rDNA Types Research Articles

Nuclear Ribosomal DNA Restriction-Site Variation in Penstemon Section Peltanthera (Scrophulariaceae): An Evaluation of Diploid Hybrid Speciation and Evidence for Introgression

Penstemon spectabilis is a putative stabilized diploid hybrid of P. centranthifolius and P. grinnellii. It is morphologically intermediate, and all three species have different pollinators. Penstemon centranthifolius and P. spectabilis have been proposed as parents of P. clevelandii, which is purportedly isolated by ecological factors. Although hybridization between the proposed parental species has been reported, hybrid swarm formation has not been observed and introgression is purported to be minimal. We tested hypotheses of diploid hybrid speciation and introgression among these species based on rDNA restrictionsite and length variation for 56 populations within and outside of the hybrid complex. Unambiguous molecular markers clearly differentiated P. centranthifolius, P. grinnellii, and P. spectabilis, whereas P. spectabilis and P. clevelandii had the same rDNA type. The P. centranthifolius rDNA type was found in some populations of P. spectabilis and P. clevelandii, but there was no evidence of the additive profile documented for other recent diploid hybrid species. In contrast, the rDNA profile of P. × parishii had a completely additive profile of its proposed parental species P. centranthifolius and P. spectabilis. Ribosomal DNA markers for P. grinnellii were restricted to populations within the species and were not found in any population of P. spectabilis. Our data did not support hybrid-speciation hypotheses, but were in accord with allozyme data that provided evidence for introgression between P. centranthifolius and P. grinnellii, P. spectabilis and P. clevelandii. These results were used to propose criteria to differentiate ancient diploid hybrid speciation from patterns of introgression.

Nuclear ribosomal DNA restriction‐site variation in Penstemon sectionPeltanthera (Scrophulariaceae): an evaluation of diploid hybrid speciation and evidence for introgression

Penstemon spectabilis is a putative stabilized diploid hybrid of P. centranthifolius and P. grinnellii. It is morphologically intermediate, and all three species have different pollinators. Penstemon centranthifolius and P. spectabilis have been proposed as parents of P. clevelandii, which is purportedly isolated by ecological factors. Although hybridization between the proposed parental species has been reported, hybrid swarm formation has not been observed and introgression is purported to be minimal. We tested hypotheses of diploid hybrid speciation and introgression among these species based on rDNA restrictionsite and length variation for 56 populations within and outside of the hybrid complex. Unambiguous molecular markers clearly differentiated P. centranthifolius, P. grinnellii, and P. spectabilis, whereas P. spectabilis and P. clevelandii had the same rDNA type. The P. centranthifolius rDNA type was found in some populations of P. spectabilis and P. clevelandii, but there was no evidence of the additive profile documented for other recent diploid hybrid species. In contrast, the rDNA profile of P. × parishii had a completely additive profile of its proposed parental species P. centranthifolius and P. spectabilis. Ribosomal DNA markers for P. grinnellii were restricted to populations within the species and were not found in any population of P. spectabilis. Our data did not support hybrid‐speciation hypotheses, but were in accord with allozyme data that provided evidence for introgression between P. centranthifolius and P. grinnellii, P. spectabilis and P. clevelandii. These results were used to propose criteria to differentiate ancient diploid hybrid speciation from patterns of introgression.

Dimorphism of rDNA organization in mating types of the zygomycete Absidia glauca

Complementary mating types of the zygomycete Absidia glauca exhibit a differential organization of the repeat units coding for ribosomal RNA. The (+) mating type contains two distinguishable forms of rDNA whereas the (-) type has only one, which — at the restriction level — is nearly identical with one of the (+) specific rDNA repeats. All three forms of rDNA were cloned in pBR325 as BamHI fragments spanning the entire repeats. Restriction maps and locations of the sequences coding for the large rRNAs are presented. Both rDNA types from the (+) strain differ by small rearrangements within the non-coding spacer region, whereas the (-) specific repeat has an additional HindIII site in the coding region for 185 RNA compared with the corresponding repeat unit of the (+) type. The gene for 5S rRNA is part of the repeat unit, but was found to be transcribed in the opposite direction. Thus, the rDNA organization of Absidia glauca resembles most closely the situation encountered in baker's yeast.

Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins

A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity.

Restriction site and length polymorphism of the rDNA unit in the cultivated basidiomycetePleurotus cornucopiae

In the ribosomal DNA unit ofPleurotus cornucopiae, the rDNA coding regions are in the order 5', 5S-18S-5.8S-25S, 3', with the 5' location of the 5S gene differing from its 3' location found in other basidiomycetes. The most discriminating probe used to study the rDNA polymorphism consisted of a fragment that included the 5S, 18S and part of the 5.8S and 25S genes flanking three intergenic sequences. A high degree of rDNA polymorphism was observed in the sevenP. cornucopiae dikaryons studied. For the first time within a basidiomycete species, the restrictions maps distinguished two types of rDNA units (I and II). In each rDNA type, length variations in the external intergenic sequence IGS 1 located between the 25S and 5S genes allowed characterization of two different rDNA units in type I and four rDNA units in type II. This suggested that theP. cornucopiae rDNA units were derived from two kinds of ancestors (type I and II) by insertion or deletion events (100-700 bp) in the IGS 1. In four dikaryotic strains, two rDNA units of the same type (I or II) differing only by the IGS 1 length, were found in a similar number of copies, and presented a meiotic segregation in homokaryotic progeny. In one progeny, some homokaryotic strains possessed two different rDNA units: one with a high copy number and another with a lower one, showing that two different rDNA units could coexist in a single nucleus.

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.

Nuclear Ribosomal DNA as a Probe for Genetic Variability in the Japanese Pear Pathotype of Alternaria alternata

A restriction fragment length polymorphism analysis of nuclear ribosomal RNA genes (rDNA) was used to measure the amount and distribution of genetic variability in populations of the Japanese pear pathotype of Alternaria alternata on both micro- and macrogeographical scales. A total of 322 isolates were obtained from 13 areas in Aichi, Gifu, and Tottori Prefectures in central and western Japan. The restriction fragment length polymorphism analysis revealed that the pathogen populations contained at least eight rDNA variants. The eight variant types differed in the lengths and in the presence of the restriction sites in spacer DNA outside the coding regions for rRNAs. A total of 271 isolates were classified into the eight types. The remaining 51 isolates were determined to have mixed rDNA types. Single pear fields typically contained two to five types of rDNA variants. The frequencies of rDNA variants in 11 populations in Tottori Prefecture were compared; in this prefecture orchards containing the susceptible pear are common. Except for one collection site, there were no significant differences in the composition of the rDNA variants among the populations. This suggests that dispersal of inocula has occurred frequently in Tottori Prefecture. In contrast, significantly different distributions were observed in the three prefectures, indicating that gene flow between prefectures might be limited by geographical isolation. DNA fingerprints resulting from hybridization with a moderately repetitive DNA sequence of the fungus revealed greater genetic variability and geographical differences in genetic population structure even within the same rDNA type.

Characterization ofListeria strains from a foodborne listeriosis outbreak by rDNA gene restriction patterns compared to four other typing methods

The rDNA gene restriction patterns of 134 isolates of Listeria species were determined with pKK3535--a pBR322 derived plasmid containing an Escherichia coli rRNA operon--used as a probe following digestion of chromosomal DNA by EcoRI endonuclease. Nineteen reference and type strains representing all species and serotypes of Listeria showed 17 distinct ribotypes. One hundred and fifteen wild strains of Listeria monocytogenes were ribotyped and the results were compared to those of serotyping, phage typing, multilocus enzyme electrophoresis (MEE) and restriction endonuclease analysis (REA). Ninety-six Listeria monocytogenes serotype 4b wild strains displayed six distinct ribotypes (I-VI), 72% (69/96) of them clustering in two very close rDNA patterns (I and II) of eight and nine bands, respectively. The same 96 strains displayed six REA patterns and eight MEE electrotypes. Among the 96 Listeria monocytogenes 4b isolates, the 34 epidemic strains defined by phage typing and by epidemiological data all belonged to one ribotype (ribotype I) representing 56% of the strains belonging to this ribotype. These same 34 epidemic strains were also grouped by REA and MEE typing in a unique profile (REA-A) and MEE electrotype (ET 1). Twenty-two Listeria monocytogenes strains of serogroup 1/2 analyzed by rDNA typing showed nine distinct ribotypes. For the 96 Listeria monocytogenes 4b strains studied, the discriminatory index was highest for phage typing and for any combination including phage typing. Ribotyping appears to be a well reproducible molecular typing method and could be a useful complement to other typing methods for the epidemiological study of listeriosis.

Inheritance of the group I rDNA intron in Tetrahymena pigmentosa.

We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+ and intron- strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron+ and intron- rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing. During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis.

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila.

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria

The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.

Evolutionary Relationships of Lentinus to the Polyporaceae: Evidence from Restriction Analysis of Enzymatically Amplified Ribosomal Dna

Evolutionary relationships of Lentinus to the Tricholomataceae and Polyporaceae were studied using restriction analysis of nuclear-encoded ribosomal RNA genes (rDNA). Five species of Lentinus, three species in the Polyporaceae and two species in the Tricholomataceae were examined. Ribosomal DNA phenotypes were determined by restriction endonuclease digestion of enzymatically amplified rDNA (PCR fingerprinting). This method generates restriction fragment length polymorphism data without Southern blotting or autoradiography. With five different four-base restriction enzymes, one hundred and one unique restriction fragments were resolved. Among the 16 individuals studied, there were twelve different rDNA types. A similarity matrix based on presence or absence ofcomigrating restriction fragments was analyzed with UPGMA, complete and single-linkage analysis (using the NTSYS computer package), and Fitch-Margoliash and KITSCH analysis (using the PHYLIP computer package). Results of all analytical approaches were highly consistent and strongly suggest that Lentinus tigrinus is more closely related to the Polyporaceae than to the Tricholomataceae. Morphological characters (dimitic hyphae and hyphal pegs) also support this hypothesis. The other species of Lentinus appear to be intermediate between the Polyporaceae and Tricholomataceae and could not be clearly assigned to either family. The results also suggest that Lentinus is paraphyletic and, therefore, that lamellae have

MOLECULAR TESTS OF THE HYPOTHESIZED HYBRID ORIGIN OF TWO DIPLOID HELIANTHUS SPECIES (ASTERACEAE).

Enzyme electrophoresis and restriction-fragment analysis of chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) were used to test the hypothesis that both Helianthus neglectus and H. paradoxus are stabilized hybrid derivatives of H. annuus and H. petiolaris. The four species are annuals, diploid outcrossers, and have the same chromosome number. Helianthus annuus and H. petiolaris had the same allele in highest frequency for 16 of the 18 isozyme loci examined and had different majority alleles for only 6-Pgd3 and Pgi2. The two species had divergent rDNAs that could be distinguished by seven diagnostic restriction site mutations and three length mutations, and their cpDNAs could be differentiated by three diagnostic restriction site mutations. The alleles observed in H. neglectus were not a combination of those observed in H. annuus and H. petiolaris. Although H. neglectus had only one unique allele, it possessed none of the three alleles specific to H. annuus. In contrast, it had four of the seven alleles specific to H. petiolaris. Furthermore, H. neglectus had the same rDNA type as H. petiolaris and had the same cpDNA as that found in two populations of H. petiolaris ssp. fallax. These data allowed us to speculate that H. neglectus may be a recent derivative of H. petiolaris ssp. fallax, rather than a stabilized hybrid derivative as originally proposed. In contrast, H. paradoxus combined the alleles of H. annuus and H. petiolaris and had no unique alleles. At Adh2, H. paradoxus was monomorphic for an allele found only in H. petiolaris ssp. fallax, whereas at 6-Pgd3 and Pgi2, it was monomorphic for high frequency H. annuus alleles. Furthermore, H. paradoxus combined the rDNA repeat types of both proposed parents and had the chloroplast genome of H. annuus. These data provide compelling evidence that H. paradoxus, in contrast to H. neglectus, was derived via hybridization.

Molecular Tests of the Hypothesized Hybrid Origin of Two Diploid Helianthus Species (Asteraceae)

Enzyme electrophoresis and restriction-fragment analysis of chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) were used to test the hypothesis that both Helianthus neglectus and H. paradoxus are stabilized hybrid derivatives of H. annuus and H. petiolaris. The four species are annuals, diploid outcrossers, and have the same chromosome number. Helianthus annuus and H. petiolaris had the same allele in highest frequency for 16 of the 18 isozyme loci examined and had different majority alleles for only 6-Pgd3 and Pgi2. The two species had divergent rDNAs that could be distinguished by seven diagnostic restriction site mutations and three length mutations, and their cpDNAs could be differentiated by three diagnostic restriction site mutations. The alleles observed in H. neglectus were not a combination of those observed in H. annuus and H. petiolaris. Although H. neglectus had only one unique allele, it possessed none of the three alleles specific to H. annuus. In contrast, it had four of the seven alleles specific to H. petiolaris. Furthermore, H. neglectus had the same rDNA type as H. petiolaris and had the same cpDNA as that found in two populations of H. petiolaris ssp. fallax. These data allowed us to speculate that H. neglectus may be a recent derivative of H. petiolaris ssp. fallax, rather than a stabilized hybrid derivative as originally proposed. In contrast, H. paradoxus combined the alleles of H. annuus and H. petiolaris and had no unique alleles. At Adh2, H. paradoxus was monomorphic for an allele found only in H. petiolaris ssp. fallax, whereas at 6-Pgd3 and Pgi2, it was monomorphic for high frequency H. annuus alleles. Furthermore, H. paradoxus combined the rDNA repeat types of both proposed parents and had the chloroplast genome of H. annuus. These data provide compelling evidence that H. paradoxus, in contrast to H. neglectus, was derived via hybridization.

Latent NORs in the species Pycnogaster cucullata (Orthoptera)

The chromosomal location of the ribosomal cistrons 18 + 26S and 5S has been determined in spermatocytes of two cytological races of Pycnogaster cucullata by in situ hybridization using molecular probes from both types of rDNA. A comparative analysis with previous results suggested the existence of more 18 + 26S rDNA sites than active NORs as shown by silver impregnation, but 5S rDNA sites are outside the NORs. Thus, different categories of latent NORs have been shown in this specialised cell type which are discussed in relation to the evolutionary process of differentiation in this species.

Use of mitochondrial and ribosomal DNA polymorphisms to classify clinical and soil isolates of Histoplasma capsulatum

We have developed an improved scheme for the classification of environmental and clinical isolates of Histoplasma capsulatum that is based on analysis of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA). Strains were initially divided into mtDNA groups according to restriction digests of whole-cell DNA and Southern hybridization with cloned mtDNA probes. Strains within a mtDNA class could be further grouped by polymorphisms in rDNA. The majority of soil and clinical isolates from the United States had identical mtDNA patterns; however, rDNA polymorphisms were common in both types of isolates. The combination of mtDNA and rDNA typing described in this report will be useful in resolving questions concerning the epidemiology of H. capsulatum infections.

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis.

The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by DNA-DNA hybridization of labeled probes to total DNA at various stages of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative difference in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentatively proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.

Ribosomal DNA inheritance and recombination in Neurospora crassa.

The genetic segregation of ribosomal DNA (rDNA) in Neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (NTS) sequences of nine laboratory wild-type strains and wild-collected strains. In an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rDNA was shown to be inherited in a simple, stable Mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rDNA types. No meiotic recombinants were detected among the progeny, indicating that non-sister-chromatid crossing over is highly suppressed in the rDNA region. The basis for this suppression of meiotic recombination is not known.

Ribosomal DNA (rDNA) spacer polymorphism in mole rats.

Genetic differentiation of ribosomal DNA (rDNA) nontranscribed-spacer (NTS) polymorphism was analyzed in 50 individuals from 13 populations among the four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of subterranean mole rats of the Spalax ehrenbergi complex in Israel. Southern blot analysis with a mouse rDNA probe and two restriction enzymes, EcoRI and BamHI, revealed various sizes of major restriction fragments. The assumption that this variation is due to length polymorphism of NTS DNA was supported by the construction of restriction-site maps. On the basis of the EcoRI and BamHi fragment lengths, we could characterize the major types of NTS rDNA repeating units in each individual. Each member of the central population in the four chromosomal groups of mole rats has a characteristic combination of the NTS types, suggesting that the karyotype groups were genetically diverged. Some near-hybrid-zone populations reflect similarities with the rDNA spectra of a neighbor chromosomal group. This might have resulted from gene flow across the hybrid zones.

The effect of mei-41 on rDNA redundancy in Drosophila melanogaster.

The recombination and repair defective mutant, mei-41, exhibits three rather striking effects on the genetic properties and chromosomal stability of rDNA in Drosophila. First, mei-41 inhibits rDNA magnification. However, mei-9, another recombination and repair defective mutation has no similar effect. This indicates that magnification requires some, but not all, of the gene products necessary for meiotic exchange. Second, under magnifying conditions, mei-41 induces interchanges between the X rDNA and either arm of the Ybb- chromosome. These interchanges occur at high frequency and are independent of rDNA orientation. Third, in mei-41 bb+/Ybb+ males, bobbed mutants in the X, but not the Y, also arise at high frequency. Evidence suggests that these events involve the rDNA type I insertion. The recombination and repair defective properties of mei-41 together with our results regarding its unusual and specific effects involving rDNA are explained in a simple model that has general implications for chromosome structure.