Systematic studies on some species of the genus Artemisia: biomolecular analysis

ABSTRACT

The internal transcribed spacers (ITS) of the ribosomal DNA gene of 11 taxa of the genus Artemisia were sequenced and compared with other 14 species taken from GenBank. The aims of this study are to clarify phylogenetic relationships for 25 taxa within the genus Artemisia, and to highlight the phylogenetic position of some species of geobotanical interest from the Alps or from other European areas. The results support the monophyly of the genus Artemisia, and the presence of the five main clades, corresponding to the morphologically based sections, Absinthium, Artemisia, Seriphidium, Dracunculus and Tridentatae. Only A. annua and A. genipi are not classified in the section in which they were traditionally included: A. annua is assigned to Seriphidium and not Artemisia, and A. genipi to Absinthium and not Artemisia. The basal structure of the tree differed in the 45 equally parsimonious MP trees, and thus appeared as a polytomy in the consensus tree. This does not allow us to completely solve the relationships among the clades. The molecular data are complementary with the morphological and biogeographical information and all are essential to draw valid conclusions on the relative closeness of the various taxa.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

Systematics of the species of the yeast genus Saccharomyces associated with the fermentation industry

Summary The so-called wine yeasts Saccharomyces cerevisiae, S. chevalieri, S. bayanus, S. italicus and S. uvarum are characterized by high ethanol tolerance and fermentation velocity. They are ecologically related, being predominantly associated with grape must and wine, and are taxonomically indistinguishable. The only significant physiological differences are between the ability to ferment certain sugars. A taxonomic revision of more than 1,000 strains isolated during the past 50 years and belonging to the above species showed extreme instability in the ability to ferment different sugars. The relationships between these yeasts were examined for DNA base composition and DNA-DNA reassociation. The G+C ranged from 37.6% to 39.0% while optical reassociation experiments defined a first group of species (Saccharomyces cerevisiae, S. chevalieri and S. italicus) exhibiting high base sequence complementarity (>90%). S. bayanus and S. uvarum also showed a high degree of relatedness. Low homology values (30%) indicate that the two groups of species are not closely related. While it is proposed to combine S. cerevisiae, S. chevalieri and S. italicus into one single species under the oldest epithet Saccharomyces cerevisiae, a study of a larger number of strains is recommended before considering the taxonomic position of S. bayanus and S. uvarum.     

Protein fingerprinting of Saccharomyces isolates with therapeutic relevance using one- and two-dimensional electrophoresis

In therapeutic products and preparations Saccharomyces cerevisiae is used because of its nutritive properties. Moreover, so called Saccharomyces boulardii yeasts are used in the prevention and treatment of several types of diarrhea. Taxonomically however, S. boulardii is not accepted as a distinct species. The protein fingerprint obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for all isolates and therefore confirmed the designation of S. boulardii to the species S. cerevisiae. In contrast, using native polyacrylamide gel electrophoresis, 12 different protein fingerprints were detected, and allowed grouping of the product isolates. The spot patterns obtained by two-dimensional electrophoresis revealed a large degree of resemblance, however, small qualitative expression differences could be detected as well. Firstly, a spot having an isoelectric point of approximately 6 and 30 kDa could not be detected in S. boulardii yeasts. Secondly, nine different formations of spots occurred in the region around 16 kDa and pH 6. Therefore, on the one hand, it could be demonstrated that all of the product isolates belong to the same species, and on the other hand, it was possible to extensively subdivide the strains. In particular, two-dimensional electrophoresis allowed clustering of so called S. boulardii strains within the species S. cerevisiae.     

Differentiation of European cattle by AFLP fingerprinting

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.     

Differentiation of selected species of Belgrandiella and the redefined genus Graziana (Gastropoda: Hydrobiidae)

Seven species of the genus Belgrandiella A. J. Wagner, 1928 ( sensu lato ), from Austria and Italy are described in detail. The genus Graziana Radoman, 1975, is redefined on a newly discovered character of the stomach, the shield caecum. This is remarkable, since hydrobiid genera are usually defined by characters of the reproductive system. Two species, B. pelerei sp. nov. and G. klagenfurtensis sp. nov. , are new to science. Allozyme electrophoresis of ten populations of these seven species revealed an unusual lack of variability within each population. This is explained as a consequence of the presumed mode of dispersal of these crenobiontic snails, i.e. aerial transport with insects. Genetic distances between conspecific populations and congeneric species fall within the expected range. The only exception is the genetic identity of G. klagenfurtensis and G. lacheineri Küster, 1853. This identity is probably due to the recent origin of G. klagenfurtensis , whose area was covered with ice during the Wtirm glaciation. The high distance values between Belgrandiella and Graziana justify the generic separation based on a character of the digestive system. This is also confirmed through the comparison with three species of the genus Hydrobia Hartmann, 1821, which differs in all systematically relevant aspects from the other two genera.     

SED1 polymorphism within the genus Saccharomyces

The SED1 gene is characterised by abundant length and sequence polymorphisms within the species Saccharomyces cerevisiae, due to the expansion and contraction of minisatellite-like sequences located within the ORF. A survey of the SED1 ORFs of 26 yeasts ascribed to the species S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus, S. cariocanus, S. kudriavzevii and S. mikatae revealed SED1 gene length and sequence variations between the species of the genus. Moreover, results obtained by Neighbour-Joining analysis of a dataset comprising the partial predicted amino acid sequences of SED1 ORFs agreed with the phylogenetic relationships of the seven species. Thus, the SED1 gene may represent a further molecular target for the identification of Saccharomyces isolates.     

Revealing constitutively expressed resistance genes in Agrostis species using PCR-based motif-directed RNA fingerprinting

Agrostis species are mainly used in athletic fields and golf courses. Their integrity is maintained by fungicides, which makes the development of disease-resistance varieties a high priority. However, there is a lack of knowledge about resistance (R) genes and their use for genetic improvement in Agrostis species. The objective of this study was to identify and clone constitutively expressed cDNAs encoding R gene-like (RGL) sequences from three Agrostis species (colonial bentgrass (A. capillaris L.), creeping bentgrass (A. stolonifera L.) and velvet bentgrass (A. canina L.)) by PCR-based motif-directed RNA fingerprinting towards relatively conserved nucleotide binding site (NBS) domains. Sixty-one constitutively expressed cDNA sequences were identified and characterized. Sequence analysis of ESTs and probable translation products revealed that RGLs are highly conserved among these three Agrostis species. Fifteen of them were shown to share conserved motifs found in other plant disease resistance genes such as MLA13, Xa1, YR6, YR23 and RPP5. The molecular evolutionary forces, analysed using the Ka/Ks ratio, reflected purifying selection both on NBS and leucine-rich repeat (LRR) intervening regions of discovered RGL sequences in these species. This study presents, for the first time, isolation and characterization of constitutively expressed RGL sequences from Agrostis species revealing the presence of TNL (TIR-NBS-LRR) type R genes in monocot plants. The characterized RGLs will further enhance knowledge on the molecular evolution of the R gene family in grasses.     

Molecular fingerprinting of isolates of the genus Peptostreptococcus using rRNA genes from Escherichia coli and P. anaerobius.

Restriction fragment length polymorphisms (RFLPs) of rRNA genes were evaluated as a tool for intra- and interspecies differentiation of Peptostreptococcus isolates. RFLPs from a collection of 20 clinical isolates and five ATCC strains representing five Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros and P. prevotii) were obtained by hybridization of Southern blots of HindIII- or EcoRI-digested genomic DNA with three probes: probe A, a 0.98 kb HindIII fragment with a partial 16S rRNA gene sequence from P. anaerobius ATCC 27337; probe B, cloned Escherichia coli rrnB operon in plasmid pKK3535; and probe C, E. coli 16S and 23S rRNA. The hybridization patterns varied, but all yielded RFLPs useful for both intra- and inter-species differentiation. RFLPs of P. asaccharolyticus clinical isolates were closely related to each other and differed significantly from those of the ATCC type strains. The profiles of P. prevotii differed from those of the other four species studied, and based on the HindIII- and EcoRI-generated RFLPs, the strains in this species are more heterogeneous than the other four species studied.     

Identification and genetic fingerprinting of Brachyspira species

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.     

Assessment of discriminatory power of three different fingerprinting methods based on killer toxin sensitivity for the differentiation of Saccharomyces cerevisiae strains

AIMS: A panel composed of 44 taxonomically certified strains of Saccharomyces cerevisiae of different origin was used to evaluate the discriminatory power of three different fingerprinting methods based on sensitivity towards 24 killer toxins. METHODS AND RESULTS: Binary data matrix (BDM), triplet data matrix (TDM) and numerical data matrix (NDM) were used as fingerprinting methods. NDM possessed the highest discriminatory power, assessed through the Simpson's, and Hunter and Gaston's indices for the measurement of diversity. The upper limits of fingerprinting ability expressed by the three above methods have been also discussed. CONCLUSIONS: NDM determined a significant increase of discriminatory power than the use of BDM or TDM, in terms of an effective amplification of their fingerprinting efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The NDM fingerprinting method could find application in control laboratories for the discrimination of yeast strains of industrial importance or covered by patent.     

The application of the acoustic spectrophonometer to biomolecular spectrometry: a step towards acoustic "fingerprinting"

A tunable acoustic biosensor for investigating the properties of biomolecules at the solid-liquid interfaces is described. In its current, format the device can be tuned to frequencies between 6.5 MHz and 1.1 GHz in order to provide a unique detection feature: a variable evanescent wave thickness at the sensor surface. The key to its successful implementation required the careful selection of antennae designs that could induce shear acoustic waves at the solid-liquid interface. This non-contact format makes it possible to recover resonant shear acoustic waves over 100 different harmonic frequencies as a result of the electrical characteristics of the spiral coil. For testing this multifrequency sensing concept the surface of a quartz disc was exposed to solutions of immunoglobulin G (IgG) to form an adsorbed monolayer, whence protein A and IgG were added again in order to form multilayers. Spectra at frequencies between 6 and 600 MHz were generated for each successive layer and revealed two characteristic phases: an initial phase at the low megahertz frequencies consistent with the conventional Sauerbrey relation, and a possible additional phase towards the high megahertz to gigahertz frequencies, that we believe relates to the structure of the biomolecular film. This two-phase behaviour evident from differences between high and low frequencies, rather than from any distinct frequency transition, was anticipated from the reduction in evanescent wave thickness down to nanometre dimensions, and thin film resonance phenomena that are known to occur for film and fluid systems. These measurements suggested that the single element acoustic biosensor we present here may form the basis from which to generate acoustic molecular spectra, or "acoustic fingerprints", in a manner akin to optical spectroscopy.