Mitochondrial Control Region Database of Hungarian Fallow Deer (Dama dama) Populations for Forensic Use.

The evidential value of an mtDNA match between biological remains and their potential donor is determined by the random match probability of the haplotype. This probability is based on the haplotype's population frequency estimate. Consequently, implementing a population study representative of the population relevant to a forensic case is vital to correctly evaluating the evidence. The emerging number of poaching cases and the limited availability of such data emphasizes the need for an improved fallow deer mtDNA population databank for forensic purposes, including targeting the entire mitochondrial control region. By sequencing a 945-base-pair-long segment of the mitochondrial control region in 138 animals from five populations in Hungary, we found four different haplotypes, including one which had not yet been described. Our results, supplemented with data already available from previous research, do not support the possibility of determining the population of origin, although some patterns of geographical separation can be distinguished. Estimates of molecular diversity indicate similarly low mtDNA diversity (Hd = 0.565 and π = 0.002) compared to data from other countries. The calculated random match probability of 0.547 shows a high probability of coincidence and, therefore, a limited capacity for exclusion. Our results indicate that despite the overall low genetic diversity of mtDNA within the Hungarian fallow deer samples, a pattern of differentiation among the regions is present, which can have relevance from a forensic point of view.

A Selection of 14 Tetrameric Microsatellite Markers for Genetic Investigations in Fallow Deer (Dama dama).

The fallow deer (Dama dama) represents significant game management value globally, and human activities are significantly impacting the species. Besides the positive effects, these activities can threaten its existence, health, and value. The aim of the authors was to develop a tetranucleotide microsatellite panel that could be clearly interpreted and used for genetic testing of fallow deer. Such a panel did not exist until now and could be particularly useful in the field of conservation genetics and forensics. A total of 99 tetrameric microsatellites, originally designed for related deer species, were tested on 20 fallow deer individuals from five Hungarian sampling areas. Original and newly designed primers were used to amplify the microsatellite regions using previously published or optimized PCR protocols. The lengths and sequences of specific amplicons were detected using capillary electrophoresis, and the rate of polymorphism was determined. Altogether, 80 markers provided PCR products of adequate quality and quantity. Among them, 15 markers proved to be polymorphic (2-5 alleles/locus), and 14 tetrameric markers were selected for further analysis. Statistical calculations showed that the selected polymorphic microsatellites can potentially enable key individualization in many areas of wildlife and population genetics, thus protecting the species.

Evaluation of Maternal Genetic Background of Two Hungarian Autochthonous Sheep Breeds Coming from Different Geographical Directions.

The aim of our research was the evaluation of the maternal genetic background of two Hungarian autochthonous sheep breeds of different geographical origin. A major argument for the preservation of endangered animal breeds is their documented past and historical importance. These also include the registration of pedigree data. This is the first study to evaluate and compare Tsigai and Cikta sheep in Hungary. Our investigation is based on two complete sequences of mitochondrial DNA (cytochrome b gene and control region). Our research was performed on these two sheep breeds with markedly different breed histories and breed characteristics to determine a possible common maternal genetic background, as ultimately the origin of both breeds can be traced back to Asia Minor. Between 2015 and 2017, a total of 203 biological samples were taken using a newly introduced founder sampling method. We found that the prevailing haplogroup B accounted for over 80% of both breeds, strengthening the common ancestral root. However, the pairwise genetic differentiation estimates (KST) calculated using the sequence-based statistics for cytochrome b gene and control region were 0.034 and 0.021, respectively (both at level p < 0.05); thus, revealing genetic differentiation in both sequences between the Tsigai and Cikta. We note that the known different history of the breeds is clearly justified by the currently studied deviations in their maternal genetic background.

Molecular Sexing and Species Detection of Antlered European Hunting Game for Forensic Purposes.

Molecular sexing techniques are widely applied in conservation biology, although the range of forensically validated methods is fairly limited. The primary aim of this work was to develop forensically validated assays, using two PCR panels for sex and species assignment for the abundant antlered European game species: red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama). Segments of the SRY and Amelogenin X/Y genes for sex determination, additionally species-specific cytochrome b regions for species detection were targeted and separately amplified in two multiplex reactions. These assays can reliably analyze trace amounts of DNA. The results of both can easily be visualized and interpreted practically, either on agarose gel or by capillary electrophoresis. These simple, fast molecular assays are able to affect the early-stage resolution of disputed or unsolved poaching cases, without the need of individualization or sequencing of forensic samples.

Comparison of mtDNA control region among descendant breeds of the extinct Zaupel sheep revealed haplogroup C and D in Central Europe.

The consideration of the descendance is indispensable in the preservation of endangered animal breeds. The authors compared mitochondrial DNA (mtDNA) control region sequence in three descendant breeds of the extinct Zaupel sheep, firstly. Their investigation was carried out in order to prove the common origin of Waldschaf (Austria), Bovec sheep (Slovenia) and Cikta (Hungary). A total of 118 biological samples were taken from non-related representatives of the three breeds between 2015 and 2017. A newly designed primer pair was also used to amplify the segment (1180 bp) to be tested. The total number of haplotypes in the whole study population was 49. The majority of which fell into haplogroup B. The significant negative value of the Fu's Fs statistic (Fs statistic = -3.296, p = 0.013) based on haplotype frequencies demonstrated a moderate foreign gene flow. As a novel observation haplogroups C and D appeared in Cikta and Bovec sheep, respectively. The Tajima D-test value in the entire study population was -0.914 (p > 0.10), meaning that the separation of the three descendant breeds did not cause genetic drift, these are collectively in genetic equilibrium. The genetic information confirmed the common origin of the breeds known from the breed history.

Family aggregation analysis shows a possible heritable background of equine grass sickness (dysautonomia) in a Hungarian stud population.

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a century ago, several factors, such as the phenotype, intestinal microbiome, environment, management and climate, have been supposed to be associated with the increased risk of dysautonomia. In this retrospective study, we examined the possible involvement of genetic factors. Medical and pedigree datasets regarding 1,233 horses with 49 affected animals born during a 23-year period were used in the analysis. Among the descendants of some stallions, the proportion of animals diagnosed with dysautonomia was unexpectedly high. Among males, the odds of dysautonomia were found to be higher, albeit not significantly, than among females. Significant familial clustering (genealogical index of familiality, P = 0.001) was observed among the affected animals. Further subgroups were identified with significant (P < 0.001) aggregation among close relatives using kinship-based methods. Our analysis, along with the slightly higher disease frequency in males, suggests that dysautonomia may have a genetic causal factor with an X-linked recessive inheritance pattern. This is the first study providing ancestry data and suggesting a heritable component in the likely multifactorial aetiology of the disease.

Being Merle: The Molecular Genetic Background of the Canine Merle Mutation.

The intensity of the merle pattern is determined by the length of the poly(A) tail of a repeat element which has been inserted into the boundary of intron 10 and exon 11 of the PMEL17 locus in reverse orientation. This poly(A) tail behaves as a microsatellite, and due to replication slippage, longer and shorter alleles of it might be generated during cell divisions. The length of the poly(A) tail regulates the splicing mechanism. In the case of shorter tails, the removal of intron 10 takes place at the original splicing, resulting in a normal premelanosome protein (PMEL). Longer tails generate larger insertions, forcing splicing to a cryptic splice site, thereby coding for an abnormal PMEL protein, which is unable to form the normal fibrillar matrix of the eumelanosomes. Thus, eumelanin deposition ensuring the dark color formation is reduced. In summary, the longer the poly(A) tail, the lighter the coat color intensity of the melanocytes. These mutations can occur in the somatic cells and the resulting cell clones will shape the merle pattern of the coat. When they take place in the germ line, they occasionally produce offspring with unexpected color variations which are different from those of their parents.

Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants.

The genetic sexing of animals having long gestation periods offers significant benefits in regard to breeding management among their populations living in captivity. In our study, a new increased-sensitivity PCR method for fetal sexing was developed and tested successfully on elephants, from only a small volume of maternal plasma. Suitable sensitivity was obtained by using short, reduced amplicon lengths with fluorescent labelling for capillary electrophoresis detection. The fundamental principle for this technique was based on the detection of two Y-specific markers (AmelY and SRY), the presence of which indicates the mother is carrying a male fetus and the absence of these markers designates a female fetus. As a reaction control, the X-chromosomal marker (PlpX) was used. To the best of our knowledge, this is the first report on this topic, confirming the presence of fetal cell-free DNA from the plasma of a pregnant captive elephant, and demonstrating a new opportunity for non-invasive assessment in fetal sex determination.

Merle allele variations in the Mudi dog breed and their effects on phenotypes.

A retrotransposon insertion in the SILV gene is associated with a peculiar phenotype of dog, known as a merle. It is characterised by various areas of their coat colour becoming diluted due to a malfunction in the eumelanin-producing pigment cells. Recent studies have shown that the exact size of the short interspersed element (SINE) insertion is in correlation with specific phenotypic attributes, but was not able to absolutely confine dogs to a certain colour pattern. Our study focused on the merle variations occurring in the Mudi breed. Altogether, 123 dog samples from 11 countries were tested and genotyped. The exact length of the merle alleles were determined by automated fluorescent capillary fragment analysis. The most frequent merle genotype in this Mudi sample collection was the 'classic' merle (m/M: 61.8%), whereas other variants, such as atypical (m/Ma and m/Ma+: 5.7%), harlequin (m/Mh: 13.8%), double merle (M/M: 0.8%) and mosaic profiles (17.9%) were also observed. The practical significance of testing this mutation is that, phenotypically, not only merle dogs are carriers of this insertion, but also the so-called hidden merle individuals (where the merle phenotype is fully covered by the pheomelanin-dominated colouration) are potentially capable of producing unintentionally homozygous 'double merle' progeny with ophthalmologic, viability and auditory impairments.

The response of prion genic variation to selection for scrapie resistance in Hungarian indigenous sheep breeds.

The authors studied the present status of Hungarian indigenous sheep breeds based on the genetic background of scrapie resistance. The aim of this investigation was to estimate the relative frequency of prion haplotypes, genotypes and risk categories, as well as to reveal the efficiency of the scrapie eradication programme achieved over the last decade. A novel approach in the characterisation of prion by using its genic variation was also implemented. The authors established that the proportion of deleterious sites (%) can be a useful indicator of the eradication programme. Based on a large sample size, it was confirmed that the scrapie resistance of the Cikta breed is low, and the classification of this breed according to risk category has not improved. However, the frequent genotype ARQ and risk category 3 can also be considered characteristic of the breed. The careful use of these genotypes is permitted and will contribute to the maintenance of breed diversity. The response of prion genic variation to selection for scrapie resistance in the other breeds (Tsigai, Milking Tsigai, White Racka, Black Racka and Gyimes Racka) was definitely successful.

Constructing STR multiplexes for individual identification of Hungarian red deer.

Red deer is the most valuable game of the fauna in Hungary, and there is a strong need for genetic identification of individuals. For this purpose, 10 tetranucleotide STR markers were developed and amplified in two 5-plex systems. The study presented here includes the flanking region sequence analysis and the allele nomenclature of the 10 loci as well as the PCR optimization of the DeerPlex I and II. LD pairwise tests and cross-species similarity analyses showed the 10 loci to be independently inherited. Considerable levels of genetic differences between two subpopulations were recorded, and F(ST) was 0.034 using AMOVA. The average probability of identity (PI(ave)) was at the value of 2.6736 × 10(-15). This low value for PI(ave) nearly eliminates false identification. An illegal hunting case solved by DeerPlex is described herein. The calculated likelihood ratio (LR) illustrates the potential of the 10 red deer microsatellite markers for forensic investigations.

Possible genetic sign of heat stress adaptation in Hungarian Grey Bos taurus breed.

The synthesis of Heat Shock Protein 70.2 mRNA is also regulated by the Upper Promoter elements of the gene. This promoter region is polymorphic in cattle. These polymorphisms have a major effect on the activity of the mRNA transcription. In a comparison of quantity of transcribed mRNA from the wild type and AP2 mutant allele the wild type can produce 2-3-fold more transcripts.The Hungarian Grey Cattle (HG) and Norwegian Red (NFR) as control breed were genotyped with PCR-RFLP method. Our results showed that the frequencies of alleles in breeds (p(wt)HG = 0.859419, p(wt)NFR = 0.5) are different. The effective response to heat stress in the Norwegian Red seems to be less important than in the Hungarian Grey breed. The extensive keeping in hot and arid region during centuries could have been proved as selection pressure for the heat tolerance.Our results combined with the global climate forecasts emphasize the role of autochthonous, well adopted, heat tolerant breeds in the near future.

Characterization of the WILMS-TF microsatellite marker in Hungarian dog populations.

Demand for correct and cost-effective genetic-based identification and parentage control has increasing importance in domestic animals, including dogs. In our study the applicability of a canine hyperpolymorphic microsatellite marker - which localized in the WILMS-TF (tumor factor) gene - was examined in mixed breed and purebred canine populations. The redesigned and shortened amplicons were genotyped using an allelic ladder which was constructed from sequence verified fragments. The nomenclature for allele calling based on repetition structures is suitable for international comparisons. Our study justified the potential use and efficiency of the marker D18S12 in parentage control.