Systematics and evolution of the Dactylorhiza romana/sambucina polyploid complex (Orchidaceae)

The European–Mediterranean–Oriental Dactylorhiza romana/sambucina polyploid complex was studied with regard to genetic and morphological variation patterns. Allozyme and morphometric data were collected from 24 and 19 populations, respectively, initially identified as D. flavescens, D. insularis, D. markusii, D. romana, D. sambucina, and an indeterminate taxon. Genetic distances were calculated and illustrated by an unweighted pair‐group method using arithmetic averages (UPGMA) dendrogram, and principal components analyses (PCAs) were used to summarize morphological variation patterns. Another PCA was performed on combined allozyme and morphometric data. On the basis of the dendrogram and the PCA plots, main groups of populations were delimited, and the probability that each morphological character would distinguish correctly between these groups was estimated. After combining morphometric interpretations with studies of herbarium material and information from the literature, the following taxa were confidently accepted: D. romana ssp. romana, D. romana ssp. guimaraesii (comb. et stat. nov.) , D. romana ssp. georgica, D. sambucina, D. cantabrica (sp. nov.) , and D. insularis. Levels of genetic diversity suggest that D. romana s.s. is the least derived member of the complex. The evolutionary divergence of the diploid species, D. romana and D. sambucina, was probably the outcome of vicariant speciation, whereas D. romana ssp. georgica and D. romana ssp. guimaraesii appear to have evolved from D. romana s.s. through incomplete vicariant and peripheral isolate speciation events, respectively. In some populations of the diploid taxa, a significant deficiency in heterozygotes was found at one to three loci. It is proposed that this pattern may indicate a Wahlund effect, hypothesizing that local populations are subdivided into demes determined by the commonly sympatric occurrence of two distinct colour morphs combined with partial morph constancy of individual pollinators (bumblebees). Several pathways are possible for the origin of the allotriploid D. insularis and the apparently allotetraploid D. cantabrica. A taxonomic revision is provided. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 152 , 405–434.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

AFLP analysis using four selective primers was performed on a set of 33 Listeria monocytogenes including strains from patients and foods implicated in outbreaks, human sporadic cases or foods. Strains were tested belonging to serovars 1/2a, 1/2b, 1/2c, 3b, and 4b. Using one of the primers, the AFLP technique generated 20 different sized DNA fragments. The 33 cultures segregated into 14 different patterns, each comprising 7-12 different fragments. Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and 1/2c, while group 2 serovars 1/2b, 3b and 4b.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

An agarose gel based single enzyme AFLP method using EcoR1 digestion of Listeria monocytogenes DNA was developed for epidemiological typing. The method was evaluated with 84 L. monocytogenes cultures, and results were compared with those obtained with serotyping, phage-typing and cadmium and arsenic resistance typing. The AFLP method was reproducible and 14 different banding patterns comprising between five and eight DNA fragments were produced. All except two of the AFLP patterns were serorype specific. Different AFLP patterns were recognised within serovar 4b (four patterns), 1/2a (two patterns), 1/2b (six patterns): single patterns were obtained from cultures of serovars 1/2c, 3a, 3b and 3c. There were associations with AFLP results and those from phage-typing and cadmium and arsenic resistance typing, although each method showed some independence. This preliminary evaluation suggests that this AFLP method will be useful for epidemiological typing of L. monocytogenes.     

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.     

Evolutionary history of the Dactylorhiza maculata polyploid complex (Orchidaceae)

Taxonomic complexity may be associated with migration history and polyploidy. We used plastid and nuclear DNA markers to investigate the evolutionary history of the systematically challenging Dactylorhiza maculata polyploid complex. A total of 1833 individuals from 298 populations from throughout Europe were analysed. We found that gene flow was limited between the two major taxa, diploid ssp. fuchsii (including ssp. saccifera) and tetraploid ssp. maculata. A minimum of three autotetraploid lineages were discerned: (1) southern/western ssp. maculata; (2) northern/eastern ssp. maculata; and (3) Central European ssp. fuchsii. The two ssp. maculata lineages, which probably pre‐date the last glaciation, form a contact zone with high genetic diversity in central Scandinavia. Intermediate plastid haplotypes in the contact zone hint at recombination. Central Europe may have been a source area for the postglacial migration for the southern/western lineage of ssp. maculata, as well as for ssp. fuchsii. The northern/eastern lineage of ssp. maculata may have survived the LGM in central Russia west of the Urals. The tetraploid lineage of ssp. fuchsii is indistinguishable from diploid ssp. fuchsii, and is probably of postglacial origin. The Mediterranean region and the Caucasus have not contributed to the northward migration of either ssp. fuchsii or ssp. maculata. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 503–525.     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

Amplified fragment length polymorphism (AFLP) markers reveal extra-pair parentage in a bird species: the bluethroat (Luscinia svecica)

We tested the use of amplified fragment length polymorphism (AFLP) to assess the frequency of extra-pair parentage in a bluethroat (Luscinia svecica namnetum) population. Thirty-six families totalling 162 nestlings were analysed. Using a combination of three primer pairs, we reached an exclusion probability of 93% for the population. This probability can reach 99% considering families independently. We revealed that extra-pair fertilizations are very common: 63.8% of all broods contain at least one extra-pair young, totalling 41.9% of all young analysed. However, with the technique and the three primer pairs used it was not possible to attribute the parentage exclusions to extra-pair paternity, maternity or both. As brood parasitism has never been reported in this species, it seems likely that the exclusions are due to extra-pair males. This study shows that dominant AFLP markers can be useful for studying the mating system of taxa for which no microsatellite primers are available. This technique allows the approximate estimation of parentage exclusions despite the fact that it is not possible to know which parent has to be excluded.     

Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam

Moringa oleifera is an important multipurpose tree introduced to Africa from India at the turn of this century. Despite limited knowledge of the levels of genetic diversity and relatedness of introduced populations, their utilization as a source of seed for planting is widespread. In order to facilitate reasoned scientific decisions on its management and conservation and prepare for a selective breeding programme, genetic analysis of seven populations was performed using amplified fragment length polymorphism (AFLP) markers. The four pairs of AFLP primers ( Pst I/ Mse I) generated a total of 236 amplification products of which 157 (66.5%) were polymorphic between or within populations. Analysis of molecular variance ( AMOVA ) revealed significant differences between regions and populations, even though outcrossing perennial plants are expected to maintain most variation within populations. A phenetic tree illustrating relationships between populations suggested at least two sources of germplasm introductions to Kenya. The high levels of population differentiation detected suggest that provenance source is an important factor in the conservation and exploitation of M. oleifera genetic resources.     

Amplified fragment length polymorphism (AFLP) markers reveal that population structure of triploid dandelions (Taraxacum officinale) exhibits both clonality and recombination

Highly variable amplified fragment length polymorphism (AFLP) fingerprints of triploid apomictic dandelions obtained from three localities in an area where diploids are lacking were analysed to infer the predominant modes of reproduction. The distribution of markers was analysed using character compatibility to infer whether many genotypes agree with a tree-like structure in the data set. The presence of incompatible character state combinations (matrix incompatibility; MI) was used as a measure of genetic exchange. The detection of overrepresented genotypes, of which some were widespread, confirmed asexual reproduction. Not all genotypes were overrepresented; approximately half of the genotypes in the three localities were found only once. Because, in terms of genotype frequencies, only a part of the genetic variation is described, more important aspects of the molecular data such as relationships between markers or genotypes have been studied. The analysis of character compatibility indicated a disagreement of the data with a clonal structure. Nearly all genotypes contributed to MI and this contribution varied considerably among genotypes in each sampled locality. A gradual decrease of matrix incompatibility upon successive deletion of genotypes showing the highest contribution to MI indicated that marker distribution of virtually all genotypes disagreed with a tree-like structure in the data. This result suggested that many genotypes were separated by one or more sexual generations. Consistent with this conclusion was the fact that markers that show a low probability of contributing to MI are different in every sampled locality, which is most easily explained as the result of recombination. Apparently, asexual reproduction has resulted in overrepresented, widespread genotypes but sexual recombination has also substantially contributed to genetic variation in the sites studied.     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998     

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

AIMS: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). METHODS AND RESULTS: Seventy-one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. CONCLUSIONS: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

Amplified fragment length polymorphism (AFLP) reveals introgression in weedy Onopordum thistles: hybridization and invasion

Onopordum L. (Compositae) is an extremely diverse genus of thistles, which includes several species that have become serious pasture weeds in several regions of the world. We present a comparison of the genetic diversity in invasive forms of Onopordum from Australia with several known native European species. A total of 108 polymorphic genetic markers was generated using amplified fragment length polymorphism (AFLP) fingerprinting. Non-metric multidimensional scaling (NMDS) revealed that Australia contained O. acanthium, O. illyricum and a full range of genetic intermediates between these species. Intermediates largely comprised segregating fragments diagnostic for European O. acanthium and O. illyricum with a low frequency of fragments that were diagnostic for other species never recorded in Australia. The current genetic patterns in Australia may be best explained by a combination of processes, both in the native and in the alien range. These include multiple introductions of seed, including hybrid material, and the continuous dispersal in Australia, leading to an increase in the contact among hybridizing taxa. Such processes appear to have produced more widespread hybridization and introgression in Australian Onopordum than is found in Europe.     

Using amplified fragment length polymorphisms (AFLP) to identify Black Cohosh (Actaea racemosa)

The rhizome ofActaea racemosa L., commonly called black cohosh, is a popular botanical dietary supplement used to treat female health concerns. The rhizomes used in black cohosh products are often collected from the wild. To ensure quality control, it is imperative that plants be correctly identified. This paper examines the use of the DNA fingerprinting technique, AFLP, as an analytical means of identifyingA. racemosa from three other closely related sympatric species. To this end, 262 AFLP markers were generated, and one unique fingerprint was identified forA. racemosa, whereas two, six, and eight unique fingerprints were identified for the closely related speciesA. pachypoda, A. cordifolia, andA. podocarpa, respectively. Two commercial black cohosh products were also subjected to AFLP analysis and shown to contain onlyA. racemosa. The results of this study suggest that AFLP analysis may offer a useful method for quality control in the botanical dietary supplements industry.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.     

Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomyrmex minutus

A PCR-based fingerprinting technique based on amplified fragment length polymorphisms (AFLP) is used to screen symbiotic fungi of the fungus-growing ant Cyphomyrmex minutus for genetic differences. AFLP fingerprints reveal several fungal ‘types’ that (a) represent distinct clones propagated vegetatively by the ant, or (b) correspond to free-living fungi that may be acquired by the ant. Fungal types identified by AFLP fingerprints correspond to vegetative-compatibility groups established previously, suggesting that vegetative compatibility can be used as a crude indicator of genetic differences between fungi of C. minutus.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Systematics of the Dactylorhiza euxina/incarnata/maculata polyploid complex (Orchidaceae) in Turkey: evidence from allozyme data

 Material of Dactylorhiza were sampled from 49 localities in Turkey and investigated for allozyme variation at ten loci (nine enzyme systems). Among diploids, the Anatolian D. osmanica and D. umbrosa were allozymically variable, but not distinct from each other or from D. incarnata. Dactylorhiza saccifera contained the same alleles as the European D. fuchsii. Dactylorhiza iberica and D. euxina were distinct from each other and the other diploids. On basis of allozyme patterns three distinct allotetraploid genotypes were distinguished, and each of them could be treated as a separate species. Dactylorhiza nieschalkiorum is similar to European allotetraploids, and may have arisen from hybridization between D. incarnata s.l. and D. saccifera. Dactylorhiza urvilleana may have arisen from parents related to present-day D. saccifera and D. euxina, but it also contains additional alleles that have not been found in any of the diploids investigated. A third allotetraploid known from four populations in the Ardahan and Kars provinces of north-eastern Turkey combines the allozyme patterns found in material of D. incarnata s.l. from the same area with those from D. euxina. It is here described for the first time as D. armeniaca. Received November 14, 2000 Accepted June 20, 2001