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Phylogenetic studies of soybean pathogen Phoma species by Bayesian analysis
53-61Views:119We carried out phylogenetic study analyzing sequences of genetic markers in the taxonomy of Phoma and Phoma-like fungi. Different species of Phoma and Phoma-like fungi occurring on soybean (Phoma pinodella, Phoma sojicola, Phyllosticta sojicola, Phoma exigua var. exigua) are difficult to identy because of their high morphological and symptomatic similarities.
Twenty-two isolates of nine different Phoma species were obtained from reference culture collections. Seven of them were isolated from soybean, the others were collected from different hosts.
The Phoma isolates were firstly characterised by morphologically, and then we employed a part of the gene responsible for the synthesis of translation elongation factor 1 subunit alpha protein (tef1), ITS region, as well as β-tubulin partial sequences as potential genetic markers to infer
phylogenetic relationships among different Phoma species..Finally, their ITS and tef1 sequences were sequenced and analysed by Bayesian approaches.
According to phylogenetic trees inferred by Bayesian analysis of tef1, ITS and β-tubulin sequences, different Phoma species can be separated proving that these phylogenetic markers are well suited for phylogenetic studies of Phoma species. However, the phylogenetic tree does not support the traditional Phoma sections based on morphological characterization.
Bayesian analyses of the 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 molecular data provide evidence for 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. -
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. -
Genetic discontinuity analysis of Collared Dove (Streptopelia decaocto)
5-11Views:160The Collared Dove conquered continent areas within a few decades. Causes and dispersion pattern of expansion has been investigated in several studies. However, the relationship between the geographic distribution and genetic structure of populations has not been researched. We used 152 individuals from 19 countries in this study. We analyze a 650 bp long mitochondrial COI sequences of each individuals. We were performed Spatial Autocorrelation Analysis, Principal Component Analysis and analysis of the genetic discontinuity in this study. Under 2500 km distance was a positive correlation between the genetic differentiation and different geographical areas. Hidden genetic barriers were found only Carpatian Basin. Could not be detected signs of genetic isolation in other regions. This will probably due to the unevenness of the sample collection, because these areas proportionally much fewer sequences were available. Therefore, is worth repeat this analysis after further sample collection, in the future.
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Mitochondrial DNA-based diversity study of Hungarian brown hares (Lepus europaeus Pallas 1778)
23-29Views:131The brown hare being an important game species which is widespread across the European continent has been in focus of many population genetic studies. However only a few comprising researches can be found on the diversity of Central-European populations.
The aim of our large scale long term ongoing study is to fill this gap of information on the species by describing the genetic history and structure of the brown hare populations of the area using both mitochondrial DNA markers and genomic skin and hair colour regulating genes.
This article gives forth a part of our results concerning the mitochondrial DNA diversity of Hungarian brown hares based on amplification of a 512 bp long D-loop sequence. N=39 tissue or hair samples have been collected from 15 sampling sites on the Hungarian Great Plain. We have described a high level of haplotype diversity (Hd=0.879±0.044) based on a 410 bp alignment of our sequences. We have found 17 haplotypes within our sample set with the nucleotid diversity of π=0.01167±0.0022. Our ongoing research shows high genetic diversity for the brown hare in the studied region and a second alignment with 156 sequences downloaded from GenBank indicates a geographic pattern of haplotypes among the studied populations though these results need confirmation by our further analyses.
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Lack of polymorphism of the agouti signaling protein (ASIP) gene among four different brown hare (Lepus europaeus Pallas 1778) populations
81-85Views:123The brown hare (Lepus europaeus Pallas 1778) is a common palearctic and a popular game species therefore it has been an obvious subject for population genetic studies since the second part of the 20th century. Among the several mitochondrial DNA studies some have been carried out concerning nuclear genes as well. The agouti signaling protein gene (ASIP) is involved in regulating the synthesis of eumelanin and pheomelanin in melanocytes of mammals. Though many studies focused on it in relation with several mammalian species, minimal information is available on this topic concerning the brown hare.
Here we present a short communication concerning the agouti signaling protein (ASIP) gene in four different country’s L. europaeus populations, namely Lithuania, Hungary, Serbia and Georgia. N=45 tissue samples have been investigated from overall 17 sampling sites of the different countries. There has not been found any polymorphism among the sequences. In an alignment with other Leporid species’ partial ASIP sequences downloaded from ENA we have found that based on a 178 base pairs long DNA sequence the haplotype of our samples contains three other Lepus species as well. This is concordant with the findings of a previous study focusing predominantly on the European rabbit (Orycto lagus cuniculus Linnaeus 1758) and the several mutations of its ASIP gene.
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Study of alternative oxidase as possible molecular marker for phylogenetic analysis of the Botrytis cinerea
127-132Views:115Botrytis cinerea (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is able to attack several economically important plants causing gray rot. Botrytis cinerea species complex includes two cryptic species (B. cinerea and B. pseudocinerea) that tolerate fungicides differently. On the basis of classical taxonomic markers, the two related species are very difficult to be distinguished; therefore, their separation is usually performed using molecular methods based on the time-consuming molecular analysis of several markers. Our goal was to find markers, which are suitable for the differentiation. Testing the nucleotide sequences of the alternative oxidase encoding gene, B. cinerea and B. pseudocinerea strains were clearly differentiated. Moreover, the analysis of the protein sequences of the enzyme with the maximum likelihood method reflected well the taxonomic relationships of the different fungi.
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Qualitative detection of genetically modified organisms in plant samples
309-313Views:93We analysed the GMO content of corn samples by polymerase chain reaction following the appropriate optimization of the reaction. The analysis included two main steps: extraction of DNA from the sample, and detection of the GMO content by polymerase chain reaction. The polymerase chain reaction is an in vitro method to multiply chromosomatic or cloned DNA (cDNA) sequences through the enzymatic pathway. The reaction is sensitive enough to produce DNA in sufficient amount for the analysis from a single DNA. We identified the PCR products by agarose gel electrophoresis. When optimizing the reaction, the MgCl2 concentration, reaction time and temperature have to be taken into consideration. The temperature of the anellation has to be increased until the highest specificity and yield is reached. If the temperature of the anellation is too high, the primer is linked to non-specific sites as well; in the gel visualization, more lines can be seen at one sample. If the temperature of the anellation is too high, the primer is insufficiently linked or is not linked at all (too few lines in the gel visualization). After optimization, the GMO content in the unknown sample can be determined along with the appropriate positive and negative controls.
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Analysis of longevity in Holstein Friesian cattle using proteomic approaches
21-25Views:124The aim of the present study was to determine marker proteins those are associated with functional longevity of dairy cattle. Holstein-Friesian cows were grouped based on their performance as follows: group 1) individuals with good longevity traits; group 2) short production life because of poor reproduction traits; group 3) short production life with low milk yield. Twelve individuals were sampled in each group, blood and milk samples were collected from cows. Blood samples were analysed with two dimensional polyacrylamide gel electrophoresis (2D PAGE), MALDI TOF/TOF and nanoLCMS/MS. The milk samples were analysed with MALDI TOF/TOF and nanoLC-MS/MS. Using the optimized gel based proteomic approach,
we have succesfully separated 143 proteins in the group1, 139 proteins in the group2 and 136 proteins in the group3, but we could not find significant differences between groups in the expression pattern. Using MALDI TOF/TOF and nanoLC-IonTrap MS, we have found eleven protein sequences those were expressed only in the samples of good longevity group. -
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. -
Molecular studies on Cryphonectria parasitica isolates from Carpathian-basin
76-80Views:81Chryphonectria parasitica, the casual agent of chestnut blight, is one of the most important fungal pathogens of chestnut (Castanea spp.) in Europe and Hungary. In this study we analyzed the diversity of 14 Cryphonectria parasitica strains isolated from different location of Carpathian-Basin. For the analyses we used the partial sequences of the translation elongation coding gene, tef1. Our results showed that the tef1 gene, contrary to other fungal species, is not suitable for the molecular analyses of C. parasitica. In the future, for the molecular studies of C. parasitica, we need to use other molecular markers like microsatellites.
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Isolation of promoters of tissue and ripening-specific strawberry genes by TAIL-PCR and bioinformatic characterization of the sequences
91-99Views:45Isolation of ripening- and tissue-specific promoters has become a very important subject of the genetic regulation and plant physiology research in recent years. It could be possible to reveal the regulation of gene expression, and it may be a very useful approach in the biotechnology. In our work, we have isolated promoter regions of genes exhibiting ripening- and tissuespecific expression in our previous experiments, and the data were
characterized by bioinformatic methods. In the sequence of the ripening-specific Spatula and ACC-oxidase promoters (ACCoxidase is one of the key-enzymes of ethylene biosynthesis, directly related to the process of ripening); we could identify auxin- and ethylene-related cis-regulatory elements. This suggests that there is an interaction between ripening and ethylene-synthesis, in case of non-climacteric strawberry, too. We investigated the promoter regions of three green receptacle-specific genes (putative nitrilaselike protein, Ring transcription factor and an aquaporin protein)
and we could identify several regulatory elements, which refer to hormonal regulation. Additionally, we could find several cisacting elements which associated with stress-responsiveness and endospermium-specific expression. -
Laboratory diagnoses of the isolates of chestnut blight disease fungus Cryphonectria parasitica (MURR. BARR)
45-52Views:84Chryphonectria parasitica, the casual agent of chestnut blight, is one of the most important fungal pathogens of chestnut (Castanea spp.) in Europe and Hungary. In this study, we analyzed the ITS region of five Cryphonectria parasitica strains isolated from different location of Hungary. The differences among the Cryphonectria parasitica isolates were not insignificant because only two sites were considered as informative for the parsimony analysis. As the differences among geographically different isolates were insignificant, we mean that the evolutionary distance by ITS sequences within Hungarian Cryphonectria parasitica isolates is too small to get well based consequences for the phylogenetic relationships.
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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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Investigation of host-specificity of phytopathogenic fungi isolated from woody plants
155-160Views:137Host-specificity is an important characteristic of fungal pathogens. Changing climate could create more appropriate environmental conditions for phytopathogens, thus formerly host-specify fungi could be able to colonize new hosts. Noxious plant pathogen fungi, which can infect several plant species are well-known worldwide. These genera may expand their range of hosts because of the appearance in new geographic areas due to climate change. This new exposure can result in serious problems in agriculture because of the lack of immunity. The susceptibility of apple tree was studied through testing pathogenicity in vitro with species isolated from walnut twigs and nuts, and identified by ITS sequences. Three of four tested species, Botryosphaeria dothidea, Diaporthe eres and Diplodia seriata colonized and necrotized the infected apple branches, while Juglanconis juglandina was not able to infect the twigs. Members of Botryosphaeriaceae were the most virulent, causing the largest lesions in the fastest way. This experiment draws attention to the threat of new host-pathogen connections, which can arise because of the favourable weather conditions and can spread between neighbouring cultures.
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Identification of Hucul mare families by mtDNA markers
75-79Views:153Hundred animal species have disappeared during the last century. By this time, approximately one-third of domestic animals have been in the endangered category. Hucul horses are also in this category; furthermore saving the genetic diversity beside the race preservation is an important challenge as well. The number of mares and stallions is only one of the expressive elements of genetic diversity; together with their quality determine the genetic variability of this breed. Beyond that, if an exact breed can originates from more founders, it can be more renewed genetically. Stud book documents these data by registering the mare families and stallions’ genealogical lineage. Molecular genetics, especially mitochondrial DNA analysis can make the precise identification of mare families possible. As a result of these molecular based methods, protection of genetic diversity, as well as breed preservation became more reliable. After the primer designing, the optimal primer pair was chosen which targets a 1092 bp length DNA sequence in the cytochrome b region. After the successful PCR optimalisation, we determined 170 Hucul mares’ sequences. According to our results, the samples compose ten haplotypes, which are much less, than the registered number of mare families in the stud book. Further investigations are needed to reach more representative results, and drawn the further consequences.
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Preliminary results of the phylogenetic analysis of European hare (Lepus europaeus)
99-104Views:123Brown hare (Lepus europaeus) is one of the most wide spread mammal in Europe. Its genetic structure is affected not only by last glacial, even by human activities (hunting, agricultural activities), isolation of such areas or competing for food resources. According to literature datas brown hare populations has different genetic variants in Europe, however its evolution, phylogenetics has not studied well.
The main goal of this work was to know genetic structure of some brown hare. Mitochondrial DNA analysis was performed in two regions (D-loop, 513 bp and cytochrome-b, 1183 bp). Genetic distance values and Network analysis were calculated. NCBI Genbank was used for further sequences. Our results showed that Italian samples differed from the Genbank samples. We found two main clades: 1: Greece without islands; Bulgaria, Italy and Central-Europe; 2: East-Macedonia,Greece with islands, Cyprus and North Israel.