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Mitochondrial DNA-based diversity study of Hungarian brown hares (Lepus europaeus Pallas 1778)
23-29Views:171The 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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Genetic diversity study of Hungarian honey bee (Apis mellifera L.) colonies – Previous announcement
61-64Views:179In Hungary, the Apismelliferacarnica is the native breed which is the only recognised and breedable honey bee in the country. It is assumed that there are a number of non-native and hybrid honey bee breeds in Hungary. The microsatellite and mitochondrial DNA surveys applied here will be utilised to easily and accurately identify the various sprads, and open up new ways in the research of honey bees. The isolation of the genomial DNS from 5 to 7 day old larvae samples was successfully carried out. In the future the plan is to carry out the measure with microsatellite markers. As an initial step the optimal annealing temperature was identified. In the mitochondrial DNA survey the COI-COII mytochonrial regional primer due to its low anneling temperature cannot be used with any normal sequencing methods. By using these method the aim of this research is the measurement of genetic variance.
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Identification of Hucul mare families by mtDNA markers
75-79Views:232Hundred 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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Q-PCR analysis of the resistance of Hungarian Botrytis cinerea isolates toward azoxystrobin
41-44Views:120The genes being in the mitochondrial DNA primarily encode the enzymes of cellular respiration. Fungicides belonging to the family of quinol oxidase inhibitors (QoIs) play on important role in the protection against several plant diseases caused by fungi. These fungicides bind to the cytochrome bc1 complex so they block electron transport between cytochrome b and cytochrome c1. This way these fungicides inhibit the ATP synthesis consequently they inhibit the mitochondrial respiration. The QoI resistance has two mechanisms. One of them is the point mutation of the cytochrome b gene (CYTB), e.g. the substitution of a single glycine by alanine at position 143 results in high-resistance. The other is the cyanide-resistant alternative respiration sustained by the alternative oxidase.
In a cell there are several mitochondria. The phenomenon when the genomes of all mitochondria in the cell are identical is called homoplazmy. If in the cell there is wild and mutant mitochondrial DNA this is called heteroplasmy. Whether the mutation in the mitochondria causes fenotypical diversity or does not depend on the dose, i.e. it depends on the percentage of the changed mitochondrials. During our work we investigated Botrytis cinerea single spore isolates which have been collected in 2008-2009 on different host plants. Our goal was to decide whether heteroplasmy influences the level of resistance. We managed to detect the change of the level of heteroplasmy, so the change the level of the resistance due to the treatment with fungicide. -
Application of molecular genetic methods for birds (literature review)
37-41Views:145Ornithology studies have been extended by molecular genetic techniques. In this paper we are dealing with the most common use of areas. Our basic aim is to give a comprehensive view of the most commonly used methods in ornithological studies, including the available results by their use. We also deal with the following areas: an essential step during examination namely the applicability of sample collecting methods, and the unique identification, also the sex determination, methodology of DNA barcoding, as well as the role of molecular methods to protect endangered species. We discussed the advantages and disadvantages of the methods, such as the current trend for each method.
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Identification of cow’s and buffalo’s milk and dairy product using a DNA-based method
279-282Views:246Aim 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.
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Genetic diversity of the Hungarian draft horse assessed by mitochondrial DNA
29-32Views:253Hungarian draft is a horse breed with a recent mixed ancestry. It was developed in the 1920s by crossing local mares with draught horses imported from France and Belgium. To genetically characterize the breed and to set up the basis for a conservation programme, we have employed a molecular marker: a 256-bp D-loop mitochondrial DNA fragment. We analyzed 124 horses representing Hungarian draft horses to assess the maternal phylogeography of the breed. Sequence analysis of a 256-bp segment revealed a total of 34 haplotypes with thirty-four polymorphic sites. High haplotype and nucleotide diversity values (Hd=0.953±0.001; π=0.024±0.001) were detected. The average number of pairwise differences were k=5.998. This breed counts 800 mares today, and only survive due to breeding programmes, this way each haplotype frequency depends on the extent to which mares are involved into the breeding. The reduced number of surviving maternal lineages emphasizes the importance of establishing a conservation plan for this endangered breed. Due to the revealed 34 polymorphic sites we could presuppose twelve maternal linages, which could be a first step for making a breeding programme.
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Application of molecular genetic methods for species Equus caballus – Literature review
21-28Views:175In this study our aim was to provide a comprihensive overview of the most commonly used methods in molecular genetic studies related to Equus caballus. Thus we are dealing with the D-loop region of mitochondrial DNA, with microsatellites and also with single nucleotid polimorphism as SNP. The advantages and drawbacks of each method were also explored.
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Sequence stability at SSR, ISSR and mtDNA loci of common millet (Panicum miliaceum) from the middle ages
10-19Views:108Seed 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. -
Lack of polymorphism of the agouti signaling protein (ASIP) gene among four different brown hare (Lepus europaeus Pallas 1778) populations
81-85Views:164The 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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Genetic diversity study in Hungarian coldblooded horses
29-34Views:288Because of the feeding technology innovation, accelerated transport and communication facilities breeds of high performance breeds replaced local autochone breeds worldwide. These latter species however have an important role in gene conservation. Hungarian cold-blooded horse breeding stock are lacking pedigree, the actual founder breed mares are not known. For this reason, it is an major priority defining the genetic backround of the existing flock, for that breeding could operate with purposeful using of origin maternal founders. In the present study 195 cold-blooded Hungarian mares tail and mane sample were analized. Our analysis was carried out between 15531–15752 base pairs in mithocrondial DNA D-loop region, which reported a total of 222 base pairs. Fourtyone polymorphic sites were determined, which resulted in 39 haplotypes (h=39). The average pairwise differences were k=6.825. High haplotype and nucleotide diversity values were observed (Hd=0.968±0.003, π=0.026±0.003). Based on the previously defined variable positions of haplotypes defined by Jansen et al (2002), we groupped our haplotypes into haplogroups. 23 percent of the studied population (45 mares) belonged to haplogroup F1. Nearly 97% of the analyzed population was classified into one of eight haplogroups defined by Jansen.et al. (2002). This study gives genetic information nearly 25% of the Hungarian population. Another possibility would be patterning more mares or involving more genetic marker in the study which will assuming the possibility of a more comprehensive analysis.
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Preliminary results of the phylogenetic analysis of European hare (Lepus europaeus)
99-104Views:151Brown 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.
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Morpometric study of Hungarian honey bee (Apis mellifera L.) colonies
59-63Views:308The honey bees are essential for the pollination of agricultural plants. The Pannonian honey bee, Apis mellifera pannonica, is native to Hungary, only these subspecies are being bread in our country. The parameters have been separated the pannon and italian honey bee subspecies, the colour of tergit, the cubital index and proboscis length. The morphometric analisys is of special importance because this, on the one hand, shows correlation with honey bee production and on the other hand, the pure morphometric charactersitics is the basis of any potential honey bee export. Mitochondrial DNA and microsatellites are the common methods to define genetic diversity and the separation of subspecies.
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Coincidences between molecular genetic and studbook data of gidrán mare families on the basis of mtDnA
69-73Views:232The traditional Hungarian horse breed, Gidran has been close to the edge of extinction several times. Despite the multiple bottleneck effect, the breed has retained a part of its genetic variability, and performed prominently in carriage driving and show-jumping competitions. Maintaining of the Gidran breed is important in the point of view of world heritage; because besides Hungary, smaller Gidran populations exist only in Bulgaria and Romania. Taking advantage of the special inheritance features of mtDNA, our study focused on two mtDNA regions of Gidran mares. Altogether, 251 hair samples from various Hungarian studs were examined. The analysis was successfully made in case of 251 samples of the cytochrome b and in case of 246 samples of D-loop regions. Because of the distinct mutation rates of the two mtDNA markers, the number of the haplotypes and the way of grouping samples into haplotypes was different. Our key finding was that most haplotypes may be compatible with mare families of the stud book; however incidental mistakes in stud book have occurred only in a few cases. Our results indicate the importance of the preservation and breeding those mare families, which are molecular genetically more diverse than the others, and are in the edge of extinction.