Amplified Fragment Length Polymorphism Profiles Research Articles

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.

Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in High-Risk Wards

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group ( r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.

Genotyping ofEdwardsiella ictaluriisolates in Japan using amplified-fragment length polymorphism analysis

The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007. Ten isolates of Edw. ictaluri in 2007-2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles. No DNA polymorphisms were found among Japanese Edw. ictaluri isolates. A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.

Molecular comparison of natural hybrids of Phytophthora nicotianae and P. cactorum infecting loquat trees in Peru and Taiwan

Natural Phytophthora hybrids (P. nicotianae × P. cactorum) infecting loquat in Peru and Taiwan were characterized with AFLP (amplified fragment length polymorphism) markers, the internal transcribed spacer (ITS) region and the phenol acid carboxylase gene (Pheca) and inheritance of the mitochondrial cytochrome oxidase I gene (coxI). AFLP profiles of two Taiwanese isolates recovered in 1995 were polymorphic in ~50% of the fragments whereas five Peruvian isolates, recovered 2002–2003 and 2007, showed no genotypic variation. Sequencing analysis of the cloned ITS region resulted in the identification of sequences with high homology to either P. nicotianae (99%) or P. cactorum (97%). Direct sequence analysis of the Pheca gene revealed 13 heterozygous sites suggesting the presence of both P. nicotianae and P. cactorum genes in P. hybrids isolates. Melting analyses of coxI suggested that all seven Phytophthora hybrids inherited the mitochondrial DNA from P. nicotianae. Our results suggest that Phytophthora hybrids from Peru might have originated from a single hybridization event and that the two isolates from Taiwan might have originated through different hybridization events. The Peruvian hybrids appear to have persisted at least 3 y at three locations. Possible factors influencing the population structure of Phytophthora hybrids infecting loquat are discussed.

Identification of AFLP fragments linked to seedlessness in Ponkan mandarin ( Citrus reticulata Blanco) and conversion to SCAR markers

Seedlessness is a desirable trait in citrus and has been an important breeding objective. In this study, we employed the amplified fragment length polymorphism (AFLP) technique to find molecular markers linked to the seedless trait in the Ponkan mandarin ( Citrus reticulata Blanco). After screening with 72 primer pairs, 5 AFLP markers were identified that putatively correlated with the target trait. Their association was also tested by analyzing the AFLP profile from pooled individual accessions. The five fragments were cloned and sequenced, and BLAST searches showed that four of the markers had high homology to functional genes, providing some promising information that may aid in understanding the molecular mechanism of seedlessness in citrus. Based on the sequence information, eight specific primers were designed and eventually fragments AFLP-2 and AFLP-5 were successfully converted into sequence characterized amplified region (SCAR) markers. Thus, the markers detected could be useful for accelerating citrus breeding programmes by enabling early screening for seedlessness mutations using marker-assisted selection.

Lactobacillus kisonensis sp. nov., Lactobacillus otakiensis sp. nov., Lactobacillus rapi sp. nov. and Lactobacillus sunkii sp. nov., heterofermentative species isolated from sunki, a traditional Japanese pickle

Thirty six Gram-positive, rod-shaped, non-spore-forming, non-motile bacterial strains were isolated from the non-salted pickle solution used in producing sunki products, a traditional Japanese pickle. The novel strains were discriminated and separated into four groups by amplified fragment length polymorphism profiling, and by analysis based on recA gene sequences. The strains were classified into four species groups belonging to the Lactobacillus buchneri species group, which consists of L. buchneri, Lactobacillus diolivorans, Lactobacillus hilgardii, Lactobacillus kefiri, Lactobacillus parabuchneri and Lactobacillus parakefiri. The phenotypic and genotypic features of the four groups demonstrated that they represented four novel species, for which the names Lactobacillus kisonensis sp. nov. (type strain YIT 11168(T)=NRIC 0741(T)=JCM 15041(T)=DSM 19906(T)), Lactobacillus otakiensis sp. nov. (type strain YIT 11163(T)=NRIC 0742(T)=JCM 15040(T)=DSM 19908(T)), Lactobacillus rapi sp. nov. (type strain YIT 11204(T)=NRIC 0743(T)=JCM 15042(T)=DSM 19907(T)) and Lactobacillus sunkii sp. nov. (type strain YIT 11161(T)=NRIC 0744(T)=JCM 15039(T)=DSM 19904(T)) are proposed.

Successful analysis of AFLPs from non-lethally sampled wing tissues in butterflies

We demonstrate the successful generation and analysis of amplified fragment length polymorphism (AFLP) profiles from small samples of wing tissue in two butterfly species. With slight modifications of commercial DNA extraction and AFLP kit protocols, we produced highly repeatable AFLP profiles from these non-destructive tissue samples. Error rates were comparable to those previously reported for AFLPs generated from lethally obtained thoracic tissue. Furthermore, AFLP profiles obtained from thoracic and wing tissues of the same individuals were identical. Our results indicate that AFLP analysis is a viable method of obtaining genetic data from threatened populations of butterflies, and potentially other insects, using small, non-destructive tissue samples.

Molecular Marker Analysis of Differentially Aged Seeds of Soybean and Safflower

Storage of seeds for extended periods causes a number of degradative changes related to the aging process such as decreased seedling vigor and reduced germination. In this study, molecular markers were used to study the aging process in seeds of two different plants species. Seeds of three differentially aged seed groups, including control (un-aged), naturally aged, and accelerated aging, from soybean (Glycine max) and safflower (Carthamus tinctorius) were evaluated for genetic variability using random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. For both plant species, naturally aged and accelerated aged groups clustered together with RAPD markers, whereas control and naturally aged seeds showed similarity in both AFLP and SSR profiles. Based on these findings, it can be concluded that observed changes in DNA profiles of seeds from different aged groups did not contribute to accumulation of genetic variations of the same magnitude. Therefore, seed of similar viability must be selected for molecular marker analysis for plant variety protection, among other comparative studies.

Species delimitation and phylogeny of a New Zealand plant species radiation

BackgroundDelimiting species boundaries and reconstructing the evolutionary relationships of late Tertiary and Quaternary species radiations is difficult. One recent approach emphasizes the use of genome-wide molecular markers, such as amplified fragment length polymorphisms (AFLPs) and single nucleotide polymorphisms (SNPs), to identify distinct metapopulation lineages as taxonomic species. Here we investigate the properties of AFLP data, and the usefulness of tree-based and non-tree-based clustering methods to delimit species and reconstruct evolutionary relationships among high-elevation Ourisia species (Plantaginaceae) in the New Zealand archipelago.ResultsNew Zealand Ourisia are shown to comprise a geologically recent species radiation based on molecular dating analyses of ITS sequences (0.4–1.3 MY). Supernetwork analyses indicate that separate tree-based clustering analyses of four independent AFLP primer combinations and 193 individuals of Ourisia produced similar trees. When combined and analysed using tree building methods, 15 distinct metapopulations could be identified. These clusters corresponded very closely to species and subspecies identified on the basis of diagnostic morphological characters. In contrast, Structure and PCO-MC analyses of the same data identified a maximum of 12 and 8 metapopulations, respectively. All approaches resolved a large-leaved group and a small-leaved group, as well as a lineage of three alpine species within the small-leaved group. We were unable to further resolve relationships within these groups as corrected and uncorrected distances derived from AFLP profiles had limited tree-like properties.ConclusionOurisia radiated into a range of alpine and subalpine habitats in New Zealand during the Pleistocene, resulting in 13 morphologically and ecologically distinct species, including one reinstated from subspecies rank. Analyses of AFLP identified distinct metapopulations consistent with morphological characters allowing species boundaries to be delimited in Ourisia. Importantly, Structure analyses suggest some degree of admixture with most species, which may also explain why the AFLP data do not exhibit sufficient tree-like properties necessary for reconstructing some species relationships. We discuss this feature and highlight the importance of improving models for phylogenetic analyses of species radiations using AFLP and SNP data.

Genetic diversity in European pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae), populations assessed using AFLP analysis

The pollen beetle (Meligethes aeneus) is one of the most important insect pests of oilseed rape (Brassica napus), and extensive use of insecticides is required to protect crop yields. To meet the challenges set by agricultural demands for more sustainable production and changing climate more information about pest biology and population genetics is needed. Using genomic Amplified Fragment Length Polymorphism (AFLP) analysis, DNA polymorphism was studied in 14 field populations of pollen beetles, collected during 2004 in six European countries (Denmark, France, Finland, Germany, Sweden, and UK). Using one primer combination 410 polymorphic DNA fragments were obtained based on analysis of single beetles. AFLP profiles were analysed with similarity measures using the Nei and Li coefficient and dendrograms generated. Dendrograms constructed from distance matrices revealed clustering by population origin and assignment analysis generally supported the genotype classification. Principal component analysis of the fourteen groups resulted in wide dispersion but also connections between some groups. Statistical analysis using AMOVA showed that the levels of genetic variation within populations explained most of the variation. Migrant analysis suggested a low level of gene flow between pollen beetle populations at different geographical locations indicating little long range dispersal of pollen beetles. However, a Mantel test found no correlation between genetic and geographical distance. Apparently genetic differentiation among populations has a complex background and may involve factors such as local adaptation and founder effects.

Can Campylobacter coli induce Guillain-Barré syndrome?

Can Campylobacter coli induce Guillain-Barré syndrome?

Amplified fragment length polymorphisms reveal genetic differentiation among strains of Xanthomonas albilineans

Xanthomonas albilineans, the causative agent of leaf scald disease (LSD), colonizes the vascular system of sugarcane ( Saccharum spp. hybrids). In this study X. albilineans strains from 28 countries were differentiated by using two methods of amplified fragment length polymorphism (AFLP). In the manual procedure, AFLP reactions were performed on 57 X. albilineans strains and after selective amplification using radiolabelled primers, the resulting products were separated using polyacrylamide gel electrophoresis. The autoradiographs were analyzed using GelCompar version 4.1 software (Applied Maths) to construct dendograms from similarity matrices. Fluorescent AFLP (FAFLP) was also employed on 52 X. albilineans strains using three fluorescently labelled primer combinations (automated AFLP). The FAFLP data was converted to a binary format using the Genemapper Software 3.7 (Applied Biosystems). Thereafter, dendograms were generated using the NTSYSpc. Software (USA). Distinct AFLP profiles were produced for the majority of the strains and were found to be useful in differentiating X. albilineans strains from various geographical locations. Fingerprints unique to each strain were reproducibly obtained and may be used to create a database for use in the identification of the various X. albilineans strains. It can be also concluded from the results obtained that the FAFLP has considerable technical advantages compared with the manual AFLP and also that the FAFLP is more sensitive than AFLP using radiolabelled primers in differentiating X. albilineans.

Analysis of wild Lactuca accessions: conservation and identification of redundancy

Germplasm accessions of wildLactucaspecies are maintained worldwide inex situcollections as gene reservoirs for quality and disease resistance traits for cultivated lettuce. Accessions of 12Lactucaspecies from 6 genebanks were compared via morphological characterization and AFLP (Amplified Fragment Length Polymorphism)-based profiling to estimate the extent of duplication. A method of assessing redundancy within very similar, but not identical accessions, is proposed, based on 352 polymorphic AFLP products. Seven duplication groups showed a high level of AFLP similarity, and one pair ofLactuca salignaaccessions displayed identical AFLP profiles. In several cases, the morphological assessment indicated that a taxonomic reclassification of accessions was necessary. Candidate duplicates were identified using population parameters and inter- and intra-accession variability. The implications of these findings on the conservation of wild species are discussed.

Cryptic speciation in a Holarctic passerine revealed by genetic and bioacoustic analyses

There has been much controversy regarding the timing of speciation events in birds, and regarding the relative roles of natural and sexual selection in promoting speciation. Here, we investigate these issues using winter wrens (Troglodytes troglodytes), an unusual example of a passerine with a Holarctic distribution. Geographical variation has led to speculation that the western North American form Troglodytes troglodytes pacificus might be a distinct biological species compared to those in eastern North America (e.g. Troglodytes troglodytes hiemalis) and Eurasia. We located the first known area in which both forms can be found, often inhabiting neighbouring territories. Each male wren in this area sings either western or eastern song, and the differences in song are as distinct in the contact zone as they are in allopatry. The two singing types differ distinctly in mitochondrial DNA sequences and amplified fragment length polymorphism profiles. These results indicate that the two forms are reproductively isolated to a high degree where they co-occur and are therefore separate species. DNA variation suggests that the initial split between the two species occurred before the Pleistocene, quite long ago for sister species in the boreal forest. Surprisingly, the two forms are similar in morphometric traits and habitat characteristics of territories. These findings suggest that sexual selection played a larger role than habitat divergence in generating reproductive isolation, and raise the possibility that there are other such morphologically cryptic species pairs in North America.

Molecular typing of Streptococcus agalactiae isolates from fish

The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphism (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 46 S. agalactiae cultures isolated from different fish species and geographic origins as well as related reference strains were included in the study. ISR-SSCP divided the S. agalactiae isolates analysed into five distinct genotypes. Genotype 1 grouped all Kuwait isolates while genotype 4 clustered the majority of non-Kuwait isolates (USA, Brazil and Honduras). AFLP analysis offered a higher resolution level by dividing the isolates into 13 different genotypes. Two different AFLP profiles were identified within the Kuwait isolates. When data from both ISR-SSCP and AFLP were combined through a multidimensional analysis (MDS), a good correlation between geographical origin and genotypes was observed. Both AFLP and ISR-SSCP revealed genetic differences between S. agalactiae isolates from fish. While AFLP offered a higher resolution, ISR-SSCP also provided valid information being a simpler and faster method.

AFLP identification of Berberis thunbergii cultivars, inter-specific hybrids, and their parental species

SummaryCultivars of Berberis thunbergii DC. are among the most popular garden shrubs in the United States. Using 174 amplified fragment length polymorphism (AFLP) markers we were able to differentiate between 43 B. thunbergii cultivars and analyse the genetic similarity of 62 B. thunbergii genotypes, B. julianae, B. koreana, B. vulgaris and B. vulgaris ‘Atropurpurea’. Seven different accessions of B. thunbergii ‘Crimson Pygmy’ from a variety of sources had identical AFLP profiles, indicating that there is only one genotype in cultivation and not a collection of similar genotypes. B. thunbergii cultivars ‘Crimson Pygmy’, ‘Crimson Dwarf’, and ‘Monomb’ Cherry Bomb™ could not be distinguished and appeared to be the same genotype. The B. thunbergii cultivars ‘Concorde’ and ‘Criruzam’ Crimson Ruby™ are most likely genotypes of B. ottawensis since they formed a cluster with known B. ottawensis cultivars. The B. thunbergii var. atropurpurea accessions analysed had Jaccard’s coefficients ranging from 0.89 – 0.99 and, therefore, were probably derived from highly inbred seed. A dichotomous identification key was developed for 42 B. thunbergii cultivars and hybrids and B. vulgaris ‘Atropurpurea’ using 23 markers.This key can be used to ensure that only legal cultivars are sold, if cultivar-specific bans are enacted, and to prevent the importation of rust-susceptible cultivars into Canada.

USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS TO DIFFERENTIATE ISOLATES OF PASTEURELLA MULTOCIDA SEROTYPE 1

Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida, kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission.

Differentiation ofLactobacillus spp. isolated from Chinese sourdoughs by AFLP and classical methods

TwentyLactobacillus strains isolated from sourdoughs that were used as starters for Chinese steamed bread were characterised by amplified fragment length polymorphism (AFLP) and classical methods. Twenty strains were divided into five clusters with AFLP profiles using similarity coefficient 0.60 (Jaccard coefficient) as a cut-off point, while three bio-clusters were formed with a numerical taxonomy classification based on phenotypic features, at a similarity level of 0.80 (Jaccard coefficient) and an average linkage algorithm (UPGMA) of clustering. A good correlation was observed between the two methods. The results suggested that the AFLP method is a useful taxonomic tool for typing of LAB, and the combination of the two methods may lead to obtain an improved understanding of the relationship between the phenotype and genotype of theLactobacillus spp. from the sourdough.

Population genetic structure of Gibberella zeae isolated from wheat in Argentina

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations—results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the world's largest wheat growing countries.