Analysis of the wild potato germplasm of the series Acaulia with AFLPs: implications for ex situ conservation

The wild potato germplasm of the series Acaulia maintained at the Centre for Genetic Resources, The Netherlands, currently consists of 314 accessions. This collection comprises seed samples of the species Solanum acaule (ssp. acaule, ssp. aemulans, ssp. palmirense and ssp. punae) and Solanum albicans collected from South America. In order to validate taxonomic classification, to investigate the extent of redundancy and to study the distribution of genetic diversity across the collection area, the entire collection was analysed with two AFLP primer pairs on two plants per accession. Within the entire sample a total number of 130 polymorphic bands were scored for the two primer pairs. An UPGMA cluster analysis grouped the majority of plants according to the species and subspecies. A total number of 16 misclassifications were identified, including four cases that did not seem to belong to the series Acaulia. Two accessions were found to consist of plants of different AFLP clusters. AFLP data also allowed the taxonomic classification of the subspecies of 97 accessions that previously were described as S. acaule only. For 126 accessions the two individuals studied displayed identical AFLP profiles. Forty six of these 126 accessions shared their profiles with both or single plants of other accessions. These were all tested for identical profiles for a third primer pair, resulting in 15 duplication groups consisting of a total number of 22 accessions and 14 single plants. Analyses of molecular variance (AMOVA) were performed to examine the distribution of genetic variation. Comparison of geographic distances between the collection site of plants and the number of AFLP polymorphisms revealed no consistent relationship between geographic distance and genetic diversity. AFLP analysis appeared to be an efficient method to verify taxonomic classification and to identify redundancies in the wild germplasm of the series Acaulia. Implications of the results for the ex situ conservation of wild potato germplasm are discussed. Received: 6 November 2000 / Accepted: 20 April 2001     

Variability for restriction fragment lengths and phylogenies in lentil

Summary Thirty accessions of domesticated (Lens culinaris ssp. culinaris) and wild (L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans ssp. ervoides and L. nigricans ssp. nigricans) lentil were evaluated for restriction fragment length polymorphisms (RFLPs) using ten relative low-copy-number probes selected from partial genomic and cDNA libraries of lentil. Nei's average gene diversity was used as a measure of genetic variability for restriction fragment lengths within subspecies and a dendrogram was constructed from genetic distance estimates between subspecies. The wild lentils L. culinaris ssp. orientalis and L. culinaris ssp. odemensis showed the greatest variability for restriction fragment lengths and were closely positioned to domesticated lentil in the dendrogram. Little variability for restriction fragment lengths was observed within accessions of L. nigricans ssp. ervoides and L. nigricans ssp. nigricans. This observation is consistent with a previously published proposal that nigricans may have been independently domesticated. Estimates of genetic variability based on RFLPs tended to be greater than estimates from isozymes.     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

Paternal plastid DNA can be inherited in lentil

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F₁ progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F₁ progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F₁ plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F₁ progeny.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.     

Potato germ plasm enhancement with disomic tetraploid Solanum acaule. II. Assessment of breeding value of tetraploid F1 hybrids between tetrasomic tetraploid S. tuberosum and S. acaule

The breeding value of tetraploid F₁ hybrids between tetrasomic tetraploid S. tuberosum and the disomic tetraploid wild species S. acaule was examined. The F₁ hybrids showed a tuber yield and appearance comparable to those of their cultivated parent, indicating a potential as acceptable breeding stocks despite the 50% contribution to their pedigree from wild S. acaule. The cytological behavior of the tetraploid F₁ hybrids was examined to determine the probability of recombination for the introgression of S. acaule genes. The majority of the meiotic configurations at metaphase I was bivalents and univalents with mean frequencies of 17.6 and 9.9, respectively. Further, a low frequency of trivalents and quadrivalents was observed. An acceptable low level of meiotic irregularities were observed at the later stages of microsporogenesis, and a reasonable level of pollen stainability was obtained. Therefore, these hybrids could likely be employed for further introgression. From the cytological observations, the following speculations were drawn: (1) some genomic differentiation exists between the S. acaule genomes, (2) at least one of the S. acaule genomes may be homoeologous to the S. tuberosum genomes, (3) intergenomic recombination would likely occur due to the nature of the genomic constitution of the hybrids, and (4) the nature of sesquiploidy of the hybrids may facilitate efficient introgression and establishment of unique aneuploid and euploid recombinant genetic stocks.     

Kritsche Betrachtungen zur Nomenklatur argentinischer Wildkartoffeln

Summary Continuing the critical publications on the nomenclature of the Argentine wild potatoes, set on in 1956, now the results of 5 years' studies of the seriesAcaulia are discussed. This series is to be found in the highlands of the Cordilleras and its vegetation limit is at about 4700 m above sea-level. The frost-resistance ofS. acaule andS. aemulans is remarkably high (up to minus 10°). ForS. acaule cytological determinations have proved again the already known number 2n=48, besides this in the province of Jujuy a species with 2n=36 has been found, being probably a hybrid ofS. acaule andS. alticolum. This species with 36 chromosomes is increasingly spreading in certain arid areas in an altitude of 4000 m. ForS. aemulans the chromosome number 2n=24 is published for the first time. Reciprocal crosses under greenhouse conditions betweenS. aemulans (2n=24) andS. acaule (2n=48) showed no success. In opposition to the view ofHawkes the author insists onS. aemulans being a good species, endemical for the arid mountains of Famatina and Ambato in the provinces of La Rioja and Catamarca. Having been collected in 1907 for the last time, nowS. aemulans has anew been found by the author in the Famatina mountains.As toS. acaule and its numerous varieties and related species the author holds the following opinion: The termsS. depexum, Schreiteri, punae, subexinterruptum, checcae, caulescens, chorruense established byBitter, Bukasov, Juzepzuk, andHawkes, are superfluous, the real range of variability is moreover much greater. To illustrate this point of view some examples were set.By its valuable resistance-genes againstRhizoctonia, frost, and Virus,S. acaule, at present, has gained considerable importance for plant breeding. For this reason we think it the more necessary to elucidate the systematical and cytological problems of the whole SeriesAcaulia.

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Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

We evaluated chloroplast DNA (cpDNA), isozymes, single to low-copy nuclear DNA (RFLPs), and random amplified polymorphic DNAs (RAPDs) in terms of concordance for genetic distance of 15 accessions each of Solanum etuberosum and S. palustre, and 4 accessions of S. fernandezianum. These self-compatible, diploid (2n=24), and morphologically very similar taxa constitute all species in Solanum sect. Etuberosum, a group of non-tuber-bearing species closely related to Solanum sect. Petota (the potato and its wild relatives). Genetic distance and multidimentional scaling results show general concordance of isozymes, RFLPs and RAPDs between all three taxa; cpDNA shows S. etuberosum and S. palustre to be more similar to each other than to S. fernandezianum. Interspecific sampling variance shows a gradation of resolution from allozyme (low) to RAPD to RFLP (high); while intraspecific comparisons graded from RFLPs (low) to RAPDs (high; lack of sufficient allozyme variability within species precluded comparisons for allozymes). Experimental error was low in RFLPs and RAPDs.Names are necessary to report factually and available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

Origin of chloroplast DNA diversity in the Andean potatoes

Summary Wide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.     

A low-copy-number Sorghum DNA sequence that detects hypervariable EcoRV fragments

A sorghum genomic DNA clone that hybridized on Southern blots in simple but different patterns to fragments produced by digestion of DNA from the parents of an F₂ mapping population was hybridized to EcoRV-digested DNA from 53 accessions. Forty-six different fragment patterns were observed, each comprised of from one to ten bands. Much less variability was detected in EcoRI than EcoRV digests of a selected subset of the accessions. Base-sequence analysis of the clone did not reveal a functional identity for the sequence and the clone does not contain repeated sequences often associated with hypervariable loci. Clones such as this will be especially useful in evaluating germplasm diversity and in identifying the potential parentage of hybrids.     

Species boundaries and interrelationships of two closely related sympatric diploid wild potato species, Solanum astleyi and S. boliviense, based on RAPDs

 The more than 200 wild and cultivated species relatives of potato (Solanum sect. Petota) present a valuable germplasm base for cultivar improvement. However, species boundaries and interrelationships within sect. Petota are controversial, inhibiting the efficient organization of the many germplasm collections of these species. One controversy involves questions of species boundaries and interrelationships of S. astleyi and S. boliviense. Solanum boliviense is narrowly endemic to two Departments in southern Bolivia, and S. astleyi is known only from one site entirely within the range of this species, where they co-occur. Both species are diploid and morphologically very similar. Artificial hybrids between them are fully fertile, and the species putatively hybridize naturally. These data have been interpreted to designate them as separate species or as S. astleyi an ecotype of S. boliviense. Putative progenitors of S. astleyi are S. boliviense, S. megistacrolobum subsp. megistacrolobum, and S. megistacrolobum subsp. toralapanum. We evaluated interrelationships among these species with random amplified polymorphic DNA’s (RAPDs) generated for 2 accessions of S. astleyi and 14 accessions of S. boliviense. These represent the entire geographic range of the former species and nearly the entire range of the latter. We also analyzed 1 accession each of S. acaule subsp. acaule, S. acaule subsp. aemulans, S. albicans, S. berthaultii, S. megistacrolobum subsp. megistacrolobum, S. megistacrolobum subsp. toralapanum, S. raphanifolium, S. sogarandinum, and S. sparsipilum. Phenetic analyses of the RAPD data show S. astleyi and S. boliviense to form two distinct groups and to be more similar to each other than to any of the other species investigated, suggesting that S. astleyi and S. boliviense are sister taxa. The divergence of S. astleyi and S. boliviense relative to other species examined suggests that they are worthy of taxonomic recognition at the subspecies, rather than species level, and we propose the new combination S. boliviense subsp. astleyi. Received: 21 November 1996 / Accepted: 18 April 1997     

Cytokinins antagonize the jasmonates action on the regulation of potato (Solanum tuberosum) tuber formation in vitro

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

An interpretation of genomic balance in seed formation in interspecific hybrids of Solanum

Summary To investigate the mechanisms of seed failure in intraspecific and interspecific crosses of Solanum two diploid, S. commersonii and Group Phureja, and one tetraploid species, S. acaule, species were crossed and the seeds were analyzed for embryo and endosperm development. Many seeds of certain crosses observed seven days after pollinations were found to contain abnormal embryos and degenerating endosperms. In some cases seeds contained an embryo with no endosperm, or an endosperm with no embryo. Other interspecific crosses which were predicted to fail actually produced seeds with normally developed embryos and endosperms. To further characterize the intra- and interspecific embryos and endosperms the nuclear DNA was measured. There are several ways to explain the ploidy levels of embryos and endosperms among the crosses, the occurrence of unreduced gametes in some cases and genomic instability in other cases. The latter resulted in chromosome loss at meiotic and mitotic divisions. Genomic balance in interspecific seeds is critical to both embryo and endosperm development.Scientific Journal Series Article No. 14636 of the Minnesota Experiment Station     

Orchardgrass (<Emphasis Type="Italic">Dactylis glomerata</Emphasis> L.) EST and SSR marker development,annotation, and transferability

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

The origin of the cultivated tetraploid potato based on chloroplast DNA

Summary By using restriction enzyme analysis of chloroplast DNA, a geographical cline from the Andean region to coastal Chile was found for the tetraploid potato (Solanum tuberosum). This supports the Andean origin of Chilean ssp. tuberosum. One of the relic cultivars of the early introduction of potato to Europe had ssp. andigena type chloroplast DNA. Its derivatives were largely lost in the mid-19th century due to the late blight epidemic and were replaced by ssp. tuberosum originally introduced from Chile. Therefore, the present common potato has the same type chloroplast DNA as Chilean ssp. tuberosum.     

Molecular diversity and relationships of North American Elymus trachycaulus and the Eurasian E. caninus species

The morphological similarity of Elymus trachycaulus to the Eurasian E. caninus has often been noted. This has lead to controversial and contradicting taxonomic treatments. Nevertheless, there has been no systematic investigation on molecular genetic similarity between E. trachycaulus and E. caninus. In this study, random amplified polymorphic DNA (RAPD) analysis was used to study the similarity between the two species. RAPD analysis of 38 samples representing E. caninus and E. trachycaulus complex yielded 111 interpretable RAPD bands. The Jaccard’s similarity values for E. caninus ranged from 0.38 between accessions H10345 and H10353 to 0.97 between accessions H8745 and H10096, with an average of 0.67. The Jaccard’s similarity values for E. trachycaulus complex ranged from 0.09 between E. trachycaulus ssp. subsecundus (PI 537321) and E. trachycaulus ssp. violaceus (PI 272612) to 0.78 between accessions PI 315368 and PI 372644, with an average of 0.43. The results from different analyses (NJ and PCA) were similar but not identical. The molecular genetic separation between E. caninus and E. trachycaulus was consistent. The PCA analysis clearly separated all E. caninus accessions from E. trachycaulus and its subspecies. The NJ analysis also showed separation between most accessions of E. caninus and E. trachycaulus. Further analysis excluding E. trachycaulus ssp. subsecundus and ssp. violaceus revealed that E. caninus species and E. trachycaulus species were clearly separated into two distinct groups. The RAPD data thus support the treatment of E. caninus and E. trachycaulus as distinct species. The analyses further indicate that E. violaceus is nested within E. trachycaulus, and more related to E. trachycaulus complex rather than to E. caninus.