Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radio-isotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLP-generated data with DNA-DNA homology data.     

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.     

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.     

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.     

Characterization of a genetic resource collection for Miscanthus (Saccharinae,Andropogoneae, Poaceae) using AFLP and ISSR PCR

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat markers were employed to characterize a genetic resource collection of Miscanthus, a grass under trial in Europe as a biomass crop. The 26 polymorphic markers produced by two ISSR fingerprinting primers were able to discriminate taxa and identify putative clones. AFLP fingerprints were fully reproducible and produced a larger number of markers for the three primer pairs tested, of which 998 were polymorphic (representing 79.3% of all bands). AFLP markers distinguished species, infra-specific taxa (varieties and cultivars) and putatively clonal material. They were also used to assess the inter-relationships of the taxa, to investigate the origin of important hybrid plants and to estimate the overall level of genetic variation in the collection. They were useful for assessing the species status of certain taxa such as M. transmorrisonensis, an endemic from Taiwan that was clearly distinct from M. sinensis; whereas other taxa of disputed species status, such as M. condensatus and M. yakushimanum were not genetically distinct from M. sinensis. The AFLP markers detected a high degree of infra-specific variation and allowed subdivisions of the genetic resource collection to be made, particularly within M. sinensis.     

Karyotype and AFLP data reveal the phylogenetic position of the Brazilian endemic Hypochaeris catharinensis (Asteraceae)

The genus Hypochaeris offers an excellent model for studies of recent adaptive radiation in the South American continent. We used karyotype analysis with chromomycin?A₃ (CMA₃)/4??,6-diamidino-2-phenylindole (DAPI) banding and fluorescence in?situ hybridization (FISH), and amplified fragment length polymorphism (AFLP) fingerprinting to investigate for the first time the Brazilian endemic H.?catharinensis and define its position within the South American group of species. Strong CMA-positive signals were seen at the end of both arms of chromosome?3 and at the end of the long arm of chromosome?4. DAPI bands were only detected in subterminal position on short arm of chromosome?4. FISH with 5S and 35S ribosomal DNA (rDNA) probes revealed a single 5S rDNA locus on short arm of chromosome?2, typical for all other South American Hypochaeris taxa analyzed to date. The 35S rDNA locus was identified at subterminal position on the short arm of chromosome?3, as reported so far for only two of the known species (H.?lutea and H.?patagonica). The AFLP study included 55 individuals, comprising nine species of the South American Hypochaeris plus their putative ancestor H.?angustifolia. Eleven AFLP primer combinations generated a total of 401 fragments, of which 388 (96.7%) were polymorphic. High genetic similarities were observed among taxa, with all South American Hypochaeris species falling into one main cluster [100% bootstrap (BS)]. Hypochaeris catharinensis is closely related to H.?lutea (82% BS), forming a well-separated subcluster within the South American species. Taken together, the karyological and AFLP data contribute to the placement of H.?catharinensis within the phylogenetic framework of South American species of Hypochaeris and allow the definition of a novel and well-resolved phylogenetic group (the Lutea group).     

AFLP characterization of natural populations of Berberis (Berberidaceae) in Patagonia, Argentina

 AFLP markers were used to analyse the intra- and interspecific relationships among 22 natural populations of 13 Patagonian species of Berberis and the relationships among the taxa belonging to homoploid and polyploid complexes. Seven primer combinations gave rise to 231 AFLP bands, of which 199 were polymorphic. Correspondence between AFLP data, morphological traits and seed protein bands was also assessed. The dendrogram inferred from AFLP fingerprints showed that, in general, populations of the same species formed closely related groups with high coefficients of similarity. Principal co-ordinates analysis showed two separate subgroups: (i) B. bidentata and their putative ancestors –B. darwinii and B. linearifolia– which form a homogamic group, and (ii) B. buxifolia, B. heterophylla and B. parodii– which could form a polyploid hybrid complex. Received March 21, 2001 Accepted September 11, 2001     

Effects in s.e. Queensland during 1967–72 of insects introduced to controlLantana camara

Seasonal fluctuations in populations of the introduced speciesTeleonemia scrupulosa Stål and damage by this insect to common taxa ofLantana camara, were observed at several field sites and experimental plots. At 2 plots effects ofT. scrupulosa populations on untreated plants were compared with plants protected by application of insecticides. On untreated plants damage varied seasonally and between taxa ofL. camara. In general attack byT. scrupulosa reduced fruiting and caused severe short term foliage damage. Combined with severe stress from some other cause heavy attack may result in long term damage or death to plants. However Common PinkL. camara was more resistant to damage than were the other taxa studied. At the time of these experiments other insect species were at very low population levels and did not have observable effects onL. camara.     

Phylogenetic relationships and genetic diversity of the Salicornieae (Chenopodiaceae) native to the Atlantic coasts of France

Phylogenetic relationships of members of the tribe Salicornieae, native to the Atlantic coasts of France, were assessed by three molecular markers: the nuclear ribosomal internal transcribed spacer (ITS), the chloroplast trnL-F and the chloroplast matK sequences. In parallel to the phylogenetic studies, a population genetic study was carried out based on randomly amplified polymorphic DNAs (RAPD). Neither the MP/ML analyses of the sequences, nor the AMOVA and NJ analyses of RAPD fingerprints confirmed the morphology-based classification at the specific level within Salicornia. Instead, our investigations are in favour of the species aggregate concept. Two sister groups were revealed in the genus: one is composed of the diploid taxa, while the other clusters the tetraploid taxa. Conflicting nuclear versus plastid phylogenetic positions of some tetraploid samples, referred to as S. fragilis, indicate that they most likely derive from a reticulate evolution.     

Dynamic grapevine clones��an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.     

The suitability ofTeleonemia harleyi for biological control ofLantana camara in Australia

The tingidTeleonemia harleyi Froeschner from Trinidad is known to destroy the flowers ofLantana camara L. and so prevent seed formation. Colonies caged on plants destroy all flowers. Studies on the host range ofT. harleyi indicate that it is highly specific, feeding and ovipositing only onL. camara. It attacks all pest taxa ofL. camara naturalized in Australia and should be a valuable addition to the biological control complex. It was approved for liberation in Australia in September 1972.     

The potential of AFLPs in biosystematics: A first application inSolanum taxonomy(Solanaceae)

Using the AFLP technique highly informative DNA fingerprints were generated from 19 taxa ofSolanum sect.Petota (potatoes) and three taxa ofSolanum sect.Lycopersicum (tomatoes). Both phenetic and cladistic analyses were conducted from the individual genotypic level to the species level. An AFLP fingerprint, using a combination of suitable AFLP primers, generated 12 to 71 scorable fragments per genotype which was sufficient for taxonomic interpretation. The classifications based on the molecular markers were generally in agreement with current taxonomic opinions. Unexpectedly,S. microdontum was associated with ser.Megistacroloba rather than with ser.Tuberosa, andS. demissum (ser.Demissa) and species of ser.Acaulia appeared closely affiliated. AFLP is an efficient and reliable technique to generate biosystematic data and therefore a promising tool for evolutionary studies.     

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.     

吴茱萸的AFLP指纹图谱的初步研究

首次报道中草药植物吴茱萸基因组DNA指纹图谱的研究。吴茱萸是贵州省内经济价值极高的中草药之一,采用扩增片段长度多态性（AFLP）技术来分析来自不同地区的石虎、疏毛、大花吴茱萸3个品种的DNA指纹图谱,从18对引物中筛选出3对引物对19份材料的DNA检测,共得到93条带,其中多态性片段57条（平均61.3%）。3对引物组合从DNA指纹图谱上将19份材料完全区分开,结果表明AFLP技术是鉴别吴茱萸相近品种的有效方法,是形态学鉴定方法的有益补充;UPGMA方法聚类分析显示19份种质材料间的相似系数为0.235～0.941,表明吴茱萸种质间的遗传多样性丰富;余庆地区种植基地的石虎和疏毛样本聚为一类,提示人工栽培影响到吴茱萸的遗传特性。     

Genetic relationship and genetic diversity among Typha taxa from East Asia based on AFLP markers

The genetic relationship and diversity among four Typha taxa in East Asia were evaluated using amplified fragment length polymorphism (AFLP) markers. Three AFLP selective primer combinations generated a total of 707 amplification products, of which 704 (99.6%) were polymorphic. The unweighted pair-group method with arithmetic mean (UPGMA) dendrogram and principal component analysis (PCA) plot confirmed the taxonomic status of four separate species. East Asian Typha taxa separated into two groups: the first with Typha angustifolia and the second with T. orientalis, T. laxmanni, and T. latifolia with a high bootstrap value for UPGMA (93%) and a low first score for PCA (25%). The two clusters corresponded with two sections based on the bracteoles in the female flower: section Bracteolatae and section Ebracteolatae. T. angustifolia showed the highest genetic diversity among the four Typha taxa (percentage of polymorphic loci [PPL] = 71%, H_o = 0.157), whereas T. latifolia had the lowest genetic diversity (PPL = 40%, H_o = 0.117). Genetic diversity was related to the presence of the gap between male and female inflorescences. A positive correlation between genetic distance and geographic distance was clearly found in the two species with continuous inflorescences (T. latifolia and T. orientalis). This positive correlation was not observed in the other species with discontinuous spikes (T. angustifolia and T. laxmanni).     

Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.     

Genome and species relationships in genus<Emphasis Type="Italic"> Avena</Emphasis> based on RAPD and AFLP molecular markers

Species and genome relationships among 11 diploid (A and C genomes), five tetraploid (AB and AC genomes) and two hexaploid (ACD genome) Avena taxa were investigated using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers. The two primer pairs used for the AFLP reactions produced a total of 354 polymorphic bands, while 187 reproducible bands were generated using ten RAPD primers. Genetic similarities amongst the entries were estimated using the Jaccard and Dice algorithms, and cluster analyses were performed using UPGMA and neighbor joining methods. Principle coordinate analysis was also applied. The highest cophenetic correlation coefficient was obtained for the Jaccard algorithm and UPGMA clustering method (r=0.99 for AFLP and r=0.94 for RAPD). No major clustering differences were present between phenograms produced with AFLPs and RAPDs. Furthermore, data produced with AFLPs and RAPDs were highly correlated (r=0.92), indicating the reliability of our results. All A genome diploid taxa are clustered together according to their karyotype. The AB genome tetraploids were found to form a subcluster within the A_s genome diploids (AFLPs), indicating their near-autoploid origin. The AC genome tetraploids are clustered to the ACD genome hexaploids. Finally, the C genome diploids form an outer branch, indicating the major genomic divergence between the A and C genomes in Avena.Communicated by J.S. Heslop-Harrison     

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.