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.     

Two different satellite DNAs in Beta vulgaris L.: evolution,quantification and distribution in the genus

Summary Two highly repeated EcoRI (0.45 × 10⁶) and BamHI (0.17 × 10⁶) fragments per haploid genome were found in sugar beet genomic DNA. Both fragments were located by 6% acrylamide-gel electrophoresis, purified and cloned in pUC18. Four of the inserts corresponding to each family were chosen for further study. Both fragment families display the main characteristics of the satellite DNA of animals and plants. The EcoRI and BamHI fragment families are arranged in long tandem arrays. Fragments of the EcoRI family (pBVE) were analyzed. They vary both in sequence and in length (158–160 nt) in comparison with the consensus sequence of 159nt. Both families are A-T rich; pBVE is 59% rich while pBVB is 69% rich. The BVESAT family is present in all the members of the section Vulgares. It is conserved in the section Procumbentes with 80% homology and the same length, but is not detectable in the Corollinae. The sequence variation rate and the variation in length (330±5 nt) are of the same order in comparison with those of the BVESAT family. However, the BVBSAT family is present in species of the section Vulgares only. As regards other plant satellite DNAs, the BVESAT family shares homology with Allium cepa satellite DNA, with three of the yeast centromeric sequences, and with three Arabidopsis thaliana sequences. The BVBSAT family is unique to the Vulgares and does not share any homology with other plant or animal satellite DNAs sequences so far.     

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.     

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.     

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.     

Molecular systematics of Brassica and allied genera in subtribes Brassicinae, Raphaninae, Moricandiinae, and Cakilinae (Brassicaceae, tribe Brassiceae); the organization and evolution of ribosomal gene families

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of ribosomal DNA (rDNA) of Brassica and allied genera. The total genomic DNA of 95 accessions of 52 species representing 16 genera was restricted with six enzymes, and the restriction fragments were probed with three ribosomal clones (pTA71, Ver 18‐6, and Ver 6‐5). Eleven repeat unit length classes were recognized. The repeat unit size classes of 8.9 kb and 9.5 kb were observed most commonly, being represented in 17 and 14 species, respectively. The restriction enzyme SacI produced three to six (generally three) bands with detectable hybridization to the probe pTA71. This probe–enzyme combination indicated a remarkable uniformity amongst Brassica and allied genera in the coding region of repeat units. By contrast, an extensive size variation in the restriction fragments could be localized in the intergenic spacer (IGS) region. Eleven IGS‐containing length variants were detected. Complex hybridization patterns, resulting from extensive repeat unit heterogeneity and taxon‐specific methylation of one or more cleavage sites, were obtained with the EcoRI + pTA71 combination. The relative homologies between the coding regions were evident from the presence of 1.5 kb in all the taxa, and 0.4‐, 1.3‐, and 1.7‐kb fragments in 33, 27, and 24 species, respectively. The SacI + pTA71 and EcoRI + pTA71 combinations were generally able to distinguish taxa both within and between genera. Three restriction endonuclease digests probed with three ribosomal clones yielded essentially identical fragmentation patterns across all the accessions within the cultivated species Brassica campestris, B. oleracea, and B. juncea. In B. napus, three and seven accessions exhibited restriction profiles similar to one and both diploid progenitor species, respectively. Overall, rDNA repeat unit length polymorphism showed good correlation with the cytodeme‐based classification of Brassica and allied genera. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 545–557.     

Mitochondrial gene variation and phylogenetic relationships in the genus Beta

Restriction fragment length polymorphisms (RFLPs) for three mitochondrial genes, coxI, coxII and atpA, were used to determine mitochondrial (mt) DNA diversity in 21 accessions of the genus Beta representing wild and cultivated species. On the basis of distribution of the RFLP patterns these Beta genotypes were assigned into six distinct chondriome groups. A high degree of heterogeneity was found to exist between the mitochondrial genomes of the sugarbeet cultivar and the wild species of Procumbentes section. The polymorphic fragments from wild Beta species were cloned and subjected to fine mapping. We found that most of the RFLPs are due to sequence rearrangements rather than point mutations. Our data also suggest that the close linkage between coxII and coxI is taxonomically localized to an evolutionary lineage that led to Vulgares and Corollinae species but not to Procumbentes species. This linkage is most likely to have arisen via the mutation(s) that inserted the DNA segment containing coxI downstream of coxII in the common ancestor of Vulgares and Corollinae species. The results are discussed with regard to the taxonomic and phylogenetic relationships of the Beta species.     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical–ecological–climatic regions of India are nodulated by Bradyrhizobium yuanmingense

Vigna mungo, Vigna radiata and Vigna unguiculata are important legume crops cultivated in India, but little is known about the genetic resources in native rhizobia that nodulate these species. To identify these bacteria, a core collection of 76 slow-growing isolates was built from root nodules of V. mungo, V. radiata and V. unguiculata plants grown at different sites within three agro-ecological-climatic regions of India. The genetic diversity of the bacterial collection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA fragments of the 16S–23S rDNA intergenic spacer (IGS) region, and the symbiotic genes nifH and nodC. One rDNA IGS type grouped 91% of isolates, but more diversity was found at the symbiotic loci (17 symbiotic genotypes). Overall, no host plant specificity was shown, the three host plant species sharing common bradyrhizobial genotypes that represented 62% of the collection. Similarly, the predominant genotypes were found at most sampling sites and in all agro-ecological-climatic regions. Phylogenies inferred from IGS sequencing and multi-locus sequence analysis of the dnaK, glnII and recA genes indicated that all isolates but one were clustered with the Bradyrhizobium yuanmingense species. The nifH phylogeny also grouped the different nif haplotypes within a cluster including B. yuanmingense, except for one infrequent nif haplotype which formed a new lineage within the Bradyrhizobium genus. These results may reflect a long history of co-evolution between B. yuanmingense and Vigna spp. in India, while intra-species polymorphism detected in the symbiotic loci may be linked with the long history of diversification of B. yuanmingense coinciding with that of its host legumes.     

PCR-RFLP analysis of the IGS region of rDNA: a useful tool for the practical discrimination between species of the genus Debaryomyces

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for␣distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.     

Ribosomal DNA variation and its phylogenetic implication in the genusBrachypodium (Poaceae)

The structure of ribosomal DNA ofBrachypodium and several other grass species was investigated using a heterologous rDNA probe from wheat. Several different rDNA families were present among perennial and annual species within the genus. In contrast to the annual species the perennial species exhibited a very low degree of repeat length variation. An extra Eco RI site and a Hin dIII site were observed in the IGS, which distinguishedBrachypodium from other grass genera. The restriction fragment length polymorphism and length variation of the repeat units have taxonomic value withinBrachypodium and are correlated with the classification ofBrachypodium derived from other data.     

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.     

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)     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997     

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.     

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

Summary The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.     

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.     

Molecular Cloning and Restriction Analysis of EcoRI-fragments of Vicia faba rDNA

EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325.Southern blot hybridization of BamHI-digests of these clonedplasmids and Vicia genomic DNA led to the determination of relativepositions of BamHl sites in the rDNA and the physical map thathad been tentatively made is corrected. (Received May 20, 1982; Accepted July 13, 1983)