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     

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

Structure and variability of nuclear ribosomal genes in the genus Helianthus

Summary The restriction map of the rDNA unit of Helianthus annuus was constructed using EcoRI, BamHI, HindIII, KpnI and SacI restriction enzymes. Variations in this map among 61 ecotypes representing 39 species of the genus Helianthus were analyzed. The sizes of the rDNA unit ranged from 9.8 to 11.0 kbp, due to a length-repeat heterogeneity of the external non-transcribed spacer by increments of 200 base pair segments. Lengthrepeat heterogeneity and restriction polymorphism were found to be characteristic of populations or species of Helianthus. Restriction patterns and thermal melting with probes of a cloned H. annuus ENTS segment allowed us to differentiate species from each other. However, most lines of the cultivated sunflower were found to be identical on the basis of the physical properties of their ribosomal DNA.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum

Summary We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.     

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.     

Difference Between Xanthomonas campestris pv. phlei and X.c pv. graminis Revealed by Genomic Fingerprinting and Restriction Fragment Length Polymorphism Patterns

Genomic DNA was prepared from 16 strains of Xanthomonas campestris pv. graminis and Xanthomonas campestris pv. phlei isolated from six species of forage grasses in four countries. The two pathovars could be distinguished clearly by genomic fingerprints generated by EcoRI, BamHI or HindIII digestion. DNA profiles produced by HindIII digestion could differentiate not only between the two pathoars but also among strains within the same pahtovar from different countries. A 1.6 kb EcoRI fragment was cloned from genomic DNA of strain LMG726 and used to detect restriction fragment-length polymorphism among the same strains. EcoRI and BamHI polymorphisms were seen between the two pathovars probed with this 1.6 kb EcoRI fragment (p726EI probe). These polymorphisms appeared to be highly conserved and unique for each pathovar, consistent with previous grouping of the strains based on other criteria.     

Mapping genetic factors controlling pollen viability in an interspecific cross in Helianthus sect. Helianthus

Segregation of 48 genetic markers, including one CMS restorer gene, one morphological character gene, six isozymes and 40 RAPD loci, was scored in a backcross progeny of an interspecific hybrid H. argophyllusxH. annuus cv RHA274. A linkage map was generated taking into account segregation distortions for 11 of the 48 loci in the frame of two different models considering locus-pair segregation in the context of either independent selection pressures or non-equilibrated parental classes. The map consists of nine linkage groups and nine isolated markers covering 390 cM. Approximately half of the plants of the BC1 were male fertile as expected for the segregation of one dominant male-fertility restorer gene; however, these displayed a large range of variation for pollen viability. About 80% of this variation was explained by three genomic regions located on linkage groups 1, 2 and 3. The observation of meiotic chromosomes revealed a significant rate of mispairing (rod bivalents and tetravalents) in tight correlation with pollen viability, indicating that chromosome rearrangements (translocations) are the preponderant factors reducing pollen viability in this progeny. Cytogenetic and mapping data suggest that the three genomic regions involved in pollen-viability variation are located close to translocation points which differentiate the parental-species karyotypes. Segregation distortion was observed for loci correlated with pollen-viability variation. These were most likely the result of two possible suggested mechanisms.     

Kuraishia MolischianaSp. Nov., the Teleomorph of Candida Molischiana

Thirty-two strains, many of them isolated from wood-associated habitats, and designated as Kuraishia (Pichia) capsulata and Candida molischiana according to their phenotype, exhibited two types of HaeIII restriction fragment patterns of their small subunit rDNA with the neighboring ITS. One fragment pattern corresponded to that of the type strain of K. capsulata, whereas the other pattern was unique to the typestrain of C. molischiana. Sequencing of the D1/D2 domain of the large subunit rDNA confirmed that the different HaeIII restriction fragment patterns of small subunit rDNA with the neighboring ITS reliably distinguished K. capsulata from C. molischiana. Ascospore formation was observed in several C. molischiana strains and K. molischiana (type strain: NCAIM Y.01725, CBS 9993) is proposed as the teleomorphic state of Candida molischiana.     

Indication for deletion of two introns in theoxi-3 gene of a respiratory-competentSaccharomyces cerevisiae strain

Physical mapping of the mitochondrial DNA of the wild-typeSaccharomyces cerevisiae strainRXII revealed that most of the restriction sites as well as the location of the apocytochromeb gene were identical in comparison with the known maps of the mitochondrial genome in otherSaccharomyces cerevisiae strains. In the middle of theSalI linearized map of theRXII mitochondrial DNA, a deletion was detected which resulted in the loss of twoEcoRI and oneBamHI restriction sites. The corresponding region, however, exists in most other laboratory strains ofSaccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that theBamHI site was located close to the aI3-A4 intron-exon junction and the distalEcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strainRXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.     

Structural organization of the nuclear ribosomal RNA genes in Cannabis and Humulus (Cannabaceae)

The structural organization of the nuclear ribosomal DNA (rDNA) of Humulus lupulus, H. japonicus and Cannabis sativa was determined by restriction site mapping. A high degree of DNA sequence similarity was evident in the coding regions of the rDNA repeats of the taxa and supports the placement of Cannabis and Humulus in one family, Cannabaceae. However, the presence of a BstEII site, an additional SacI site, absence of the SpeI site and positional differences of the SspI sites in the 25 S gene distinguished H. japonicus from H. lupulus. Humulus lupulus has an additional EcoRV site in the IGS region. A XhoI site in the 18S region of C. sativa distinguishes it from the two hop species. The diagnostic differences in the IGS of C. sativa include the EcoRI, HindIII and XhoI sites. These sites were not detected in the IGS of the two hop species.     

Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco,Calocedrus decurrens (Torr.), and Pinus taeda L.

Summary The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis.     

Identification of an RFLP marker tightly linked to theHt1 gene in maize

Summary We have identified tight linkage of an RFLP marker to theHt1 gene of maize that confers resistance to the fungal pathogenHelminthosporium turcicum race 1. This was accomplished by the use of four pairs of near isogenic lines (NILs; B73, A619, W153R, and CM105), each differing by the presence or the absence of the geneHt1. SinceHt1 maps to chromosome 2, 26 clones already mapped to this chromosome were labeled and probed against Southern blots of these NILs DNA digested with three restriction enzymes:EcoRI,BamHI, andHindIII. Six markers exhibited an RFLP for at least one pair of NILs. Presumptive linkage was further tested by analyzing the segregation of five of the six markers (one was monomorphic in the cross studied) and resistance toH. turcicum race 1 on 95 F₂ individuals from the cross DF20 × LH146Ht. The results indicate a tight linkage between one of the DNA markers,UMC150B, and theHt1 gene.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

Ribosomal DNA variations in Erianthus, a wild sugarcane relative (Andropogoneae-Saccharinae)

Variation at the 18S+26S and 5S ribosomal DNA loci was assessed on 62 Erianthus Michx. clones, representing 11 species, and 15 clones from two Saccharum L. species used as a reference. Genus-specific markers for Erianthus Michx. sect. Ripidium Henrard (Old World species) were identified. Ribosomal DNA units in Erianthus sect. Ripidium exhibited an additional BamHI site compared to Saccharum, and 5S units showed length and restriction-site differences between Erianthus and Saccharum. These markers will be useful to follow introgression in Saccharum x Erianthus hybrids. Six ribosomal units (for 18+26S genes) were revealed in Erianthus sect. Ripidium, differing by restriction-site positions and/or length. These results provided new information on species relationships and evolution within the genus Erianthus. The Indonesian and Indian forms of E. arundinaceus (Retz.) Jeswiet gave different restriction patterns, which were similar to those of E. bengalense (Retz.) R. C. Bharadwaja and E. procerus (Roxb.) Raizade, respectively. The two 2n=20 species, E. ele-phantinus Hook.f. and E. ravennae (L.) P. Beauv., could also be differentiated at this locus. Two of the New World Erianthus species studied, E. rufipilus (Steud.) Griseb. and E. longisetosus Andersson, appeared more like Erianthus sect. Ripidium, whereas E. trinii Hack, and E. brevibardis Michx. showed patterns consistent with Miscanthus sinensis Andersson and S. spontaneum L., respectively. Finally, the comparison of rDNA restriction maps among Erianthus sect. Ripidium, Saccharum, sorghum and maize, led to unexpected conclusions concerning the relationships between the different genera and the position of Erianthus in the Saccharum complex.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Restriction site patterns in the ribosomal DNA of Camelidae

The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI, BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group.     

The Organization of rRNA Genes in Vicia faba

Vicia faba DNA was digested with restriction endonucleases andfractionated on 1% agarose gels. The physical map for EcoRI,BamHI and XbaI cleavage sites in V. faba cytosol rDNA was determinedusing the Southern blot hybridization technique. XbaI and BglIIdigestion or partial EcoRI digestion showed that the lengthof a major repeat unit of V. faba rDNA is 6.7 x 10⁶ daltons.EcoRI and BamHI cleaved this unit into two and five DNA fragments,respectively. (Received April 23, 1981; Accepted July 20, 1981)     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.