Amplified Fragment Length Polymorphism Data Research Articles

A call for more transparent reporting of error rates: the quality of AFLP data in ecological and evolutionary research

Despite much discussion of the importance of quantifying and reporting genotyping error in molecular studies, it is still not standard practice in the literature. This is particularly a concern for amplified fragment length polymorphism (AFLP) studies, where differences in laboratory, peak-calling and locus-selection protocols can generate data sets varying widely in genotyping error rate, the number of loci used and potentially estimates of genetic diversity or differentiation. In our experience, papers rarely provide adequate information on AFLP reproducibility, making meaningful comparisons among studies difficult. To quantify the extent of this problem, we reviewed the current molecular ecology literature (470 recent AFLP articles) to determine the proportion of studies that report an error rate and follow established guidelines for assessing error. Fifty-four per cent of recent articles do not report any assessment of data set reproducibility. Of those studies that do claim to have assessed reproducibility, the majority (~90%) either do not report a specific error rate or do not provide sufficient details to allow the reader to judge whether error was assessed correctly. Even of the papers that do report an error rate and provide details, many (≥23%) do not follow recommended standards for quantifying error. These issues also exist for other marker types such as microsatellites, and next-generation sequencing techniques, particularly those which use restriction enzymes for fragment generation. Therefore, we urge all researchers conducting genotyping studies to estimate and more transparently report genotyping error using existing guidelines and encourage journals to enforce stricter standards for the publication of genotyping studies.

Molecular evidence for Neotyphodium fungal endophyte variation and specificity within host grass species

Host specificity of Neotyphodium species symbiotic with three grass species, Festuca arundinacea, Festuca pratensis and Lolium perenne, was studied based on comparisons of amplified fragment length polymorphisms (AFLP) between hosts and their corresponding endophytes. Endophytic fungi were isolated from 24 accessions of host plants. Neotyphodium identity was determined based on morphological characteristics observed in cultures and polymerase chain reaction analysis using specific primers. The results of AFLP data analysis revealed high genetic variation in plant and fungal endophyte species. Plant AFLP genotypes from different species clustered in three distinctive groups, congruent with species. A cluster analysis of AFLP data grouped endophytic isolates according to their host species and secondarily according to their host geographic distribution. The result of the AMOVA on AFLP data accounted for a large and significant proportion of genetic variation due to differences among plant and endophyte species. Phylogenetic groups of isolates corresponded to their respective host genotypes based on maximum parsimony phylograms. Comparisons of the two phylograms illustrated a significant congruence between nodes and branches of host and endophyte clades. These results strongly suggest host specificity of Neotyphodium fungal endophytes with their geographically distant host grasses within each species.

Analysis of genetic diversity in farmers’ rice varieties in Sierra Leone using morphological and AFLP® markers

The objective of the current research was to investigate the status of rice genetic resources in post-war Sierra Leone using both morphological and amplified fragment length polymorphism (AFLP) data. Specifically, we aimed at investigating farmers’ rice genetic resources for homogeneity and differentiation, and at examining the genetic identity of similarly named varieties, including varieties within Sierra Leone, and between Sierra Leone and Guinea. This research was also motivated by the assumption that genetic erosion might have occurred as a result of the civil war. To determine the level of diversity and genetic relationships among farmers’ varieties of rice recently collected in Sierra Leone, two methods were used using subsets of the collected samples: (1) Using morphological data, 74 samples of 29 different varieties were analysed to investigate the relationship between (a) varieties grown in two districts in Sierra Leone, and (b) the two main cultivated species, Oryza sativa and Oryza glaberrima. A dendrogram largely clustered the varieties according to region and to the species to which the varieties belonged. (2) Using AFLP data, three separate investigations were conducted: (a) 33 samples of 10 varieties were investigated to evaluate diversity within and between varieties. The results indicated that the rice varieties possess different levels of intra-variety variation, whereas inter-variety diversity was high enough to distinguish one variety from the other. In particular, an AMOVA analysis revealed that 38 % of the total variation occurred within varieties, and 62 % between varieties. (b) 37 samples of 18 different varieties were investigated to determine the consistency of naming of varieties by farmers. The results showed that there was consistency in the naming by farmers of traditional varieties, but inconsistency in the naming of newly acquired varieties and cultivars. (c) 12 samples were investigated to check the identity of varieties carrying identical names collected in two separate regions, Sierra Leone and the neighbouring country of Guinea. The results indicated no close genetic relationships between the varieties found in Sierra Leone and Guinea despite similarities in the names given to these varieties by farmers, indicating the influence of different cultivation practices in the two countries.

Genetic variation among natural isolates of the ectomycorrhizal hypogenous fungus, Rhizopogon roseolus from Japanese pine forests inferred using AFLP markers

Genetic variation among 45 Rhizopogon roseolus isolates from 21 different regions of Japan were inferred using amplified fragment length polymorphism (AFLP) markers. Using three primer pair combinations, AFLP analysis reproducibly produced a total of 223 DNA fragments, 74.4% of which were polymorphic. Pairwise dissimilarity of AFLP patterns between isolates ranged from 0.043 to 0.228. Cluster analysis and principal coordinate analysis of AFLP data generally showed four major clusters from geographically distinct areas. The findings suggested that the Japanese populations of R. roseolus from different geographical regions can be distinguished based on AFLP characters.

Phylogenetic relationships and species delimitation of New Zealand bluebells (Wahlenbergia, Campanulaceae) based on analyses of AFLP data

Phylogenetic and phenetic analyses of amplified fragment length polymorphism (AFLP) data were used to compare 38 individuals of 17 species of Australasian Wahlenbergia, with a focus on the New Zealand representatives. Overall, support values were slightly higher in the AFLP analyses than in previously published DNA sequence analyses, indicating the marginally better phylogenetic resolution of this technique. However, higher support was generally found towards the tips of the branches, whereas deeper relationships were equally poorly supported using either AFLPs or DNA sequence data. AFLP analyses are useful for species delimitation in New Zealand Wahlenbergia. The four members of the lowland radicate Wahlenbergia gracilis complex may all belong to the same species. We also present evidence that Wahlenbergia vernicosa should not be considered a subspecies of the Australian Wahlenbergia littoricola. The morphologically distinctive Wahlenbergia matthewsii and Wahlenbergia congesta subsp. haastii were recovered as being distinct using AFLP analyses, but members of the alpine rhizomatous Wahlenbergia albomarginata/ Wahlenbergia pygmaea complex could not be distinguished from each other.

AFLP, Pathogenicity, and VCG Analyses of Fusarium oxysporum and Fusarium pseudocircinatum from Acacia koa.

Acacia koa (koa), a native tree in Hawaii, suffers from a dieback caused by Fusarium oxysporum. Pathogenicity tests, vegetative compatibility group (VCG) tests, and amplified fragment length polymorphisms (AFLP) analyses were conducted on Fusarium isolates recovered from diseased koa. Koa seedling mortality with individual strains ranged from 0 to 85%, with 42% of the strains killing no seedlings. Thus, strains of F. oxysporum recovered from dying koa trees may or may not be virulent. In addition to F. oxysporum, F. pseudocircinatum strains were isolated from diseased koa; however, they were either nonvirulent or had weak virulence. This is the first report of F. pseudocircinatum in Hawaii. The 46 strains of F. oxysporum and F. pseudocircinatum were grouped into 16 VCGs, but 86% of the highly virulent strains belonged to VCG 2. In AFLP analyses, strains from the same VCG generally clustered with one another. Identification of the same set of strains using VCG, AFLP, and pathogenicity tests showed that the highly virulent strains are genetically close and that high virulence toward koa is not a property of all strains of F. oxysporum. Thus, VCG 2 with the corresponding AFLP data is a significant biological entity for which we propose the name F. oxysporum f. sp. acaciae to reflect its virulence on koa.

The incomplete history of mitochondrial lineages between two rockfishes, Sebastes longispinis and Sebastes hubbsi (Scorpaeniformes: Scorpaenidae)

The genetic divergence between two closely related rockfishes, Sebastes longispinis and Sebastes hubbsi, was inferred from both mitochondrial DNA (mtDNA) sequence variations and amplified fragment length polymorphism (AFLP) markers. The two species were placed into two distinct clades in a neighbour-joining tree based on the AFLP data, clearly indicating that they represented separate species. Although this evidence, together with a previous morphological study, revealed clear differences between the two species, no obvious clustering of haplotypes by species was detected in the minimum spanning network inferred from sequence variations in the mtDNA control region (c. 500 base pairs). In fact, the significant Φ(ST) estimates indicated only a restriction of gene flow between the two species. Uncorrected pairwise sequence differences in mtDNA between two species were small (1·8% at maximum, on the lower end of the range of control region divergence between previously studied sister species pairs), suggesting their speciation event as having been fairly recent. The incongruent results of AFLP and mtDNA phylogenies suggested incomplete lineage sorting and introgression of mtDNA in the course of the evolution of the two species. Differences in their main distributional ranges and the small level of sequence divergence in mtDNA suggests that speciation and dispersal may have been associated with glacio-eustatic sea level fluctuations between the Japanese Archipelago and the Korean Peninsula during the past 0·4 million years.

Phylogeographic analysis and environmental niche modeling of widespread shrub Rhododendron simsii in China reveals multiple glacial refugia during the last glacial maximum

Abstract The phylogeography of common and widespread species can help us to understand the history of local flora and vegetation. Here, we study the semi‐evergreen shrub Rhododendron simsii Planch., which is found in most areas of current evergreen broad leaved forest in China. Two noncoding chloroplast DNA (cpDNA) regions (rpl20‐rps12 and trnL‐F) and three amplified fragment length polymorphism (AFLP) primer sets (E‐AAC/M‐CTA, E‐AGC/M‐CTA and E‐AGG/M‐CAT) were used to examine the phylogeographic pattern in relation to past (last glacial maximum) and present distributions based on ecological niche modeling. The cpDNA data revealed four phylogeographic groups (East, South, West, and North groups) corresponding to geographic regions. Molecular dating suggests that lineage diversification within species likely occurred during the mid‐to‐late Pleistocene. In contrast, the four main cpDNA phylogeographic groups were not supported by the AFLP dataset. The highest likelihood of the AFLP data was obtained when samples were clustered into three groups (K= 3). However, these groupings did not correspond to separate geographic regions supported by cpDNA data. Both mismatch distribution analysis and environmental niche modeling (ENM) indicated that multiple glacial refugia were maintained across the range of Rhododendron simsii during the last glacial maximum, contrary to the previous hypothesis that subtropical broad leaved evergreen forests were forced to retreat southward as far as 25° N. The discordance between the patterns revealed by cpDNA and AFLP data indicate that localized postglacial range expansions may facilitate extensive gene flow between the major glacial refugia.

Genetically diverse but with surprisingly little geographical structure: the complex history of the widespread herb Carex nigra (Cyperaceae)

AbstractAim Based on extensive range‐wide sampling, we address the phylogeographical history of one of the most widespread and taxonomically complex sedges in Europe, Carex nigra s. lat. We compare the genetic structure of the recently colonized northern areas (front edge) and the long‐standing southern areas (rear edge), and assess the potential genetic basis of suggested taxonomic divisions at the rank of species and below.Location Amphi‐Atlantic, central and northern Europe, circum‐Mediterranean mountain ranges, central Siberia, Himalayas.Methods A total of 469 individuals sampled from 83 populations, covering most of the species’ range, were analysed with amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) markers. Bayesian clustering, principal coordinates analysis, and estimates of diversity and differentiation were used for the analysis of AFLP data. CpDNA data were analysed with statistical parsimony networks and maximum parsimony and Bayesian inference of phylogenetic trees.Results Overall genetic diversity was high, but differentiation among populations was limited. Major glacial refugia were inferred in the Mediterranean Basin and in western Russia; in addition, there may have been minor refugia in the North Atlantic region. In the southern part of the range, we found high levels, but geographically quite poorly structured genetic diversity, whereas the levels of genetic diversity varied among different areas in the north. North American populations were genetically very similar to the European populations.Main conclusions The data are consistent with extensive gene flow, which has obscured the recent history of the taxon. The limited differentiation in the south probably results from the mixing of lineages expanding from several local refugia. Northward post‐glacial colonization resulted in a leading‐edge pattern of low diversity in the Netherlands, Belgium, Scotland and Iceland, whereas the observed high diversity levels in Fennoscandia suggest broad‐fronted colonization from the south as well as from the east. The patterns found in the American populations are consistent with post‐glacial colonization, possibly even with anthropogenic introduction from Europe. Our data also suggest that the tussock‐forming populations of C. nigra, often referred to as a distinct species (Carex juncella), represent an ecotype that has originated repeatedly from different populations with creeping rhizomes.

Analysis of genetic diversity and population structure of Pleuronectes yokohamae indicated by AFLP markers

Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity and variation of two populations of Pleuronectes yokohamae from China and North Korea. A total of 228 loci ranging in size were detected from 63 individuals, of which 139 were polymorphic. The number of bands per primer combination varied from 37 to 51 and the percentage of polymorphic bands per primer combination ranged from 43.14% to 72.97%.The proportion of polymorphic loci, the Nei's genetic diversity and Shannon genetic diversity index of China and North Korea populations were 55.09% and 55.71%, 0.1205 and 0.1917, 0.1370 and 0.2129, respectively. The results showed that the genetic diversity of these two populations was at the same level. Dominant gene frequency revealed that these two populations have the same genetic population structure. F-st value of the two populations was 0.0986 and AMOVA analysis indicated that 80.75% genetic variation came from individuals within populations, and there were also 19.25% genetic differentiation occurred between the two populations. There was significant genetic difference between two populations. Exact-p test showed there was no random genetic communication between two populations. The UPGMA and NJ trees based on AFLP data supported this result. (c) 2012 Elsevier Ltd. All rights reserved.

Pattern of genetic differentiation in Gentiana pannonica Scop.: did subalpine plants survive glacial events at low altitudes in Central Europe?

The molecular population structure of 20 populations of the subalpine plant Gentiana pannonica was studied by use of amplified fragment length polymorphism (AFLP) and sequencing of non-coding regions of plastid DNA. Of the populations sampled, 18 were native (11 were from the Eastern Alps, which is the distribution centre of the species, and seven were from the Bohemian Forest, which is on the margin of the distribution range), and two were from the Giant Mts and of unclear status. No plastid DNA polymorphisms were found within the entire 6,185 bp investigated. The AFLP data revealed grouping of populations at the regional level. However, differentiation at the regional level (10.3 %) and at the interpopulation level (14.2 %) was low. Even though current populations are isolated and contain small numbers of individuals, the within-population variation (75.511 %) was high. Genetic variation was higher for alpine populations than for Bohemian Forest populations, probably because of fundamental differences in historical changes in population size between these regions. Within-population variation was intermediate for populations in the Giant Mts. The results indicate the possibility of a large distribution of species in the unglaciated areas of Central Europe, irrespective of altitude, during the late Pleistocene and early Holocene. Our results do not confirm that G.pannonica was introduced in the Giant Mts, and native status in the Giant Mts is possible.

Lack of Evidence for Recombination or Spatial Structure in Phoma ligulicola var. inoxydabilis Populations from Australian Pyrethrum Fields.

Ray blight, caused by Phoma ligulicola var. inoxydabilis, causes substantial annual losses in Australian pyrethrum fields. Fifty-nine P. ligulicola var. inoxydabilis isolates were randomly selected from fields in three distinct geographical regions in Tasmania, Australia. Genetic diversity was characterized using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Based on genetic similarities of less than 99%, 56 distinct genotypes (putative clones) were observed. Mean haploid gene diversity of clone-corrected populations ranged between 0.05 and 0.31, and 0.11 and 0.32, for the RAPD and AFLP data sets, respectively. Cluster analysis indicated two distinct groups of isolates supported by all bootstrap replicates. The first cluster contained all but four isolates with representatives from all three populations. The second cluster contained two isolates from the Western and Central populations, respectively, while the remaining isolates were not able to be grouped with any distinct cluster. Analysis of the population structure suggested no evidence for spatial autocorrelation at the smallest distance classes. The presence of linkage disequilibrium was indicated regardless of population scale. Collectively, these findings provided further evidence for the absence or minor role of the teleomorph in the epidemiology of ray blight in Australian pyrethrum fields.

Genome wide AFLP markers support cryptic species in Coniophora (Boletales)

Numerous fungal morphospecies include cryptic species that routinely are detected by sequencing a few unlinked DNA loci. However, whether the patterns observed by multi-locus sequencing are compatible with genome wide data, such as amplified fragment length polymorphisms (AFLPs), is not well known for fungi. In this study we compared the ability of three DNA loci and AFLP data to discern between cryptic fungal lineages in the three morphospecies Coniophora olivacea, Coniophora arida, and Coniophora puteana. The sequences and the AFLP data were highly congruent in delimiting the morphotaxa into multiple cryptic species. However, while the DNA sequences indicated introgression or hybridization between some of the cryptic lineages the AFLP data did not. We conclude that as few as three polymorphic DNA loci was sufficient to recognize cryptic lineages within the studied Coniophora taxa. However, based on analyses of a few (three) sequenced loci the hybridization could not easily be distinguished from incomplete lineage sorting. Hence, great caution should be taken when concluding about hybridization based on data from just a few loci.

Application of the unified species concept reveals distinct lineages for disjunct endemics of the Brassica repanda (Brassicaceae) complex

Species delimitation is an important issue in terms of conservation priorities, especially for narrow endemics under threat of extinction. The Alpine endemics Brassica repanda subsp. glabrescens and subsp. baldensis belong to a highly polymorphic species complex, although their disjunct distribution suggests favourable conditions for independent evolution. In the present study, we applied the unified species concept to test whether the endemics form distinct evolutionary lineages, both from one another and also from the remainder of the complex. Compliance with the criteria of monophyly, diagnosability, and genotypic clustering was examined, primarily by making use of amplified fragment length polymorphism data. Both endemics were indicated as monophyletic by phylogenetic analyses, and diagnostic characters were found for both taxa. Population structure analyses showed clear genetic discontinuity for each of the endemics, with little admixture among the clusters. This evidence indicates that the endemics have acquired multiple properties that satisfy each of the species criteria considered. Hence, we suggest the taxonomic recognition of B. baldensis and B. glabrescens as separate species. Comparative population genetics analyses show the lack of marked genetic structuring within either taxon, as well as low levels of heterozygosity. Conclusions on the status of threat and on recommended conservation actions are drawn. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 482–497.

Genetic and Virulence Analysis of Rhizoctonia spp. Associated with Sugar Beet Root and Crown Rot in the Northeast Region of Iran

Rhizoctonia spp. are the main causal agents of root and crown rot on sugar beet. In this study, isolates of Rhizoctonia spp. were obtained from diseased sugar beet in Iran over 2 years. Of 68 isolates, 61 were R. solani and 7 were R. cerealis. The anastomosis group (AG) of all isolates was determined on glass slides against the testers. Characterization of intraspecific groups (ISGs) of R. solani isolates revealed that, of 61 isolates, 43 were AG2-2 IIIB and 18 were AG2-2 IV. Amplified fragment length polymorphism (AFLP) analyses were used to investigate genetic structure of Rhizoctonia populations. Principal coordinate plots and cluster analysis differentiated R. solani from R. cerealis isolates and separated the R. solani isolates belonging to different ISGs. AFLP data indicated that the R. solani and R. cerealis populations are not clonal. Analysis of molecular variance in AG2-2 IIIB isolates showed that geographic region was the main factor determining genetic structure of the populations. Sampling year had no significant effect on the genotypes. Pathogenicity tests on Beta vulgaris 'FD0432' revealed that R. solani AG2-2 IIIB and AG2-2 IV isolates were more virulent than R. cerealis.

Development and comparison of RAPD, SCAR and AFLP markers for distinguishing some medicinally important species of the genus Phyllanthus

Random amplified polymorphic deoxyribonucleic acid (RAPD), amplified fragment length polymorphism (AFLP) and sequence characterized amplified region (SCAR) markers were employed for proper identification of five medicinally important species from the genus Phyllanthus, thus allowing these species to be used in herbal drug industry straight after bringing them from the field. RAPD data segregated the herb P. urinaria from the remaining species, whereas AFLP data successfully distinguished all the herbs, i.e., P. amarus Schum., P. fraternus Webster and P. urinaria L. A SCAR marker for one of the herbaceous species, P. urinaria, was also developed. In this study, AFLP came out as a better polymorphic marker than RAPD.

MultiLocus Sequence Analysis- and Amplified Fragment Length Polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species

MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain.

AFLPMax: a user‐friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm.

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.

Molecular Linkage Mapping and Marker-Trait Associations with NlRPT, a Downy Mildew Resistance Gene in Nicotiana langsdorffii.

Nicotiana langsdorffii is one of two species of Nicotiana known to express an incompatible interaction with the oomycete Peronospora tabacina, the causal agent of tobacco blue mold disease. We previously showed that incompatibility is due to the hypersensitive response (HR), and plants expressing the HR are resistant to P. tabacina at all stages of growth. Resistance is due to a single dominant gene in N. langsdorffii accession S-4-4 that we have named NlRPT. In further characterizing this unique host-pathogen interaction, NlRPT has been placed on a preliminary genetic map of the N. langsdorffii genome. Allelic scores for five classes of DNA markers were determined for 90 progeny of a “modified backcross” involving two N. langsdorffii inbred lines and the related species N. forgetiana. All markers had an expected segregation ratio of 1:1, and were scored in a common format. The map was constructed with JoinMap 3.0, and loci showing excessive transmission distortion were removed. The linkage map consists of 266 molecular marker loci defined by 217 amplified fragment length polymorphisms (AFLPs), 26 simple-sequence repeats (SSRs), 10 conserved orthologous sequence markers, nine inter-simple sequence repeat markers, and four target region amplification polymorphism markers arranged in 12 linkage groups with a combined length of 1062 cM. NlRPT is located on linkage group three, flanked by four AFLP markers and one SSR. Regions of skewed segregation were detected on LGs 1, 5, and 9. Markers developed for N. langsdorffii are potentially useful genetic tools for other species in Nicotiana section Alatae, as well as in N. benthamiana. We also investigated whether AFLPs could be used to infer genetic relationships within N. langsdorffii and related species from section Alatae. A phenetic analysis of the AFLP data showed that there are two main lineages within N. langsdorffii, and that both contain populations expressing dominant resistance to P. tabacina.