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.     

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria

Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA ⁺ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.     

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.     

Generalized linear mixed model for segregation distortion analysis

Background

Concerted evolution refers to the pattern in which copies of multigene families show high intraspecific sequence homogeneity but high interspecific sequence diversity. Sequence homogeneity of these copies depends on relative rates of mutation and recombination, including gene conversion and unequal crossing over, between misaligned copies. The internally repetitive intergenic spacer (IGS) is located between the genes for the 28S and 18S ribosomal RNAs. To identify patterns of recombination and/or homogenization within IGS repeat arrays, and to identify regions of the IGS that are under functional constraint, we analyzed 13 complete IGS sequences from 10 individuals representing four species in the Daphnia pulex complex.

Results

Gene conversion and unequal crossing over between misaligned IGS repeats generates variation in copy number between arrays, as has been observed in previous studies. Moreover, terminal repeats are rarely involved in these events. Despite the occurrence of recombination, orthologous repeats in different species are more similar to one another than are paralogous repeats within species that diverged less than 4 million years ago. Patterns consistent with concerted evolution of these repeats were observed between species that diverged 8-10 million years ago. Sequence homogeneity varies along the IGS; the most homogeneous regions are downstream of the 28S rRNA gene and in the region containing the core promoter. The inadvertent inclusion of interspecific hybrids in our analysis uncovered evidence of both inter- and intrachromosomal recombination in the nonrepetitive regions of the IGS.

Conclusions

Our analysis of variation in ribosomal IGS from Daphnia shows that levels of homogeneity within and between species result from the interaction between rates of recombination and selective constraint. Consequently, different regions of the IGS are on substantially different evolutionary trajectories.     

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.     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

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.     

Fine structure and evolution of the rDNA intergenic spacer in rice and other cereals

Summary The intergenic spacer of a rice ribosomal RNA gene repeating unit has been completely sequenced. The spacer contains three imperfect, direct repeated regions of 264–253 bp, followed by a related but more highly divergent region. Detailed analysis of the sequence allows the presentation of an evolutionary scenario in which the 264–253-bp repeats are derived from an ancestral 150-bp sequence by deletion and amplification. Comparison of the rice sequence with those of maize, wheat, and rye shows that, despite considerable divergence from the ancestral sequence, several regions have been highly conserved, suggesting that they may play an important role in the structure and/or expression of the ribosomal genes.Abbreviations IGS ribosomal gene intergenic spacer - rDNA ribosomal DNA - rRNA ribosomal RNA Offprint requests to: M. Delseny     

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.     

Aspects of nonrandom turnover involved in the concerted evolution of intergenic spacers within the ribosomal DNA of Drosophila melanogaster

Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage hot spots within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family. Correspondence to: G. A. Dover     

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.     

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.Non-commun abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer     

Host Selectivity and Genetic Variation of Discula umbrinella Isolates from Two Oak Species: Analyses of Intergenic Spacer Region Sequences of Ribosomal DNA

Discula umbrinella, a fungal endophyte of oak species, colonizes and reproduces on leaves of Quercus alba and Q. rubra in forest ecosystems. Twenty-nine isolates collected from leaves of both oak species (16 from Q. alba and 13 from Q. rubra) were assayed for oak species preference and genetic variation based on primer-specific polymerase chain reactions for the intergenic spacer region (IGS) of ribosomal DNA. DNA sequencing of the polymerase chain reaction products revealed a 10-bp insertion (237–247 bp) at the 3′ end of the IGS region present in nine isolates and absent in 20 of the isolates. Phylogenetic analysis of the IGS region using the neighbor-joining method identified IGS groups (groups I–V) based on single nucleotide sequence differences. Host selectivity and geographic origin of isolates were correlated in some instances with the IGS groups. Isolates within each IGS group were further analyzed for nucleotide polymorphisms to confirm genotype identity and genotype diversity. Ten different genotypes (V_a–V_j) were identified among the isolates analyzed. Genotype diversity was greatest in IGS groups I, IV, and V. Seventy percent of the genotypes (V_c, V_d, V_e, V_f, V_g,V_i, and V_j) contained isolates with single tree species preferences.     

Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba

Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.by D. Schweizer     

Intraspecific Concerted Evolution of the rDNA ITS1 in Anopheles farauti Sensu Stricto (Diptera: Culicidae) Reveals Recent Patterns of Population Structure

We examined the intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) of Anopheles farauti to determine the level of divergence among populations for this important malarial vector. We isolated 187 clones from 70 individuals and found regional variation among four internal tandem repeats. The data were partitioned prior to analysis given the presence of a paralogous ITS2 sequence, called the 5'-subrepeat, inserted in the ITS1 of most clones. A high level of homogenization and population differentiation was observed for this repeat, which indicates a higher rate of turnover relative to the adjacent 'core' region. Bayesian analysis was performed using several substitutional models on both a combined and a partitioned data set. On the whole, the ITS1 phylogeny and geographic origin of the samples appear to be congruent. Some interesting exceptions indicate the spread of variant repeats between populations and the retention of ancestral polymorphism. Our data clearly demonstrate concerted evolution at the intraspecific level despite intraindividual variation and a complex internal repeat structure from a species that occupies a continuous coastal distribution. A high rate of genomic turnover in combination with a high level of sequence divergence appears to be a major factor leading to its concerted evolution within these populations.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.