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.     

Sequence comparison of distal and proximal ribosomal DNA arrays in rice (Oryza sativa L.) chromosome 9S and analysis of their flanking regions

Rice (Oryza sativa ssp. japonica cv. Nipponbare) harbors a ribosomal RNA gene (rDNA) cluster in the nucleolar-organizing region at the telomeric end of the short arm of chromosome 9. We isolated and sequenced two genomic clones carrying rice rDNA fragments from this region. The rice rDNA repeat units could be classified into three types based on length, which ranged from 7,928 to 8,934 bp. This variation was due to polymorphism in the number of 254-bp subrepeats in the intergenic spacer (IGS). Polymerase chain reaction (PCR) analysis suggested that the rDNA units in rice vary widely in length and that the copy number of the subrepeats in the IGS ranges from 1 to 12 in the rice genome. PCR and Southern blot analyses showed that most rDNA units have three intact and one truncated copies of the subrepeats in the IGS, and distal (telomere-side) rDNA units have more subrepeats than do proximal (centromere-side) ones. Both genomic clones we studied contained rDNA-flanking DNA sequences of either telomeric repeats (5′-TTTAGGG-3′) or a chromosome-specific region, suggesting that they were derived from the distal or proximal end, respectively, of the rDNA cluster. A similarity search indicated that retrotransposons appeared more frequently in a 500-kb portion of the proximal rDNA-flanking region than in other subtelomeric regions or sequenced regions of the genome. This study reveals the repetitive nature of the telomeric end of the short arm of chromosome 9, which consists of telomeric repeats, an rDNA array, and a retrotransposon-rich chromosomal region.Sequence accession numbers in DDBJ assigned for OSJNOa063K24 and OSJNBb0013K10 are AP009051 and AP008245, respectively.     

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.     

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Methylated and undermethylated rDNA repeats are interspersed at random in two higher plant species

The organization of methylated rDNA repeats of radish and pea is described and it is shown that methylated repeats and non-methylated repeats are interspersed one with another. Methylated arrays are not much longer than 100 kb, or about 10 repeat units in length.     

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.     

Variation of nuclear ribosomal RNA genes in Eragrostis tef (Zucc.) Trotter.

Variation in the ribosomal RNA genes (rDNA) was examined to assess the genetic variability among 314 plants representing 28 accessions of Eragrostis tef, an important food crop. A restriction site map was constructed for the species by localization of the BamHI, BglII, DraI, EcoRI, EcoRV, NdeI, SacI, SpeI, XbaI, and XhoI sites. A comparison of this map with those of other grasses showed conservation of sites, especially in the coding region. However, a unique EcoRI site combined with a BamHI site in the 18S region may be of diagnostic value for the species. A BamHI fragment that spans the intergenic spacer was used as an indicator of length variation of rDNA repeat units. rDNA repeat units in E. tef ranged in size from 8.4 to 11.07 kbp. Considerable size variation of rDNA repeats was present among accessions, between individual plants within some accessions, and within single plants. A total of 19 spacer length (sl) phenotypes was observed in 16 accessions in which 11-42 plants were analyzed. A single restriction site polymorphism was detected in PI442115 that was also distinguished by having a single sl variant. Variation in the rRNA genes is a useful indicator of genetic diversity in E. tef germplasm.     

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.     

Investigations of 5S rDNA of Vitis vinifera L.: sequence analysis and physical mapping.

Here we report the first results of a study of 5S rDNA of Vitis vinifera. 5S rDNA sequences from seven genotypes were amplified by PCR, cloned, and sequenced. Three types of repeats were found. Two variants, denominated long repeat and short repeat, appeared to be the main components of the 5S rDNA of this species, since they were found in all genotypes analyzed. They differed markedly from each other in both the length and the nucleotide composition of the spacers. The third variant, classified as DEL short repeat, differs from the short repeat owing to a large deletion in the spacer region. It appears to be the most recent repeat type, since it was identified in only one genotype. The organization of the 5S rDNA repeat unit variants was investigated by amplifying the genomic DNA with primers designed on the sequence of the long and short spacers. The PCR-amplified fragments showed that the long repeat is associated with the other two repeats, indicating that in V. vinifera different repeat units coexist within the same tandem array. FISH analysis demonstrated that 5S rRNA genes are localized at a single locus. The variability of 5S rDNA repeats is discussed in relation to the putative allopolyploid origin of V. vinifera.     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.     

Relationships among Nicotiana species revealed by the 5S rDNA spacer sequence and fluorescence in situ hybridization

To investigate phylogenetic relationships in Nicotiana, the intergenic spacer sequences of 5S rDNA were analyzed in species with 2n=18, 20 or 24, and amphidiploid species with 2n=48. The chromosomal localization of the 5S rDNA was determined by fluorescence in situ hybridization (FISH). In species with 2n=24 and their descendants, a major 5S rDNA-specific PCR fragment of 400–650 bp was obtained. The amphidiploid species contained similar length of 5S rDNA units derived from putative diploid progenitors. Among the five clones from each representative PCR fragment, some nucleotide exchanges and length heterogeneity were observed. The latter was due to variation in the spacer region, such as differences in the length of poly A and/or poly T tracts as well as insertions/deletions. Interspecific comparisons of each 5S rDNA sequence demonstrated that the spacer sequence could be divided into three regions. Excluding gaps from the aligned spacer sequences of 5S rDNA, phylogenetic trees were constructed. Each phylogenetic tree showed an almost identical topology even if different algorithms were applied. The chromosomal locations of the 5S rDNA in each species correlated with the phylogenetic topology. The phylogenetic trees were generally in agreement with the current classification. Received: 15 January 2001 / Accepted: 15 February 2001     

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed,using the Arabidopsis genome sequence and radish genetic mapping

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H. C. BeckerS. Giancola and S. Marhadour contributed equally to this work     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Sister chromatid exchange and the evolution of rDNA spacer length

The structures of rDNA spacers from several species have been characterized and virtually all have internally repeated sequences. Different numbers of these internal repeats are responsible for most spacer length variation. Because unequal recombination between these internal repeats will cause new length variation, while unequal exchange between rDNA copies will homogenize the variants, we modeled the interaction of these two processes. Two models were used to simulate both types of unequal exchange at the sister chromatid level. Both models indicate that a narrow range of relative recombination frequencies is required to produce levels of variability comparable to those published. One model puts a lower limit on the number of internal repeats, and the other puts both a lower and upper limit on the number of repeats. The model with both maximum and minimum constraints produces a distribution closer to actual spacer distributions. These results imply that small changes in recombination rates can generate the differences in numbers of length variants observed in different species.     

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.     

The wheat ribosomal DNA spacer region: Its structure and variation in populations and among species

Summary The wheat rDNA clone pTA250 was examined in detail to provide a restriction enzyme map and the nucleotide sequence of two of the eleven, 130 bp repeating units found within the spacer region. The 130 bp units showed some sequence heterogeneity. The sequence difference between the two 130 bp units analysed (130.6 and 130.8) was at 7 positions and could be detected as a 4 °C shift in Tm when heterologous and homologous hybrids were compared. This corresponded to a 1.2% change in nucleotide sequence per Tm of 1 °C. The sensitivity of the Tm analysis using cloned sequences facilitated the analysis of small sequence variations in the spacer region of different Triticum aestivum cultivars and natural populations of T. turgidum ssp. dicoccoides (referred to as T. dicoccoides). In addition spacer length variation was assayed by restriction enzyme digestion and hybridization with spacer sequence probes.Extensive polymorphism was observed for the spacer region in various cultivars of T. aestivum, although within each cultivar the rDNA clusters were homogeneous and could be assigned to particular chromosomes. Within natural populations of T. dicoccoides polymorphism was also observed but, once again, within any one individual the rDNA clusters appeared to be homogeneous. The polymorphism, at the sequence level (assayed by Tm analysis), was not so great as to prevent the use of spacer sequence variation as a probe for evolutionary relationships. The length variation as assayed by restriction enzyme digestion did not appear to be as useful in this regard, since its range of variation was extensive even within populations of a species.