ITS Polymorphism Research Articles

Multiple Origins of Lima Bean Landraces in the Americas: Evidence from Chloroplast and Nuclear DNA Polymorphisms

ABSTRACTCrop wild relatives that have experienced multiple and independent domestication events provide an excellent model for understanding adaptation processes in crop populations and a first and relevant aspect to investigate is the geographic origin of landraces. The aim of this research was to establish the origin of Mesoamerican and Andean Lima bean (Phaseolus lunatus L.) landraces by analyzing chloroplast DNA and ITS polymorphisms in a sample of 59 wild and 50 landrace accessions. According to seed size, genetic distance analyses, and haplotype networks, at least two independent domestication events are proposed. The first one would have taken place in the Andes of southern Ecuador–northwestern Peru and would have given rise to the large‐seeded landraces collectively known as the “Big Lima” cultivars. The second one would have taken place in central–western Mexico, more likely in the area to the north and northwest of the Isthmus of Tehuantepec. This event, along with post‐domestication migrations of landraces in South America, would have given rise to the great variety of small‐seeded Mesoamerican landraces that exist today. We did not find any evidence supporting the existence of two discrete groups within Mesoamerican landraces that might correspond to the previously proposed “Sieva” and “Potato” cultigroups. A severe reduction in genetic diversity because of domestication, known as the “founder effect”, was detected, which may have implications for the conservation of genetic resources in this species.

Diversity of Saccharomyces and non- Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non- Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.

High nrDNA ITS polymorphism in the ancient extant seed plant Cycas: Incomplete concerted evolution and the origin of pseudogenes

Molecular studies of six species from the ancient extant seed plant Cycas, covering a wide range of its morphological diversity and all major areas of distribution, revealed a high level of intra-individual polymorphism of the internal transcribed spacer (ITS1, 5.8S, and ITS2) region, indicative of incomplete nrDNA concerted evolution. Through a range of comparisons of sequence characteristics to functional cDNA ITS copies, including sequence length and substitution variation, GC content, secondary structure stability, the presence of a conserved motif in the 5.8S gene, and evolutionary rates, the PCR amplified divergent genomic DNA ITS paralogs were identified as either putative pseudogenes, recombinants or functional paralogs. This incomplete ITS concerted evolution may be linked to the high number of nucleolar organizer regions in the Cycas genome, and the incomplete lineage sorting due to recent species divergence in the genus. Based on the distribution of a 14bp deletion, an early evolutionary origin of the pseudogenes is indicated, possibly predating the diversification of Cycas. Due to their early origin combined with the unconstraint evolution of the ITS region in pseudogenes, they accumulate high levels of homoplastic mutations. This leads to random relationships among the pseudogenes due to long-branch attractions, whereas the phylogenetic relationships inferred from the functional ITS paralogs grouped the sequences in species specific clades (except for C. circinalis and C. rumphii). The findings of our extensive study will have a wide significance, for the evolution of these molecular sequences, and their utilization as a major marker for reconstructing phylogenies.

Multiple patterns of rDNA evolution following polyploidy in Oryza

Ribosomal ITS sequences are commonly used for phylogenetic reconstruction because they are included in rDNA repeats, and hence are ubiquitous and present in high copy number. Ribosomal rDNA repeats often undergo rapid concerted evolution within and between arrays. Interspecific hybridization merges divergent repeat types in a single nucleus, setting in motion evolutionary processes leading to coexistence, maintenance of paralogs, origin of novel sequence variants, loss of arrays, or inter-array sequence homogenization via concerted evolution. Here we examined ITS polymorphism within and among six Oryza tetraploids of varying genomic composition to infer the extent and direction of concerted evolution following allopolyploid speciation. We demonstrate that different polyploids have experienced varying fates, including maintenance or homogenization of divergent arrays, even among allopolyploids having the same genomic origins but in different geographic locations. Bidirectional concerted evolution, in which arrays become homogenized to alternative progenitor diploid types in different allopolyploid derivatives, is evident among species in one clade. Our results exemplify the panoply of outcomes for ribosomal DNA evolution following allopolyploid speciation.

Detection of rDNA ITS polymorphism in Rhizoctonia solani AG 2-1 isolates

The sequence variability of the ribosomal internal transcribed spacer regions ITS1 and ITS2, including the 5.8S gene, was investigated for Rhizoctonia solani isolates of anastomosis group (AG) 2-1. During PCR RFLP analysis of eight isolates, the restriction patterns of four isolates showed an excess of bands after restriction with the enzymes AvaII and/or HincII, which suggested the presence of more than one ITS region. By cloning the ITS region of six isolates sequence heterogeneity was detected in the isolates that showed an excess of bands in the PCR RFLP analysis; up to nine different ITS regions were identified within one isolate. The same level of diversity was found within the same isolate as among isolates. In the phylogenetic tree based on the rDNA ITS sequences of several AG 2-1 isolates, sequences derived from the same isolate did not form distinct clusters, questioning the relevance of further subdivision of heterogeneous AG 2-1 isolates based on the ITS region.

Internal Transcribed Spacer Polymorphism in Pseudo-nitzschia multistriata (Bacillariophyceae) in the Gulf of Naples: Recent Divergence or Intraspecific Hybridization?

The planktonic diatom Pseudo-nitzschia multistriata is a potentially toxic species recorded during late summer-autumn in the Gulf of Naples (Tyrrhenian Sea, Italy). We analysed the genetic structure by amplifying the internal transcribed spacer (ITS-1-5.8S-ITS-2) region of the ribosomal DNA of 44 strains isolated along 2 years. Polymorphism in the ITS region was detected by direct sequencing and the PCR-products from selected strains were thus cloned to assess intra-strain ITS variability. Strains clustered into three main types: type A and B - differing by 0.6% sequence divergence - and type A/B, showing both A and B variants within the same genome. The three types showed no differences in the large subunit sequences (LSU) of the rDNA, ultrastructure of the valve, secondary structure of ITS-1 and ITS-2, ploidy level and they were sexually compatible. Based on the results of these multiple approaches, we can state that the three ITS-types belong to the same reproductive unit (or "species" sensu Mayr [(1942). Systematics and the Origin of Species. Columbia University Press, New York]). We suggest that ITS polymorphism in P. multistriata may be related to the contemporary occurrence of different but still inbreeding populations which either diverged recently or originated in different geographic areas and became sympatric in the studied area.

Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus

Molecular studies of 19 species of the genus Pyrus L. revealed different degrees of intra-individual polymorphism of the internal transcribed spacer (ITS 1, 5.8S rDNA and ITS 2) region due to the existence of putative non-functional copies (pseudogenes), putative recombinants and non-concerted evolution among functional copies. Different types of ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions. Functional ITS copies were successfully obtained in all investigated accessions under the modified PCR conditions. All pseudogenes were highly divergent from their corresponding functional copies and formed a monophyletic group in the phylogenetic tree based on all paralogs, indicating they were of relatively early origin. Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence, existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism, while certain types of pseudogenes and some relict pseudogenes offered more credible clues for the evolutionary history of Pyrus species.

Differentiation of pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.

Coding of intraspecific nucleotide polymorphisms: A tool to resolve reticulate evolutionary relationships in the ITS of beech trees (Fagus L., Fagaceae)

The internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA (rDNA) have recently been found to display remarkable intraspecific polymorphism, a feature suggested as limiting their value for phylogenetic reconstructions. A comparative study of oligonucleotide motives and intraindividual nucleotide variability across all species of the tree genus Fagus (beech) shows, however, that this intraspecific ITS polymorphism follows a particular pattern, which can be used to detect reticulation and ancient polymorphism within the genus. Coding ITS polymorphisms as phylogenetically informative characters, moreover, resulted in better‐resolved phylogenies than traditional ‘base‐per‐base’ maximum parsimony and maximum likelihood analyses.

Distinguishing homokaryons and heterokaryons in Phellinus sulphurascens using pairing tests and ITS polymorphisms

Phellinus sulphurascens Pilát causes laminated root rot of coniferous species in both western North America (WNA) and Asia. Accurate somatic incompatibility tests for mapping population structures have been difficult to conduct for P. sulphurascens because no single, unambiguous criterion has allowed differentiation of homokaryotic and heterokaryotic isolates. In a population study of P. sulphurascens in WNA, two types of ITS sequences were found in the single spore and vegetative isolates. All single spore isolates (SSIs) had either ITS type-1 or type-2 whereas some vegetative isolates had both ITS types. The segregation pattern for inheritance of ITS, which we observed in SSIs from eight basidiocarps, suggested that each ITS type occurred in a different nucleus and that each basidiospore inherited only one ITS type. In four SSIs from Russia and eight heterokaryotic isolates from Japan, nine different ITS types, referred to as type-3 to -11, were detected. A variety of pairing tests conducted between known Asian and WNA homokaryon and heterokaryon isolates did not always give consistent results with respect to fungal mat morphologies and formation of demarcation lines. However, the ITS types that occurred after pairing tests did follow consistent patterns. Thus, using ITS polymorphisms and pairing tests between Asian tester isolates and 49 vegetative isolates from WNA, we were able to accurately distinguish between homokaryotic and heterokaryotic isolates.

Morphological and molecular diversity and phylogenetic relationships among anuran trypanosomes from the Amazonia, Atlantic Forest and Pantanal biomes in Brazil

We examined for the presence of trypanosomes in blood samples from 259 anurans (47 species from 8 families), the majority of which were from the Brazilian Amazonia, Atlantic Forest and Pantanal biomes. Trypanosomes were detected by a combination of microhaematocrit and haemoculture methods in 45% of the anurans, and 87 cultures were obtained: 44 from Hylidae, 22 from Leptodactylidae, 15 from Bufonidae, 5 from Leiuperidae and 1 from an unidentified anuran. High morphological diversity (11 morphotypes) was observed among blood trypanosomes from anurans of different species and of the same species as well as among trypanosomes from the same individual. Conversely, morphologically similar trypanosomes were found in anurans from distinct species and biomes. ITS and SSU rDNA polymorphisms revealed high diversity among the 82 isolates examined. Twenty-nine genotypes could be distinguished, the majority distributed in 11 groups. Phylogenetic relationships based on rDNA sequences indicated that isolates from more phylogenetically related anurans are more closely related. Comparison of anuran trypanosomes from Brazil and other countries revealed several new species among the isolates examined in this study. Phylogenetic relationships suggest that host restriction, host switching and overall ecogeographical structure may have played a role in the evolution of the anuran trypanosomes.

Congruence of morphological, reproductive and ITS rDNA sequence data in some Australasian Eunotia bilunaris (Bacillariophyta)

Clones identified as Eunotia bilunaris were isolated from two freshwater habitats in New Zealand and one in Tasmania. Experimental studies of sexual reproduction showed that the mating system was strictly heterothallic. All clones were morphologically similar and, in mixtures of compatible clones, were able to produce a viable F1, regardless of geographical origin. There was nevertheless some geographically related variation in ITS rDNA sequences, suggesting that dispersal and concerted evolution are unable to prevent allopatric divergence. Isolates from two of the three localities also showed significant ITS polymorphisms. Auxosporulation followed a similar pattern to that in other Eunotia species, being isogamous with a single functional gamete per gametangium; plasmogamy was effected via two fusing papillae. Details of auxospore structure, initial cell size, and vegetative valve metrics (width, striation densities) show that conspecificity with a recently studied European E. bilunaris is doubtful. The first SEM observations of the perizonium of Eunotia are presented and show that the central transverse band is apparently a split ring as in araphid pennate diatoms, in contrast to other raphid diatoms, except some Bacillariaceae.

Wax plants disentangled: A phylogeny of Hoya (Marsdenieae, Apocynaceae) inferred from nuclear and chloroplast DNA sequences

Hoya (Marsdenieae, Apocynaceae) includes at least 200 species distributed from India to the Pacific Islands. We here infer major species groups in the genus based on combined sequences from the chloroplast atpB-rbcL spacer, the trnL region, and nuclear ribosomal DNA ITS region for 42 taxa of Hoya and close relatives. To assess levels of ITS polymorphism, ITS sequences for a third of the accessions were obtained by cloning. Most ITS clones grouped by species, indicating that speciation in Hoya usually predates ITS duplication. One ITS sequence of H. carnosa, however, grouped with a sequence of the morphologically similar H. pubicalyx, pointing to recent hybridization or the persistence of paralogous copies through a speciation event. The topology resulting from the combined chloroplast and nuclear data recovers some morphology-based sections, such as Acanthostemma and Eriostemma, as well as a well-supported Australian/New Guinean clade. The combined data also suggest that morphological adaptations for ant-symbiosis evolved at least three times within Hoya.

Low variability of Internal Transcribed Spacer rDNA andtrnL (UAA) intron sequences of several taxa in the Festuca ovina aggregate (Poaceae)

Identification and classification of numerous Festuca species is still a difficult problem due to the close morphological resemblance. The most difficult fine fescues to identify belong to the Festuca ovina aggregate, which is the largest group in the genus Festuca. Many taxons are considered to be separate species based on quantitative taxonomic characters, differences in ploidy level or the structure of sclerenchyma cells. In order to evaluate the taxonomic value of DNA-based markers, sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and the chloroplast trnL (UAA) intron was performed in the ten most problematic fine fescues belonging to the Festuca ovina aggregate. Intraspecific ITS variants were found in a single case while in other cases only intragenomic ITS polymorphisms were detected with 1-2 ambiguous positions. Among the sequences of the trnL (UAA) intron even intragenomic polymorphisms were not detected in any of the Festuca species studied. Thus, the results do not support the species status of these ten taxa.

Genetic characterization of selected Trifolium species as revealed by nuclear DNA content and ITS rDNA region analysis

Thirty-one clover species with significant agronomic value, originating from Eurasia, Africa and America, were analyzed to reveal their nuclear DNA content and propidium iodide/4,6-diamidino-2-phenylindole (PI/DAPI) ratio. Chromosome numbers of the same accessions were determined for calculation of 1Cx genome size values, and the relationships between species were studied based on ITS-rDNA analysis. The nuclear DNA content of the majority of species was determined for the first time and revealed large variations among investigated species. The somatic nuclear DNA content ranged from 0.688 pg ( T. ligusticum) to 7.375 pg ( T. burchellianum), exhibiting a 10.7-fold difference. Since polyploidy is characteristic of several species of the genus Trifolium, 1Cx values provided the most informative measure of interspecific DNA content variation. Analysis of nuclear DNA content based on 1Cx-values within the Lotoidea section revealed that the basic genome size values of six African and American species were twice those of the six Eurasian species of the same section. Measurements of nuclear DNA content using propidium iodide/DAPI staining highly correlated ( r = 0.99), the average PI/DAPI ratio of 29 species was 1.043. Clustering based on ITS polymorphisms showed high relationship with botanical sections with the only exemption in the section Lotoidea, which was divided mainly according to its origin (American, African or Eurasian). Genome size data and the ITS clustering were highly correlated in sections Chronosemium, Involucrarium and Vesicaria and varied approximately two-fold within sections Trifolium, Lotoidea (African origin) and Lotoidea (Eurasian origin).

ITS polymorphisms at inter- and intra-strain levels of Trametes versicolor

ITS polymorphisms at inter- and intra-strain levels of Trametes versicolor

Patterns of molecular and morphological differentiation in Fagus (Fagaceae): phylogenetic implications

To study phylogenetic relationships among species of Fagus, the internal transcribed spacer regions ITS1 and ITS2 of the nuclear ribosomal DNA and morphological data were analyzed. Both molecular and morphologically based phylogenies suggest that Eurasian species of Fagus subgenus Fagus are basal to the North American Fagus grandifolia. The subgenus Fagus is a paraphyletic group basal to three East Asian species forming the subgenus Engleriana. Due to a considerably large amount of DNA polymorphism, relationships among basal species of Fagus could not be entirely resolved when analyzing ITS sequences with standard methods. Morphological trees helped to resolve more clearly relationships within the subgenus Fagus. The East Asian F. hayatae is suggested to be basal to the rest of the genus. This hypothesis is further supported by distinctive patterns of nucleotide variability found for ITS regions, allowing for basic and derived types to be distinguished. The high degree of ITS polymorphism within Fagus can be explained by (1) the complex evolutionary behavior of this marker, (2) the stenoecious ecological characteristic of Fagus with respect to its continuous geographic range throughout much of the Cenozoic, and (3) the absence of major radiations into further habitats as occurred in other Fagaceae.

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25–35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7–13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.

Non-homogenized ITS regions in the parasitic nematode Cooperia oncophora.

Here, the validity of the assumption of concerted evolution of ribosomal regions in larval and adult Cooperia oncophora was assessed. In each of 4 individuals of this parasitic nematode, at least 78% of the sequences comprised different ITS variants. This implies that concerted evolution is not acting, which is corroborated by the scarcity of signatures of gene conversion and recombination. Mis-incorporation of nucleotides and illegitimate PCR-induced recombination turned out to be unlikely, and positions with substantial frequencies of alternative nucleotides corresponded to ambiguous positions in published ITS2 sequences of this and other Cooperia species based on direct sequencing. The ITS regions of each individual C. oncophora displayed a significant excess of unique mutations in agreement with expansion of the ribosomal gene family. Interesting corollaries of the inferred size changes of this gene family are genomic rearrangements that occur during larval development such as multiple rounds of endoduplication (in Rhabditidae), chromatin diminution (in Ascaris), and non-compensatory mutations on the secondary structure of the ITS2. It is yet unknown which process is important in trichostrongylids. Finally, although it can not be rigorously assessed in Cooperia, the ITS polymorphisms can readily be envisioned to affect phylogenetic reconstructions of closely related nematodes.

Genetic diversity in potato field populations of Thanatephorus cucumeris AG-3, revealed by ITS polymorphism and RAPD markers

DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability in relation to agronomic and regional factors among 60 isolates of Thanatephorus cucumeris (anamorph Rhizoctonia solani) collected from lesions on potato stems or sclerotia of potato tubers. Based on comparative sequence analysis it was shown that all isolates belonged to anastomosis group 3 subgroup Potato Type (AG-3 PT). ITS1 sequence polymorphisms were found within 45 of the 60 isolates showing that different types of the ITS-region are present in individual isolates. Cloning and sequence analysis of the ITS1 region from three selected isolates with sequence polymorphism showed that two different ITS1-types were present in each isolate. RAPD analysis identified 51 RAPD-phenotypes among the 60 investigated isolates indicating a high level of diversity within the subgroup AG-3 PT. Putative clonal isolates with identical RAPD- and ITS1-types were identified within fields, and in one case the same phenotype was found in two different fields separated by several hundred kilometers. Population subdivision analysis based on phenotypic as well as genotypic diversities showed differentiation among populations from different fields when isolates were sampled from tubers, indicating restricted gene flow among soil populations. Low differentiation was seen among field populations sampled from stems, indicating that gene flow is taking place. The population structure was not influenced by the previous crop in the rotation nor by the two cultivars 'Sava' and 'Bintje'.