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SNP analysis of ITS1-5.8S-ITS2 rDNA loci in modern and ancient melons (Cucumis melo)
120-124Views:89ITS (internal tanscribed spacer) profiles of the aDNA (ancient DNA) of seed remains extracted from an extinct sample recovered from the 15th century (Budapest, Hungary) were compared to 31 modern melon cultivars and landraces. An aseptic incubation followed by ITS analysis was used to exclude the exogenously and endogenously contaminated (Aspergillus) medieval seeds and to detect SNPs in ITS1-5.8S-ITS2 region of rDNA (ribosomal DNA). SNPs were observed at the 94–95 bp (GC to either RC, RS or AG) of ITS1; and at 414 bp (A-to-T substitution), 470 bp (T to Y or C), 610 bp (A to R or G) of ITS2. The results facilitate the final aim of molecular and morphological reconstructions of ancient melon tpyes.
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The effect of β-glucan, carotenoids, oligosaccharides and anthocyanins on bacteria groups of excreta in broiler chickens
15-20Views:175This study was conducted to examine the effect of natural compounds, such as β-glucan, carotenoids, oligosaccharides, and anthocyanins in the diet on bacteria gropus of excreta in Ross 308 broiler chickens. Chickens were fed 5 diets: control (basal) diet, a diet supplemented by β-glucan at 0.05%, and diets supplemented by carotenoids, oligosaccharides, or anthocyanins at 0.5% of each compound. On experimental day 19, excreta were collected to determine the proportion of Lactobacillus, Bifidobacterium, Campylobacter, Clostridium, and Escherichia coli. Samples were collected aseptically and snap-frozen in liquid nitrogen. Bacterial DNA was isolated from samples, then polymerase chain reaction using primer pairs designed to the 16S rDNA of bacterial groups were applied to define the proportion of the mentioned bacteria. Another universal primer pair was used to amplify a region of 16S rDNA of all the examined bacteria. Proportion of each bacterial groups was determined relatively to the intensity of universal PCR product band by gel documenting system and ImageLab software. Based on the results, carotenoids and anthocyanins increased the proportion of Bifidobacterium, which might imply the beneficial effects of the mentioned compounds on the bacteria composition of excreta.
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Sequence stability at SSR, ISSR and mtDNA loci of common millet (Panicum miliaceum) from the middle ages
10-19Views:86Seed remains of medieval millet, recovered from a 15th century layer (King’s Palace, Budapest, Hungary), showed reddish yellow grain color after rehydrating on tissue culture medium that was close to grain color of modern cultivar Omszkoje. aDNA of medieval c. millet was extracted successfully, analyzed and compared to modern common millets by ISSR, SSR, CAPS and mtDNA. Analyses of fragments and sequences revealed
polymorphism at seven ISSR loci (22 alleles) and at the 5S-18S rDNA locus of mtDNA. CAPS analysis of the 5S-18S rDNA fragment revealed no SNPs in the restriction sites of six endonucleases TaqI, BsuRI, HinfI, MboI, AluI and RsaI. Sequence alignments of the restriction fragments RsaI also revealed
consensus sequence in the medieval sample compared to a modern variety. Morphological characterization of twenty common millet (Panicum miliaceum L., 2n=4×=36) cultivars and landraces revealed four distinct clusters which were apparently consistent with the grain colors of black, black and brown, red, yellow, and white. In the comparative AFLP, SSR and mtDNA analysis modern millet cv. ‘Topáz’ was used. AFLP analysis revealed that extensive DNA degradation had occurred in the 4th CENT. ancient millet resulting in only 2 (1.2%) AFLP fragments (98.8% degradation),
compared to the 15th CENT. medieval millet with 158 (40%) fragments (60% degradation) and modern millet cv. ‘Topáz’ with 264 fragments (100%). Eight AFLP fragments were sequenced after reamplification and cloning. Microsatellite (SSR) analysis at the nuclear gln4, sh1, rps28 and rps15 loci of the medieval DNA revealed one SNP (single nucleotide polymorphism) at the 29th position (A to G) of rps28 locus compared to modern millet.
Mitochondrial (mtDNA) fragment (MboI) amplified at the 5S-18S-rDNA locus in the medieval millet showed no molecular changes compared to modern millet. The results underline the significance of survived aDNA extraction and analysis of excavated seeds for comparative analysis and molecular reconstruction of ancient and extinct plant genotypes. An attempted phenotype reconstruction indicated that medieval common millet showed the closest morphological similarity to modern millet cultivar Omszkoje. -
Isolation and identification of endophytic fungi connected to Grapevine Diseases, from the Tokaj wine region, Hungary
61-66Views:149Grapevine Trunk Diseases (GTD) is one of the most important diseases in vineyards worldwide, which can be found in Hungarian vineyards as well. In Hungarian wine regions there is very little information about the occurrence of pathogens which cause GTD, in case of Tokaj wine region there is no knowledge about that, what kind of pathogens can be found in the vineyards.
The objective of our research is to assess the situation and occurrence of GTD in Tokaj wine region in cooperation with local specialists, as well as identification of pathogens which were isolated from the diseased trunks by morphological and genetic basis.
We were able to isolate endophytic fungi from all sampled grape trunk. The majority of them were determined as Diplodia seriata not only based on colony morphology, but also determined by rDNA sequences.
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Phylogenetic studies of Phoma species by maximum likelihood analysis
37-46Views:104The cosmopolitan Phoma genus contains mainly phytopathogenic, opportunistic parasite, and saprophyte fungal species. Up to now the characterization of Phoma species and other taxa of Phoma has so far been determined on the basis of morphology on standardized media, and gene sequence analysis was only used as a confirmative or distinctive complement.
In this study we have tried to study phylogenetic relationships by maximum likelihood method in the Phoma genus. We employed a part of the gene responsible for the synthesis of translation elongation factor 1 subunit alpha protein (tef1) containing both introns and exons, a part of the gene responsible for synthesis of tubulin protein and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA as potential genetic markers to infer phylogenetic relationships among different Phoma taxa. Twenty-four isolates of eleven different Phoma species were firstly characterised by morphologically, and then their tef1, tubulin and ITS sequences were sequenced and analysed by maximum likelihood method carried out by PAUP*4.0b program. According to constructed phylogenetic trees, the different Phoma taxons are well separated. However these trees do not support the traditional Phoma sections based on morphological characterization.
The maximum likelihood analyses of all three sequences confirmed that the Phyllosticta sojicola species is clustered with the Phoma exigua var. exigua group and the Phoma sojicola is grouped with Phoma pinodella group. The experienced molecular evidences initiate the demand of reclassification of formerly mentioned soybean pathogens. -
Phylogenetic analysis of Phoma species
100-107Views:92The cosmopolitan Phoma genus contains mainly phytopathogenic, opportunistic parasites, and saprophyte fungal species. Up to now, the characterization of Phoma species and other taxa of Phoma has been determined on the basis of morphology on standardized media, and gene sequence analysis was only used as a confirmative or distinctive complement.
In this study, we tried to find molecular markers which can be used as phylogenetics markers in the molecular based classification in the Phoma genus.
We employed a part of the translation elongation factor 1 subunit alpha (EF-1α=tef1) containing both introns and exons and ITS region containing the internal transcribed spacer regions 1 and 2 and the 5.8S rDNA, as potential genetic markers to infer phylogenetic relationships among different Phoma taxa. Twelve different Phoma species sequences were analysed together with the closely related Ascochyta ones. The constructed phylogenetic trees, based on tef1 and ITS sequences, do not support the traditional Phoma sections based on morphological characterization. However, we managed to distinguish between the Phoma strains and Ascochyta species by comparing their tef1 sequences through parsimony analysis. We proved that a tef1 can be a useful phylogenetic marker to resolve phylogenetic relationships at species level in Phoma genus.
Both parsimony sequence analyses confirmed that the Phyllosticta sojicola species is identical to the Phoma exigua var. exigua species as Kövics et al. (1999) claimed. However, the evolutionary distance by ITS sequences within Phoma species is too small to get well based consequences for the phylogenetic relationships of Phoma genus.
Further investigations would be necessary to clarify whether the tef1 and ITS sequences as phylogenetic molecular markers are well suited for the classification of Phoma species.