Abstract Amplified Fragment Length Polymorphism Research Articles

Genetic Variation and Phylogeny of Wabisuke Camellias by Amplified Fragment Length Polymorphism (AFLP) Analysis

Amplified fragment length polymorphism (AFLP) analysis was conducted on the wabisuke camellia and its relative camellia species. Genetic polymorphism was identified among the ‘Uraku’ camellia, its offspring ‘Tosa-uraku’ and Camellia japonica, whereas the two accessions of the old ‘Uraku’ showed monomorphism in all the fragments. The results suggested that the two old ‘Uraku’ trees are asexually-propagated clonal strains. The genetic distance between wabisuke cultivars and Chinese camellias and between wabisuke camellias and C. sinensis was much further than that between wabisuke cultivars and Camellia japonica. It has also been suggested that wabisuke camellias can be classified into two subgroups, I-1 and I-2, and that Subgroup I-2 originated from C. japonica, while Subgroup I-1, including ‘Uraku’ (synonym: ‘Tarokaja’), was developed by the repeated hybridization of C. japonica to interspecific hybrids with the Chinese camellias, e.g., C. pitardii var. pitardii, or by the involvement of related species not investigated in this study.

Amplified fragment length polymorphism analysis to assess genetic diversity in rice

Amplified Fragment Length polymorphism (AFLP) was studied involving 48 diverse rice genotypes, comprising of indica, japonica, and Tongil type (indica × japonica) varieties to assess the genetic diversity. Initially 64 primer pair combinations were screened of which 5 were found to be suitable producing clear fragments (35–500 bp). In total 700 amplicons (bands) were produced with an average of 140 bands per primer pair. Maximum (207) from EcoRI AC*/MseI-CAC and minimum (75) amplicons from EcoRI TG*/MseI-CTT primer pairs were observed. Minimum (0.973) and maximum (0.990) PIC values recorded for primer pair EcoRI AC*/MseI-CAC and EcoRI AC*/MseI-CAT, respectively. Pairwise genetic similarity estimates ranged in between 0.6412 and 0.943 with a mean of 0.797. Triguna, an indica high yielding variety (HYV), and a new plant type (NPT) rice, IR 7946-46-1-3-2 having indica x japonica parentage showed minimum genetic similarity coefficient (0.641) with maximum genetic divergence, whereas ADT 41 and Sasyasree, two indica HYVs displayed maximum genetic similarity coefficient—0.953 with lowest genetic distance. All accessions could be clearly identified by using five primer pairs, offers immense scope of AFLP in assessing genetic diversity in rice. The present study also identified prospective varieties for use in selective hybridization and productive progeny selection. Most of the varieties shared distinct clusters based on varietal types, race, parentage and growing area where those are cultivated traditionally albeit with a few exceptions. The dendrogram could demarcate the varieties irrespective of race with unique traits, albeit with exceptions, which warrants further experimentation involving more number of primers in future.

The genetic divergence and genetic structure of two closely related fish species Lateolabrax maculatus and Lateolabrax japonicus in the Northwestern Pacific inferred from AFLP markers

Amplified fragment length polymorphism markers were used to estimate the genetic divergence and genetic structure of Lateolabrax maculatus, L. japonicus and their possible hybrid populations in the Northwestern Pacific. Ninety one individuals were sampled from nine localities including two putative hybrid samples across the distribution range of the two species. Five primer combinations generated a total of 423 loci among 91 individuals of L. maculatus and L. japonicus, 266 of which were polymorphic (62.88 %). The percentage of polymorphic loci in three populations of L. maculatus and five populations of L. japonicus ranged from 31.96 % (Beihai) to 37.94 % (Weihai), 31.05 % (Ishikawa) to 49.29 % (Yatsushiro Sea), respectively. The UPGMA tree constructed with individuals of both species indicated reciprocal monophyletic relationship between L. japonicus and L. maculatus. Hybrid individuals between the two species in Ariake Sea and Yatsushiro Sea were identified by UPGMA tree and principal component analysis (PCA). The sympatry of two clades in the Yatsushiro Sea gave the first evidence of possible reproductive isolation between hybrid and L. japonicus. AMOVA analysis and pairwise FST revealed significant genetic differentiation within both species, indicating limited dispersal ability in two species. Historical isolation coupled with low dispersal ability was responsible for the genetic divergence between species and significant genetic structure in the two species.

Genetic variation of Chlamys farreri, Patinopecten yessoensis and their hybrids

Amplified fragment length polymorphism(AFLP) technology was used to analyze the genetic variation and genetic structure among three populations of Chlamys farreri(♀),Patinopecten yessoensis(♂)and their off-spring F1.In total,315 loci were amplified with five primer pairs,and 311 of these loci were polymorphic,acco-unting for 98.73%.Shannon genetic diversity indices were 0.477 1±0.260 6,0.423 9±0.277 7 and 0.548 8±0.220 9,respectively for the female parent,the male parent and F1,while Nei's gene diversities were 0.329 1±0.185 0,0.289 7±0.195 9 and 0.382 8±0.161 2,respectively.The genetic distance between C.farreri and P.yessoensis was 0.411 6,while it was 0.131 6 between F1 and their female parents,and 0.278 0 between F1 and their male parents.The dendrograms showed that the three population gathered independently,which indicated that each population was independent.AMOVA analysis indicated that 30.47% of variance was among populations and 69.53% vari-ance was within populations,which suggested rich genetic diversity level within populations.In all,the expression of heterosis has a correlation with the genetic variation.

Molecular phylogeny of Chinese snakegourd (Trichosanthes kirilowiiMaxim) based on cytological and AFLP analyses

Amplified fragment length polymorphism (AFLP) marker and cytological analyses were employed to evaluate the genetic relationships and diversity of Chinese snakegourd. According to the cytological analyses, accessions 1, 2 and 3 were diploid (2n = 22), and accessions 4 to 12 were octaploid (2n = 88). Fourteen AFLP primer combinations generated a total of 645 reproducible loci among 12 Chinese snakegourd accessions studied, of which 628 loci were polymorphic with an average of 44.9 polymorphic bands per primer combination. Genetic similarities were obtained using Nei and Li similarity coefficients, and a dendrogram reflecting the relationship of 12 accessions was made using the unweighted pair-group method with arithmetic averages (UPGMA) clustering method. Based on the cluster analyses, 12 accessions were classified into two primary groups, in which group one included accessions 1, 2 and 3 that were diploid, and the other nine octaploid accessions formed group two. Analysis of molecular variance (AMOVA) re...

AFLP-based molecular characterization of 63 populations of Jatropha curcas L. grown in provenance trials in China and Vietnam

Abstract Amplified fragment length polymorphism (AFLP) fluorescence primers were used to characterize the genetic variation among 63 populations of Jatropha curcas from 10 countries in Asia, Africa and Mexico grown in provenance trials in China and Vietnam. Four primer combinations were used to generate a total of 89 bands of which 87 were polymorphic. Total genetic diversity (Ht) was low (0.15). AMOVA partitioned 31% of variability among populations and 69% within populations. Gene flow (Nm) was found to be low, 0.44, suggesting limited gene exchange among populations. Polymorphic loci (Np = 16), the percentage of polymorphic loci (Pp = 18%), Nei’s diversity index ( H = 0.07) and Shannon’s information index (I = 0.10) all indicated low genetic diversity among populations. The populations from Mexico were exceptional as they all displayed high genetic diversity. The UPGMA-based dendrogram, with corroborative indications from Principal Coordinates Analysis, divided the populations into 4 major clusters. Subpopulations from a wide geographic range within India formed a major cluster suggesting a common origin for these subpopulations; the same was indicated for many of the Chinese subpopulations. Material from a wide range of countries in Southeast Asia and accessions from Africa formed another cluster. The Mexican populations formed a fourth, separate group, relatively distant from the other three clusters, indicating that the Asian material probably does not originate from Mexico. Despite generally low genetic diversity of cultivated Jatropha curcas in Asia, this study identified 6 Asian populations with genetic diversity considerably higher than overall population mean. Though the impact of inbreeding on jatropha fitness is not yet well-understood, it is probable that broadening the genetic base will benefit breeding programs in Asia, guarding against inbreeding depression and providing a range of material with the genetic diversity to adapt to a wide range of planting sites. Drawing from the more-diverse populations identified in Asia as well as from wild material originating in Mexico and other parts of the natural range would be a sound strategy when applied in concert with further research on the species’ breeding system, quantitative trait variation and environmental plasticity.

Development of large-scale AFLP markers in jute

Amplified fragment length polymorphism (AFLP) analysis was conducted to investigate the level of polymorphism in four jute genotypes including two genotypes (JRC 321 and CMU 010) of Corchorus capsularis (the white jute) and two genotypes (JRO 524 and PPO4) of Corchorus olitorius (the tossa jute). Out of 1024 primer combinations that were tried, as many as 281 combinations of selective primers (13 EcoRI and 64 MseI) were selected, which produced a total of 9092 amplicons, including 752 (8.3%) polymorphic bands in C. capsularis and a total of 8856 amplicons including 1477 (16.7%) polymorphic bands in C. olitorius. The average number of bands/primer combination was 32.3 for C. capsularis and 31.5 for C. olitorius. For C. capsularis, highest polymorphism of 56.6% was shown by primer combination E35M50, while for C. olitorius highest polymorphism of 50% was shown by E41M91. In C. olitorius, 30–50% polymorphism was observed with 27 primer combinations, but in C. capsularis only 3 primer combinations gave this level of polymorphism. Similarly, in C. capsularis <10% polymorphism was detected by 115 primer combinations while in C. olitorius, <10% polymorphism was shown by only 56 primer combinations. These results indicate a higher level of polymorphism in C. olitorius relative to that in C. capsularis. The occurrence of such a large number of polymorphic AFLP markers will facilitate preparation of molecular maps and QTL analysis in jute.

Amplified Fragment Length Polymorphism Reveals Specific Epigenetic Distinctions between Mycobacterium avium Subspecies paratuberculosis Isolates of Various Isolation Types

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

Genotyping diptera using amplified fragment length polymorphism (AFLP): development of a genetic marker system for species in the families Calliphoridae and Sarcophagidae

Amplified Fragment Length Polymorphism (AFLP) analysis of entomological material provides a means of obtaining a reproducible DNA profile in a relatively short period of time. AFLP profiles were obtained using larval samples from Cochliomyia macellaria, Phormia regina and Sarcophaga bullata. Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (preamplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. Species specific profiles were developed for all three species. The results indicate that the AFLP technique is a viable and valuable technique for identification of entomological material.

Genomic variability among high pristinamycin-producing recombinants of Streptomyces pristinaespiralis revealed by amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze genomic variability between high pristinamycin-producing recombinants of Streptomyces pristinaespiralis produced by genome shuffling and their ancestral strain. The AFLP fingerprints obtained with two restriction enzyme combinations of ApaI/TaqI and PstI/SacII showed together that there was no major polymorphism (less than 10%) between these high yield recombinants and their ancestor. However, the unique polymorphic bands, which might be related to the yield increasing of pristinamycin, could be distinguished from all the recombinants. Clustering analysis further indicated that the recombinants with similar ability of pristinamycin production had similar genomic variability.

The Genetic and Morphological Evidence for Sympatric Speciation Pattern of Paris from Emei Mountain

Amplified Fragment Length Polymorphism(AFLP) was used to analyze the differentiation of 2 natural species(Paris polyphylla Smith var.minora S.F.Wang P.thibetica Franch) of Paris of Emei Mountain.AFLP analysis indicated that there were relatively high genetic comparability among the two species and high genetic differentiation of the P.thibetica Franch and P.polyphylla var.minora with in a geographical region.The flower of morphological diversity was detected among the two species of Paris in Emei Mountain by morphological analysis.The results suggested that high genetic differentiation and morphological differentiation of the P.thibetica and P.polyphylla var.minora in a geographical region.

Genetic Diversity of Actinobacillus pleuropneumoniae Assessed by Amplified Fragment Length Polymorphism Analysis

Amplified fragment length polymorphism (AFLP) was evaluated as a method for genotypic characterization and subtyping within the bacterial species Actinobacillus pleuropneumoniae. A total of 155 isolates of A. pleuropneumoniae, representing the serotypic variation described to occur within this species, were analyzed. In order to elucidate the species boundaries, six strains of the phylogenetically closely related species Actinobacillus lignieresii were also included. Furthermore, the ability of AFLP to subtype was studied using 42 isolates of serovar 2 and the performance compared to that obtained by pulsed-field gel electrophoresis (PFGE). AFLP analysis provided a clear separation of A. lignieresii and A. pleuropneumoniae and divided the isolates of A. pleuropneumoniae into 20 clusters. Most of the serovars of A. pleuropneumoniae were represented by single and quite homogeneous clusters. The exceptions were serovars 10, K2:O7, and K1:O7, which were represented by two clusters each. In the cases where the serovars were represented by more than one cluster, the existence of these clusters was supported by additional phenotypic or genotypic properties. Furthermore, AFLP typing was able to allocate serologically nontypeable isolates to appropriate genetic groups within the species. Further investigations are needed to determine whether some of the clusters revealed through AFLP analysis represent additional serovars. When evaluated as a method for subtyping within serovar 2 of A. pleuropneumoniae, AFLP was found to achieve a degree of separation among isolates superior to that obtained by PFGE. However, a higher degree of separation between serovar 2 isolates was obtained by a combination of the two methods.

AFLP analysis for examining genetic differences in cultivated strains and their single-spore isolates and for confirming successful crosses in Agaricus blazei

Amplified fragment length polymorphism (AFLP) analysis was used to examine genetic differences in Agaricus blazei cultivated strains and their single-spore isolates (SSIs). AFLP analysis with five primer combinations identified a total of 267 AFLP bands from nine cultivated strains (one from Brazil and eight from Japan), of which 165 were polymorphic between the nine strains. An AFLP data dendrogram grouped the eight Japanese strains, with the Brazilian strain acting as an outlier, suggesting that the Brazilian and Japanese strains are genetically quite different. Twelve SSIs derived from each of four cultivated strains were subjected to AFLP analysis. All the AFLP bands detected in the cultivated strains were also found in at least one SSI, but some unique bands were detected in SSIs. The total number of AFLP bands from individual SSIs was clearly less than those from their parental strains, and many of polymorphic AFLP bands from the parental strains segregated in SSIs at a ratio of 1 : 1, suggesting that the SSIs are homokaryotic. Distance values based on presence or absence of individual AFLP bands among SSIs from different strains were clearly higher than those among SSIs from a single strain. In addition, AFLP analysis was shown to be useful in confirming hybrid formation in crosses between SSIs.

Comparative analysis of genetic relationship and diagnostic markers of several taxa of Guizotia Cass. (Asteraceae) as revealed by AFLPs and RAPDs

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) were employed to examine the genetic relationship between Guizotia taxa, to suggest the taxonomic status of some of these taxa, and to identify their diagnostic markers. Results from AFLPs and RAPDs share some features in common, both revealing G. scabra ssp. schimperi as the most closely related taxon to G. abyssinica, and indicating that G. arborescens and G. zavattarii are the most divergent taxa. Most of the diagnostic markers revealed in this study were specific to G. arborescens and G. zavattarii. Our analysis suggests that G. scabra ssp. scabra, G. scabra ssp. schimperi, Chelelu and Ketcha are separate species. In this study, AFLP was found to be superior to RAPD in detecting genetic variation, in internal consistency of the data and in the fitness of its clusters to genetic similarity data. AFLPs revealed genetic relationship between Guizotia taxa that is more inline with the cytogenetic and hybridization studies than that revealed by RAPDs.

Toxin Profiles and Resistances to Macrolides and Newer Fluoroquinolones as Epidemicity Determinants of Clinical Isolates of Clostridium difficile from Warsaw, Poland

Amplified fragment length polymorphism genotypes, antibiotic resistance profiles, and toxin profiles of Clostridium difficile strains from Warsaw were determined. The isolates segregate in six major genotypes, coinciding with toxin profiles. Most of the toxin A-negative toxin B-positive toxin CDT-negative strains possess ermB, and several strains were resistant to fluoroquinolones. Resistograms and toxin types of C. difficile strains are epidemicity determinants.

A Bayesian Model of AFLP Marker Evolution and Phylogenetic Inference

Amplified Fragment Length Polymorphism (AFLP) markers are formed by selective amplification of DNA fragments from digested total genomic DNA. The technique is popular because it is a relatively inexpensive way to produce large numbers of reproducible genetic markers. In this paper, we describe a Bayesian approach to modeling AFLP marker evolution by nucleotide substitution and an MCMC approach to estimate phylogeny from AFLP marker data. We demonstrate the method on species in Carex section Ovales, a group of sedges common in North America. We compare the results of our analysis with a clustering method based on Nei and Li's restriction-site distance and a two-state Bayesian analysis using MrBayes.

Amplified Fragment Length Polymorphism (AFLP) is useful for finding markers associated with QTL for architectural trait in Hedysarum coronarium L.

Amplified fragment length polymorphism (AFLP) has been proposed as a valuable tool for finding molecular markers linked to QTL controlling architectural trait. Segregation of polymorphic AFLP fragments was followed in F2 offsprings Hedysarum coronarium derived from two native wild accesions crossing: Jebel Zit x El Haouaria, which had previously characterised by morphological and AFLP markers. A comparison of profiles from the morphologically contrasted parental individuals and F2 offspring has been assessed using five AFLP primer combinations. These generated 178 bands revealed by silver-stained denaturing polyacrylamide gels, of which 150 were polymorphic. Several AFLP markers appeared to be implied in the orthotropic form that can assist the selection and improve Hedysarum forage crop.

Identification and characterization of an AFLP marker associated with carcass composition in the pig (Brief report)

Abstract. Amplified Fragment Length Polymorphism (AFLP) is a DNA fingerprinting technique that allows genotyping of DNA polymorphisms, mainly single nucleotide substitutions (SNP), across the whole genome with high multiplex ratio and without the need of their prior knowledge. We have successfully combined AFLP with selective genotyping for mapping of quantitative trait loci (QTL) in a F2 resource population based on Duroc and Berlin Miniature pig (DUMI) (WIMMERS et al., 2002). Here we report application of this approach for the identification of loci associated with carcass composition in a herd of commercial German Landrace pigs.

Getting the most out of fluorescent amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP™) is one of the most widely applied molecular marker detection systems used today. Among the reasons for its popularity are its reproducibility, capacity to generate large numbers of data points in a single assay, and “off-the-shelf” universal applicability. The original AFLP protocol was developed using radioactive detection. The transfer of this technique to fluorescent detection on automated DNA fragment analysers not only removed the undesirable requirement for radioactivity but also provided the possibility for increased effectiveness and detection throughput. Unfortunately, a number of problems are frequently encountered with fluorescent AFLPs, particularly failure to amplify high molecular-weight fragments and generation of nonuniform peak distributions. Here, we describe an improved generic protocol for fluorescent AFLPs achieved mainly thorough optimization of the multiplexed selective amplification reaction. This improved protocol gives increased production of valuable high molecular-weight markers and uniform peak intensities, facilitating unambiguous scoring. The protocol has been successfully applied, without further optimization, to species of Salix and Populus (Salicaceae), Melampsora (Melampsoraceae, rust fungi) and Heracleum (Apiaceae), as well as sugar beet ( Beta vulgaris L. subsp. vulgaris , Amaranthaceae), the endangered species Ranunculus kadzunensis Makino (Ranunculaceae), and to Aphidius ervi Haliday (Braconidae), a parasitoid wasp.

Little genetic differentiation across Europe between early-flowering and late-flowering populations of the rapidly declining orchid Neotinea ustulata

Amplified fragment length polymorphism (AFLP) was used to characterize genetic diversity of the endangered Burnt Orchid, Neotinea (formerly Orchis) ustulata. Fingerprinting of Estonian and British populations revealed surprisingly little genetic differentiation between populations but larger amounts of diversity within populations, especially in Britain. The resulting mean Fst value of 0.51 is unusually high for an orchid species. Much of the variation follows a west–east cline across Europe, whereas the much-discussed early- and late-flowering taxa of N. ustulata are considered insufficiently distinct to be viewed as separate subspecies. The later flowering N. ustulata var. aestivalis probably evolved independently on two or three occasions, each time diverging from the earlier flowering nominate race. The identity of the genes underpinning phenology in the species, and the potential selective advantages of phenological divergence, merit further study. Overall genetic diversity within populations is sufficiently high to render impoverishment an unlikely cause of their recent, precipitous decline. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 13–25.