Characterization of two types of yeast ribosomal DNA genes.

The intragenic organization of ribosomal DNA from a diploid strain of Saccharomyces cerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 X 10(6) daltons), A (1.77 X 10(6) daltons), B (1.48 X 10(6) daltons), C (1.22 X 10(6) daltons), D (0.39 X 10(6) daltons), E (0.36 X 10(6) daltons), F (0.22 X 10(6) daltons), and G (0.17 X 10(6) daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: (formula: see text) in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered.     

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     

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S-23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five different restriction enzymes, namely CfoI, HaeIII, RsaI, MspI and TaqI. Restriction digestion of the PCR product using CfoI enabled differentiation between 9 of the 13 mycobacterial species, whereas the remaining four enzymes differentiated between 7 of these 13 species. None of the five enzymes distinguished between different isolates of Mycobacterium tuberculosis or between species within the M. tuberculosis complex i.e., M. tuberculosis, M. bovis, M. bovis BCG and M. africanum. Although ARDRA analysis of the 16S-23S rDNA does not seem to have a potential for intraspecies differentiation, it has proven to be a rapid and technically relatively simple method to recognise strains belonging to the M. tuberculosis complex as well as to identify mycobacterial species outside this complex.     

Nuclear ribosomal DNA monophyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive

In two very closely related but reproductively isolated mite species, Tetranychus urticae and T. turkestani, we found nucleotide diversity to be extensive for mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI) (3-4%) but extremely reduced for nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS2) (less than 0.5%). By contrast, ITS2 was shown to evolve much faster than COI between species of this genus. Furthermore, we found that these two species are polyphyletic for mtDNA but monophyletic for rDNA. Thus it appears that despite its biparental transmission and multiplicity of copies in the genome, nuclear rDNA has a smaller effective population size than mtDNA in these species. The conjunction of efficient concerted evolution and/or gene conversion in the rDNA cluster, the haplodiploidy of these species and their female-biased sex ratio could account for this apparent contradiction.     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.     

The 5S DNA units ofAcacia species (Mimosaceae)

The DNA sequence structure of 5S DNA units inAcacia species, including representatives from the three subgenera ofAcacia, have been determined. The data was interpreted to suggest that at least three lineages of 5S DNA sequences exist inAcacia and the proposal was made that the lineages be named5S Dna-1, 5S Dna-2, and5S Dna-3. The5S Dna-1 lineage was represented by units fromA. boliviana andA. bidwilli, the5S Dna-2 lineage by units fromA. melanoxylon, A. pycnantha, A. ulicifolia, A. boliviana, A. bidwillii, andA. albida, and the5S Dna-3 lineage by units fromA. bidwillii, A. boliviana, andA. senegal. Based on this interpretation of the sequence data, the Australian species of subg.Phyllodineae grouped together as a cluster, quite separate from the subgeneraAculeiferum andAcacia. As expected from the analyses of morphological characters, the 5S DNA units fromAcacia albida (syn.Faidherbia albida) were quite separate from the otherAcacia spp.     

A phylogenetic analysis of Prunus and the Amygdaloideae (Rosaceae) using ITS sequences of nuclear ribosomal DNA

The economically important plum or cherry genus (PRUNUS:) and the subfamily Amygdaloideae of the Rosaceae have a controversial taxonomic history due to the lack of a phylogenetic framework. Phylogenetic analysis using the ITS sequences of nuclear ribosomal DNA (nrDNA) was conducted to construct the evolutionary history and evaluate the historical classifications of PRUNUS: and the Amygdaloideae. The analyses suggest two major groups within the Amygdaloideae: (1) PRUNUS: s.l. (sensu lato) and MADDENIA:, and (2) EXOCHORDA:, Oemleria, and PRINSEPIA: The ITS phylogeny supports the recent treatment of including EXOCHORDA: (formerly in the Spiraeoideae) in the Amygdaloideae. MADDENIA: is found to be nested within PRUNUS: s.l. in the parsimony and distance analyses, but basal to PRUNUS: s.l. in the maximum likelihood analysis. Within PRUNUS:, two major groups are recognizable: (1) the AMYGDALUS:-PRUNUS: group, and (2) the CERASUS:-LAUROCERASUS:-PADUS: group. The clades in the ITS phylogeny are not congruent with most subgeneric groups in the widely used classification of PRUNUS: by Rehder. A broadly defined PRUNUS: is supported.     

Physical mapping of ribosomal DNA sites in Festuca arundinacea and related species by in situ hybridization.

The positions of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of diploid, tetraploid, and hexaploid Festuca species by in situ hybridization. The number and position of the rDNA sites in the species were compared. The results confirm some of the earlier phylogenetic studies of these species but suggest that some structural rearrangements have occurred and that sites have been lost during polyploidization. Keywords: Festuca, in situ hybridization, phylogeny, physical mapping, rDNA.     

Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species

The structure and variation of nuclear ribosomal DNA (rDNA) units of Picea abies, (L.) Karst. was studied by restriction mapping and Southern hybridization. Conspicuous length variation was found in the internal transcribed spacer (ITS) region of P. abies, although the length of this region is highly conserved both within and among most of the plant species. Two types of ITS variants (A and B), displaying a size difference of 0.5 kb in the ITS2 region, were present within individuals of P. abies from Sweden, Central Europe and Siberia. A preliminary survey of 14 additional Eurasian and North American species of Picea suggested that length variation in the ITS region is widespread in this genus. Alltogether three length variants (A, B and C) were identified. Within individuals of eight Picea species, two length variants were present within the genome (combinations of A and B variants in P. glehnii, P. maximowiczii, P. omorika, P. polita and P. sitchensis and variants B and C in P. jezoensis, P. likiangensis and P. spinulosa). Within individuals from five species, however only one rDNA variant was present in their genome (variant A in P. aurantiaca, P. engelmannii, P. glauca, P. koraiensis and P. koyamai; variant B in P. bicolor). The ITS length variation will be useful as a molecular marker in evolutionary studies of the Picea species complex, whose phylogeny is controversial. The presence of intraindividual variation in, and shared polymorphism of the, ITS length variants raises questions about the occurrence of interspecific hybridization during the evolutionary history of Picea.     

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae)

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.     

Distinct geographical structure across species units evidenced by chloroplast DNA haplotypes and nuclear ribosomal ITS genotypes of Corylopsis (Hamamelidaceae) in the Japanese islands

The geographical distribution of chloroplast DNA (cpDNA) haplotypes and nuclear ribosomal internal transcribed spacer (nrITS) genotypes of Japanese Corylopsis (Hamamelidaceae), which consists of four species, was investigated. Two hundred and five individuals belonging to four species from 30 populations, covering the entire geographical range, were studied. Based on approximately 1108 bp of the three non-coding regions of cpDNA, nine haplotypes were detected, and each was distinguished from adjacent haplotypes by one substitution. Based on approximately 507-bp nrITS sequences, 47 genotypes were detected, for which three clades were identified in the phylogenetic analysis. There was inconsistency between the cpDNA haplotypes, nrITS genotypes, and classification of Corylopsis taxa, possibly because of incomplete lineage sorting or introgressive hybridization. The distribution of the haplotypes was highly structured geographically, and N _ST (0.893) was significantly greater than G _ST (0.819), implying that the current distribution of Corylopsis species was structured phylogeographically during Quaternary climatic oscillations. The haplotype composition and results of analysis of molecular variance showed that the populations in Hokuriku were highly divergent, suggesting that they are long-term persistent populations arising from refugia during the Quaternary climatic oscillations. Refugial populations in Chugoku and Shikoku may have lost genetic diversity because of a bottleneck resulting from a small population size, followed by post-glacial range expansion. Pre-existing refugia may have been so small that the subsequent range expansion replaced the pre-existing genetic structure.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 501–518.     

Comparison of the nuclear ribosomal units of five Chlamydomonas species

The organization of the nuclear ribosomal units of five different species of Chlamydomonas has been examined by hybridization of their nuclear DNA fragments produced by several restriction endonucleases with a radioactively labelled probe consisting of the two cloned BamHI ribosomal fragments of C. reinhardii. The results indicate that a) the ribosomal units of these five species are structurally related, b) changes in the non transcribed spacer occur in C. eugametos and in C. globosa, c) the rDNA unit of C. intermedia contains either an enlarged internal transcribed spacer or a ribosomal intervening sequence, d) the rDNA units of C. reinhardii and C. callosa are indistinguishable.     

Diversity in the yeast Cryptococcus albidus and related species as revealed by ribosomal DNA sequence analysis

Evidence accumulated from studies based on physiological, biochemical, and molecular characteristics has pointed to the heterogeneity of the ubiquitous anamorphic basidiomycetous yeast species Cryptococcus albidus (Saito) Skinner, with its current varieties and synonyms. The taxonomic status of this species has not been reappraised because different studies, mostly involving limited numbers of strains, have not been integrated. To assess species diversity within the clade containing Cryptococcus albidus and other phylogenetically related Cryptococcus and Filobasidium species, we determined ribosomal DNA (rDNA) sequences of 69 strains from the 5' end of the 26S gene, D1/D2 region, and in some cases, the non-coding ITS2 region. Analysis of the sequence data together with available physiological, biochemical, and molecular characteristics, showed the segregation of C. albidus into at least 12 species, leading to the elevation of former varieties to the rank of species (C. aerius, C. diffluens), the reinstatement of synonyms (C. liquefaciens, C. terricola), and the proposal of new species (C. arrabidensis, C. chernovii, C. cylindricus, C. oeirensis, C. phenolicus, C. saitoi, C. uzbekistanensis, C. wieringae). The overall analyses of the results argue in favour of the use of rDNA sequence data to improve species delineation when integrated with other available physiological and molecular characteristics.     

Characterization of microsatellite loci in two closely related Lissotriton newt species

We have developed eight di- and tetranucleotide Lissotriton microsatellite markers. Eight loci were polymorphic in the palmate newt Lissotriton helveticus and six were polymorphic in the smooth newt L. vulgaris. Polymorphism detected in 33 and 37 individuals per species ranged from 3 to 15 alleles. These markers are suitable for the investigation of population structure, genetic variation and taxonomic identification in the two focal species, and may also be of use in other Lissotriton–Triturus species.     

Characterization of chloroplast DNA microsatellites from Saccharum spp and related species

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.     

Mitochondrial and ribosomal DNA variation among members of the Anopheles quadrimaculatus (Diptera: Culicidae) species complex.

The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.     

Isolation and analysis of the total genomic DNA from the date palm (P. dactylifera L.) and related species

The objective of this study was to adapt a plant DNA preparation procedure for the isolation of biologically active DNA and DNA with a high molecular weight from the date palm and other related palms. Mature leaf tissue extractions of the date palm, Phoenix dactylifera L., the coconut tree, Cocos nucifera, and the Mexican Fan Palm, Washingtonia robusta, were characterized for total genomic DNA yield, purity, integrity, as well as restriction digestion and ligation capabilities. It is demonstrated here that the DNA isolation procedure, modified for use with various palm leaf tissues, met the criteria for simplicity and low costs, and yielded DNA of high molecular weight (～50 Kbp) and of sufficient purity suitable for molecular studies.     

Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.