Applications of DNA genetic markers to the study of plant growth and development

DNA genetic markers, such as restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA markers (RAPDs), are powerful tools for studying the genetics of plant growth and development. DNA markers are defined sequences of DNA that can be used in traditional linkage mapping. Using DNA marker technology, scientists can uncover relationships between cloned cDNA sequences and classically characterized genes. DNA markers make it possible to dissect the contributions of multiple genetic loci underlying complex developmental processes. Moreover, changes in genome organization that occur during development or in response to environmental signals can be monitored using RFLP technology. In the future, it may be possible to clone any gene based solely on its map position. This will involve the use of tightly linked DNA markers as entry points for chromosome walking, in which a series of overlapping genomic clones reaching from the tightly linked DNA marker to the gene of interest are identified.     

Comparisons of Isolates of Fusarium avenaceum from White Lupin and Other Crops by Pathogenicity Tests,DNA Analyses and Vegetative Compatibility Tests

Isolates of Fusarium avenaceum, mostly from crops of white lupin or wheat, were tested for pathogenicity on white lupin and wheat plants and compared by DNA tests and, in a limited study, vegetative compatibility. Most of the 80 isolates were pathogenic on both plant species after inoculation on shoot bases. Disease severity was greater at higher incubation temperatures that ranged from 15/10°C to 25/20°C (day/night temperatures). Isolates from lupin crops tended to be more pathogenic, on average, on lupins than on cereals. Polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer region of the rDNA distinguished two groups of isolates that occurred in different proportions among isolates from lupins and cereal crops. Random amplified polymorphic DNA (RAPD)‐PCR analyses indicated considerable genetic variation among isolates, but there was some similarity among groups of isolates from populations in the same field. Genetic diversity was confirmed by a high degree of vegetative incompatibility among 20 isolates using nitrate nonutilizing mutants. There were no relationships among pathogenicity, RFLP group, RAPD group and vegetative compatibility group.     

Biochemical and molecular identification of Enterococcus spp. from red pitaya

Red pitaya is a popular fruit worldwide due to its unique appearance and high nutrient contents. Many studies about the nutritional content and the physico-chemical characteristics of red pitaya have been performed but microorganisms that occurred naturally in the fruit remained unknown. Therefore, this study was conducted to characterize the enterococci present in red pitaya. Enterococci were isolated from red pitaya fruit and characterized with biochemical tests, randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) of the 16S rDNA region. A total of 30 bacteria were isolated from red pitaya, of which 20 isolates were confirmed as enterococci, genus Enterococcus. 13 isolates were further confirmed as Enterococcus casseliflavus and 7 were identified as Enterococcus gallinarum.     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Patterns of genetic variation detected by RAPDs suggest a single origin with subsequent mutations and long-distance dispersal in the apomictic fern Dryopteris remota (Dryopteridaceae)

Debates on speciation processes in pteridophytes have revived. In order to study the evolutionary origin of an apomictic fern species, we investigated the genetic variation in the strictly agamosporous Dryopteris remota. We determined the genotypes of 22 individuals from many different locations within the species' European distribution and of 20 individuals from a Swiss population. A previous study on isozyme variation showed no intraspecific genetic variation in a similar sample set (Schneller and Holderegger, 1994, American Fern Journal 84: 94-98). In contrast to this, four out of 12 random amplified polymorphic DNA (RAPD) primers tested revealed low genetic diversity among individuals of D. remota from different locations. Intrapopulational genetic variation was also very low, but in the single population studied, a unique multiband genotype could be detected. The geographic distribution of genetic variation found in D. remota was best explained by the assumption of a single origin, the accumulation of somatic mutations during spread, and occasional, but effective, events of dispersal over large distances. The present study thus stresses the importance of long-distance dispersal in evolutionary processes and biogeography of ferns.     

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

RAPD and RFLP markers tightly linked to the locus controlling carnation (Dianthus caryophyllus) flower type

 Flower doubleness as a breeding characteristic is of major importance in carnation (Dianthus caryophyllus), one of the major cut-flowers sold worldwide, since flower architecture is of the utmost value in ornamentals. Based on the number of petals per flower, carnations are grouped into “single”, “semi-double” and “double” flower types. The first have five petals and are easily distinguishable, but of no economic value to the carnation industry. Flowers of standard and spray varieties, which constitute the largest market share, are usually of the double and semi-double type, respectively. These flower types are not easily distinguishable due to phenotypic overlaps caused by environmental conditions. To study the inheritance of this trait, several progeny segregating for flower type were prepared. Based on the number of single-flower type fullsibs among the offspring, we found that this phenotype is expressed only in plants homozygous for the recessive allele and that a dominant mutation in this allele causes an increase in petal number. Using random decamer primers, we identified a random amplified polymorphic DNA (RAPD) marker which is tightly linked to this recessive allele. The RAPD marker was cloned and used to generate a restriction fragment length polymorphic (RFLP) marker. This RFLP marker could discriminate with 100% accuracy between the semi-double and double- flower phenotypes in carnations of both Mediterranean and American groups. The advantages of RFLP over RAPD markers and their applicability to markerassisted selection in carnation are discussed. Received: 11 August 1997 / Accepted: 22 August 1997     

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme Not I in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15×2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M. Johnston     

Allotetraploid origin ofAllium altyncolicum (Alliaceae, Allium sect.Schoenoprasum) as investigated by karyological and molecular markers

The tetraploidAllium altyncolicum (2n = 4x = 32) is considered to be of hybrid origin, because most of its morphological characters are intermediate between those of its putative parents,A. schoenoprasum andA. ledebourianum. In the present work an attempt has been made to ascertain its parentage by several methods: Giemsa C-banding, genomic in situ hybridization (GISH), PCR-RFLP of cpDNA, restriction enzyme mapping of the rDNA, and RAPDs. C-banding and GISH indicates clearly thatA. altyncolicum is a segmental allopolyploid.Allium schoenoprasum andA. ledebourianum are the most likely the parental species and the larger part of the genome ofA. altyncolicum (26 chromosomes) is derived fromA. schoenoprasum. The low genetic divergence between these three species was confirmed by the lack of sequence variation in the ITS sequences of nuclear rRNA genes and of the plastid rbcL-atpB intergenic spacer. Both parental species andA. altyncolicum could be distinguished by RFLP of the rDNA repeats. The geographic origin of the putative parental species was investigated using RAPDs.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers

To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers.     

Genetic variation of Usnea filipendula (Parmeliaceae) populations in western Germany investigated by RAPDs suggests reinvasion from various sources

Random amplified polymorphic DNA (RAPD) markers were characterized for 25 specimens of Usnea filipendula to evaluate the genetic diversity of populations reinvading formerly uninhabited regions in Northrhine-Westphalia due to decreasing sulfur dioxide levels. With six 10-mer randomly amplified polymorphic DNA (RAPD) primers, a 66 character by 25 specimens matrix was generated. Phenetic analysis (UPGMA) showed no obvious groupings. The reinvading populations are distributed over the phenogram and are not genetically closely related. The results suggest that the reinvading populations of this usually sterile species are derived from different sources and do not consist of a particular clone capable of re-entering the area.     

Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radio-isotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLP-generated data with DNA-DNA homology data.     

Phylogenetic analysis of the hard pines (Pinus subgenus Pinus,Pinaceae) from chloroplast DNA restriction site analysis

Phylogenetic analysis of plastid DNA restriction site and rearrangement mutations suggests a number of major revisions to taxonomy and phylogenetic concepts in the hard pines. Total genomic DNA from 18 species that sampled all nine subsections was digested with 19 restriction enzymes, blotted, and probed with 70% of the Douglas-fir (Pseudotsuga menziesii) chloroplast genome, or, with clones encompassing the entire chloroplast genome of Pinus contorta. A total of 204 site mutations and five rearrangement mutations were generated, of which 126 were phylogenetically informative. Wagner parsimony analyses revealed 11 clades that were strongly supported by bootstrap and decay index analyses. All North American species except P. resinosa formed a distinct monophyletic group that was strongly separated from the Eurasian species. Within the Eurasian clade subsect. Sylvestres was polyphyletic; its Mediterranean species were closely allied with members of sect. Pinea. Sect. Pinea appeared polyphyletic as well; both species of its subsect. Leiophyllae showed a close affinity to Mesoamerican pines of subsect. Oocarpae in sect. Pinus. Within the North American pines subsects. Ponderosae and Oocarpae were polyphyletic. Despite its shallow fossil record, subsect. Contortae emerged as a sister group to all of the North American pines apart from P. resinosa, which was allied with Eurasian species of subsect. Sylvestres. The remaining North American subsections formed two groups: a poorly resolved clade with subsects. Ponderosae and Sabinianae, and sequentially nested clades represented by: P. radiata; P. taeda; representatives of subsects. Oocarpae and Ponderosae from Mesoamerica, and subsect. Leiophyllae. We present estimates of divergence times for each of these major clades based on molecular clocks calibrated using two hard pine fossil observations.     

Possible causes of morphological variation in an endemic Moroccan groundsel (Senecio leucanthemifolius var. casablancae): evidence from chloroplast DNA and random amplified polymorphic DNA markers

Genetic variation was assessed in Senecio leucanthemifolius var. casablancae (Compositae), a Moroccan Atlantic coast endemic, in order to examine possible causes of atypical leaf morphology in three populations south of the known range. Evidence for introgression from S. glaucus ssp. coronopifolius and/or divergence was investigated with molecular markers. Both random amplified polymorphic DNA (RAPD) and chloroplast (cp) DNA restriction fragment length polymorphism (RFLP) differentiated the species well. Some evidence that hybridization may have occurred between the two species was provided by cpDNA markers. However, biparentally inherited RAPD markers failed to provide any support for the hypothesis that intermediate leaf morphologies in atypical populations arose through hybridization. Consequently, they are most likely to have arisen via divergence caused by drift and/or selection. Genetic distances among populations of S. leucanthemifolius were significant in all but one case. Isolation by distance was indicated by a significant positive correlation between genetic and geographical distances (r = 0.68, P = 0.01, Mantel test). These results suggest that long-distance achene dispersal is rare, despite the presence of a well-developed pappus. The observed loss of pappus at achene maturity may explain this unexpected result. Due to the morphological distinction of var. casablancae from other varieties of S. leucanthemifolius, we suggest elevation to species rank and treatment of the atypical material at infraspecific rank.     

Intraspecific differentiation of Allium wallichii (Amaryllidaceae) inferred from chloroplast DNA and internal transcribed spacer fragments

In the present study, we used two chloroplast DNA (cpDNA) fragments (trnL-F and rps16) and the nuclear ribosomal internal transcribed spacer (ITS) sequence data to examine intraspecific differentiation and phylogeographical history of Allium wallichii. Based on wide scale sampling (28 populations and 174 individuals) across the entire distribution range of this species, 33 cpDNA haplotypes and 25 ITS ribotypes were detected in our investigation. These cpDNA haplotypes were divided into three major lineages, which was further supported by the ITS phylogenetic results. High haplotype/ribotype diversity and population differentiation, together with most of the haplotypes/ribotypes being exclusive to single populations, implied restricted gene flow among populations and significant geographical isolation. Nearly all populations with high haplotype/ribotype diversity were found in the Hengduan Mountain Region (HMR), whereas the populations of the Himalayas and Nanling Mountains showed a lower level, suggesting the HMR might serve as a potential divergence center for A. wallichii. The main lineages of A. wallichii diverged from each other between Mid–Late Pliocene and Early Quaternary based on two sets of molecular markers, indicating that the Quaternary climatic fluctuation could not have contributed greatly to the divergence of the main lineages of A. wallichii. Instead, the intricate topography and heterogeneous habitats resulting from the drastic uplift of the Qinghai–Tibet Plateau from the Late Pliocene could be responsible for the intraspecific differentiation of A. wallichii. The present study further highlights the importance of geographic isolation and habitat heterogeneity in shaping and maintaining high species diversity within the HMR.     

Rapid evolution in the Nebria gregaria group (Coleoptera: Carabidae) and the paleogeography of the Queen Charlotte Islands

Morphological differentiation in the ground beetles of the Nebria gregaria group, found on the Queen Charlotte Islands, has been used as support for the glacial refugium proposed for the northwest coast of North America. Two members of this species group, N. charlottae and N. louiseae, are restricted to cobble beaches in this archipelago. A third, N. haida, is found only in alpine regions of the archipelago and the adjacent mainland. The remaining two species of the gregaria group, N. lituyae and N. gregaria, show highly restricted distributions in the mountains of the Alaska panhandle and on the beaches of the Aleutian Islands, respectively. To determine the relationships of the five species, we conducted phylogenetic analyses on nucleotide sequence data obtained from five regions of the mitochondrial DNA. In total, 1835 bp were analyzed. The results suggest that one species, N. lituyae, does not belong in the gregaria group, and that only seven mutations separated the two most divergent of the four remaining species. We also conducted random amplified polymorphic DNA fingerprinting analyses on genomic DNA extracted from the five species. Analyses of genetic diversity revealed a lack of molecular differentiation among the Queen Charlotte species, suggesting that these populations may be postglacial in origin and that together N. gregaria, N. charlottae, N. louiseae, and N. haida might represent local variations of a single species. These results are consistent with conclusions derived for the morphological and genetical differentiation among Gasterosteus populations in the archipelago.     

Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow

Geographic patterns of genetic variation in chlorolast (cpDNA) and nuclear ribosomal (nrDNA) DNA were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis in the Great Basin of western Nevada. Noncoding DNA from the trnL-trnF intergenic spacer and the trnL intron of the chloroplast genome was sequenced from seven populations of J. osteosperma and four populations of J. occidentalis sampled over a large proportion of their respective ranges. An adenine nucleotide at position 436 in the aligned sequence and within a Tru 9I restriction site was found to be present in individuals of J. osteosperma sampled from western Colorado and central Utah, but absent in sequences of J. osteosperma sampled from central and western Nevada and all sequences of J. occidentalis. Two hundred fourteen individuals from 34 populations of J. osteosperma and J. occidentalis were then screened for cpDNA haplotype by Tru 9I digestion of the trnL-trnF polymerase chain reaction (PCR) product. Two cpDNA haplotypes were evident, each consisting of restriction fragment profiles that differed solely with respect to the presence or absence of the Tru 9I site encompassing the adenine nucleotide at position 436. One of these haplotypes was monomorphic in J. occidentalis and exhibited a decreasing frequency in J. osteosperma with increasing geographic distance from J. occidentalis in west-central Nevada. Geographic patterns in nuclear ribosomal DNA (nrDNA) variation were examined by restriction fragment analysis and, although spatially more restricted, exhibited patterns of clinal variation similar to those observed in cpDNA haplotype. Genetic relationships based on DNA sequences and geographic patterns of genetic variation in chloroplast and nuclear ribosomal DNA are consistent with morphology in suggesting interspecific gene flow between J. occidentalis and J. osteosperma.     

The use of random amplified polymorphic DNA (RAPD) for hybrid detection in Scirpus from the river Schelde (Belgium)

Along the unique freshwater tidal zone of the river Schelde (Belgium), plants of Scirpus species occur primarily in small and fragmented populations. The majority of these are native Scirpus lacustris, S. pungens, S. triqueter, S. tabernaemontani and intermediate morphological forms. The distribution area of S. triqueter is even restricted to this tidal habitat. However, several cultivated S. tabernaemontani strains have recently been introduced. The latter species is often used to stabilize riverbanks. To determine the existing genetic diversity among these species, stems from plants from 44 different locations were subjected to random amplified polymorphic DNA (RAPD) analysis, using 22 decanucleotide primers. Data analysis of the amplified DNA fragments enabled us to unambiguously differentiate among these Scirpus taxa. Hybridization between S. triqueter and S. tabernaemontani was documented, and the studied hybrids were always genetically more similar to S. triqueter than to S. tabernaemontani . Among the introduced clones, at least two different origins could be distinguished. Several of the introduced S. tabernaemontani clones were clearly different from individuals of the same species that were native to this region. As there were different hybrid genotypes owing to different hybrid events or to introgression, this complex should be safeguarded from genetic pollution. New S. tabernaemontani genotypes with markers not previously observed in that hybrid complex of the unique tidal freshwater habitat should be avoided in replantation projects.     

The origin of a novel form of Senecio (Asteraceae) restricted to sand dunes in southern Sicily

The taxonomy of diploid Mediterranean Senecio sect. Senecio (Asteraceae) is complex, owing to a recent species radiation, high morphological plasticity and occasional interspecific hybridization. A study was conducted to resolve the origin of a novel form of Senecio restricted to sand dunes in southern Sicily, Italy. This has been described previously as morphologically intermediate to Senecio gallicus and Senecio glaucus ssp. coronopifolius, indicating a possible hybrid origin, or as a variant of Senecio leucanthemifolius. Plants of this form grown in a glasshouse were morphologically intermediate to S. glaucus and S. leucanthemifolius, but were also similar to some cultivated individuals of S. gallicus. No evidence for a hybrid origin was obtained from a survey of random amplified polymorphic DNA variation; instead the plants surveyed were most closely allied to Tunisian S. glaucus. They were also polymorphic for the same set of cpDNA haplotypes present in Tunisian S. glaucus. We conclude that the Sicilian Senecio is a variant form of North African S. glaucus ssp. coronopifolius, which most probably dispersed to sand dunes in southern Sicily in the relatively recent past. The presence of several cpDNA haplotypes in this material indicates that there have been multiple introductions of the species to Sicily.