Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Chromosome and nucleotide sequence differentiation in genomes of polyploid Triticum species

Summary The nature of genome change during polyploid evolution was studied by analysing selected species within the tribe Triticeae. The levels of genome changes examined included structural alterations (translocations, inversions), heterochromatinization, and nucleotide sequence change in the rDNA regions. These analyses provided data for evaluating models of genome evolution in polyploids in the genus Triticum, postulated on the basis of chromosome pairing at metaphase I in interspecies hybrids.The significance of structural chromosome alterations with respect to reduced MI chromosome pairing in interspecific hybrids was assayed by determining the incidence of heterozygosity for translocations and paracentric inversions in the A and B genomes of T. timopheevii ssp. araraticum (referred to as T. araraticum) represented by two lines, 1760 and 2541, and T. aestivum cv. Chinese Spring. Line 1760 differed from Chinese Spring by translocations in chromosomes 1A, 3A, 4A, 6A, 7A, 3B, 4B, 7B and possibly 2B. Line 2541 differed from Chinese Spring by translocations in chromosomes 3A, 6A, 6B and possibly 2B. Line 1760 also differed from Chinese Spring by paracentric inversions in arms 1AL and 4AL whereas line 2541 differed by inversions in 1BL and 4AL (not all chromosomes arms were assayed). The incidence of structural changes in the A and B genomes did not coincide with the more extensive differentiation of the B genomes relative to the A genomes as reflected by chromosome pairing studies.To assay changing degrees of heterochromatinization among species of the genus Triticum, all the diploid and polyploid species were C-banded. No general agreement was observed between the amount of heterochromatin and the ability of the respective chromosomes to pair with chromosomes of the ancestral species. Marked changes in the amount of heterochromatin were found to have occurred during the evolution of some of the polyploids.The analysis of the rDNA region provided evidence for rapid fixation of new repeated sequences at two levels, namely, among the 130 bp repeated sequences of the spacer and at the level of the repeated arrays of the 9 kb rDNA units. These occurred both within a given rDNA region and between rDNA regions on nonhomologous chromosomes. The levels of change in the rDNA regions provided good precedent for expecting extensive nucleotide sequence changes associated with differentiation of Triticum genomes and these processes are argued to be the principal cause of genome differentiation as revealed by chromosome pairing studies.     

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.     

Ribosomal DNA spacer-length variation inSecale spp. (Poaceae)

Variation in ribosomal DNA spacer length was analysed in 23 populations of 12Secale spp. by restriction enzyme analysis. Digestion of rDNA with Taq I endonuclease enzyme yields spacer fragments that include the subrepeat array and the non-repetitive region downstream of the array. Extensive spacer length variation existed in most species with Taq I fragment lengths ranging from 0.9–3.1 kb. These length variants have been attributed to the differences in number of 134 bp spacer subrepeats within rDNA arrays.S. silvestre was the only species to exhibit a unique spacer length variant of 0.9 kb and this was shown to result from the presence of an extra Taq I site in the spacer. rDNA spacer length frequencies were determined for the species. These frequencies were used to derive phenetic relationships between the species by numerical taxonomic methods. In plots constructed fromGower's distance matrices,S. silvestre appeared well separated from the major cluster consisting of the other species. On the basis of morphological and cytogenetic criteria,S. silvestre is considered the most ancient species. The rDNA data is consistent with this interpretation as it shows a clear differentiation ofS. silvestre from all the other species based on length and nucleotide sequence composition of the spacer region.     

Intraspecific and interspecific variation in the second internal transcribed spacer sequence for Metastrongylus (Nematoda: Metastrongyloidea) detected by high resolution PCR-linked restriction fragment length polymorphism

Metastrongylus species are important parasites of free-range pigs and wild boar, but little is known about the genetic make-up of natural populations. This study was undertaken to examine sequence variation in internal transcribed spacer 2 of ribosomal DNA within and among three species of Metastrongylus using PCR-linked restriction fragment length polymorphism analysis. In contrast to many other species of bursate nematodes, significant intraspecific variation was detected in restriction fragment length polymorphism profiles among individual worms. In spite of this, it was possible to identify the three species by their distinctive restriction profiles. The findings suggest that the internal transcribed spacer 2 region should be useful for analysing population variation within Metastrongylus species.     

Relative rates of divergence of spacer and gene sequences within the rDNA region of species in the Triticeae: Implications for the maintenance of homogeneity of a repeated gene family

Summary The relative rates of divergence of 11 regions of the wheat rDNA cloned in pTA250 were estimated by measuring sequence change in 6 Triticum species. The Tm analysis of ³²P probes synthesized from the pTA250 regions and hybridized to DNA from the Triticum species provided an estimate of sequence change relative to T. aestivum. The results revealed a region of 1.2 kb preceding the 18S rRNA gene which was more conserved than the rest of the spacer. In addition the transcribed spacer between the 18S and 26S rRNA genes was shown to be poorly-conserved; the genes, as expected, were highly conserved. A model which proposes RNA as a co-factor in gene conversion is suggested to account for the observations.     

Photosynthetic performance in wild emmer wheat,Triticum dicoccoides: ecological and genetic predictability

Summary In a twin study, we have shown that wild emmer wheat, Triticum dicoccoides, the progenitor of all cultivated wheats, harbours important genetic variation (Vg) in photosynthetic characteristics. This Vg resides within and between populations and ecogeographical regions in Israel, which is the center of origin and diversity of wild emmer wheat. Here we analyzed, by univariate and multivariate methods, the significant differentiation of variation in photosynthetic characteristics of 107 genotypes from 27 populations of wild emmer in Israel, distributed in three ecogeographical regions including central, xeric (northern cold and eastern warm) marginal, and mesic (western) marginal populations. The highest photosynthetic efficiency was displayed by populations of the xeric marginal region, but most variation for photosynthetic capacity occurs between accessions within ecogeographical regions and populations. Genotypes and populations of T. dicoccoides having high photosynthetic capacity can be identified by climatic factors and isozyme markers. The identification by genetic markers, if substantiated by testcrosses, can facilitate the maximization of conservation, in situ or ex situ, and utilization of these photosynthetic genetic resources for improvement of hexaploid wheat (T. aestivum).     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Repeated DNA sequences inTriticum (Poaceae): Chromosomal mapping and its bearing on the evolution of B and G genomes

The somatic chromosomes ofTriticum turgicum var.durum cv. Langdon andT. dicoccoides (AABB tetraploids),T. timopheevii, andT. araraticum (AAGG tetraploids) were assayed for distribution patterns of a highly repeated 120bp DNA sequence by in situ hybridization. The repeated sequence appears to be an ancient sequence shared withSecale andAegilops. The distribution patterns of the chromosomes were compared to the patterns of the A and B genome chromosomes ofT. aestivum cv. Chinese Spring (AABBDD hexaploid).T. turgidum andT. dicoccoides were observed to have identical in situ hybridization patterns. In both species, nine chromosomes with a total of 21 sites of hybridization were observed. The pattern, with few exceptions, was identical to that of Chinese Spring.T. araraticum andT. timopheevii were observed to have different patterns. InT. araraticum, six chromosomes with 21 total hybridization sites are present while inT. timopheevii nine chromosomes with 19 total sites exist. Major differences in hybridization patterns were observed between the B and G genomes. The divergence of the tetraploid wheats in this study appears to have resulted in changes in location, not in amount, of the ancient repeated sequence.     

Heterogeneity of the internal transcribed spacer 1 (ITS1) inTulipa (Liliaceae)

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana Christmas Marvel to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.     

Wheat phylogeny determined by RFLP analysis of nuclear DNA. 2. Wild tetraploid wheats

Intra- and inter-specific variations in the nuclear DNA of Triticum dicoccoides Körn. (2n = 28, genome constitution AABB) and T. araraticum Jakubz. (2n = 28, AAGG), wild species, respectively, of the Emmer and Timopheevi group, were studied by restriction fragment length polymorphism (RFLP) analysis. Total DNAs of 32 T. dicoccoides and 24 T. araraticum accessions, collected from throughout the distribution areas of these species, were treated with two 6-bp cutters and hybridized with 30 nuclear DNA clones as probes to detect RFLPs. A total of 167 hybrid bands were observed per accession. All the enzyme-probe combinations showed RFLPs between accessions. The average genetic distance between the T. dicoccoides accessions was 0.0135 ± 0.0031 and that between the T. araraticum accessions 0.0036 ± 0.0015, indicative of about a four-fold intraspecific variation in T. dicoccoides as compared to T. araraticum in terms of genetic distance. No significant genetic differentiation was found for the geographical populations of these species, the genetic distance between the two species being 0.0482 ± 0.0022. The interspecific divergence corrected for intraspecific divergence was 0.0395, about three times that for T. dicoccoides and 11 times that for T. araraticum. The results show that in the wild state the Emmer and Timopheevi groups are clearly differentiated and that T. dicoccoides has much greater variation than T. araraticum, suggesting a relatively recent origin for the latter and therefore a diphyletic origin for these species.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 535     

The internal transcribed spacer 1 (ITS-1), a controversial marker for the genetic diversity of Trypanosoma evansi

Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338–342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3–3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8–14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Evolution of restriction sites of ribosomal DNA in natural populations of the field mouse,Apodemus speciosus

An analysis by restriction endonuclease digestion of ribosomal DNA (rDNA) was carried out in natural populations of Apodemus speciosus, a field mouse that is endemic to Japan. Two restriction sites, the EcoRI (E3) and DraI (D4) sites, in the nontranscribed spacer region downstream from the gene for 28S RNA showed polymorphism within and between individuals in the populations from the Japanese main islands. By contrast, populations from the small adjoining islands which are thought to have separated from the main islands 1–2 × 10⁴ years ago showed relatively low levels of polymorphism within and between individuals, i.e., one of the polymorphic bands in the case of each enzyme was predominant in these populations, irrespective of the variants. These results indicate that the rate of fixation of site variations depends on population size and that the direction of fixation is random. Furthermore, each polymorphic restriction site seems to be fixed independently.Correspondence to: H. Suzuki     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

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.