Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

Genetic variability in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) and in the <Emphasis Type="Italic">Helianthus</Emphasis> genus as assessed by retrotransposon-based molecular markers

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon’s and Jaccard’s indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Characterization of the nuclear ribosomal DNA units and phylogeny of Beta L. wild forms and cultivated beets

Summary The nuclear rDNA units of species belonging to the genus Beta were characterized using heterologous probes of flax (entire unit and 25S) and sunflower (6.1-kb Eco fragment containing the 18S, the entire intergenic spacer (IGS) and a small piece of the 25S). The physical maps of one species from each section of the genus was constructed by localization of the EcoRI, BamHI, HindIII, KpnI and SacI restriction sites. For each species a single individual was used to obtain total DNA. The major unit length is 11 kb, but variant length units at 10.4, 10.7 and 11.3 kb were found as minor forms. However, some individuals carried the 10.4-kb or the 10.7-kb variant length unit as the major form. For the variant length units of one species the restriction sites were conserved, so that the variation in length occurred in the IGS. The EcoRI fragment corresponding to the intergenic spacer appeared to be the best indicator of variation. The variable sequence in the IGS sometimes generated new restriction sites for the Corollinae and mainly, did so, for the Vulgares relative to the Procumbentes. The variable sites were able, to differentiate the three sections and species within the sections. Corollinae species belong to two different groups according to the absence or the presence of the BamHI (B4) site. The Vulgares species contain several unit types. We proposed that all the unit types derived from a unique unit, V-11-2.3, by unequal crossing-overs or conversion. We also supposed a homogenization mechanism because we found individuals homogeneous for every unit type. Among the cultivated beets, all the root beets contain only one rDNA unit type, V-11-2.9. Thus, we supposed that the common unit type of cultivated beets either brings a physiological advantage or is strictly linked to a favorable allele. It is likely that the rDNA unit of B. maritima were eliminated from sugar beet by the breeding process since they were not recovered. Whatever the process, we deduced that all the cultivated forms of beets likely originated in a unique plant ascendant.A phylogenic tree of the genus is proposed, based on the nuclear rDNA maps, and subsequently discussed relative to the systematic tree and other molecular phylogenies.This work was supported by grant No. 9157A between INRA and the companies Deleplanque et Cie, SES France, Maribo France, Graines Franco Suédoises, KWS France and Van der Have France     

Characterization of diploid,tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA₃ (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x=17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Phylogenetic analysis of Helianthus (Asteraceae) based on chloroplast DNA restriction site data

 Chloroplast DNA (cpDNA) restriction site data were used to analyze phylogenetic relationships within Helianthus. Data from new samples were consistent with previous results and showed the genus to be a well-defined (11 site changes) lineage within subtribe Helianthinae. The affinities of 2 species, Viguiera similis and V. phenax (V. ludens) that have sometimes been included in Helianthus were shown to lie outside the genus. The two species of Phoebanthus, however, formed a phylogenetic ingroup to the perennial Helianthus. Within the genus, cpDNA data indicated that there are four distinct phylogenetic lineages. Two of the lineages each contained a single, annual species (H. agrestis and H. porteri, respectively). The remaining annual species collectively formed a third lineage (sect. Helianthus). The fourth lineage contained all of the perennial species, including those of Phoebanthus. Within the perennial lineage, there was little variation in restriction site characters. The 3 species of series Pumili formed a paraphyletic group at the base of the perennial lineage, and the 3 species of series Ciliares formed a group that was supported by a single restriction site character. Received: 2 September 1996 / Accepted: 25 October 1996     

The wheat ribosomal DNA spacer region: Its structure and variation in populations and among species

Summary The wheat rDNA clone pTA250 was examined in detail to provide a restriction enzyme map and the nucleotide sequence of two of the eleven, 130 bp repeating units found within the spacer region. The 130 bp units showed some sequence heterogeneity. The sequence difference between the two 130 bp units analysed (130.6 and 130.8) was at 7 positions and could be detected as a 4 °C shift in Tm when heterologous and homologous hybrids were compared. This corresponded to a 1.2% change in nucleotide sequence per Tm of 1 °C. The sensitivity of the Tm analysis using cloned sequences facilitated the analysis of small sequence variations in the spacer region of different Triticum aestivum cultivars and natural populations of T. turgidum ssp. dicoccoides (referred to as T. dicoccoides). In addition spacer length variation was assayed by restriction enzyme digestion and hybridization with spacer sequence probes.Extensive polymorphism was observed for the spacer region in various cultivars of T. aestivum, although within each cultivar the rDNA clusters were homogeneous and could be assigned to particular chromosomes. Within natural populations of T. dicoccoides polymorphism was also observed but, once again, within any one individual the rDNA clusters appeared to be homogeneous. The polymorphism, at the sequence level (assayed by Tm analysis), was not so great as to prevent the use of spacer sequence variation as a probe for evolutionary relationships. The length variation as assayed by restriction enzyme digestion did not appear to be as useful in this regard, since its range of variation was extensive even within populations of a species.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae

Summary The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a T_m of 80.6°C and the second phase has a T_m of 87.3°C, corresponding to GC contents of 28 and 44%, respectively. The T_m of the nonribosomal nuclear DNA, called DNA, is 85.7°C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6×10⁶ dalton rDNA SmaI restriction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions.A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.     

Variability in giant fennel (Ferula communis,Umbelliferae): Ribosomal RNA nuclear genes

The rDNA of five accessions of the giant fennel (Ferula communis, Umbelliferae) was analyzed. The restriction map of Bam H 1, Eco R 1 and Hind III sites was established for one of them. Variation between the five accessions was observed at several levels. Three have a homogeneous repeat size, whereas the two others are heterogeneous, one presenting an additional site heterogeneity. However, the general pattern of organization is very similar and there is much greater similarity between theFerula accessions than with the pattern observed for carrot, a plant from the same family. Variation was also observed in the copy number of the rDNA repeats, which ranges from 900 to 3 500. The results demonstrate that the five accessions can be clearly differentiated by molecular analysis of their DNA although they belong to the same species or subspecies. They also demonstrate that different isolated populations of a species evolve independently, thus shedding light on the molecular mechanisms of speciation.     

Cloning of ribosomal RNA genes from an Indian isolate ofGiardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation ofGiardia from other enteric pathogens

The ribosomal RNA genes from an Indian isolate ofGiardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning ofGiardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed severalSmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies ofGiardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis ofGiardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.     

rDNA cloning and rapid hybrid identification inPopulus spp. (Salicaceae)

Ribosomal DNA genes fromP. deltoides have been cloned and specific sequences of the 25 S and 18 S rDNA region, labelled by digoxigenin, have been used to determine the rDNA structure ofPopulus tremula, P. fremontii, P. maximowiczii, P. yunnanensis, P. nigra, P. wislizenii, P. alba. The restriction maps of the coding region appeared to be similar among the examined species and with those ofP. deltoides andP. trichocarpa, reported in a previous paper. Inter- and intraspecific variation in rDNA repeat unit length have been revealed after EcoRI digestions. SstI and XbaI restriction sites have been found at different positions in the IGS of some species. The polymorphic fragments generated by SstI digestion allowed the identification of the hybrid origin of some genotypes. The number of rDNA genes in the genome ofP. deltoides has been estimated to be about 2 000 copies. Finally, the usefulness of these studies inPopulus spp. taxonomy and forestry genetics is discussed.Ribosomal RNA gene structure in somePopulus spp. (Salicaceae) and their hybrids 2.     

Low genetic variation in Swedish populations of the rare speciesVicia pisiformis (Fabaceae) revealed with rflp (rDNA) and RAPD

Nine Swedish populations, 1–5 individuals/population, and one cultivated individual of the rare speciesVicia pisiformis were investigated for genetic variation. In hybridizations with two rDNA probes using 8 restriction enzymes, only two individuals belonging to one population were polymorphic. A map of the rDNA gene cluster was constructed for four of the restriction enzymes used. Two of the polymorphic sites were mapped and were found to be located outside regions coding for rRNA, presumably caused by single point mutations or small deletions. The repeat length of the rDNA region was c. 10,000 bp, which corresponds well with the size found for other species belonging toFabaceae. No length polymorphism was found in the intergenic spacer, contrary to the situation found for most other plant species investigated for rDNA variation. The haplotype diversity for the species (Hsp Shannon) was very low (0.055). Within-population values (Hpop) was 0 for all populations except the variable one, which had 0.301. PCR amplification with 6 random primers also revealed very low levels of genetic diversity. A polymorphism was observed in a limited number of individuals for four populations. Hsp was 0.065 and was 0.050. The average D value (Wetton) for the PCR haplotypes was 0.99.