DNA sequence analyses of the ribosomal spacer regions in theTriticeae

Two regions of the ribosomal DNA (rDNA) were sequenced from a range of species from the tribeTriticeae. One region, the central spacer, was found to be more divergent in sequence than the other, the 18 S-spacer junction. Both regions contained sequences 20–30 bp long which were more highly conserved than the remainder of the region and their possible significance in rDNA expression is discussed. Phenetic relationships based on the sequence data were generally consistent with the relationships based on other criteria. Species possessing the S, E, J₁J₂, D, and B genomes clustered together, with the H genome species being the most distinct of those examined. The R, P, and V genome species occupy an intermediate position in the overall pattern of relationships. Some relationships differed in detail from those established by other parameters, for example the position of the N genome species, and explanations for discrepancies of this type are discussed.     

Evolutionary change at the5S Dna loci of species in theTriticeae

Previous studies have shown that two separate loci for 5S DNA sequences may exist within a species. Two size classes have been tentatively assigned in the ranges 327–468 bp (chromosome 1), and 469–500 bp (chromosome 5) and the entire data-base was subjected to various numerical taxonomic analyses. The results confirm the existence of two lineages of 5S DNA sequences represented by the two size classes.Hordeum vulgare is separate from the species known also asCritesion and, together withDasypyrum, occupy an intermediate position between the two size classes. The 5S DNA units fromTriticum spp. that have also been namedAegilops spp. in other classifications appear as a distinct group withinTriticum spp. at both loci. The consensus 5S DNA sequence fromPsathyrostachys is remote fromHordeum s.l. in contrast to opinions based on morphology. Other aspects are detailed and discussed, including the inadequacy of the computing methods used as well as the need for more data in view of the large amount of homoplasy. The merit of using the spacer for phylogenetic inference is also discussed.     

Evolution and systematic relationships in theTriticeae (Poaceae)

The evolution and taxonomic relationships in theTriticeae are discussed with the view to highlight aspects of this agronomically important group of plants, which may be of interest to molecular biology. Some of these aspects are addressed in more detail in adjoining papers in which specific genomic loci have been examined at the DNA sequence or isozyme level. Aspects discussed include the systematics and geographic distribution of theTriticeae species, isozyme and chromosome pairing studies on some of the species as well as more recent developments in DNA analyses. A survey of the systematics of theTriticeae indicated that the genomic system ofLöve is probably the most useful starting point for interpreting molecular data even though the system has many problems from a taxonomic point of view. The geographical distribution ofTriticeae species, using both published and unpublished data, suggested that information of this type taken together with the theory of continental drift provides a broad time-span for considering data from DNA sequence studies. The significance, and modes of analyses, of isozyme studies were assessed because they often provide valuable characters in determining relationships between species. The main character underlyingLöve's andDewey's analyses of theTriticeae, namely chromosome pairing, is discussed with particular reference to isozyme studies to show that in some cases, such as species ofHordeum sensu lato, consistent relationships are obtained. Finally, new developments in understanding chromosome structure are considered in relation to the above variables in the taxonomy and evolution of theTriticeae.     

Amplification and dispersion of repeated DNA sequences in theTriticeae

Four representatives of a family of dispersed repetitive sequences which were prominent and dispersed in the E genome ofThinopyrum elongatum but poorly represented in wheat, were studied in detail. The 1.4kb sequences were present both as part of tandem and more complex arrays and appeared to have resulted from repeated amplification of the sequence and their dispersion throughout the genome. Subcloning of sections of the 1.4 kb sequences resulted in probes which improved the resolution of the E genome from the genomes in wheat and enabled identification of single E genome chromosomes introduced into wheat. The generality of these types of sequences in the tribeTriticeae was confirmed by isolating analogous sequences from the R (rye,Secale cereale), V (Dasypyrum villosum), and N (Psathyrostachys juncea) genomes. — The cloned repetitive sequences from the R, V, and N genomes each showed characteristic fluctuations in amount within the grasses examined in addition to being virtually absent from wheat. It is thus possible that these sequences may provide useful taxonomic indicators for establishing relationships within theTriticeae, as well as valuable probes for tracing alien chromatin introduced into wheat.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (llpl.l) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5S rDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.     

The 5S RNA genes inPinus radiata and the spacer region as a probe for relationships betweenPinus species

The 5S RNA genes inPinus radiata occur in two size classes of about 850 and 525 bp in length. Representatives from both the long and short size classes were cloned and sequenced. The primary difference in the two size classes was shown to be a 330 bp insertion in the spacer region of the long units. Within an individual breeding clone ofP. radiata there was some sequence heterogeneity between representatives of the short class. The 5S RNA genes in thirty pine species were characterised using either a clone of the short size class or a subclone of the 330 bp insertion characterizing the long size class. Eleven species of subg.Strobus were examined and all lacked the long type of unit of radiata pine. The New World species of subg.Pinus all had both short and long units whereas the Old World species had long units. The implications of these results for the evolution of the 5S DNA sequences and the phylogenetic relationships withinPinus are discussed.     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Phylogenetic evaluation of 5S ribosomal RNA gene and spacer in theCallistachys group (Fabaceae:Mirbelieae)

Sequences of 5S rDNA from an Australian group of papilionoid legumes were evaluated for phylogenetic informativeness. Twenty four sequences were sampled from ten species in five closely related genera:Brachysema, Callistachys, Jansonia, Nemcia andPodolobium. These sequences fell into two size classes, 200bp and 600bp, which appear to represent paralogous copies of the 5S unit at different loci. As in previous studies, the 5S rRNA gene and both ends of the inter-gene spacer are found to be conserved and almost uninformative about phylogeny at this taxonomic level. By contrast, the middle part of the spacer is highly variable, both in substitutions at individual sites and in length. The short sequences contain virtually no duplications. The long sequences contain numerous short repeats (c.20 bp) which form a regular pattern in the middle section. Phylogenies estimated from these data support monophyly of the study group, and of species. Relationships among species and genera are less clear, perhaps because divergence of the 5S spacer between species is too great. However, non-monophyly ofNemcia andBrachysema, indicated by other data, is corroborated.     

Structure and evolution of 5S rRNA genes and pseudogenes in the genusAspergillus

Summary We have cloned and determined the nucleotide sequence of 18 DNA fragments hybridizing to 5S rRNA from twoAspergillus species-A. wentii andA. awamori. Four of the analyzed sequences were pseudogenes. The gene sequences of these two species were very similar and differed fromAspergillus nidulans at both constant and microheterogeneous sites.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis

Summary The nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis has been determined to be: G- _A ^C -G-U-A-C-G-G-C-C-A-U-A-C-U-A-C-C-G-G-G-A-A-U-A-C-A-C-C-U-G-A-A-C-C-C-G--U-C-G-A-U-U-U-C-A-G-A-A-G-U-U-A-A-G-C-C-G-G-G-U-U-A-G-G-C-C-C-A-G-U-U-A-G-U-A-C-U-G-A-G-U-G-G-G-C-G-A-C-C-A-C-U-U-G-G-G-A-A-C-A-C-U-G-G-G-U-G-C-U-G-U-A-C-G-C-U-Up. This RNA is 119 nucleotides long and the sequence of a probable tRNA-binding site is GAUU (position 41–44 from the 5-terminus), which is the same as that of a trypanosoma species,Crithidia fasci-culata. TheEuglena 5S rRNA has a pseudouridine residue at position 38 and 3-terminus is phosphorylated. The 5S rRNA sequence ofEuglena resembles those of several other protozoa and higher animals rather than plants.On leave from Department of Zoology, Hiroshima University, Hiroshima, Japan     

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.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

Intraspecific 5S rRNA gene variation in flax,Linum usitatissimum (Linaceae)

A relatively large proportion of the flax genome (~ 3%) is comprised of 5S rRNA genes. This study focuses on the intraspecific sequence variation among five distinct groups of 5S rRNA genes. The results indicate that group 1 and 2 5S rRNA genes most closely resemble other angiosperm 5S genes, while groups 3–5 are highly divergent. Sequence variation is higher in the spacer region compared to the transcribed region for all pairwise comparisons. The large degree of sequence variation observed in this study is discussed with respect to genome organization and proposed models for repetitive sequence maintenance.