In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp p...rimers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.
The aim of this study was to identify and biologically analyse some Monilinia species from Hungary. 146 M. fructigena and 28 M. laxa species out 174 infectious fruit from all over the country were used for the study. For further study 10 isolates were used and apple fruit was inoculated with them according to Koch postulate. 1–2 mm ochre exog...en stromas were observbed on infectious plant parts and growing signs on culture of all isolates were identical to M. fructigena. To affirm classical identification, isolates with molecular biological method were also prepared using PCR reaction. Control isolates of M. laxa, M. fructigena and M. fructicola were used. The size of PCR product showed that all isolates had a 415 bp band which was typical of M. fructigena. Results support the previous observation that M. fructigena and M. laxa species occure all over Hungary.
Pituitary adenylate cyclase-activating polypeptide is a neuropeptide expressed primarily in the hypothalamus and in other tissues, which divergent and extensive physiological functions are proved. Large number of SNPs in PACAP are published involved SNP that is associated with phenotypic trait of cattle as well. Hungarian Grey, Hungarian Simmen...tal, Holstein, Charolais and Angus cattle breeds were involved in this study to search for polymorphism in exon 5 1–391 bp and G/A transition. Our results of PCR RFLP and PCR SSCP did not prove the occurrence neither G/A transition, nor any other SNP in cattle breeds involved.
The aim of this study was the detection of polymorphism in the bovine growth hormone and leptin genes using the PCR-RFLP method. A
polymorphic site of the growth hormone gene (Alul loci) that results in amino acid change at position 127 of the protein chain (leucine, L to
valine, V) has been linked to differences in circulating metabolite
gene is situated in the intron between two exons, which results in an amino acid change at position 2059 of the protein chain (cytosine, C to
thymine, T). The polymorphisms were studied in a group of 58 bulls of the Slovak spotted breed. A strategy employing PCR was used to amplify 428 bp (GH gene) and 422 bp (LEP gene) products from blood samples. Digestion of PCR products with restriction enzymes AluI and Sau3AI revealed alleles: L and V; A and B for GH gene and LEP gene, respectively. The growth hormone gene is a candidate gene for body weight gain in cattle, since it plays a fundamental role in growth regulation. Leptin plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle; therefore, animals with higher leptin gene expression will probably have lower daily weight gain than others with similar forage offer and nutritional condition and will also likely have longer calving intervals.
Aim of our study was the optimization of a DNA method, that is appropriate for reliable, low cost identification of animal species in milk and dairy product (cheese) and to determine the ratio of species. Mitochondrial DNA was used in our work to analyse buffalo/cow milk mixtures contained different ratio of bovine milk such as 0.1%, 0.5%, 1%,...1.5%, 2%, 5%, 10%, 15% (v/v%). Buffalo cheese were produced using buffalo and cows milk (0%, 2%, 5%, 10%, 15% – v/v% cows milk in buffalo milk). In case of milk mixtures, using species specific primers, the PCR assay showed a 0.5 v/v% detection limit. Cattle, in the buffalo/cows milk 99.9/0.1 v/v% mixture, was not detectable. The identification of buffalo and cows DNA in cheese was successful. The intensity of eletroforetic PCR fragment indicated the increase of cow milk ratio in milk and cheese samples as well.
We 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.
Hundred 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 onl...y 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.
Staphylococcus aureus is a very important pathogen for dairy farms and milk processing plants. Subclinical mastitis is often caused by this species, and it can contaminate bulk tank milk when milking cows are suffering from mastitis. Additionally, thermostable enterotoxins (SE) produced by some types of this bacterium can cause food poisoning.<...br>The aim of our research was to examine the number of S. aureus in bulk tank milk in two dairy farms and the enterotoxin-producing ability, genetic relation (pulsotype) and antibiotic resistance of S. aureus strains from different sources (bulk tank milk, udder quarter milk and environment).
The results show that the mean number of S. aureus of bulk tank milk of two farms significantly differed (P<0.05). Fourteen isolates were selected for further molecular genetic studies (five isolates were from bulk tank milk and nine isolates were from udder quarter milk). S. aureus was not recovered from the environmental samples. Three of the fourteen isolates (21.4%) tested by multiplex PCR were positive for SE genes. Two isolates carried one gene (seb) and one isolate carried two genes (seg and sei). The fourteen strains were classified into three pulsotypes and two subtypes at 86% similarity level. Isolates from bulk tank milk (n=5), were divided into 2 pulsotypes (A, C) and one subtype (C1). The isolates from udder quarter milk (n=9) belonged to three different pulsotypes (A, B, C) and two subtypes (A1, C1). The distribution of pulsotypes in the present study revealed genetic relationship between S. aureus isolated from udder quarter milk and bulk tank milk. This could be explained by the fact that in farms with a high number of infected cows, these cows could represent the main source of contamination. The results of the antibiotic resistance investigations show, that all strains were susceptible to methicillin, cefoxitin, lincomycin, tetracycline, erythromycin and sulfamethoxazole/trimethoprim. Thirteen out of fourteen strains were resistant to penicillin (A and C pulsotypes, A1 and C1 subtypes) and just one isolate was susceptible (B pulsotype) to all antibiotics tested.
In the mitochondrion of eukaryotes, cytochrome b is a component of respiratory chain complex III. Cytochrome b is encoded by the
cytochrome b (CYTB) gene located in the mitochondrial genome. The fungicidal activity of QoIs relies on their ability to inhibit mitochondrial respiration by binding at the so-called Qo site (the outer quinol-oxida
Monospore B. cinerea field isolates has been collected during 2008-2009 from different hosts in Hungary. PCR fragment length analysis
indicated the high frequency presence of type large intron in the isolates while in a few strains G143A substitution could also be detected.
These results indicated the heterogeneity of CYTB in the Hungarian B. cinerea populations, which possibly involve the heteroplasmy of this
mitochondrial gene, moreover indicates the existence op azoxystrobin resistant populations in Hungary.
This work was supported by NKFP-A2-2006/0017 grant. Erzsébet Fekete is a grantee of the János Bolyai Scholarship (BO/00519/09/8).
The polymorphism in leptin (LEP 2548A) seems to influence obesity among others genes. The aim of this study is to investigate the effect of the G2548A polymorphism on body mass index. We included 79 people from Slovakia with some genetic relatedness and used barrels kit to isolate the genomic DNA from an adenoblast swab- from the salivary. PCR...products were amplified by pursued polymorphisms and G2548A, we restriction-analyzed them and then we identified the specific fragments describing the presence of chosen SNP polymorphism by the agarose electrophoresis, to analyze SNP polymorphism by PCR-RFLP method.
The LEP gene had increased frequency of G allele (0.5506). The most common genotype occurring in the gene LEP was heterozygous genotype (AG) and the least frequent genotype in LEP was AA (0.1899). Taking the age into account the BMI is higher if the G allele occurs in the LEP gene. Moreover, if the G allele genotype was situated in dominant form, then the highest average BMI was present.
According to the results we can assume that the AA genotype (LEP) has a protective effect on the prevalence of obesity compared to the other genotypes.
Etiology of pepper fruit melanotic ringspot (FMRS) disease (Salamon, 2009) was studied on fruit samples collected in forced pepper populations. It was noticed that in spite of heavy thrips (Frankliniella occidentalis) infestations and of TSWV epidemy detected in the forcing houses, FMRS occurred only in plants having healthy foliage. Symptomato...logical surveys strongly suggested that FMRS appeared exclusively in specific pepper genotypes. The size of melanotic ringspots has been observed to grow at room temperature during postripening of diseased fruits. A mechanically transmitted plant virus was isolated from symptomatic parts of 9 white pepper fruits affected by FMRS. On test plants each of the virus isolates caused systemic symptoms characteristic to TSWV. Using cDNA/PCR technique and TSWV N-gene specific primers a ca. 300 bp long DNA fragment has been amplified from total nucleic acid extracted from symptomatic tissues but never from asymptomatic parts of the fruits showing FMRS. Plant progenies grown from seeds of FMRS diseased fruits segregated in respect of resistance and/or susceptibility to TSWV infection. TSWV was also detected in and isolated from three fruits showed non-melanotic yellow rings (one of them was infected with a tobamovirus, too). Seedlings derived from these fruits proved to be susceptible to TSWV. Based on the above results we could conclude that the FMRS disease developed on fruits of “cecei” type white peppers that carry a TSWV resistance gene, most likely the Tsw gene in heterozygous form. These fruits were infected with thrips transmitted TSWV and FRMS appeared as a hypersentive reaction (HR) manifested in fruits.
The genotypes for kappa-casein locus were established using PCR-RFLP. Genome DNA for PCR amplification was isolated from hair roots in cows (30 Schwitz individuals), and from frozen semen in bulls (42 bulls of different breeds).
A higher frequency of the B allele in Schwitz (pA = 0.45 and qB = 0.55 in cows and pA = 0.25 and qB = 0.75 in bull
The majority of stone fruit species are self-incompatible, a feature that is determined by a specific recognition mechanism between the S-ribonuclease enzymes residing in the pistils and the F-box proteins expressed in the pollen tubes. Failure in the function of any component of this bipartite system resulted in self-compatibility (SC) in many... cultivars of Prunus species. Peach (Prunus persica (L.) Batsch.) is the only species in the Prunoideae subfamily that is traditionally known to be self-compatible, but its molecular background is completely unknown. Isoelectric focusing and S-gene specific PCR revealed that SC is not due to functional inability of pistil ribonucleases. We hypothesize that SC may be a consequence of a kind of pollen-part mutation or the action of one or more currently unknown modifier gene(s). Only two S-alleles were identified in a set of peach genotypes of various origin and phenotypes in contrast to the 17–30 alleles described in self-incompatible fruit trees. Most important commercial cultivars carry the same S-allele and are in a homozygote state. This indicates the common origin of these cultivars and also the consequence of self-fertilization. According to the available information, this is the first report to elucidate the role of S-locus in the fertilization process of peach.
During our research we aimed at finding an answer as to what extent the different concentrations of 17-alpha methyl testosterone incorporated in the diet of common carp fries can influence the production parameters of the species, as well as how efficient their sexreversal can be with the use of this method. To this end, an aquarium experiment...was conducted in the course of which four different hormone treatments were set and monitored. The fish feed was enriched with 17-alpha methyl testosterone in 50 ppm, 75 ppm, 100 ppm, 500 ppm dosages.
The obtained figures revealed that the hormone treatments had no influence on the production parameters and conservation of the common carp fries. Further on, our team is to determine the sex of the fish through the examination of gonads during autopsy when they reach the 500 g average weight.
Furthermore, a male specific test method which was supposed to be of great help in our attempt to select the sex-reversed specimens in the subsequent processes was also put to the trial. During the experiment the DNA-isolation of different sample types (muscle tissue, fin, mucus) of common carp with identified sex was successfully carried out. The extracted PCR product was examined with agarose gel. Our results indicated that the ccmf2 marker was applicable, however, the obtained figures were not reliable.