA sequences, nuclear DNA content and chromosome numbers

We explored the circumpolar phylogeographic history of the arctic-alpine Juncus biglumis using amplified fragment length polymorphisms (AFLPs), sequences of cpDNA, relative nuclear DNA content and chromosome numbers. The analyses of the AFLP and cpDNA data gave congruent results and revealed three distinct clades. One of them, represented by a single population from the Taymyr peninsula in northern Siberia, had approximately fourfold larger genome size than the other samples and produced an AFLP pattern that was too aberrant to be analysed together with the rest of the data set. The two other clades represented different ploidy levels (2 n = 60 and 120) as judged from chromosome counts of selected populations but differed only in c. 6% relative DNA content. Based on the AFLP and partly also on the cpDNA data, each of the two main clades was further subdivided into two well-supported subgroups. Three of the subgroups were widespread and exhibited largely overlapping distribution patterns. The fourth subgroup seems to be absent from the North Atlantic region and from western Siberia. We suggest that the four subgroups diverged during isolation in different glacial refugia during the Quaternary. Interestingly, individuals of both main clades were encountered in geographically close populations in eastern Greenland and even within a single population from Svalbard, indicating that both areas were colonised at least twice. The different genome sizes and ploidy levels strongly suggest that the three main clades represent distinct gene pools and act as cryptic species.

Genetic Relationships of Spathiphyllum Cultivars Analyzed by AFLP Markers

Peace lily (Spathiphyllum Schott) is one of the most popular tropical ornamental foliage plants and is used worldwide for interiorscaping. However, little information is available on the genetic relationships of cultivars. Using amplified fragment length polymorphism (AFLP) markers with near-infrared fluorescence-labeled primers, this study analyzed genetic relatedness of 63 commercial cultivars and breeding lines. Forty-eight EcoRI + 2/MseI + 3 primer set combinations were initially screened, from which six primer sets were selected and used in this investigation. All cultivars were clearly differentiated by their AFLP fingerprints, and the relationships were analyzed using the unweighted pair-group method of arithmetic average cluster analysis (UPGMA). The 63 cultivars were divided into four clusters. All commercial cultivars or breeding lines resulted from crosses of some of the cultivars, a total of 45, were positioned in cluster I with Jaccard's similarity coefficients between 0.61 and 0.88. There was only one cultivar in cluster II. Cluster III contained 16 cultivars; they are either species or breeding lines generated from interspecific hybridization. Cluster IV had one unknown species. This study provides genetic evidence as to why cultivars from cluster I and III are not readily crossable because the Jaccard's similarity coefficient between the two clusters was only 0.35. Results also indicate that commercial cultivars are genetically close. Strategies for increasing genetic diversity of cultivated peace lily should be sought for future breeding efforts.

Genetic diversity inferred from AFLP fingerprinting in populations of Onosma echioides (Boraginaceae) from serpentine and calcareous soils

Onosma echioides is a non-obligate serpentinophytic borage occurring discontinuously on calcareous and serpentine outcrops at the northwest limit of its range. Mean concentrations of Ca, Mg and heavy metals in root and shoot samples of eight populations from the two soil types were first determined. Subsequently, the genetic polymorphism of the same accessions was estimated by means of Amplified Fragment Length Polymorphism (AFLP) fingerprinting technique. Root and shoot samples from serpentine outcrops showed higher levels of Ni, Cr and Mg, and lower Ca/Mg ratios compared with those from calcareous soils. Based on 353 polymorphic AFLP bands, the two edaphic groups of populations showed comparable levels of genetic diversity. A remarkable genetic differentiation between populations and a high level of within-population genetic variance were found. Results of Mantel's test supported a significant correlation between genetic and geographical distances, while no difference in relation to the edaphic factor was detected. Molecular data suggested isolation as the key factor shaping the infraspecific genetic structure of O. echioides, which may be in relation with the short-distance, zoochorous systems of seed dispersal and pollination of this species.

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.

AFLP fingerprinting of Colombian Clostridium spp strains, multivariate data analysis and its taxonomical implications

Amplified Fragment Length Polymorphism (AFLP) analysis was used for characterising 13 native Colombian Clostridium spp strains. The DNA extraction method was optimised and the use of cetyl trimethyl ammonium bromide (CTAB) and sodium chloride (NaCl) was incorporated. All strains could be typed in these conditions. The AFLP profiles obtained were submitted to multivariate analysis and compared with previous pulsed field gel electrophoresis (PFGE) results. The results suggested that the set of native strains could correspond to two new species different to those having been described to date. It is proposed that DNA–DNA hybridisation analysis should be done to produce complementary information for describing the new species.

Amplified Fragment Length Polymorphism, Serotyping, and Quinolone Resistance of Campylobacter jejuni and Campylobacter coli Strains from Chicken-Related Samples and Humans in Taiwan

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni isolates from chicken-related samples (n = 32) and humans (n = 27) as well as between Campylobacter coli isolates from chicken-related samples (n = 27) and humans (n = 5). These isolates were collected between 1994 and 2003 in Taiwan. All C. jejuni and C. coli isolates showed highly heterogeneous fingerprints. C. jejuni isolates were separated in two distinct genetic clusters (A and B) at 40% genetic similarity and 42 different AFLP types at 90% similarity. However, three clusters at 40% genetic similarity and 33 different AFLP types at 90% similarity were observed in C. coli isolates. These results showed that AFLP analysis could be used to identify individual isolates of two Campylobacter species. Among C. jejuni isolates, the predominant AFLP type 1 was observed in five (7.9%) isolates, and types 5 and 12 in four (6.3%) isolates each. Cluster B consisted of 10 isolates, while the majority of isolates (n = 53) belonged to cluster A. In some AFLP types (1, 5, 12, 14 and 31), AFLP fingerprints of chicken-related isolates were closely related genetically to those of isolates from humans with gastroenteritis. The predominant serotypes in C. jejuni isolates were B:2 and Y:37. All isolates belonging to serotype O:19 grouped into one single AFLP type. Some chicken samples yielded multiple isolates of Campylobacter harboring simultaneously quinolone-resistant and quinolone-sensitive isolates attributed to the same species, or harboring C. jejuni and C. coli that have the characteristics of quinolone resistance.

TOPAAS, a Tomato and Potato Assembly Assistance System for Selection and Finishing of Bacterial Artificial Chromosomes

We have developed the software package Tomato and Potato Assembly Assistance System (TOPAAS), which automates the assembly and scaffolding of contig sequences for low-coverage sequencing projects. The order of contigs predicted by TOPAAS is based on read pair information; alignments between genomic, expressed sequence tags, and bacterial artificial chromosome (BAC) end sequences; and annotated genes. The contig scaffold is used by TOPAAS for automated design of nonredundant sequence gap-flanking PCR primers. We show that TOPAAS builds reliable scaffolds for tomato (Solanum lycopersicum) and potato (Solanum tuberosum) BAC contigs that were assembled from shotgun sequences covering the target at 6- to 8-fold coverage. More than 90% of the gaps are closed by sequence PCR, based on the predicted ordering information. TOPAAS also assists the selection of large genomic insert clones from BAC libraries for walking. For this, tomato BACs are screened by automated BLAST analysis and in parallel, high-density nonselective amplified fragment length polymorphism fingerprinting is used for constructing a high-resolution BAC physical map. BLAST and amplified fragment length polymorphism analysis are then used together to determine the precise overlap. Assembly onto the seed BAC consensus confirms the BACs are properly selected for having an extremely short overlap and largest extending insert. This method will be particularly applicable where related or syntenic genomes are sequenced, as shown here for the Solanaceae, and potentially useful for the monocots Brassicaceae and Leguminosea.

Lactobacillus nantensis sp. nov., isolated from French wheat sourdough

A polyphasic taxonomic study of the bacterial flora isolated from traditional French wheat sourdough, using phenotypic characterization and phylogenetic as well as genetic methods, revealed a consistent group of isolates that could not be assigned to any recognized species. These results were confirmed by randomly amplified polymorphic DNA and amplified fragment length polymorphism fingerprinting analyses. Cells were Gram-positive, homofermentative rods. Comparative 16S rRNA gene sequence analysis of the representative strain LP33T indicated that these strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus paralimentarius and Lactobacillus mindensis. DNA-DNA reassociation experiments with the three phylogenetically closest Lactobacillus species confirmed that LP33T (= DSM 16982T = CIP 108546T = TMW 1.1265T) represents the type strain of a novel species, for which the name Lactobacillus nantensis sp. nov. is proposed.

Cytogenetic and AFLP Fingerprints for Five Species of Thai Macaques

Genetic studies of 5 species of macaques using G-banding patterns were investigated. Blood samples of them were examined using lymphocyte culture technique. The results indicate that they have identical data including the number of diploid chromosome is 42; the types of autosomes are metacentrics and submetacentrics viz. 6 large metacentrics, 4 medium metacentrics and 8 small metacentrics, and 8 large submetacentrics, 12 medium submetacentrics and 2 small submetacentrics; the short arm of chromosome pair 13 is a satellite chromosome. A few difference is X and Y chromosome. The X and Y chromosomes are medium submetacentrics and medium metacentrics, and small submetacentric, medium submetacentric and small telocentrics, respectively. The cytogenetic studies show that all 5 species Thai macaques have a common evolutionary relationship. For studying in depths, molecular markers were assessed using the Amplified Fragment Length Polymorphism (AFLP) method. The AFLP fingerprints from 7 primer combinations were compared. A total of 1754 bands, with 890 (50.71%) polymorphic bands, were generated. Seven monomorphic bands (0.78%) were found in all individuals of macaques. The average genetic distance (D) between species based on AFLP analysis shows 0.269 to 0.380. The developing of specific cytogenetic and AFLP fingerprints of a species is possible further benefit for humanity.

Assessment of genetic diversity in Turkish oregano (Origanum onites L.) germplasm by AFLP analysis

SummaryOriganum onites (L.) is one of the most widely traded culinary herb species in Turkey. Despite limited knowledge on the level of genetic diversity, its use as a source of seed for planting is widespread. To initiate a selective breeding programme, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the oregano germplasm collection of the Aegean Agricultural Research Institute of the Ministry of Agriculture and Rural Affairs, Turkey. Forty-four Origanum accessions were analysed with ten Eco RI-Mse I primer combinations. In total, 365 unambiguous, selectively amplified polymorphic fragments were detected and 294 polymorphic bands were scored for analysis. Seven main groups were identified by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering using Jaccard’s pairwise similarity coefficient. When this genetic analysis was compared with the sites of collection of the accessions, there was no close genetic similarity among accessions related to their growing region. Relatively low genetic similarity (0.396 – 0.725%) between accessions indicated that the rate of gene flow between Origanum species was high, as a result of cross-pollination. The results of this study indicate that a broad range of genetic variability exists between accessions of oregano and that future breeding programmes could be improved, based on these data, by selecting materials with maximally different AFLP fingerprints.

Genetic variation, taxonomy and mountain-hopping of four bipolar Carex species (Cyperaceae) analysed by AFLP fingerprinting

The bipolar sedges Carex canescens, C. echinata, C. lachenalii and C. magellanica (including C. paupercula) were analysed by amplified fragment length polymorphisms (AFLPs). The genetic variation of Carex populations from northern Europe was compared with those from the farthest locations in the Southern Hemisphere (i.e. Australia, New Zealand and Chile). This DNA fingerprinting of 152 plants yielded 223 AFLP bands, which were scored as present/absent and converted to Jaccard’s dichotomy coefficients. Unweighted pair-group methods using arithmetic averages (UPGMA), neighbour-joining (NJ), and principal coordinate analysis (PCA) were performed. We obtained AFLP-based separations of the four Carex species that were in full agreement with previous morphology-based taxonomy. A large number of species-specific bands occurred. Hemisphere-specific bands were not identified, but all analyses showed a clear distinction between populations collected from the Northern and Southern Hemispheres. The results are discussed in relation to the mountain-hopping hypothesis, which is one possible mechanism underlying the bipolar distribution of those species.

Studies on genetic changes in rye samples (Secale cereale L.) maintained in a seed bank

The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.

Genetic diversity of bacterial strains isolated from soils, contaminated with polycyclic aromatic hydrocarbons, by 16S rRNA gene sequencing and amplified fragment length polymorphism fingerprinting

In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.

Development of a Multilocus Sequence Typing Scheme for Characterization of Clinical Isolates of Acinetobacter baumannii

In this study a multilocus sequence typing (MLST) scheme for Acinetobacter baumannii was developed and evaluated by using 40 clinical A. baumannii isolates recovered from outbreaks in Spanish and German hospitals during the years 1990 to 2001, as well as isolates from other European hospitals and two DSMZ reference strains of A. baumannii. For comparison, two isolates of Acinetobacter species 13 (sensu Tjernberg and Ursing), two clinical isolates, and three DSMZ strains of A. calcoaceticus (both belonging to the A. calcoaceticus-A. baumannii complex) were also investigated. Primers were designed for conserved regions of housekeeping genes, and 305- to 513-bp internal fragments of seven such genes-gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD-were sequenced for all strains. The number of alleles at individual loci ranged from 6 to 12, and a total of 20 allelic profiles or sequence types were distinguished among the investigated A. baumannii strains. The MLST data were in high concordance with the epidemiologic typing results generated by pulsed-field gel electrophoresis and amplified fragment length polymorphism fingerprinting. The MLST scheme provides a high level of resolution and an excellent tool for studying the population structure and long-term epidemiology of A. baumannii.

(285) DNA Fingerprinting of Flowering Dogwood Cultivars

Flowering dogwood (Cornus florida L.) is an important tree of forests and urban landscapes in the eastern United States. Currently, there are over 100 cultivars of flowering dogwood commercially available. An identification process based on genotype would be of use to researchers, breeders, and nurserymen, as many cultivars are similar phenotypically. Molecular markers offer a promising way of definitively identifying flowering dogwood cultivars. Amplified fragment length polymorphism (AFLP) is a technique that can be used to generate DNA fingerprints. DNA was isolated from leaves of 17 common cultivars of dogwood and AFLP fingerprints were generated by a Beckman Coulter CEQ™ 8000. Fingerprints were converted to binary data and verified manually. Two drafts of a cultivar identification key were generated based on the corrected, verified binary data and cultivar-specific peaks. Six primer combinations were used to construct all keys and were tested with seven unknown dogwood cultivar samples. Six unknown samples were correctly identified using the keys. Only one unknown, `Cherokee Brave', was unidentifiable with any key. In all cases, some intracultivar variation was observed. A similarity index was calculated and visualized with a tree of genetic relatedness using NTSYSpc. Intracultivar variation was observed in the similarity index as well. This database for cultivar-specific molecular markers will serve as a starting point to which other cultivars can be added and also can be used in breeding applications, patent application and other projects, such as mapping the C. florida genome.

DNA Methylation Polymorphisms in Somaclonal-derived Cultivars of Ornamental Aroids

Plant tissue culture can induce a variety of genetic and epigenetic changes in regenerated plantlets, a phenomenon known as somaclonal variation. Such variation has been widely used in the ornamental foliage plant industry as a source for selection of new cultivars. In ornamental aroids alone, at least 63 somaclonal-derived cultivars have been released. In addition to morphological differences, many somaclonal aroid cultivars can be distinguished by amplified fragment length polymorphism (AFLP) analysis. However, a few cultivars have no detectable polymorphisms with their parents or close relatives by AFLP fingerprints. It is postulated that DNA methylation may be involved in the morphological changes of these cultivars. In this study, methylation-sensitive amplification polymorphism (MSAP) technique was used to study DNA methylation in selected somaclonal cultivars of Alocasia, Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium. Results showed that polymorphisms were detected in the somaclonal cultivars, suggesting that DNA methylation polymorphisms may associate with tissue culture-induced mutation in ornamental aroids. This is the first study of methylation variation in somaclonal variants of ornamental foliage plants. The results clearly demonstrate that the MSAP technique is highly efficient in detecting DNA methylation events in somaclonal-derived cultivars.

Analysis of genetic variation in selected stocks of hatchery flounder, Paralichthys olivaceus, using AFLP markers

Amplified fragment length polymorphism (AFLP) analysis was performed in order to evaluate genetic characteristics of one common population and two selective hatchery populations of flounder Paralichthys olivaceus. A group of 60 genotypes belonging to three populations was screened using 10 different AFLP primer combinations. A total of 491 loci were produced in the three studied populations. The loci of 65.78%, 61.47% and 60.92% were polymorphic over all the genotypes tested in common, susceptible and resistant populations, respectively. The number of polymorphic loci detected by single primer combination ranged from 21 to 43. The average heterozygosity of common, susceptible and resistant populations was 0.1656, 0.1609 and 0.1586, respectively, which showed no significant difference. Compared with the common population, the two selective hatchery populations, susceptible and resistant, showed significant genetic differences including a smaller ( P < 0.05) number of total loci, a smaller ( P < 0.05) number of total polymorphic loci and a smaller ( P < 0.05) percentage of low frequency (0–0.2) polymorphic loci. AFLP banding pattern was transformed into binary data and matrices were processed with POPGENE and TFPGA software. Similarity relationships were described graphically by a dendrogram, which clustered the three populations. The AFLP fingerprinting technique was confirmed to be a reproducible and sensitive tool for the study of population genetics of flounder. The present study confirmed that it was important to detect the genetic variability of the selective hatchery populations for the conservation of natural flounder resources.

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.

Assessment of the genetic diversity of geographical unrelated Microcystis aeruginosa strains using amplified fragment length polymorphisms (AFLPs)

Molecular marker analysis is becoming increasingly capable of identifying informative genetic variation. Amplified fragment length polymorphism markers (AFLPs) are among the recent innovations in genetic marker technologies, and provide a greater capacity for genome coverage and more reproducible results than previous technologies. We have investigated the usefulness of AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate between geographical unrelated Microcystis strains. In total 23 strains were subjected to the AFLP fingerprinting. After analysis of the data on the basis of the average linkage method, known as the Unweighted Pair Group Method using Arithmetic averages (UPGMA), a dendrogram with four clusters was obtained. Cluster 1 consisted mainly of the NIES strains that originated from Japan, while in cluster 2 the European strains grouped together. The South African strains that originated from the northern part of the country group together in cluster 3, while the strains collected from the central and southern regions group together with the US strains in cluster 4. The study had reveals extensive evidence for the applicability of AFLP in cyanobacterial taxonomy, and furthermore clearly demonstrates the superior discriminative power of AFLP towards the differentiation of geographical unrelated Microcystis aeruginosa strains that belong to the same species, as well as highlighting the potential of this fingerprinting method in evolutionary studies. African Journal of Biotechnology Vol. 4 (5), pp. 389-399, 2005