Ribosomal DNA Polymorphism Research Articles

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

Species Identification of Five Penaeid Shrimps Using PCR-RFLP and SSCP Analyses of 16S Ribosomal DNA

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCRRFLP of 16S rDNA(560) whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S rDNA(560). Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA(560) of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S rDNA(312) was as effective as that of 16S rDNA(560). Differentiation of all shrimp species were successfully carried out by SSCP analysis.

Molecular Taxonomy of Cupped Oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand Based on COI, 16S, and 18S rDNA Polymorphism

Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids ( Dactylorhiza maculata group)

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or “spotted marsh-Orchids”. These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.

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%.

Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

Organization of genetic variation in individuals of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) are thought to be the oldest group of asexual multicellular organisms. They colonize the roots of most land plants, where they facilitate mineral uptake from the soil in exchange for plant-assimilated carbon. Cells of AM fungi contain hundreds of nuclei. Unusual polymorphism of ribosomal DNA observed in individual spores of AM fungi inspired a hypothesis that heterokaryosis--that is, the coexistence of many dissimilar nuclei in cells--occurs throughout the AM fungal life history. Here we report a genetic approach to test the hypothesis of heterokaryosis in AM fungi. Our study of the transmission of polymorphic genetic markers in natural isolates of Glomus etunicatum, coupled with direct amplification of rDNA from microdissected nuclei by polymerase chain reaction, supports the alternative hypothesis of homokaryosis, in which nuclei populating AM fungal individuals are genetically uniform. Intrasporal rDNA polymorphism contained in each nucleus signals a relaxation of concerted evolution, a recombination-driven process that is responsible for homogenizing rDNA repeats. Polyploid organization of glomeromycotan genomes could accommodate intranuclear rDNA polymorphism and buffer these apparently asexual organisms against the effects of accumulating mutations.

Ribosomal DNA polymorphism and its association with geographical and climatic variables in 27 wild barley populations from Jordan

Ribosomal DNA (rDNA) repeat unit length polymorphism was examined in 285 accessions of wild barley, Hordeum spontaneum C. Koch, which were collected from 27 locations across Jordan. As many as 19 spacer length variants (slvs) or rDNA alleles were available, which formed 70 slv phenotypes. The two missing alleles (098, 099) of the series (097, 100–118) and one additional allele 119 were also discovered in the present study thus raising the number of ribosomal slvs in barley to 24. Relatively more frequent rDNA alleles were analyzed in detail, and it was shown that they occurred non-randomly at locations with different environmental factors (annual rainfall, highest and lowest temperatures, altitude, longitude, latitude) and exhibited association with specific environments. Ecogeographical factors, rather than geographical factors per se, seem to affect the distribution of rDNA alleles. The present study thus demonstrates that rDNA repeat unit length polymorphism in some cases can be adaptive in nature.

Eubacterial community structure and population size within the soil light fraction, rhizosphere, and heavy fraction of several agricultural systems

The hypothesis that soil light fraction and heavy fraction harbor distinct eubacterial communities and have differing numbers and sizes of bacterial cells was tested in three agronomic cropping systems. This hypothesis would imply that these soil fractions are distinct microbial habitats. Shoot residue and rhizosphere soil were also included in the analysis. Terminal restriction fragment length polymorphism (T-RFLP) of 16S ribosomal DNA was used to assay eubacterial community structure. T-RFLP profiles were affected by both soil fraction and cropping system, accounting for 35–50% of the variance in the profiles. T-RFLP profiles separated samples into two distinct eubacterial habitats: soil heavy fraction, which includes the mineral particles and associated humified organic matter, and soil light fraction/shoot residue and rhizosphere, which includes particulate soil organic matter. Differences were not based on organic C content of fractions alone; T-RFLP profiles were also differentiated by cropping system and by rhizosphere versus light fraction/shoot residue. Heavy fraction communities had the least amount of random variability in T-RFLP profiles, resulting in the clearest cropping system effects, while rhizosphere and shoot residue communities were the most variable. Profiles from organically managed corn soil were more variable than for either conventionally managed corn or alfalfa. The log number of bacterial cells per gram fraction was affected by soil fraction but not cropping system, being highest in the light fraction. The percentage of cells >0.18 μm 3 was also greater in the light fraction than in other fractions. While bacterial cell density was generally correlated with C content of the soil fraction, heavy fraction did have a significantly greater number of cells per μg C than other soil fractions. The results show that habitat diversity in soil, related both to the amounts and types of organic matter, as well as other potential factors, are important in maintaining the high soil bacterial species diversity and evenness that is found in soil.

Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.

Molecular Analysis of Exophiala Species Using Molecular Markers

Genetic relatedness of medically important Exophiala species such as E. dermatitidis, E. mansonii, and three E. jeanselmei varieties: jeanselmei, lecanii-corni, and heteromorpha was examined using PCR-RFLP (restriction fragment length polymorphism) of ribosomal DNA, M-13, (GTG)5, and nucleotide sequences of ribosomal ITS (internal transcribed space) II regions. Three E. jeanselmei varieties showing distinct band patterns for each DNA markers as well as different nucleotide sequences of ribosomal ITS II regions could be considered as a separate species. E. dermatitidis and E. mansonii demonstrated the identical band patterns of RFLP of ribosomal DNA, M-13, and (GTG)5 markers. However, nucleotides sequences of ribosomal ITS II region were different between these two species.

Identification of Colletotrichum acutatum from rubber using random amplified polymorphic DNAs and ribosomal DNA polymorphisms

The fungal pathogen responsible for Colletotrichum leaf disease of Hevea , develops three different disease symptoms: raised spots, anthracnose and papery lesions. These diseases have been attributed to Colletotrichum gloeosporioides (teleomorph Glomerella cingulata ) and the fungi involved are morphologically indistinguishable. Twenty-five Colletotrichum isolates, which originated from three different disease symptoms, were characterized initially using RAPD markers. Two major RAPD profiles were detected which were related to the type of disease symptom developed. Colletotrichum isolates causing raised spot symptom were easily distinguished from isolates originating from either anthracnose or papery lesions. Restriction analysis of the PCR amplified 5.8S ribosomal DNA (rDNA), including both the flanking internal transcribed spacers (ITS) of representative isolates from the three different disease symptoms, also revealed two distinct RFLP patterns reflecting the same groupings as detected through RAPDs. Both molecular approaches suggested that there were two species of Colletotrichum associated with Hevea inciting the development of three different symptoms: Colletotrichum acutatum causing raised spot symptom, and C. gloeosporioides causing both anthracnose and papery lesions. This is the first record of Colletotrichum acutatum on Hevea in India.

A comparative study of genetic diversity of populations of Nectria galligena and N. coccinea var. faginata in North America

Genetic variability was investigated in North American populations of the tree pathogenic fungi Nectria galligena and N. coccinea var. faginata. Genetic diversity was estimated by analysing RAPD and ribosomal DNA polymorphisms. The number of haplotypes was the maximum value in N. galligena (51), whereas 17 different haplotypes were detected among the 23 isolates of N. coccinea var. faginata studied. The frequency of polymorphic loci was greater in N. galligena (92%) than in N. coccinea var. faginata (35%). The estimate of genotypic diversity was a maximum in N. galligena while it represented only 59% of the maximum value for N. coccinea var. faginata. Gene diversity was also higher in the former (21%) than in the latter (6%). Moreover, four distance indexes calculated from the same genetic distance matrix gave similar results and showed that distances among isolates of N. galligena were four to nearly six times larger than distances among N. coccinea var. faginata isolates. An analysis of molecular variance based on genetic distances among haplotype frequencies confirmed that host origin (beech vs other species) was not the source of the genetic variability in N. galligena since less than 1% of the variability was attributable to the host factor. Overall, our results are consistent with the hypothesis that N. galligena is a pathogen native to North America while N. coccinea var. faginata may be a recent introduction.

Genotypic diversity of symbiotic dinoflagellates associated with hermatypic corals from a fringing reef at Sesoko Island, Okinawa

The genotypes of zooxanthellae associated with eight scleractinian corals and one hydrocoral from a fringing reef of Sesoko Island (Okinawa, Japan) were determined using the single stranded conformational polymorphism (SSCP) of large subunit ribosomal DNA (28S rDNA). Preliminary RFLP analysis of small subunit (18S) rDNA showed that four pocilloporid corals harbored zooxanthellae of clade C, except one branch of Stylophora hystrix kept at 8% light which contained zooxanthellae of clade A and C. The hydrocoral Millepora intricata harbored zooxanthellae of clade A and C. RFLP analysis failed to distinguish three morphophysiological types described by Titlyanov et al. (2001a, b). SSCP analyses of 28S rDNA showed that zooxanthellae from the nine coral species gave at least fourteen distinct SSCP patterns. While zooxanthellae from S. hystrix and S. caliendrum had identical SSCP patterns, SSCP patterns of zooxanthellae from other coral species studied were distinct from each other. In six out of nine coral species, branches from the same colonies kept under different light conditions contained zooxanthellae that showed different SSCP patterns. However, no clear relationship was found between zooxanthellae genotypes and light intensity. The present results suggest that three morphophysiological types are not unique but that each type consists of zooxanthellae of several genotypes.

Differentiation of Brewing and Related Yeasts Based on PCR Amplification and Restriction Fragment Length Polymorphism of Ribosomal DNA

A study to differentiate commercially applied brewing yeasts selected from the culture collection of the University of the Free State from related yeasts of the genus Saccharomyces using PCR amplification and RFLP of the internal transcribed spacers region was conducted. Differentiation was dependent on the restriction enzymes used to digest the amplified rDNA. Digestion with Hae III, Cfo I, Sau 3AI and Msp I divided representatives of the genus Saccharomyces into several unique groups. With Msp I the DNA patterns for the two brewing strains were similar, but could be differentiated from Sacch. cerevisiae and other species tested. It was also possible to distinguish some members of the Saccharomyces sensu stricto group i.e. Sacch. bayanus and Sacch. pastorianus from Sacch. cerevisiae and Sacch. paradoxus using Hae III as well as Sacch. paradoxus from the other sensu stricto members using Msp I digestion.

The genus Olea: molecular approaches of its structure and relationships to other Oleaceae

Ribosomal DNA RFLP and RAPD polymorphisms were examined in taxa belonging to Oleaceae genera. The objective of this study was to determine the taxonomic position of the genus Olea and of its components: subg. Olea section Olea, subg. Olea section Ligustroides, subg. Paniculatae and subg. Tetrapilus. Ribosomal DNA polymorphisms supported that O. subg. Tetrapilus was separated from other Olea species and appeared close to other Oleaceae genera as Nestegis, Chionanthus and Phillyrea. Moreover, RAPDs enabled us to distinguish the four groups of the genus Olea and O. subg. Tetrapilus was also closer to Nestegis and Chionanthus than to the other Olea species. Thus, Tetrapilus should be considered as a genus. Our results sustained the common origin of the sections Ligustroides and Olea.

Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5'-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH).

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA3-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n = 50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA3-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants - (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.

Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis.

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses. html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5' terminus of the user-specified primer to the 3' terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.