Ribosomal DNA variation in the grasshopper, Dichroplus elongatus.

We report an RFLP analysis of ribosomal DNA variation in natural populations of the grasshopper, Dichroplus elongatus, previously analyzed for mitochondrial DNA variation. DNA samples were digested with five restriction enzymes, BamHI, EcoRI, HindIII, PstI, and XbaI. BamHI was the only enzyme that showed no variation. The remaining enzymes showed fragment size variation at both intra- and interpopulation levels. Stepwise regression analysis revealed that the average number of length variants per individual is significantly associated with altitude. Moreover, the same analysis indicated that the frequency of some restriction variants exhibits a significant regression on both geographic and climatic variables. The intra- and interpopulation variability of rDNA was analysed by Lynch's and Hedrick's similarity indices using presence or absence of a fragment and band intensities, respectively. The corresponding neighbour-joining (N-J) trees based on Lynch's and Hedrick's genetic distances resulted in similar topologies. However, these trees were not in agreement with the N-J dendrogram obtained from mtDNA data previously reported by Clemente et al. (2000). The disagreement between mtDNA and rDNA phenograms along with the observed correlation between rDNA variability and geographical and climatic variables suggest some form of selection, besides genetic drift and migration, is involved in the pattern of rDNA variation.     

Intraspecies polymorphism for the structural organization of ribosomal gene cluster in Blattella germanica

Intraspecific HindIII restriction fragment length polymorphism (RFLP) for the structural organization of the ribosomal gene cluster was described in Blattella germanica. In this species, homologous chromosomes were shown to contain ribosomal DNA (rDNA) repeats that differ in structure. The pattern of inheritance was determined for various structural variants of rDNA.     

Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta:Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A reverse similar 1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5' end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.     

Biased gene conversion and asymmetrical introgression between subspecies

Data from chromosomal in situ hybridization and restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) obtained from the same individuals of two subspecies of the Australian grasshopper Caledia captiva, suggest the occurrence of biased exchange of DNA sequences in hybrid individuals. Thus, there are a disproportionate number of Torresian individuals that possess Moreton ribosomal RNA gene sequences. This bias correlates with the previously described pattern of asymmetrical introgression of Moreton rDNA into Torresian populations. The present findings demonstrate the potential effect of biased gene conversion on the frequencies of introduced gene variants.     

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.     

Restriction enzyme analysis of variation and taxonomy in the kelp genus Laminaria (Laminariales,Phaeophyta)

High levels of phenotypic variation in kelp species necessitate the use of taxonomic markers that are independent of morphology. Restriction fragment length polymorphism (RFLP) analysis of nuclear DNA can provide such markers. In this paper we present the results of an RFLP analysis of cytoplasmic ribosomal DNA (rDNA) in three Laminaria species (L. agardhii, L. digitata, L. groenlandica). Comparison of the restriction maps of the nontranscribed spacer (NTS) in the rDNAs suggests that this method should be useful for the differentiation of these taxa. These results are discussed, as are the applications of RFLP mapping to the identification of field-collected, morphologically variable plants.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Mitochondrial DNA restriction map for the mediterranean fruit fly,Ceratitis capitata

Molecular genetic research on the Mediterranean fruit fly,Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variableEcoRV andXbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene regions based on polymerase chain reaction-RFLP and sequence analyses. A partial sequence of the mitochondrial 16S ribosomal RNA gene is reported.     

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.      

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.     

Mitochondrial and ribosomal DNA variation among members of the Anopheles quadrimaculatus (Diptera: Culicidae) species complex.

The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.     

Ribosomal DNA non-transcribed spacer polymorphism in subterranean mole rats: genetic differentiation,environmental correlates and phylogenetic relationships

Summary An analysis is presented of genetic differentiation in the non-transcribed spacers of ribosomal DNA (NTS rDNA). Diversity, environmental correlates and the phylogenetic relationships are examined within and between species of the actively speciating subterranean mole rat, superspeciesSpalax ehrenbergi (2n=52, 54, 58, 60) in Israel. This analysis is based on a previous study of the geographic distribution of restriction fragment length polymorphisms of NTS rDNA. Here we present results indicating that NTS rDNA diversity exists mostly (66%) within populations, while 20% is between populations within species, and 14% between species. Multivariate discriminant analysis succeeded in separating 10 of the 13 populations (77%) into their correct chromosomal species, on the basis of the combination of three NTS rDNA repetypes. The phylogenetic relationships suggest that the complex involves two pairs of closely related species (2n=52–54 and 2n=58–60). NTS rDNA diversity, as well as the decrease southward in frequency of repetype C, are correlated with climatic factors of humidity and temperature. These data are discussed in terms of the evolutionary forces of migration and selection which may cause NTS rDNA differentiation. Climatic selection appears to be the major differentiating factor of NTS rDNA.     

Change in ribosomal DNA spacer-length composition in maize recurrent selection populations. 2. Analysis of BS10, BS11, RBS10, and RSSSC

Four maize (Zea mays L.) populations selected for grain yield (BS10, Iowa Two-ear Synthetic; BS11, formerly Pioneer Two-ear Composite; RBS10, Illinois strain of BS10; and RSSSC, Illinois strain of Iowa Stiff Stalk Synthetic) were assayed for molecular variation in the ribosomal DNA (rDNA) intergenic spacer (IGS) at initial and advanced cycles of selection. RSSSC and RBS10 underwent reciprocal recurrent selection with an inbred tester in a high-yield environment, whereas BS10 and BS11 were subjected to full-sib reciprocal recurrent selection. Maize rDNA, which encodes the ribosomal RNA genes, is highly repetitive and shows IGS length variation within and among individuals. Five different ribosomal spacer-length variants (rslvs) and a polymorphic SstI restriction site in the IGS were detected in the four populations. The five rslvs and the polymorphic restriction fragment were observed in 20 different combinations or hybridization fragment patterns (HP). RSSSC, RBS10, and BS11 showed significant changes in the overall rslv and HP frequencies between cycle 0 and the advanced cycle of selection, whereas BS10 did not. In general, two specific HPs were more frequent in the majority of the advanced cycles of the four populations. The frequency changes between initial and advanced cycles were more dramatic for HPs than rslvs. These results are consistent with earlier findings and further support the hypothesis that certain rDNA HPs and/or linked loci may be responding to selection for grain yield and may be associated with a selective advantage in US Corn Belt environments.     

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.     

Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases. SmaI, HinfI and HhaI, provided one or two different bands between two species, although the hybridization patterns generated with the others. HindIII, XbaI, StyI and HaeIII, were the same in both. RFLPs in the digested profiles with SmaI, HinfI and HhaI could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.     

Heritability and Variability in Ribosomal RNA Genes of Vicia faba

We have compared the restriction patterns and copy numbers of ribosomal RNA genes (rDNA) between and within individuals of Vicia faba . While the EcoRI blot-hybridization patterns changed only after one to two generations, copy number changes were found among different tissues of the same plant. Copy number differences among individuals in the population were as great as 95-fold, whereas as much as a 12-fold variation was seen among tissues of the same plant. Among individual F(1) progeny from genetic crosses, nearly an 8-fold variation was seen, and among individuals of the F(2) generation a spread of 22-fold was measured. Among individual siblings of self-pollinated parents, up to 7-fold variation was observed. However, changes in copy number did not necessarily indicate changes in rDNA EcoRI blot-hybridization pattern, and vice versa. Furthermore, nearest neighbor analysis of R-loop experiments showed that the arrangement of members of the "nontranscribed" spacer (NTS) size classes along the chromosome was not random, but some clustering was indicated. The data are consistent with the hypothesis that sister chromatid exchange in somatic cells of V. faba is the primary mechanism for altering the rDNA copy number as well as causing the extreme variation observed in the NTS. Variation among individuals in rDNA blot-hybridization pattern was also observed for Vicia villosa, Vicia dasycarpa, Vicia benghalensis and Vicia pannonica.     

Multiple gene analyses reveal extensive genetic diversity among ‘Candidatus Phytoplasma mali’ populations

This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north‐western Italy, where apple proliferation (AP) disease is widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR‐1, PR‐2 and PR‐3 non‐ribosomal region, ribosomal protein genes rplV‐rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north‐western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage‐specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.     

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Frequency in hypertensives of alleles for a RFLP associated with the renin gene

The genetic basis of primary hypertension is not known. Renin is important in blood pressure and volume control and a HindIII restriction fragment length polymorphism (RFLP) is present within the human renin gene locus. To examine whether there is a relationship between this RFLP and primary hypertension, DNA and renin analyses were performed on leukocytes and plasma from hypertensive and normotensive individuals. In hypertensives the frequencies of alleles for the HindIII RFLP were found to be 0.55 and 0.45, compared with 0.60 and 0.40 in the total population of 231 subjects examined, a difference that was not statistically significant. There also appeared to be no significant difference in renin activity in plasma for hypertensive patients of each genotype, nor in their pre- or post-treatment blood pressures. We thus conclude that, within the limits of the present study, the suspected genetic abnormalities associated with primary hypertension in man do not appear to be related to a HindIII RFLP in the renin gene.     

Nuclear rDNA variability in laboratory strains of the German cockroach Blattella germanica L. (Blattellidae)

The polymorphism of nuclear ribosomal DNA has been studied in male German cockroaches. The RFLP analysis has been used to identify seven types of HindIII fragments, the variation of which may serve as the basis for the differentiation of laboratory strains with respect to some population indices, including the average number of fragments per animal (N), the proportion of polymorphic loci (P), and the average pairwise similarity (APS). The interpopulation variation (FST) calculated for nine haplotypes is 53.85%.