Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs

Summary Ninety-three accessions representing 21 species from the genus Oryza were examined for restriction fragment length polymorphism. The majority (78%) of the accessions, for which five individuals were tested, were found to be monomorphic. Most of the polymorphic accessions segregated for only one or two probes and appeared to be mixed pure lines. For most of the Oryza species tested, the majority of the genetic variation (83%) was found between accessions from different species with only 17% between accessions within species. Tetraploid species were found to have, on average, nearly 50% more alleles (unique fragments) per individual than diploid species reflecting the allopolyploid nature of their genomes.Classification of Oryza species based on RFLPs matches remarkably well previous classifications based on morphology, hybridization and isozymes. In the current study, four species complexes could be identified corresponding to those proposed by Vaughan (1989): the O. ridleyi complex, the O. meyeriana complex, the O. officinalis complex and the O. sativa complex. Within the O. sativa complex, accessions of O. rufipogon from Asia (including O. nivara) and perennial forms of O. rufipogon from Australia clustered together with accessions of cultivated rice O. sativa. Surprisingly, indica and japonica (the two major subspecies of cultivated rice) showed closer affinity with different accessions of wild O. Rufipogon than to each other, supporting a hypothesis of independent domestication events for these two types of rice. Australian annual wild rice O. meridionalis (previously classified as O. rufipogon) was clearly distinct from all other O. rufipogon accessions supporting its recent reclassification as O. meridionalis (Ng et al. 1981). Using genetic relatedness as a criterion, it was possible to identify the closest living diploid relatives of the currently known tetraploid rice species. Results from these analyses suggest that BBCC tetraploids (O. malampuzhaensis, O. punctata and O. minuta) are either of independent origins or have experienced introgression from sympatric C-genome diploid rice species. CCDD tetraploid species from America (O. latifolia, O. alta and O. grandiglumis) may be of ancient origin since they show a closer affinity to each other than to any known diploid species. Their closest living diploid relatives belong to C genome (O. eichingeri) and E genome (O. Australiensis) species. Comparisons among African, Australian and Asian rice species suggest that Oryza species in Africa and Australia are of polyphyletic origin and probably migrated to these regions at different times in the past.Finally, on a practical note, the majority of probes used in this study detected polymorphism between cultivated rice and its wild relatives. Hence, RFLP markers and maps based on such markers are likely to be very useful in monitoring and aiding introgression of genes from wild rice into modern cultivars.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

Analysis of tetraploid Elymus species using wheat microsatellite markers and RAPD markers.

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.     

Mitochondrial microsatellite variability in common wheat and its ancestral species

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.     

Simple sequence repeat diversity in diploid and tetraploid Coffea species.

Thirty-four fluorescently labeled microsatellite markers were used to assess genetic diversity in a set of 30 Coffea accessions from the CENICAFE germplasm bank in Colombia. The plant material included one sample per accession of seven East African accessions representing five diploid species and 23 wild and cultivated tetraploid accessions of Coffea arabica from Africa, Indonesia, and South America. More allelic diversity was detected among the five diploid species than among the 23 tetraploid genotypes. The diploid species averaged 3.6 alleles/locus and had an average polymorphism information content (PIC) value of 0.6, whereas the wild tetraploids averaged 2.5 alleles/locus and had an average PIC value of 0.3 and the cultivated tetraploids (C. arabica cultivars) averaged 1.9 alleles/locus and had an average PIC value of 0.22. Fifty-five percent of the alleles found in the wild tetraploids were not shared with cultivated C. arabica genotypes, supporting the idea that the wild tetraploid ancestors from Ethiopia could be used productively as a source of novel genetic variation to expand the gene pool of elite C. arabica germplasm.     

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.     

AFLP,RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> Boiss. and <Emphasis Type="Italic">Aegilops variabilis</Emphasis> Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.     

Genetic distance detected with RAPD markers among selected Australian commercial varieties and boron-tolerant exotic germplasm of pea (Pisum sativum L.)

The optimisation of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in pea was investigated and the results were applied to an analysis of five representative Australian varieties and five selected boron-tolerant accessions derived from different geographical regions. Genotypes were compared using 34 random primers (Operon Technologies, Alameda, CA) which generated 180 polymorphic bands. Genetic similarity among genotypes was estimated on the basis of the percentage of common bands between genotypes and a dendrogram was constructed by the unweighted pair grouping method. A pattern of RAPD reaction corresponding to two main groups was discerned. The genetic divergence between Australian varieties and the boron-tolerant accessions suggests an intensive back-crossing programme would be required to transfer boron tolerance to a locally adapted genetic background. Our results show RAPD to be useful for clarifying phylogenic relationships within a species and also to provide useful genetic markers for varietal identification in pea.     

Genome- and species-specific markers and genome relationships of diploid perennial species in Triticeae based on RAPD analyses.

Eight different genomes (E, H, I, P, R, St, W, and Ns) represented by 22 diploid species of the tribe Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique. The genome relationships were obtained based on 371 RAPD fragments produced with 30 primers. The four species of the genus Psathyrostachys (having various Ns genomes) were closely related. The genomes Ee and Eb had a similarly close relationship and were distinct from all other genomes analyzed. Genomes P, R, and St were grouped in one cluster and genomes H and I in another. Genome W had a distant relationship with all other genomes. These results agree with the conclusions from studies of chromosome pairing and isozyme and DNA sequence analyses. Twenty-nine and 11 RAPD fragments are considered to be genome- and species-specific markers, respectively. One to six genome-specific markers were identified for each genome. These RAPD markers are useful in studies of genome evolution, analysis of genome composition, and genome identification.     

Genetic diversity in a world germplasm collection of tall fescue

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs.     

Genetic variation in monoploids of diploid potatoes and detection of clone-specific random amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers have been used to study the genetic variation among androgenetic monoploids of diploid Solanum species. Cluster analysis of pairwise genetic distances was used to construct a genetic relationship among anther donor and anther-derived potato plants. The clustering based on Rogers' distances resembled classifications based on parental origins and hybrid combinations. Six of the 32 RAPD primers used resulted in the selective amplification of DNA fragments which were polymorphic between the two S. phureja parental clones, 1.22 and A95. It should be possible to construct a genetic linkage map, without making crosses, using monoploids derived from a single heterozygous diploid clone and RAPD markers.     

Evolutionary history and speciation modes in the cyprinid genus Barbus.

Phylogenetic relationships and evolutionary patterns in the genus Barbus were examined through the analysis of the complete sequences of three mitochondrial genes: ATPases 8 and 6, which overlap slightly, and cytochrome b. This complex genus includes diploid as well as tetraploid and hexaploid species that are distributed throughout the Palaearctic, Ethiopian and Asiatic biogeographical regions. Given that genome duplication is an important evolutionary mechanism in eukaryotes, in the present report we attempt to describe its role in the evolution of the genus Barbus, as well as drawing systematic and phylogenetic conclusions. The phylogenetic results indicated the splitting of the current Barbus genus into five main mitochondrial lineages corresponding to (i) the genus Barbus sensu stricto (tetraploid, which is subdivided into the subgenera Barbus and Luciobarbus), (ii) the hexaploid species, (iii) the Ethiopian tetraploid species, (iv) the African diploid species, and (v) the Asian diploid species. The branching of 'foreign' genera as sister groups of some of these monophyletic assemblages (such as Aulopyge is to Barbus sensu stricto or Varicorhinus is to the hexaploid barbels) demonstrates the polyphyly of the group. Moreover, the relationships between the proposed lineages also show that genome duplication may be considered as a homoplasic character since it must have occurred over at least three independent periods and/or in three independent areas. In relation to the possible saltational evolutionary model for the polyploid species examined here, it was found that, although feasible at the nuclear level, the mitochondrial markers looked at do not appear to have undergone this type of evolution. Rather, they seem to have experienced more or less constant change through time.     

Genetic diversity and evolutionary relationships ofOryza species with the B- and C-genomes as revealed by SSR markers

Genetic diversity and evolutionary relationships of 72 accessions representing six species with the B-, C-, and BC-genomes in the genusOryza were investigated by seven microsatellite markers. Of four diploid species,Oryza officinalis maintained the highest diversity (P=71.4%, He=0.565), followed by Oryza eichingeri (P=57.1%, He=0.376), Oryzapunctata (P=57.1%, He=0.272) and Oryza rhizomatis (P=42.9%, He=0.222). In comparison, a higher level of genetic diversity was revealed in the tetraploid (P=71.4%, He=0.461-0.637). UPGMA dendrograms based on genetic distance revealed an obvious genetic differentiation between Asian and African races ofO. eichingeri. Three BBCC species clustered with different accessions of the diploidO. punctata, suggestive of their multiple origins. The results inferred from the dendrogram suggested that diploid species,O. officinalis and AfricanO. eichingeri might be the C-genome donors for tetraploid species,Oryza minuta andO. punctata, respectively, while the C-genome ancestor ofOryza malampuzhaensis seemed to be eitherO. rhizomatis or the Sri LankanO. eichingeri species. The genetic relationship among the CC and BBCC species further indicated that the tetraploid species with the BC-genome have originated independently, at least three times in history. In addition, we have demonstrated successful cross-species amplification of seven rice SSR loci acrossOryza species with B-and C-genomes.     

Population genetic diversity in the polyploid complex of wheatgrasses using isoenzyme and RAPD data

Thirty five bands (alleles) from six enzyme systems and fifty seven random amplified polymorphic DNA (RAPD) fragments were selected to analyse the genetic diversity of 33 polyploid wheatgrasses (Triticeae) populations of species Thinopyrum junceiforme and Elytrigia pycnantha, and two hybrids, one pentaploid and one novel 9-ploid. Dice’s similarity coefficient, the UPGMA-derived phenograms from RAPD, and allozymes markers showed that the clustering of wheatgrass populations was based on ploidy level. These markers had similar levels of diversity between populations, with high genetic similarity within the same ploidy-level and within population’s individuals. The tetraploid Th. junceiforme populations are closely related, with a large similarity distances varied from 0.8 to 1. Based on the isozyme and RAPD analyses, diploid taxa are related to polyploids with similarity coefficients 0.4.     

Genetic Variability ofLepidium meyeniiand other AndeanLepidiumSpecies (Brassicaceae) Assessed by Molecular Markers

A phylogenetic survey based on similarity levels was performedfor 29 cultivated accessions of maca (Lepidium meyeniiWalp.)and 27 accessions of wild species ofLepidiumfrom Ecuador, Peruand Bolivia, with RAPD markers. Chromosome counts for each accessionwere also performed. The similarity tree matrix separated intwo main branches: cultivated and wild species. The similaritylevel among cultivated accessions was high (0.952 or higher)indicating a low level of polymorphism. Within the wild species,two main secondary branches could be resolved, of which onewas subdivided into two tertiary branches. Morphological evaluationof the wild species accessions within each main group identifiedthree wild species: (1)L. bipinnatifidum, consisting mostlyof tetraploids and a single octoploid accession; (2)L. kalenbornii,consisting only of tetraploid accessions; and (3)L. chichicara,consisting mostly of octoploid and a few tetraploid accessions.Clustering by principal coordinates analysis supported the resultsobtained by the similarity tree matrix. These results indicatethat none of the three wild species is related enough to beconsidered ancestral to the cultivatedL. meyenii. Three accessionsof intermediate position may be of hybrid origin. None of thewild species was found to be diploid, which suggests that polyploidyhas been an important adaptation to high altitude habitats inthese species.Copyright 1998 Annals of Botany Company Lepidum meyenii, Lepidium peruvianum, maca, DNA markers, phylogeny.