Anna Trakovická

Genomic characterization of Pinzgau cattle: genetic conservation and breeding perspectives

A genome-wide scan of Slovak Pinzgau cattle was prepared for the first time in order to estimate their genetic diversity at a more detailed level compared to previously published studies. The aim of this study was to describe the genetic diversity based on the runs of homozygosity (ROHs), linkage disequilibrium (LD) and effective population size (NeLD) using genome-wide data. Moreover, Bayesian clustering algorithms and multivariate methods were used to detect the population structure, potential admixture level and relationship between Austrian and Slovak Pinzgau cattle with respect to a large meta-population consisting of 15 European cattle breeds. The proportion of ROH segments ranged from 0.43 to 1.91% in Slovak Pinzgau, depending on the minimum size of an ROH. The genomic inbreeding coefficients were higher than the pedigree ones possibly due to the limited number of available generations in pedigree data. The observed NeLD was close to the limit value characterizing the endangerment status, based both on genomic and pedigree data. Population structure within analyzed breeds based on the Wright’s FST index, Nei’s genetic distances, and unsupervised as well as supervised analysis has been established. Overall, these analyses clearly distinguished populations based on their origin. A detailed analysis of the introgression of each breed into the Pinzgau breeds prepared using a Bayesian approach showed that the contribution of Holstein cattle in Austrian as well as Slovak Pinzgau was larger than contribution of beef breeds. A possible reason is the recent usage of Holstein sires to increase milk production. There are considerable differences between well-defined regions that clearly distinguish Austrian and Slovak Pinzgau, despite their close common history. Generally, the breeding program of Austrian Pinzgau is more focused on meat production than Slovak Pinzgau, which was clearly reflected in the obtained autozygosity islands. Considering the genetic establishment of Slovak Pinzgau population the genetic potential of the breed is insufficiently used. On a long term, more global breeding program including very close populations will be more efficient providing higher genetic progress and diversity. Established methodology how to distinguish genealogically close populations on high-throughput molecular information based of Slovak and Austrian Pinzgau can be proposed as general for analysis of differences in all highly related breeds.

Identification of cryptic allele for merle patterning in dogs by molecular genetics methods.

Abstract Merle patterning in dogs, caused by the insertion of a short interspersed element (SINE) in the genetic structure of SILV gene, is characterized by patches of diluted pigment intermingled with normal melanin. Sequencing analyses of SINE element localized in the canine SILV gene discovered a variability of the poly (A)-tail length which is responsible for the different expression of merle pattern. The SINE element with the length of poly(A)-tail between 91 - 101 nucleotides is responsible for the merle phenotype with all characters of merle pattern. On the contrary the dogs which have SINE element with the shorter length of poly(A) tail between 54-65 nucleotides are referred as cryptic merles without expresion of Merle pattern. The aim of this study was to improve molecular genetics method for the detection of cryptic allele for merle patterning in dogs. A total of 40 dogs of four breeds - Border collie, Shetland sheepdog, Australian Shepherd dog, and Chihuahua were used in this study. Canine genomic DNA was isolated from samples of whole blood and buccal cells by commercial column kit. Detection of merle (M), cryptic merle (Mc) and non-merle (m) alleles was done using M13-tailed primer protocol and two different allele-sizing methods for the verification of the electrophoresis result. In the analyzed population of dogs were detected 20 dogs with non-merle genotype mm, 17 dogs with merle genotype Mm, 2 dogs with double merle genotype MM and one dog with merle phenotype but with the presence of cryptic merle allele Mc with the consequential genotype MMc. Mramorirana šara pasa, nastala umetanjem kratkih ponovaca elemenata (SINE) u genetsku strukturu SILV gena, karakteriše se prisustvom slabije pigmentisanih polja pomešanih sa poljima normalne pigmentacije. Analizom SINE elementa lokalizovanog u SILV genu pasa sekvencioniranjem otkrila je varijabilnost poli(A) repa koji je odgovoran za različitu ekspresiju mramorirane šare. SINE element sa poli(A) repom dužine od 91 do 101 nukleotida je odgovoran za mramorirani fenotip sa svim karakterima mramorirane šare. Nasuprot tome, psi koji poseduju SINE element sa kratkim poli(A) repom dužine od 54 do 65 nukleotida su opisani kao prikriveni mramorirani bez ekspresije mramorirane šare. Cilj ove studije je poboljšanje metoda molekularne genetike za detekciju skrivenog alela za mramoriranu šaru pasa. Ukupno 40 pasa četiri rase: border koli, šetlandski ovčar, australijski ovčar i čivava bilo je uključeno u studiju. Genom DNK pasa bio je izolovan iz uzoraka pune krvi i ćelija sluznice usne duplje bio je izolovan pomoću komercijalnih kitova. Detekcija mramoriranog (M), prikriveno mramoriranog (Mc) i ne-mramoriranog (m) alela je izvršena pomoću „M13-tailed“ prajmera protokola i dve različite metode određivanja veličine alela za verifi kaciju rezultata elektroforeze. U analiziranoj populaciji pasa otkriveno je 20 pasa sa ne - mramoriranim genotipom mm, 17 pasa sa mramoriranim genotipom Mm, 2 psa sa duplim mramoriranim genotipom MM i jedan pas sa mramoriranim fenotipom, ali sa prisustvom prikrivenog mramoriranog alela Mc sa posledičnim genotiom MMC.

GENETIC MARKERS AS ONE OF TOOLS FOR PRODUCTION OF TENDERNESS MEAT IN CATTLE

Meat tenderness is one of the major characteristic quality of beef not only for consumers but for breeders of beef cattle too. Selection of cattle focussed on an increment of meat tenderness is complicated because this trait has large variability not only between different breeds but between individuals of equal breed too. Similarly a measurement of meat tenderness is expensive because it make after slaughter of animal and ageing of meat post mortem. Therefore it was developed a several methods, by the help of which is possible increase tenderness of meat. However still exist variance in values of meat tenderness which are caused by distinctness genetic base of animal. By using molecular genetic methods was described the most significant candidate genes (CAPN1, CAST) coding formation of the calpains-calpastatin proteolytic system, which exercise an influence on tenderness. The single nucleotide polymorphisms (SNPs) in these genes were using to design commercially genetic marker panels GeneSTAR Tenderness and Igenity Tender-GENE. By help this commercially test is possible to make genotyping and selection of animals for production of tenderness beef meat in meat industry. doi:10.5219/102

POLYMORPHISMS IN CANDIDATE GENES FOR BEEF QUALITY IN PINZGAU CATTLE

The aim of present study was to identify the polymorphisms in genes encoding calpastatin (CASTUoG), calpain (CAPN1, CAPN2), diacylglycerol Oacyltransferase (DGAT1), thyroglobulin (TG5), and Stearoyl-CoA Desaturase (SCD) in order to analyse genetic structure of Pinzgau cattle. The genomic DNA for genotyping was obtained from in total 56 blood samples of Pinzgau bulls. After extraction, the concentration of DNA was controlled by the spectrophotometry measurement. The genotyping of each individual was carried out by using PCRRFLP methods. The average value of observed (0.37±0.05) and expected heterozygosity (0.39±0.06) clearly indicated the prevalence of homozygous individuals. Observed Wrights fixation indexes showed positive values across all loci (0.03±0.06), which confirmed slight deficiency of heterozygote animals compared to the Hardy-Weinberg equilibrium expectations. The Hardy-Weinberg equilibrium was found in population, which signalizes only slight impact of factors such as selection, migration or inbreeding. The effectiveness of loci allele impact in populations has been described also by effective allele numbers (1.68±0.13) that expressed the decrease of allele activity in population. The loss of heterozygosity in analysed population was found across all of genetic markers. Each of the evaluated indicators clearly points to the need of genetic diversity monitoring. Moreover, the analyses of single nucleotide polymorphisms in genes significantly involved in control of economically important production traits are still very usable methods for identification of genetic markers that can be used in marker assisted selection of cattle.

Effect of selected polymorphisms of genes lep, MTHFR and FTO to BMI level and gender-specificity

The aim of this study is to investigate the effect of selected polymorphisms LEP (G2548A), MTHFR (C677T) and FTO ( rs1121980 ) on body mass index in humans. In the study participated 79 people from Slovakia with some genetic relatedness. Genomic DNA was isolated from the buccal swabs using a commercial kit Qiagen DNA Mini Kit (Qiagen). The detection of SNP polymorphisms in human genes LEP, MTHFR and FTO was performed using molecular genetics methods such as PCR-RFLP and ARMS. The most common genotypes in all 3 polymorphism were found in heterozygous form (for LEP AG = 0.5190, for MTHFR CT = 0.519, for FTO CT =0.4051). The LEP gene had increased frequency of G allele (0.5506), the MTHFR gene T allele (0.6646) and FTO gene T allele (0.50635). The least frequent genotype in LEP was AA (0.1899), in MTHFR was TT (0.0759), in FTO it was CC (0.2911). According to the results we can assume that the genotypes AA (LEP G2548A), CC (FTO rs1121980 ) and TT (MTHFR C677T) in case of women have a protective effect on the prevalence of obesity, based on BMI, compared to the other genotypes and this polymorphism is gender-specific. Added anthropometric measurements, blood test and extension of the group in the future evaluation could increase the statistical relevance in relation to obesity and gender-specificity.

Genomic variation across cervid species in respect to the estimation of red deer diversity

Abstract The aim of this study was to assess the genetic variations and relationships across evolutionary related cervid species in order to estimate the genetic diversity of the Red deer population that inhabits the forest area in the south-western part of Slovakia. The study was based on the application of cross-species SNP genotyping. The genomic data were obtained from a total of 86 individuals representing six genera (Axis, Dama, Cervus, Alces, Rangifer, and Odocoileus) using Illumina BovineSNP50 BeadChip. From 38.85% of successfully genotyped loci up to 1,532 SNPs showed polymorphism and were informative for subsequent analyses of the diversity and interspecific genetic relationships. Generally, a good level of observed heterozygosity was found across all species. The value of FIS (0.23±0.13) signalised the increase of a homozygous proportion within them. The application of molecular variance analysis to the hierarchical population structure showed that most of the variation was conserved within separate species (96%). The performed diversity analysis of Slovak Red deer population and comparative analysis of their phylogenic relationships among subspecies from genus Cervus did not identify a remarkable loss of genetic variability. Also, were not identified any degree of admixture that could be due to the historical background of deer farming in Slovakia or reintroduction and hybridisation by other species from genus Cervus (C. canadensis, and C. nippon) which are the major risk of loss of autochthonous Red deer populations in many areas of Central Europe. The analysis of individual’s ancestry showed consistent results with patterns of evaluated group differentiations which means low migration rates among all species.

Extent of genome-wide linkage disequilibrium in Pinzgau cattle

The aim of this study was to describe the extent of linkage disequilibrium (LD) based on genotyping data in Pinzgau cattle population. DNA samples were obtained from 19 bulls of active Pinzgau cattle population in Slovakia. Genotyping was carried out in commercial lab using an Illumina BovineSNP50 BeadChip. The genotyping array contained 54,609 single nucleotide polymorphisms (SNPs). After quality control SNPs localized on sex chromosomes were excluded. A total of 42,248 (79.97%) SNPs were found to be polymorphic. The distribution of SNPs over entire genomes of all chromosomes was not uniform. The minor allele frequencies across autosomal loci ranged from 0.266 (BTA4) to 0.280 (BTA23) with average value 0.273±0.133. Only adjacent SNPs with distance less than 5 Mb and LD (r2) values from 0.01 to 0.99 were used in the characterization of LD extent. After filtering, the genome coordinates of 31,063 SNP markers covered all regions of autosomes on the length 2519232 kb. The spacing across genome between adjacent SNPs was in average 46.89±47.48 kb and within autosomes ranged from 43.65 kb (BTA25) to 52.59 kb (BTA5). The distribution of SNPs on different chromosomes ranged from 633 (79.13%) on BTA29 to 1123 (83.62%) on BTA14. The average LD between adjacent markers within autosomes reached values from 0.189 to 0.234 for chromosomes BTA 29 and 21, respectively. Division of r2-values over all 29 autosomes were performed according to their physical intermarker distances and averaged within the groups for analysis of distance effect on LD. The results of our study indicated the rapid decay of LD with increasing distance between markers. Further investigation will be oriented on evaluation of effective population size based on LD data. This could allow improvement of our knowledge about genetic diversity and its use for breed preservation of original phenotype supported by the current selection schemes and breeding programmes.

SNPs analyses of the bovine LEP and PIT-1 genes by multiplex PCR-RFLP method and their effect on milk performance traits in Slovak Simmental cattle

The aim of this study was detection of polymorphisms in leptin (LEP) and pituitary specific transcription factor (Pit-1) genes and their evaluation as a genetic markers affected milk performance traits in population of Slovak Simmental cows. The analyzed SNP of LEP gene is located in intron 2 on bovine chromosome 4 and SNP of Pit-1 gene in exon 6 on bovine chromosome 1. The total numbers of blood samples were taken from 288 samples of Slovak Simmental cows. Bovine genomic DNA was isolated by phenol-chloroform extraction method and used in order to estimate LEP/Sau3AI and Pit-1/HinfI genotypes by means of MULTIPLEX PCR-RFLP method with using Sau3AI restriction enzyme for LEP and HinfI restriction enzyme for Pit-1. In the population of Slovak Simmental cattle were detected genotype AA (390 and 32 bp), genotype AB (390, 303, 88 and 32 bp), genotype BB (303, 88 and 32 bp) for LEP gene and genotype AA (260 bp), genotype AB (260, 190 and 70 bp), genotype BB (190 and 70 bp) for Pit-1 gene. The predominant alleles were A with observed frequency 0.8385 and B with 0.7743 for LEP and Pit-1, respectively. In both analyzed polymorphisms were frequent animals with dominant homozygous genotype. Frequency of SNP LEP/Sau3AI AA genotype was 0.7014 and SNP Pit-1/HinfI BB genotype was 0.6007. The statistical analysis show significant effect only between LEP/Sau3AI genotype and milk yield in standard length of lactation (P≤0.05), with A as a desirable allele. In genotype comparison produced AA genotype cows significantly more milk, with difference amounting 457.9 and 293.9 kg and therefore this animals might have potentially positive effect on milk yield. The other results from the statistical association analysis between LEP/Sau3AIand Pit-1/HinfI genotypes and milk production parameters – milk, protein and fat yield (kg) in standard length of lactation were not significant.

Protein kinases required for proper segregation of chromosomes during meiosis.

Cellular division involves complex processes in which protein phosphorylation plays crucial role. As chromosome segregation is a key process in both mitosis and meiosis, its proper progression is essential for cell growth and viability. Although numerous studies investigating the role of protein kinases on chromosome segregation have been performed,1-3 no systematic analysis of their involvement in meiosis has been performed so far. The fission yeast Schizosaccharomyces pombe, a widely used model organism for studying eukaryotic biology, possesses more then one hundred protein kinases. From these, 96 are non-essential and can be deleted from the yeast genome without affecting cell viability. Kovacikova et al. analyzed chromosome segregation during meiosis in yeast strains carrying the S. pombe non-essential kinase knockout alleles and found seven mutants with apparent defect in meiosis.4 Strong defect in meiotic chromosome segregation was observed in bub1∆ and mph1∆ strains, while the remaining strains, hhp2∆, ppk24∆, mug27∆, spo4∆ and atg1∆, showed various alterations, such as weak missegregation phenotype, asci with more than four DNA masses or lagging chromosomes. In their study, Kovacikova et al. focused on the analysis of mph1∆ and spo4∆/spo6∆ mutations, as phenotypes of remaining strains, except for ppk24∆, have previously been described.5,6 First, authors analyzed cells lacking the Mph1 protein kinase. They marked chromosome II in mph1∆ knockout cells with GFP dots and tested chromosome segregation. Analysis of mph1∆ mutant cells carrying homozygous cen2-GFP dots revealed 10% homolog non-disjunction during first meiotic division. When GFP dots were present only on one copy of the chromosome II (heterozygous cen2-GFP), no major defect in sister chromatid segregation was observed when compared with wild-type cells. This indicates that the fission yeast Mph1, similar to other components of the spindle assembly checkpoint, plays a crucial role in homolog disjunction during meiosis I. The other question was if Spo4/Spo6, a meiosis-specific kinase complex that was shown to be important for progression of the second meiotic division,7 and its ortholog Dbf4-dependent Cdc7 kinase, required for DNA replication in most eukaryotes,8 is also required for proper sister chromatid segregation during meiotic divisions. Kovacikova and co-authors analyzed spo4∆ and spo6∆ mutant cells, where one copy of the first chromosome was marked with GFP (lys1-GFP). Although, most of cells arrested at the binucleate stage, a small fraction went through both meiotic divisions. These cells were able to produce asci with four nuclei. Scoring GFP dots in such asci showed that more than 40% contained GFP dots in both halves, suggesting missegregation of sister centromeres during meiosis I. Unexpectedly, missegregation of sister chromatids was not observed when the authors analyzed anaphase I cells. Importantly, Kovacikova et al. noticed that anaphase II spindles in spo4∆ and spo6∆ cells were significantly longer as compared with wild-type spindles. Live-cell imaging showed that these spindles pushed sister nuclei apart, leading to abnormal position of spore in mutant asci. This defect could be rescued only with the wild-type allele and not the “kinase dead” allele of Spo4 (Spo4K95A), which indicates that altered Spo4 kinase activity is responsible for abnormal meiosis II spindle elongation. Moreover, duration of anaphase II in spo4∆ cells was more than twice as long as compared with wild-type cells. This implies that Spo4 is required for proper timing of anaphase II. Interestingly, similar results were observed with another sporulation-defective strain, spo5∆, which points out the possible link between sporulation and proceeding of anaphase II. Concomitant inhibition of the S. pombe CDK kinase, Cdc2, together with spo4 deletion caused reduction of the spo4∆ mutant phenotype to approximately one-half, suggesting that altered Cdc2 activity is responsible for abnormal spindle elongation in cells lacking Spo4. Taken together, Kovacikova et al. showed that protein kinases such as Mph1 or Spo4 play important roles during meiosis, and detailed studies of protein kinases and reversible protein phosphorylation are essential for our understanding of processes ensuring proper chromosome segregation.

GENOTYPES OF FIVE BLOOD PROTEINS’ POLYMORPHISM IN VARIOUS PRODUCTION AGE OF DAIRY COWS

In this work were analysed samples of blood from 314 dairy cows Slovak spotted breed by the method of starch gel electrophoresis. The aim was to identify the biochemical polymorphic markers in blood plasma and in extract of erythrocytes. The polymorphism of hemoglobin (AA+AB+BB), amylasa (AA+AB+BB), transferrin (AA+AD1+AD2+AE+D1D1+D1E), albumin (AA+AB+BB) and postalbumin (AA+AB+AC+BB+BC+CC) were proved. File of dairy cows was divided to fi ve groups by age. The fi rst group create of dairy cows in age 25-48 months, second 49-72 months, third 73-96 months, fourth 97-120 months and the last over 121 months. According to evaluation of heterozygotes genotypes were detect decrease into of AB genotype in polymorphism of hemoglobin from primary 46.8 % (25-48 month) to 11.1 % in the last age group of dairy cows up 121-th months. Heterozygotic combinations in transferrin locus (AD1+AD2+AE+D1E genotypes) culminated from the beginning 14.2 % to 38.9 % at the fi rst group and last one. The postalbumin polymorphism had the high rate of heterozygotes because of AB+AC+BC genotypes. Rate of heterozygous genotypes AB in the polymorphism system amylasa variance between 15.4 - 23.7 %. Heterozygotic genotypes AB in albumin locus was according to our analyse at dairy cows in the fi rst evaluated of age group 52.9 %, second one only in fi fth groups at intervals 71.8 - 75.0 %. Among tested heterozygotes (AB genotype) and homozygotes (AA+BB genotypes) we found the signifi cant differences only in albumin locus 2,18+ (+P≤0,05).