Multiscale landscape genomic models to detect signatures of selection in the alpine plant Biscutella laevigata

Plant species are known to adapt locally to their environment, particularly in mountainous areas where conditions can vary drastically over short distances. The climate of such landscapes being largely influenced by topography, using fine‐scale models to evaluate environmental heterogeneity may help detecting adaptation to micro‐habitats. Here, we applied a multiscale landscape genomic approach to detect evidence of local adaptation in the alpine plant Biscutella laevigata. The two gene pools identified, experiencing limited gene flow along a 1‐km ridge, were different in regard to several habitat features derived from a very high resolution (VHR) digital elevation model (DEM). A correlative approach detected signatures of selection along environmental gradients such as altitude, wind exposure, and solar radiation, indicating adaptive pressures likely driven by fine‐scale topography. Using a large panel of DEM‐derived variables as ecologically relevant proxies, our results highlighted the critical role of spatial resolution. These high‐resolution multiscale variables indeed indicate that the robustness of associations between genetic loci and environmental features depends on spatial parameters that are poorly documented. We argue that the scale issue is critical in landscape genomics and that multiscale ecological variables are key to improve our understanding of local adaptation in highly heterogeneous landscapes.     

Tracking genes of ecological relevance using a genome scan in two independent regional population samples of Arabis alpina

Understanding the genetic basis of adaptation in response to environmental variation is fundamental as adaptation plays a key role in the extension of ecological niches to marginal habitats and in ecological speciation. Based on the assumption that some genomic markers are correlated to environmental variables, we aimed to detect loci of ecological relevance in the alpine plant Arabis alpina L. sampled in two regions, the French (99 locations) and the Swiss (109 locations) Alps. We used an unusually large genome scan [825 amplified fragment length polymorphism loci (AFLPs)] and four environmental variables related to temperature, precipitation and topography. We detected linkage disequilibrium among only 3.5% of the considered AFLP loci. A population structure analysis identified no admixture in the study regions, and the French and Swiss Alps were differentiated and therefore could be considered as two independent regions. We applied generalized estimating equations (GEE) to detect ecologically relevant loci separately in the French and Swiss Alps. We identified 78 loci of ecological relevance (9%), which were mainly related to mean annual minimum temperature. Only four of these loci were common across the French and Swiss Alps. Finally, we discuss that the genomic characterization of these ecologically relevant loci, as identified in this study, opens up new perspectives for studying functional ecology in A. alpina, its relatives and other alpine plant species.     

Variation in Heavy Metal Accumulation and Genetic Diversity at a Regional Scale Among Metallicolous and Non-Metallicolous Populations of the Facultative Metallophyte Biscutella laevigata subsp. laevigata

Biscutella laevigata is a facultative metallophyte, with populations on non-metalliferous and metalliferous soils. Some of its metallicolous populations have been shown to hyperaccumulate thallium or lead in nature. Only Tl hyperaccumulation has been experimentally confirmed. We aimed to compare the patterns of metal (hyper)accumulation and genetic diversity among populations of B. laevigata subsp. laevigata in NE Italy.

None of the populations exhibited foliar hyperaccumulation of Cu, Zn, or Pb. The root-to-shoot accumulation rates for these metals were unchanged or decreased rather than enhanced in the metallicolous populations, in comparison with the non-metallicolous ones. Hyperaccumulation of Tl was confined to the population of the Cave del Predil mine. This population was genetically very distinct from the others, as demonstrated by AFLP-based cluster analysis. The two other mine populations did not surpass the threshold for Tl hyperaccumulation, but showed enhanced foliar Tl concentrations and root-to-shoot translocation rates, in comparison with the non-metallicolous populations. Genetic analysis suggested that adaptation to metalliferous soil must have been independently evolved in the metallicolous populations.     

Spatial scale of local adaptation and population genetic structure in a miniature succulent, Argyroderma pearsonii

Explicit understanding of the spatial scale of evolutionary processes is required in order to set targets for their effective conservation. Here, we explore the spatial context of neutral and adaptive divergence in the species-rich Knersvlakte region of South Africa. Specifically, we aimed to assess the importance of erosional drainage basins as spatial units of evolutionary process. We used amplified fragment length polymorphism (AFLP) and reciprocal transplants to investigate genetic differentiation in Argyroderma pearsonii, sampled from sparse and dense quartz habitats within each of three drainage basins. This design allowed assessment of differentiation at two distinct spatial scales; between habitats within basins, and between basins. We found near-perfect concordance between genetic clusters and basin occupancy, suggesting restricted interbasin gene flow. In addition, transplants reveal adaptive divergence between basins on the dense quartz habitat. We have shown that neutral and adaptive differentiation occurs between basins, but not between habitats within basins, suggesting that conservation plans aimed at conserving multiple interconnected drainage basins will capture an important axis of evolutionary process on the Knersvlakte.     

Genetic analysis and conservation of 31 surviving individuals of a rare New Zealand tree, Metrosideros bartlettii (Myrtaceae)

Metrosideros bartlettii (Myrtaceae) is a distinctive and extremely rare tree, endemic to New Zealand, first discovered in 1975. Prior to this study, a total of 19 adult individuals of the species had been reported; these are located in three small forest remnants in the far north of the North Island of New Zealand. Here we describe a total of 31 adult M. bartlettii at the three sites, including 12 individuals newly discovered by us. We analyse the genetic diversity of the species, using microsatellites to examine the chloroplast genome and amplified fragment length polymorphisms (AFLPs) to monitor nuclear variation. The results clearly demonstrate that M. bartlettii is a unique species, distinct from its two closest relatives M. robusta and M. excelsa. Analysis of genetic diversity within the 31 remaining individuals of M. bartlettii showed an average heterozygosity (< H >) of 0.18 and a proportion of polymorphic genes (< P >) of 0.44. Population structure, as shown by 286 AFLP loci, varied between the three geographical sites; the site with fewest individuals, containing two trees, showed some separation from the populations at the other two locations. These two latter sites, by contrast, had highly overlapping AFLP population diversity profiles. The implications of these results for conservation of the species are discussed.     

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.     

A spatial analysis method (SAM) to detect candidate loci for selection: towards a landscape genomics approach to adaptation

The detection of adaptive loci in the genome is essential as it gives the possibility of understanding what proportion of a genome or which genes are being shaped by natural selection. Several statistical methods have been developed which make use of molecular data to reveal genomic regions under selection. In this paper, we propose an approach to address this issue from the environmental angle, in order to complement results obtained by population genetics. We introduce a new method to detect signatures of natural selection based on the application of spatial analysis, with the contribution of geographical information systems (GIS), environmental variables and molecular data. Multiple univariate logistic regressions were carried out to test for association between allelic frequencies at marker loci and environmental variables. This spatial analysis method (SAM) is similar to current population genomics approaches since it is designed to scan hundreds of markers to assess a putative association with hundreds of environmental variables. Here, by application to studies of pine weevils and breeds of sheep we demonstrate a strong correspondence between SAM results and those obtained using population genetics approaches. Statistical signals were found that associate loci with environmental parameters, and these loci behave atypically in comparison with the theoretical distribution for neutral loci. The contribution of this new tool is not only to permit the identification of loci under selection but also to establish hypotheses about ecological factors that could exert the selection pressure responsible. In the future, such an approach may accelerate the process of hunting for functional genes at the population level.     

Conservation of Britain's biodiversity: the case of Hieracium cyathis (Asteraceae) and its relation to other apomictic taxa

Hieracium cyathis (Ley) W. R. Linton (Asteraceae), Chalice Hawkweed, is a rare species endemic to Britain. It is a distinct member of Hieracium Section Oreadea , characterized by a short acladium and out-curved tips to the phyllaries. Historic records and current field survey indicate it has occurred in two localities in SW England and seven in South Wales. It is still present in all sites except for one Welsh locality, is significantly threatened at Cheddar Gorge, and two Welsh sites have only three plants each. Comparative morphology, cultivation experiments and amplified fragment length polymorphism (AFLP) analysis do not support the reported differences between material from SW England and Wales. AFLP analysis showed that every plant in the field was genetically similar but different, and minor genetic variation was found in one progeny array. It is a triploid (2 n  = 27) with apomictic seed production, and 26% potentially viable pollen. It qualifies as 'Near Threatened' under the 2001 IUCN threat criteria. General guidelines for conservation of microspecies of Hieracium are discussed.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 669–680.     

Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae)

The origin of polyploid Veronica cymbalaria (Plantaginaceae) was investigated using DNA sequence data and amplified fragment length polymorphism (AFLP) fingerprints to reveal the parentage of this taxon. The use of AFLP fingerprints in phylogenetic analysis is problematic and various methods have therefore been compared. DNA sequence data (for the internal transcribed spacer (ITS) region and the plastid trnL-F region (trnL intron, 3'exon, and trnL-F spacer)) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ITS region suggested a reliable hypothesis for the evolution of the V. cymbalaria complex. This hypothesis allowed evaluation of the effect of different distance measures (Jaccard and Nei-Li) in phenetic, character-state weighted parsimony, and Bayesian analyses of AFLP markers. The study establishes that tetraploid V. cymbalaria originated at least twice in the eastern Mediterranean, with one parent differing in the two separate origins. Hexaploid V. cymbalaria originated even more often. The results illustrate that even subtle differences in the analyses of AFLP markers can lead to drastically different conclusions. The study reveals multiple origins of a Mediterranean polyploid species. Furthermore, it demonstrates that the analysis of a complex marker system such as AFLP fingerprints using only one type of analysis can easily be misleading.     

Construction of a genetic linkage map of Thlaspi caerulescens and quantitative trait loci analysis of zinc accumulation

Zinc (Zn) hyperaccumulation seems to be a constitutive species-level trait in Thlaspi caerulescens. When compared under conditions of equal Zn availability, considerable variation in the degree of hyperaccumulation is observed among accessions originating from different soil types. This variation offers an excellent opportunity for further dissection of the genetics of this trait. A T. caerulescens intraspecific cross was made between a plant from a nonmetallicolous accession [Lellingen (LE)], characterized by relatively high Zn accumulation, and a plant from a calamine accession [La Calamine (LC)], characterized by relatively low Zn accumulation. Zinc accumulation in roots and shoots segregated in the F3 population. This population was used to construct an LE/LC amplified fragment length polymorphism (AFLP)-based genetic linkage map and to map quantitative trait loci (QTL) for Zn accumulation. Two QTL were identified for root Zn accumulation, with the trait-enhancing alleles being derived from each of the parents, and explaining 21.7 and 16.6% of the phenotypic variation observed in the mapping population. Future development of more markers, based on Arabidopsis orthologous genes localized in the QTL regions, will allow fine-mapping and map-based cloning of the genes underlying the QTL.     

Disjunct distribution of Hypericum nummularium L. (Hypericaceae): molecular data suggest bidirectional colonization from a single refugium rather than survival in distinct refugia

Hypericum nummularium has a strongly disjunct, bi‐areal distribution in Europe: it is abundant in the Pyrenees and grows in a very restricted part of the Alps, more than 1000 km away. My aim was to estimate the genetic divergence between these areas and to identify the factors responsible for the disjunction: glacial relicts, bidirectional colonization from a common refugium, long‐distance dispersal and/or human introduction? Internal transcribed spacers (ITS) sequencing (680 bp) and amplified fragment length polymorphism (AFLP) fingerprinting (104 polymorphic markers) showed very low differentiation between populations in the Alps and the Pyrenees, indicating that H. nummularium probably survived in a single refugium. Moreover, levels of genetic diversity were similar in the two areas, making human introduction and long‐distance dispersal unlikely. Thus, the species probably survived in one refugium, subsequently colonizing both areas more or less simultaneously. The comparison of genetic and geographical distances suggested a step by step migration in the Alps (isolation by distance), whereas random dispersal events were more likely in the Pyrenees. Finally, I discuss possible causes for the restricted distribution area of H. nummularium in the Alps (e.g. unsuitable habitat, low dispersal capacities) and conclude that strong human disturbance is probably the major limit to the expansion of the species in this region. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 437–447.     

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.     

Complete exclusion of nonsires in an analysis of paternity in a natural plant population using amplified fragment length polymorphism (AFLP)

The utility of the new polymerase chain reaction (PCR)-based multilocus DNA fingerprinting technique amplified fragment length polymorphism (AFLP) for paternity analysis in natural plant populations was assessed. In a natural population of 25 plants of Persoonia mollis (Proteaceae), three AFLP primer pairs generated 147 dominant loci. Of these, 125 (85%) were polymorphic, with a mean recessive allele frequency of 0.735. The theoretical expected percentage of offspring for which all males except the true father can be excluded ( P _ET) was 99.9% for this population. The estimates of P _ET drop marginally to 99.6% and 97.6% for larger populations of 100 and 1000 individuals, respectively. A preliminary investigation confirmed the power of AFLP for paternity analysis by assigning paternity, or excluding all known potential sires, for 242 of 252 (96.0%) naturally pollinated seeds. Ambiguous paternity for the remaining 10 seeds was quickly resolved by utilizing two further AFLP primer pairs, ultimately generating over 200 polymorphic loci and resulting in the exclusion of all nonsires for all 252 (100%) seeds. This study highlights the utility of AFLP for paternity analysis because: (i) it generates sufficiently large numbers of highly reproducible polymorphic loci, that are (ii) quickly and accurately scored using an automated DNA sequencer and dedicated software, and (iii) unlike microsatellites, requires no sequence knowledge so it is more easily applied to new study species.     

Adaptive sympatric speciation of polychromatic "roundfin" sailfin silverside fish in Lake Matano (Sulawesi)

The significance of sympatric speciation is one of the most controversial topics in evolutionary biology. Theory suggests that different factors can lead to speciation in full geographical contact, including selection and nonrandom mating. Strict criteria have been established for assessing sympatric speciation, which have been met in only a very few cases. Here, we investigate differentiation among sympatric morphospecies and color morphs of "roundfin" sailfin silversides (Telmatherinidae), small freshwater fish endemic to ancient Lake Matano in Central Sulawesi (Indonesia). Morphospecies are distinct according to body shape (geometric morphometrics), population structure (population-level amplified fragment length polymorphism [AFLP] markers), ecology, and mating behavior (habitat transects, stomach contents). Explorative genome scans based on AFLPs indicate that divergent selection affects only 1.3-4.2% of the analyzed loci, suggesting an early stage of speciation. Transect data demonstrate strong assortative mating and adaptive niche differentiation. However, we find no restrictions in gene flow among the conspicuous male color morphs. In summary, our data are consistent with a sympatric mode of divergence among three morphospecies under conditions effectively ruling out allopatric scenarios. Substantial, but incomplete, reproductive isolation suggests an early stage of speciation, most likely due to ecological selection pressure.     

Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors

Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.     

Correlations between Genetic,Morphological, and Chemical Diversities in a Germplasm Collection of the Medicinal Plant Origanum vulgare L.

In total, 42 accessions of Origanum vulgare L., mostly originating from Europe, were evaluated, to detect molecular, quantitative morphological, and chemotype polymorphisms and to discover possible correlations between them. Twelve traits related to morphological characteristics were measured. The components in the essential oils were identified by GC/MS analysis, and the oil contents of 18 major compounds were determined. A total of 477 molecular polymorphisms including 214 AFLP (amplified fragment length polymorphism) and 263 SAMPL (selectively amplified microsatellite polymorphic loci) were used for genotyping. Euclidean distances of morphological and chemotypic data and genetic distances (1 – Dice's similarity) of molecular markers were compared by applying Mantel tests to ascertain the congruencies between them. A relatively high correlation between chemotypic patterns and genetic markers was identified, while a lower correlation was found between the morphological and genetic matrices. Pairwise analyses of correlation among all traits showed that the stem diameter was correlated to the essential‐oil yield and the carvacrol content. Cluster analysis, population inference, and principal component analysis revealed a broad genetic and chemical variation among the accessions. The knowledge of these diversities, found in this study, will allow a plant improvement of Origanum vulgare related to pharmaceutical and spice uses.     

Comparative Chemical and Molecular Variability of Cananga odorata (Lam.) Hook.f. & Thomson forma genuina (Ylang‐Ylang) in the Western Indian Ocean Islands: Implication for Valorization

Cananga odorata (Lam.) Hook .f. & Thomson forma genuina (Annonaceae) is a tropical tree, grown for the production of ylang‐ylang essential oil, which is extracted from its fresh and mature flowers. Despite its economic and social importance, very little information is available on its variability and the possible factors causing it. Therefore, the relationship between the genetic structure, revealed by amplified fragment length polymorphism (AFLP), and the essential oil chemical composition, determined by GC/MS analysis, of ylang‐ylang grown in semi‐managed systems in three Indian Ocean islands (Grande Comore, Mayotte, and Madagascar) was investigated. Our results revealed a low genetic variation within plantations and contrasted situations between islands. Variations of the chemical composition could be observed within plantations and between islands. The genetic differentiation pattern did not match the observed pattern of chemical variability. Hence, the chemical variation could not be attributed to a genetic control. As Grande Comore, Madagascar, and Mayotte present different environmental and agronomic conditions, it can be concluded that the influence of these conditions on the ylang‐ylang essential oil composition is consistent with the patterns observed. Finally, several strategies were proposed to valorize the chemical composition variations.     

Genetic diversity and geographical differentiation of Dipteronia Oliv. (Aceraceae) endemic to China as revealed by AFLP analysis

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry, both of them are rare and endangered. AFLP markers were used to characterize the genetic diversity and geographical differentiation of the genus. Eight out of 32 PstI + 3/MseI + 3 selective primer combinations screened were applied to the analysis on 142 individuals of 17 D. sinensis and 4 D. dyeriana populations, respectively. A total of 324 fragments with 316 polymorphic were amplified. The proportion of polymorphic loci (PPB) was 97.53%. The Nei's gene diversity in D. sinensis and D. dyeriana was 0.3319 and 0.3047, respectively. About 43.6% (G_ST = 0.4356) of the genetic variation occurred among the populations, indicating a relatively high genetic differentiation among the populations. Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into three subgroups corresponding to their geographical distributions. Correlation analysis revealed a significant correlation (p < 0.05) between geographical distance and genetic distance of these populations, suggesting that the relatively high genetic differentiation among the populations of D. sinensis might be caused by geographical isolation.     

Morphometric and population genetic analyses elucidate the origin,evolutionary significance and conservation implications of Orchis × angusticruris (O. purpurea × O. simia), a hybrid orchid new to Britain

We report the first confirmed occurrence in Britain of Orchis × angusticruris Franch. ex Rouy, a hybrid between two closely related orchid species of anthropomorphic Orchis (O. purpurea Huds. × O. simia Lam.) that hybridize frequently in Continental Europe. Seven individual hybrids, most likely F1 plants representing a single interspecific pollination event, first flowered with both parents in May 2006 at a nature reserve in the Chiltern Hills near Goring, Oxfordshire. Univariate and multivariate morphometric analyses (43 characters plus 12 indices), internal transcribed spacer sequencing, plastid microsatellites and amplified fragment length polymorphism (AFLP) analyses together readily separate the parents and confirm that O. purpurea was the ovule parent and O. simia the pollen parent, presumably reflecting the greater frequency and/or later flowering period of the latter at the site. This study reinforces a more general observation that, in most orchids, the ovule parent contributes substantially more to the hybrid phenotype than does the pollen parent, perhaps reflecting cytoplasmic inheritance. In contrast, the hybrids are placed closer to O. simia than to O. purpurea in the AFLP tree. Apparently recent arrivals, the few O. purpurea plants at Goring contrast genetically with the two other small populations of this species known in the Chilterns, but rather are consistent with relatively uncommon Continental populations. This suggests that the plants may have been deliberately introduced at Goring by man, although transport from the Continent in high‐level air currents cannot be ruled out. The Goring population of O. simia is likely to have become genetically impoverished through (1) preferential removal of many relatively fit plants to herbaria in the 19^th century and/or (2) a catastrophic population crash in the first half of the 20^th century. However, both our re‐examination of herbarium specimens and our population genetic data indicate past hybridization among anthropomorphic Orchis species occurring naturally in the Chilterns. Thus, we tentatively recommend retention of the hybrid plants at Goring, despite their likely anthropogenic origin from Continental material and the partial viability of their pollen and seeds, which offers opportunities for future introgression. Although the Goring hybrids broadly resemble morphologically O. militaris, another anthropomorphic Orchis still found at two Chiltern localities, sufficient morphological and molecular differences were observed to strongly refute our initial hypothesis that O. militaris could have originated through hybridization between ancestors that resembled O. purpurea and O. simia. The comparatively complex genetic properties evident in both O. simia and O. purpurea merit further study. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 687–711.     

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean ( Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene. Received: 20 November 1997 / Accepted: 6 January 1998