Intraspecific ITS polymorphism in Scutellospora castanea (Glomales, Zygomycota) is structured within multinucleate spores.

Polymorphism of the internal transcribed spacers (ITS) of the ribosomal DNA in Scutellospora castanea (Glomales, Zygomycota) and its organization among spores were evaluated. Polymerase chain reaction (PCR) amplification with ITS1/ITS4 primers yielded several fragments of different lengths, even from single spores. Fragments produced from multisporal DNA were cloned and grouped into 6 ITS types by PCR-RFLP and sequence analysis. Five type-specific primers were designed. Spores were then analyzed by PCR and amplification profiles revealed that they were qualitatively different one from another due to the presence or absence of some ITS types. Intrasporal segregation of ITS variant length types was also shown, by PCR experiments, utilizing diluted fractions of nuclear suspensions from single spores. The results demonstrate the mainly multikaryotic condition of the spores of S. castanea.     

Phylogenetic analysis of a dataset of fungal 5.8S rDNA sequences shows that highly divergent copies of internal transcribed spacers reported from Scutellospora castanea are of ascomycete origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.     

Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta.

A highly repeated DNA sequence from the genome of an arbuscular mycorrhizal fungus has been isolated and characterized. This 1,202-bp sequence (SC1) represents about 0.24% of the Scutellospora castanea genome, estimated to be 1 pg by flow cytometry. The sequence was shown to be a Scutellospora-specific probe in Southern blots and dot blot hybridizations. After complete sequencing of SC1, PCR primers were generated and used to amplify a 907-bp fragment from spores of S. castanea or from colonized Allium porrum roots. No amplification products were obtained with DNA from either spores or mycorrhizal root of other species of arbuscular mycorrhizal fungi. These primers were sufficiently specific for unequivocal detection of S. castanea in planta.     

Levels of diversity in endomycorrhizal fungi (Glomales,Zygomycetes) and their role in defining taxonomic and non-taxonomic groups

Diversity in glomalean fungi is manifested at the molecular, morphological, and ecological levels. Characters at any of these levels can be ordered into hierarchical patterns defining taxonomic groups if they are conserved enough to be heritable through geologic time in all descendants of a common ancestor. At present, only morphological characters associated with mode of spore formation and in subcellular structure of spores are sufficiently stable and diverse to recognize at least 150 species. Ontogenetic comparisons indicate that species integrity, despite asexual reproduction, is the result of rigid internal constraints imposed on variation during the process of spore subcellular differentiation. Epigenetic factors dominate because the differentiation sequence is linear and each new stage is causally linked to preceding stages. Some morphological characters of the fungal mycelium also exist, but they define more inclusive groups at the family level and above. Most diversity in the mycorrhizae consists of life-history traits associated with abundance and architecture of fungal components, their rate of formation and longevity, and their cost in the symbiosis. These characters participate in processes at the molecular and ecological levels, so they are autonomous from morphological determinants. They often are labile or affected by external environmental conditions, so fewer stable taxonomic characters are likely to be discovered. Instead, molecular and ecological diversity has greater potential to define; (a) niche specificity of organisms/populations and (b) causal processes linked to host-fungus compatibility and mycorrhizal efficiency. Any taxonomic characters that relate to mycorrhizal functions will come only from comparative studies involving organisms from shared habitats rather than those having shared spore morphologies.     

DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus,Scutellospora castanea

A technique has been developed to efficiently extract purified, restrictable genomic DNA from spores of different arbuscular mycorrhizal fungi in order to begin detailed investigations of the genome of the Glomales. The protocol yielded variable amounts of DNA depending on the fungal species; for Scutellospora castanea and Gigaspora rosea it reached values of 1.5–2 ng/spore. EcoRI digests of DNA from S. castanea were cloned into pUC18 and about 1000 recombinant DNA clones were obtained. Of those screened, 50 contained inserts of 500–7000 bp. Selected inserts detected DNA sequences from S. castanea spores or roots infected by this fungus, but not from nonmycorrhizal roots. This is the first report of a partial genomic library from an arbuscular mycorrhizal fungus.     

Molecular analysis of Gigaspora (Glomales, Gigasporaceae)

This work presents a cooperative effort to integrate new molecular (isozyme and SSU analyses) characters into the morphological taxonomy of the genus Gigaspora (Glomales). Previous analyses of published Gigaspora SSU sequences indicated the presence of a few polymorphic nucleotides in the region delimited by primers NS71-SSU 1492'. In our study, the SSU of 24 isolates of arbuscular mycorrhizal (AM) fungi from the Gigasporaceae were amplified and the NS71-SSU 1492' region was directly sequenced. The corresponding sequences of four more isolates of AM fungi from Gigasporaceae, already published, were also included in our analyses. Three Gigaspora groups were identified on the basis of a 6 nucleotide-long 'molecular signature': Gigaspora rosea group ( G. rosea + G. albida ), Gigaspora margarita group ( G. margarita + G. decipiens ) and Gigaspora gigantea , which constituted a group by itself. The isozyme profiles (malate dehydrogenase, MDH) of 12 of these 28 isolates, and seven other isolates not sequenced, were compared. The results obtained further supported the grouping of isolates provided by the SSU analysis. Both SSU and MDH analysis indicated that two out of the 35 isolates had been misidentified, which was confirmed when their morphology was reassessed. The use of the Gigaspora intrageneric molecular signature as a quick, unambiguous and objective method to recognize Gigaspora isolates under any (field or laboratory) experimental conditions is suggested.     

Ancestral lineages of arbuscular mycorrhizal fungi (Glomales)

Using new and existing 18S rRNA sequence data, we show that at least five species of glomalean fungi lie outside the previously defined families and diverged very early in the evolution of that group. These five fungi would have been missed by many previous ecological studies because their sequences are not well matched to available taxon-specific primers and they do not stain well with the standard reagents used for morphological analysis. Based upon spore morphology, these species are currently assigned to Glomus and Acaulospora, and two of the species are dimorphic, exhibiting spore stages of both genera. This suggests that dimorphic spores are the ancestral state for the order and that one or the other morphology was lost in various lineages. Our analyses also show that Geosiphon pyriforme, a symbiont with cyanobacteria, is not necessarily a sister group of the Glomales; instead, it may be derived from mycorrhizal ancestors.     

Subtelomeric expression regions of Borrelia hermsii linear plasmids are highly polymorphic

Borrelia hermsii, a relapsing fever agent, undergoes multiphasic antigenic variation to evade its host's immune response. Serotype specificity is determined by variable membrane lipoproteins, Vmps, which are expressed from genes located near the end of a linear plasmid. Using the polymerase chain reaction and primers representing the promoter of the active vmp and a conserved telomeric sequence, we characterized the subtelomeric expression regions of the 25 known serotypes of strain HS1. The distance from the promoter to the telomere fell into three size classes of approximately 1.0, 1.5, and 2.5 kilobases. In the sequenced serotypes the size differences were accounted for by variable lengths of the vmp genes and intervening sequences between 3' end of the vmp gene and the start of a downstream homology block. The degree of nucleotide identity between different vmp genes, or between the different 3' flanking DNA varied from 39-78%. Thus, there is length and sequence variability not only between vmp genes themselves but also between the 3' flanking regions of vmp genes.     

Isolation and characterization of highly polymorphic microsatellites in tea (Camellia sinensis)

Relatively little is known about the diversity and origins of tea. The highest value tea products are sold on the basis of their region of origin but there are currently no methods available to verify the claims made on packages. We have developed 15 microsatellite loci for tea. These have been evaluated for polymorphism in a set of tea clones to determine their usefulness for authentication purposes. The majority of the microsatellites developed proved to be highly polymorphic both between and within different geographical origins and offer the potential to investigate the population genetics and genetic origins of tea.     

Isolation and characterization of highly polymorphic microsatellites in the aquatic plant,Nymphoides peltata (Menyanthaceae)

Ten microsatellite loci were described for conservation design of a threatened clonal aquatic plant, Nymphoides peltata. The microsatellite loci obtained through the construction of an enriched library were polymorphic (2–6 alleles per locus) and exhibited high levels of observed (0.333–0.889) and expected (0.284–0.765) heterozygosities. All microsatellite loci were expected to be useful for identification of genets and evaluation of genetic diversity of this species.     

Arbuscular fungi and mycorrhizae (Glomales) of the Hel Peninsula,Poland

In the years 1985–1989, the occurrence of arbuscular fungi and mycorrhizae on the Hel Peninsula (Poland) was investigated with the help of 45 soil and root samples collected under 20 plant species of eight families. Except for Zea mays, the other plant species were from uncultivated sites. All soil samples contained spores of arbuscular fungi, of which about 45% were of the genus Glomus. Acaulospora spp. preferred members of the Cupressaceae. Spores of Gigaspora occurred rarely and only in two plant families. Glomus spp. were most frequently associated with plants of the Rosaceae, and species of Scutellospora were found at markedly higher frequencies among roots of plants of the Gramineae and Cupressaceae. A total of 29 spore-forming species and Glomus tenue (a fungus recognizable by its distinctive infections) were found. The most frequently recovered fungus, Glomus tenue, was present in roots of 56.8% of examined plants. Of the spore-forming fungi, the most frequently isolated spores were those of Scutellospora dipurpurascens, then Glomus constrictum, Acaulospora 61, and Glomus microcarpum. The overall spore density in examined samples averaged 99.8 in 100 g dry soil in the range 1 to 547, and was highest in a sample taken from around roots of Festuca arundinacea. The dominant fungi forming spores in sampled soils were Glomus constrictum, Glomus microcarpum, and Scutellospora dipurpurascens. The average species density was 3.9 in 100 g dry soil in the range 1 to 10, and was highest in Corynephorus canescens, Rosa canina, and Thuja occidentalis. Levels of colonization by arbuscular fungi ranged from 0.0 to94.0% (mean 23.3%) of the root length and were highest in Festuca arundinaceae and Zea mays.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Two highly polymorphic CA repeats in the Menkes gene (ATP7A)

Two highly polymorphic CA repeats have been identified in the Menkes gene (ATP7A). These repeats should be useful for prenatal diagnosis and carrier detection in families with Menkes disease and X-linked cutis laxa. The observed heterozygosity for these two repeats was 0.778 and 0.60 in Centre d'Etude du Polymorphisme Humaine (CEPH) families.     

Additional highly polymorphic microsatellite (STR) loci for estimating kinship in rhesus macaques (Macaca mulatta)

Thirty-four short tandem repeat (STR) loci, not previously studied in rhesus macaques, were amplified by PCR. About one third of these were found to clearly and reliably amplify and exhibit high levels of genetic heterogeneity even in relatively inbred populations. These loci, together with 11 loci previously studied, were sufficiently informative to discretely differentiate between related and unrelated pairs and, in most cases, between parent/offspring and other relative pairs. An even greater number of hypervariable STR loci might be required to distinguish between half-sib and full-sib pairs in most rhesus populations.     

Large restriction fragments containing poly-TG are highly polymorphic in a variety of vertebrates.

Southern blots of genomic DNA from a variety of species digested by restriction endonucleases having a four-bp specificity, were probed with a bovine genomic clone consisting of seven tandem poly-TG stretches separated by a 29bp linker sequence. Highly variable DNA 'fingerprint' patterns were obtained in chicken, sheep, and horse, moderately variable DNA 'fingerprints' in mouse and man, and a monomorphic pattern in Drosophila. In chicken, horse and man a (TG)10 synthetic oligonucleotide probe gave results identical to those given by the bovine probe. Furthermore, in chicken the DNA fingerprint variation showed typical Mendelian inheritance and differed from the fingerprints obtained with Jeffreys 33.6 and M13 minisatellite probes. Thus, for a variety of vertebrate species, poly-TG-containing probes can uncover useful genetic variation.     

Isolation and characterization of highly polymorphic microsatellite loci in the bladder campion,Silene vulgaris (Caryophyllaceae)

This study reports the isolation and characterization of seven highly polymorphic microsatellite loci in Silene vulgaris (Caryophyllaceae). The loci were isolated from two libraries constructed from genomic DNA enriched for CA and GA repeats. These markers yielded nine to 40 alleles per locus (mean 22.1) in a survey of 45 individuals from a single population located in the western Swiss Alps. Average observed heterozygosity ranged from 16.2 to 77.4%. These microsatellite loci should be valuable tools for studying fine‐scale genetic structure.