AFLP Profiles Research Articles

Population structure of poplar rust Melampsora larici‐populina in the UK inferred from AFLP

AFLP profiles were generated from poplar leaf rust (Melampsora larici‐populina) collected from Populus trichocarpa cv. Trichobel at six sites in the UK in 2001. Of a total of 145 isolates, 121 were unique AFLP phenotypes. On one occasion, the same AFLP phenotype was found at two sites. The analysis indicated that 81·88% of the total variation was attributable to differences among individuals within replicate plots, 3·47% to differences among plots within sites and 14·65% to differences among sites. Weir & Cockerham's θ was calculated as 0·138 and θB, the Bayesian analogue of FST, as 0·146. Overall, there was sufficient gene flow among different sites to prevent genetic drift, as Nm was calculated as 1·562 using θ and as 1·462 using θB. Nei's genetic distance and population‐assignment test indicated that the rust from a site in north‐east England was differentiated from those from other sites. There was no significant correlation between genetic and geographical distances. The results indicate that sexual reproduction has a major influence on the relative lack of population structure of M. larici‐populina in the UK.

The use of AFLP and morphological markers to study Iranian grapevine germplasm to avoid genetic erosion

SummaryMolecular and morphological analyses have been used to define and clarify the relationships among the 54 most representative grapevine cultivars in Iran. A total of 459 scorable AFLP fragments, across all the accessions analysed, were detected. Among these, 185 (40.3%) were polymorphic. Values of genetic diversity, calculated by the Nei and Li index, ranged from 0 – 0.3. Our results highlight cases of different cultivars displaying the same AFLP profile and similar morphological characteristics, proving the occurrence of synonyms in Iranian germplasm. Combining AFLP results and morphological descriptions, two cases of wrong attribution were also observed: ‘Saghal Solian-2’ was renamed ‘Mosli’; and ‘Khalili Qermez’ was now called ‘Khalili Sefid’. Morphological characterisation, conducted using the IPGRI descriptors, showed large variability among the accessions analysed, mainly in young shoot and leaf colour, and in bunch size. Although synonyms were detected, and genomic similarities were verified, genetic differences and the richness observed in Iranian grapevine germplasm were not high, in contrast with their morphological characteristics. We hypothesise that many Iranian morphotypes have been derived from mutation events in a few ancient genotypes; however, the system of selection may result in the biodiversity of Iranian grapevine being endangered. For this reason, our molecular and morphological characterisation will help in the development of conservation plans and in the re-organisation of local germplasm collections.

AFLP Analysis of Genetic Diversity in Indian Soybean [Glycine max (L.) Merr.] Varieties

AFLP technique was used to assess genetic diversity in 72 soybean varieties under cultivation in India. Selected 12 AFLP primer pairs produced 1319 products of which 1257 were polymorphic (95%). Wide variations were observed for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles of the 72 soybean varieties were subjected to UPGMA cluster analysis. The dendrogram generated revealed four major clusters, which were strongly supported by the high bootstrap values obtained from analyses of 1000 bootstrap samples. In addition, the Mantel's test for cophenetic correlation with r = 0.955 indicated very good fit of the varieties to a group in the cluster analysis. Some correspondence between the clustering pattern and the pedigree, place of release or target area of the variety was observed. Overall moderately high genetic diversity was observed which appears to be due to the higher genetic diversity prevalent in 12 of the varieties included in three diverse clusters and was indicative of the need to include more diverse germplasm in the soybean improvement programs.

Efficacy of the R2 resistance gene as a component for the durable management of potato late blight in France

Many race‐specific resistance genes to potato late blight are overcome in France, but the disease appears later on genotypes carrying the R2 gene. This study examined whether R2 could contribute to durable late‐blight control in France, and analysed the conditions of its performance. Plants grown from tubers of different physiological ages showed no difference in R2 expression in field and climate‐chamber experiments, demonstrating that the delay in epidemic onset provided by R2 was not the result of gene inactivation in old plants. Among isolates collected at one site, those virulent on R2 were classified into three AFLP profiles. AFLP‐VII comprised exclusively isolates virulent to R2, whereas AFLP‐IV and AFLP‐V included both virulent and avirulent isolates. No significant aggressiveness differences were observed between virulent and avirulent isolates from AFLP‐V; however, isolates from AFLP‐VII were significantly less aggressive than virulent isolates from AFLP‐V. These results indicate that: (i) the delayed onset of epidemics on R2 cultivars is the result of the breakdown of R2 by virulent isolates; (ii) aggressiveness of isolates virulent to R2 depends primarily on the genetic background of the pathogen where the mutation to virulence occurs; and (iii) this mutation does not lead per se to lower pathogenic fitness. It is suggested that R2 is unlikely to make a lasting contribution to late‐blight control in France, and that diversification strategies such as cultivar mixtures might not considerably increase its durability.

Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.

Intraspecific and interspecific genetic and phylogenetic relationships in the genus Populus based on AFLP markers

Although Populus has become the model genus for molecular genetics and genomics research on forest trees, genetic and phylogenetic relationships within this genus have not yet been comprehensively studied at the molecular level. By using 151 AFLP (AFLP is a registered trademark of Keygene) markers, 178 accessions belonging to 25 poplar species and three interspecific hybrids were analyzed, using three accessions belonging to two willow species as outgroups. The genetic and phylogenetic relationships were generally consistent with the known taxonomy, although notable exceptions were observed. A dendrogram as well as a single most parsimonious tree, ordered the Populus sections from the oldest Leuce to the latest Aigeiros, a pattern consistent with their known evolutionary relationships. A close relationship between Populus deltoides of the Aigeiros section and species of the Tacamahaca section was observed and, with the exception of Populus wilsonii, between the species of the Leucoides, Tacamahaca, and Aigeiros sections. Populus nigra was clearly separated from its consectional P. deltoides, and should be classified separately from P. deltoides. The AFLP profiles pointed out to the lack of divergence between some species and revealed that some accessions corresponded with interspecific hybrids. This molecular study provides useful information about genetic relationships among several Populus species and, together with morphological descriptions and crossability, it may help review and update systematic classification within the Populus genus.

Genetic Diversity of Pyrenophora teres Isolates as Detected by AFLP Analysis

AbstractAmplified fragment length polymorphism (AFLP) analysis has been used to analyse mainly 83 Czech isolates of Pyrenophora teres, P. graminea, P. tritici‐repentis and Helminthosporium sativum. Each species had distinct AFLP profiles. Using 19 primer combinations 948 polymorphic bands were detected. All main clusters in dendrogram correspond to the studied species. Even the two forms of P. teres–P. teres f. teres (PTT) and P. teres f. maculata (PTM) – formed different clusters. Genetic diversity, with regard to the locality and the year of the sample's collection, was analysed separately within the AFLP‐based dendrogram cluster of PTT and PTM. Unweighted pair‐group method (UPGMA) analysis of the 37 isolates of PTT and 30 isolates of PTM, using 469 polymorphic bands, showed that the variability seemed to have been influenced more by the year of sampling than by the geographic origin of the isolate. The presence of intermediate haplotypes with a relatively high number of shared markers between the two groups indicated that hybridization between the forms of P. teres could happen, but it is probably often overlapped by selection pressure or genetic drift.

Somaclonal variation in Asparagus officinalis plants regenerated by organogenesis from long-term callus cultures

Somaclonal variation in plants regenerated by organogenesis from long-term cultured calluses of two diploid staminate genotypes of Asparagus officinalis cv. Argenteuil was characterized by plant phenotype, ploidy, meiotic behavior, pollen viability, fruit and seed set, and AFLP profiles. Phenotypic deviations from the donors were detected in foliage color, flower size, and cladode and flower morphology. Ploidy changes were observed in 37.8% of the 37 regenerants studied. Meiotic alterations in 12 out of 21 regenerants included laggards, dicentric bridges, micronuclei, restitution nuclei and polyads. Of the 408 AFLP markers screened in 43 regenerants and the donors, 2.94% showed polymorphism. High pollen viability was observed in the 22 regenerants analyzed. All crosses between one pistillate plant and 35 regenerants, as well as the controls, produced fruits and seeds; however, no plump seeds resulted in 35.3% of the crosses with regenerants, and no seeds germinated in 12.5% of those with apparently normal seeds. Fruit and seed set was similar in crosses with diploid regenerants with normal meiosis and the controls but was lower in crosses with diploid and polyploid regenerants with abnormal meiosis. Our results show that the regenerated plants exhibited conspicuous somaclonal variation that could be eventually exploited for in vitro selection systems.

Highly sensitive DNA fingerprinting of orchid pollinaria remnants using AFLP

Numerous Australian terrestrial orchid species in the genus Diuris may be pollinated by food source mimicry. In our field studies, direct observations of orchid–pollinator interactions were rare, but native bees were frequently captured carrying orchid pollinaria, or pollinaria remnants. Sometimes, pollinaria remnants were minimal and included only the viscidium, a sticky pad that was often highly persistent. Confirmation of such tissue as being of orchid source, and attributing them to a particular species can aid pollination studies. DNA-based methods that may identify more or less intact orchid pollinaria are available, but extremely small and degraded samples can pose technical challenges. We have developed an AFLP protocol for such difficult samples that offers some significant advantages over direct PCR-based analysis. We simulated AFLP profiling of very low-DNA samples using DNA template from serial dilutions. A DNA sample range from 6.4 picograms to at least as high as 100 nanograms (15 500-fold range) all yielded AFLP fingerprints. The practical application of this inherent sensitivity of AFLP is demonstrated by the identification of remnants of orchid pollinaria sampled from bees, presented here as a case study. It is expected that this approach will find many applications where sample DNA is limiting, or possibly where pollen of similar appearance may comprise species mixtures.

Corrigendum to: Highly sensitive DNA fingerprinting of orchid pollinaria remnants using AFLP

Numerous Australian terrestrial orchid species in the genus Diuris may be pollinated by food source mimicry. In our field studies, direct observations of orchid.pollinator interactions were rare, but native bees were frequently captured carrying orchid pollinaria, or pollinaria remnants. Sometimes, pollinaria remnants were minimal and included only the viscidium, a sticky pad that was often highly persistent. Confirmation of such tissue as being of orchid source, and attributing them to a particular species can aid pollination studies. DNA-based methods that may identify more or less intact orchid pollinaria are available, but extremely small and degraded samples can pose technical challenges. We have developed an AFLP protocol for such difficult samples that offers some significant advantages over direct PCR-based analysis. We simulated AFLP profiling of very low-DNA samples using DNA template from serial dilutions. A DNA sample range from 6.4 picograms to at least as high as 100 nanograms (15�500-fold range) all yielded AFLP fingerprints. The practical application of this inherent sensitivity of AFLP is demonstrated by the identification of remnants of orchid pollinaria sampled from bees, presented here as a case study. It is expected that this approach will find many applications where sample DNA is limiting, or possibly where pollen of similar appearance may comprise species mixtures.

DNA-based analysis for authenticity assessment of monovarietal pasta

Because of the presence on the market of high-value monovarietal pasta, the aim of this work was to evaluate the potential of DNA profiling to test its authenticity. AFLP profiles obtained from eight monovarietal pasta samples and from the corresponding varieties were compared. The possibility of identifying the variety of an unknown monovarietal pasta by referring to a durum wheat molecular markers database is proposed.

One or more species in the arctic grass genus Dupontia? – a contribution to the Panarctic Flora project

The grass family (Poaceae) is the largest family of vascular plants in the Arctic. The family includes several species complexes that show high ploidy levels, predominantly reproduce asexually, are poorly morphologically delimited, and have a possible origin within the Arctic area by reticulate evolution. Challenged by work on the Panarctic Flora project and apparent discrepancies in recent taxonomic treatments, we investigated the arctic grass genus Dupontia by molecular and cytogenetic methods. Identical plastid DNA (trnL–F) sequences and very similar AFLP profiles indicated that Dupontia and Arctophila are closely related and that the latter might have been involved in a possible intergeneric hybrid origin of Dupontia. Despite the presence of intraindividual rDNA (ITS) variation in Dupontia indicating an allopolyploid hybrid origin of the genus, it was not possible to predict the second parental species from sequence data or from fluorescent in situ hybridization (FISH) experiments using genomic probes. Due to lack of sequence divergence within Dupontia, AFLP fingerprinting was applied to investigate whether any genetic subdivision exists within the genus. The data sorted Dupontia first of all according to geography and not to ploidy or morphological characters that have previously been used to discriminate between proposed taxa. The results indicate that the recognition of a single polymorphic species is the best solution for the genus Dupontia.

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

AbstractAmplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.

Analysis of Genetic Diversity in Cultivated Jute Determined by Means of SSR Markers and AFLP Profiling

Genetic improvement of the cultivars of jute (Corchorus olitorius L. and Corchorus capsularis L.) is needed to broaden the genetic base of new cultivars. All the cultivars in use have been evolved through pure line selection from a few common accessions. The objective of this study was to evaluate the genetic diversity available in the cultivated species of jute. Genetic diversity was evaluated by simple sequence repeat (SSR) marker loci and an AFLP assay. A total of 305 polymorphic products were detected by AFLP analysis using 10 pairs of primers (EcoRI and MseI) to amplify template DNA from 49 genotypes of the two jute species. Additionally, polymorphism with two to four allelic lengths was detected with each pair of chloroplast microsatellite primers developed from Nicotiana tabacum L. Results from both evaluations showed that the level of variation between species is high. The two species indeed are distantly related and their maternal origins may be different. On the contrary, genetic variability present at the intraspecific level is low. The resulting dendrogram showed common ancestral origin for many accessions. A few major Indian cultivars of both the species, used as an internal check, were closely related to the wild accessions. Nevertheless, RG, an Indian accession and two Kenyan accessions, KEN/BL/17 and KEN/DS/35C, among the C. olitorius genotypes and CHN/FJ/69 of C. capsularis revealed phenetic distinctiveness from the rest of the genotypes studied. The results indicate that enough diversity exists to broaden the genetic base of new jute cultivars.

<i>Arcobacter butzlieri </i>strains from poultry abattoir effluent in Nigeria

To investigate the prevalence, species distribution and genetic diversity of zoonotic Arcobacter species. Prospective study. Drainage system of a cosmopolitan chicken abattoir in Lagos, Nigeria. One hundred and fifty drainage water samples were enriched in a minimal antibiotics-containing medium at room temperature and bacteria then isolated by use of a membrane filtration method. Twenty six (14%) of samples were positive for Arcobacter spp. Of these, 20 were examined by a comprehensive probabilistic identification scheme for Epsilobacteria and all strains identified as A. butzleri. AFLP analysis of these strains revealed considerable genetic diversity among the strains, with 12 genotypes defined at the 90% similarity level. The prevalence of A. butzleri in Nigerian poultry abattoir effluent indicates this species may constitute a public health problem in this country. AFLP profiling could be a useful tool for molecular epidemiological and population genetic studies of this organism. This is the first known report of A. butzleri in Nigeria, and first application of AFLP analysis for genotyping the species.

Analysis of Genetic Diversity in Cultivated Jute Determined by Means of SSR Markers and AFLP Profiling

Genetic improvement of the cultivars of jute (Corchorus olitorius L. and Corchorus capsularis L.) is needed to broaden the genetic base of new cultivars. All the cultivars in use have been evolved through pure line selection from a few common accessions. The objective of this study was to evaluate the genetic diversity available in the cultivated species of jute. Genetic diversity was evaluated by simple sequence repeat (SSR) marker loci and an AFLP assay. A total of 305 polymorphic products were detected by AFLP analysis using 10 pairs of primers (EcoRI and MseI) to amplify template DNA from 49 genotypes of the two jute species. Additionally, polymorphism with two to four allelic lengths was detected with each pair of chloroplast microsatellite primers developed from Nicotiana tabacum L. Results from both evaluations showed that the level of variation between species is high. The two species indeed are distantly related and their maternal origins may be different. On the contrary, genetic variability present at the intraspecific level is low. The resulting dendrogram showed common ancestral origin for many accessions. A few major Indian cultivars of both the species, used as an internal check, were closely related to the wild accessions. Nevertheless, RG, an Indian accession and two Kenyan accessions, KEN/BL/17 and KEN/DS/35C, among the C. olitorius genotypes and CHN/FJ/69 of C. capsularis revealed phenetic distinctiveness from the rest of the genotypes studied. The results indicate that enough diversity exists to broaden the genetic base of new jute cultivars.

Application of AFLP® technology to radiation hybrid mapping

We have investigated the use of AFLP technology as a tool for the high throughput enrichment of Radiation Hybrid (RH) maps. The 3000 rad TM112 bovine RH panel was assayed with 37 EcoRI/TaqI AFLP primer combinations. The number of selective nucleotides used during PCR was increased to seven, to reduce the complexity of the AFLP profile and minimise the overlap between hamster and bovine bands co-amplified from hybrid cell clones. Seven-hundred-forty-seven bovine AFLP bands were amplified that could be distinguished following electrophoresis. Repeatability was tested within and between laboratories on independent template preparations and an error rate of 1.3% found. Two-point linkage analysis clustered 428 AFLP fragments in 39 linkage groups of at least 4 markers. Multi-point maps were constructed for 5 sample linkage groups. The study demonstrated that the AFLP approach could be used to rapidly screen for the most informative clones during panel construction and to increase the number of markers on RH maps, which could be useful for joining linkage groups formed by other markers. The use of AFLP markers as anchor points between existing RH maps and other physical maps, such as BAC contigs, is also discussed.

An intraspecific genetic map of velvetbean (Mucuna sp.) based on AFLP markers.

Velvetbean ( Mucuna sp., n=11), a self-pollinated species, is an important legume used in tropical agricultural systems in rotation with other crops for nematode management and/or soil improvement. A genetic map of velvetbean was constructed in order to identify potential molecular markers linked to important morphological and agronomic traits that would be particularly useful for developing and improving the species. Traits such as seed coat color, pod color, and pod pubescence were among the main parameters observed in a process of genetic diversity estimation. Two slightly divergent velvetbean accessions, PI364362 and 'Edgar Farm White', a land race from Alabama, were used to make an intraspecific F(1) hybrid. Amplified fragment length polymorphism analysis (AFLP) detected an average of six polymorphic fragments per primer pair between the two parents. As expected for dominant markers, the sum of all AFLP bands from both parents was generally observed to be present in the AFLP profiles of the F(1) progeny, indicating full penetrance and the dominant nature of AFLP markers. An F(2) population was generated by self-pollinating a single F(1) plant. Using 37 AFLP primer pairs, we detected 233 polymorphic markers of which 164 (70.4%) segregated in 3:1 Mendelian ratios, while the remaining 69 (29.6%) both segregated and were scorable. The genetic linkage map constructed from this population comprised 166 markers, including two morphological traits (pod color and pod pubescence). Twenty linkage groups were found with an average distance between markers of 34.4 cM, covering a total of 687.9 cM. The linkage groups contained from 2 to 12 loci each and the distance between two consecutive loci ranged from 0 to 21.8 cM. The newly designated morphological traits pod color ( pdc) and pod pubescence ( pdp) co-segregated with each other at a distance of 4.2 cM. Two DNA markers designated ACGCAG2 and ACTCTG1 were located in the same group as pdc and pdp. The AFLP linkage map provides opportunities for use in marker-assisted selection and in the detection of loci controlling morphologically important traits.

Analysis of Species Relationship Among Seven Small Millets Using Molecular Markers

Genomic DNA of twenty four accessions belonging to seven small millet species were analyzed for RAPD, RFLP and AFLP profiles for a comprehensive understanding of the level of genetic diversity within the species and relationships between them. Thirty random primers generated a total of 115 amplification products of which 70 were polymorphic across all species.Twenty-five probe enzyme combinations were used for RFLP analysis that revealed 87 loci of which 62 were polymorphic across the species.AFLP analysis was done at inter-specific level using 12 primer combinations.This generated a total of 869 fragments of which 821 were polymorphic across the species analyzed. Species-specific AFLP profiles were obtained in 10 of the 12 primer combinations tested. It was noticed that the intra-specific variability in all the RAPD and RFLP marker systems was negligible. Species-specific polymorphic loci were observed for all the marker systems.The results are discussed in relation to the genetic relationship among the seven species analyzed.

Toward positional cloning of Vgt1, a QTL controlling the transition from the vegetative to the reproductive phase in maize.

Vgt1 (Vegetative to generative transition 1) is a quantitative trait locus (QTL) for flowering time in maize (Zea mays L.). Vgt1 was initially mapped in a ca. 5-cM interval on chromosome bin 8.05, using a set of near-isogenic lines (NILs) in the genetic background of the late dent line N28, with the earliness allele introgressed from the early variety Gaspé Flint. A new large mapping population was produced by crossing N28 and one early NIL with a ca. 6-cM long Gaspé Flint introgression at the Vgt1 region. Using PCR-based assays at markers flanking Vgt1, 69 segmental NILs homozygous for independent crossovers near the QTL were developed. When the NILs were tested in replicated field trials for days to pollen shed (DPS) and plant node number (ND), the QTL followed a Mendelian segregation. Using bulk segregant analysis and AFLP profiling, 17 AFLP markers linked to the QTL region were identified. Statistical analysis indicated a substantial coincidence of the effects of Vgt1 on both DPS and ND. Vgt1 was mapped at ca. 0.3 cM from an AFLP marker. As compared to DPS, the higher heritability of ND allowed for a more accurate assessment of the effects of Vgt1. The feasibility of the positional cloning of Vgt1 is discussed.