Kompetitive Allele Specific PCR Genotyping of 89 SNPs in Romanian Spotted and Romanian Brown Cattle Breeds and Their Association with Clinical Mastitis.

Mastitis is the most common production disease in the dairy sector worldwide, its incidence being associated with both cows' exposure to bacteria and the cows' genetic make-up for resistance to pathogens. The objective of our study was to analyse 89 missense SNPs belonging to six genes (CXCR2, CXCL8, TLR4, BRCA1, LTF, BOLA-DRB3), which were found to be associated with genetic resistance or susceptibility to mastitis. A total of 298 cattle (250 Romanian Spotted and 48 Romanian Brown) were genotyped by Kompetitive Allele Specific PCR (KASP) and a chi-squared test was used for genetic association studies with clinical mastitis. A total of 35 SNPs (39.3%) among the selected 89 SNPs were successfully genotyped, of which 31 markers were monomorphic. The polymorphic markers were found in two genes: TLR4 (rs460053411) and BOLA-DRB3 (rs42309897, rs208816121, rs110124025). The polymorphic SNPs with MAF > 5% and call rates > 95% were used for the association study. The results showed that rs110124025 in the BOLA-DRB3 gene was significantly associated with mastitis prevalence (p ≤ 0.05) in both investigated breeds. Current results show that the SNP rs110124025 in the BOLA-DRB3 gene can be used as a candidate genetic marker in selection for mastitis resistance in Romanian dairy cattle.

Genetic polymorphism in European and African sheep breeds reared in Hungary based on 48 SNPs associated with resistance to gastrointestinal parasite infection using KASP-PCR technique.

This pilot study used an alternative and economically efficient technique, the Kompetitive Allele-Specific Polymerase Chain Reaction (KASP-PCR) to examine 48 SNPs from 11 parasite-resistance genes found on 8 chromosomes in 110 animals from five sheep breeds reared in Hungary; Hungarian Tsigai, White Dorper, Dorper, Ile de France, and Hungarian Merino. Allele and genotype frequencies, fixation index, observed heterozygosity, expected heterozygosity, F statistic, and their relationship with the Hardy-Weinberg equilibrium (WHE) and the polymorphic information content (PIC) were determined, followed by principal component analysis (PCA). As much as 32 SNPs out of the 48 initially studied were successfully genotyped. A total of 9 SNPs, 4 SNPs in TLR5, 1 SNP in TLR8, and 4 SNPs in TLR2 genes, were polymorphic. The variable genotype and allele frequency of the TLRs gene indicated genetic variability among the studied sheep breeds, with the Hungarian Merino exhibiting the most polymorphisms, while Dorper was the population with the most SNPs departing from the HWE. According to the PIC value, the rs430457884-TLR2, rs55631273-TLR2, and rs416833129-TLR5 were found to be informative in detecting polymorphisms among individuals within the populations, whereas the rs429546187-TLR5 and rs424975389-TLR5 were found to have a significant influence in clustering the population studied. This study reported a moderate level of genetic variability and that a low to moderate within-breed diversity was maintained in the studied populations.

Simultaneous Occurrence of Hypospadias and Bilateral Cleft Lip and Jaw in a Crossbred Calf: Clinical, Computer Tomographic, and Genomic Characterization.

Congenital abnormalities in animals, including abnormalities of the cleft lip and jaw and hypospadias have been reported in all domesticated species. They are a major concern for breeders due to the increased economic loss they entail. In this article, we described a congenital bilateral cheilognathoschisis (cleft lip and jaw) with campylognathia in association with penile hypospadias and preputial hypoplasia with failure of preputial fusion in a Bos taurus crossbred Piedmontese × Wagyu calf. Clinical examination, computed tomography, and whole genome sequencing were performed to describe and identify a possible cause of the abnormalities. Clinical examination revealed a bilateral cheilognathoschisis of approximately 4 cm in length and 3 cm in width in the widest part, with computer tomography analyses confirming the bilateral absence of the processus nasalis of the incisive bone and the lateral deviation of the processus palatinus towards the left side. Genomic data analyses identified 13 mutations with a high impact on the products of the following overlapped genes: ACVR1, ADGRA2, BHMT2, BMPR1B, CCDC8, CDH1, EGF, F13A1, GSTP1, IRF6, MMP14, MYBPHL, and PHC2 with ADGRA2, EGF, F13A1, GSTP1, and IRF6 having mutations in a homozygous state. The whole genome investigation indicates the involvement of multiple genes in the birth defects observed in this case.

Polymorphism of the Prolactin (PRL) Gene and Its Effect on Milk Production Traits in Romanian Cattle Breeds.

In the present study, we investigated one polymorphism of the PRL gene (rs211032652 SNP) and assessed its influence on milk production and chemical composition in two Romanian cattle breeds. A total of 119 cattle from two breeds reared in Western Romania (64 Romanian Spotted and 55 Romanian Brown) were included in the research herd. A PCR-RFLP genotyping assay was used for the identification of the rs211032652 SNP variants. Shapiro's test and Levene's test were used to verify ANOVA assumptions and ANOVA and Tukey's test were employed to test the associations between PRL genotypes and five milk traits. Among the studied breeds, our results showed that PRL genotypes were significantly associated (p < 0.05) with fat and protein percentage in the milk of Romanian Brown cattle. The AA genotype was associated with a higher fat percentage in milk (4.76 ± 0.28) compared to the GG genotype (4.04 ± 0.22, p = 0.048), as well as a higher protein percentage (3.96 ± 0.32% vs. 3.43 ± 0.15%, p = 0.027) in Romanian Brown cattle. Moreover, the PRL locus favored a significantly higher fat (p = 0.021) and protein (p = 0.028) percentage in the milk of Romanian Brown cattle compared to the Romanian Spotted breed, with a difference of 0.263% and 0.170%, respectively.

Validation of SNP markers for thermotolerance adaptation in Ovis aries adapted to different climatic regions using KASP-PCR technique.

A study on 51 SNPs belonging to 29 genes related to heat stress was carried out in 720 sheep from 17 different breeds adapted to different climates from Hungary, Bosnia and Herzegovina, Morocco and Romania, using Kompetitive Allele-Specific Polymerase Chain Reaction. Genotype frequency and the Hardy-Weinberg equilibrium were calculated, followed by a clustering using the Principal Component Analysis. We analyzed the polymorphisms in the following genes analyzed: HSPA12A, HSP90AA1, IL33, DIO2, BTNL2, CSN2, ABCG1, CSN1S1, GHR, HSPA8, STAT3, and HCRT. We emphasized on HSPA12A and HSPA8 genes as they were successfully genotyped in all studied flocks in which genotype frequency patterns were identified. Contrary to previous findings, the A allele for HSPA8 SNP was not observed in the heat tolerant breeds, being found exclusively in cold-tolerant breeds. The principal component analysis could not clearly differentiate the breeds, while plot concentration was slightly varied among the three groups, with HSP90AA1 and IL33 SNPs' loading values significantly contributing to PC1 and PC2. We confirmed previous works that the HSPA12A, HSPA8, HSP90AA1 and IL33 SNPs are potential candidate markers for thermotolerance adaptation in sheep. This research contributes to the genetic variability of SNPs for thermotolerance adaptability in sheep.

Assessing Phenotypic Variability in Some Eastern European Insular Populations of the Climatic Relict Ilex aquifolium L.

Through its natural or cultivated insular population distribution, Ilex aquifolium L. is a paramount species which is exceptionally suitable for studying phenotypic variability and plasticity through the assessment of morphological, physiological, biochemical and genomic features with respect to acclimation and/or adaptation efficiency. The current study is focused on four insular populations of Ilex aquifolium from Eastern Europe (i.e., in Romania, Hungary, Serbia and Bulgaria), and presents an initial evaluation of phenotypic variability in order to conclude our research on phylogenetic relationships and phytochemical profiles, including several descriptive and quantitative morphological traits. Taken together, the data from different methods in this paper indicate that the Bulgarian and Romanian populations can be distinguished from each other and from Serbian and Hungarian populations, while the latter show a higher level of resemblance with regards to their quantitative morphological traits. It is likely that these morphological traits are determined through some quantitative trait loci implicated in stress responses generated by light, temperature, soil water, soil fertility and salinity conditions that will need to be analysed in terms of their physiological, genomic and metabolomics traits in future studies.

Genome-Wide Association Studies for Milk Somatic Cell Score in Romanian Dairy Cattle.

Mastitis is one of the most frequently encountered diseases in dairy cattle, negatively affecting animal welfare and milk production. For this reason, contributions to understanding its genomic architecture are of great interest. Genome-wide association studies (GWAS) have identified multiple loci associated with somatic cell score (SCS) and mastitis in cattle. However, most of the studies have been conducted in different parts of the world on various breeds, and none of the investigations have studied the genetic architecture of mastitis in Romanian dairy cattle breeds up to this point in time. In this study, we report the first GWAS for SCS in dairy cattle breeds from Romania. For GWAS, we used an Axiom Bovine v3 SNP-chip (>63,000 Single Nucleotide Polymorphism -SNPs) and 33,330 records from 690 cows belonging to Romanian Spotted (RS) and Romanian Brown (RB) cattle. The results found one SNP significantly associated with SCS in the RS breed and 40 suggestive SNPs with -log10 (p) from 4 to 4.9 for RS and from 4 to 5.4 in RB. From these, 14 markers were located near 12 known genes (AKAP8, CLHC1, MEGF10, SATB2, GATA6, SPATA6, COL12A1, EPS8, LUZP2, RAMAC, IL12A and ANKRD55) in RB cattle, 3 markers were close to ZDHHC19, DAPK1 and MMP7 genes, while one SNP overlapped the HERC3 gene in RS cattle. Four genes (HERC3, LUZP2, AKAP8 and MEGF10) associated with SCS in this study were previously reported in different studies. The most significant SNP (rs110749552) associated with SCS was located within the HERC3 gene. In both breeds, the SNPs and position of association signals were distinct among the three parities, denoting that mastitis is controlled by different genes that are dependent according to parity. The current results contribute to an expansion in the body of knowledge regarding the proportion of genetic variability explained by SNPs for SCS in dairy cattle.

Evaluation of single nucleotide polymorphisms identified through the use of SNP assay in Romanian and Chinese Holstein and Simmental cattle breeds.

Simmental and Holstein cattle, being among the most widely distributed breeds worldwide, have been subjected to continuous selection for distinct purposes. In the current study, we evaluated the levels of SNPs identified through the use of SNP assay in Romanian Holstein and Romanian Simmental cattle, which then were compared to the data from Chinese Holstein and Chinese Simmental cattle. In total, 282 animals were genotyped: Romanian Holstein (n=30), Romanian Simmental (n=22), Chinese Holstein (n=96) and Chinese Simmental cattle (n=136), using 39,724 common SNPs to analyze minor allele frequency, genetic variability and level of SNPs. Among studied breeds, the average percentage of polymorphic markers was 90.84%, with the highest value in Chinese Simmental (91.37%) and lowest in Romanian Simmental cattle (90.31%). The average HO ranged from 0.426 in Romanian Holstein to 0.416 in Romanian Simmental, and from 0.425 in Chinese Holstein to 0.422 in Chinese Simmental. The distribution of SNPs was homogenous across the breeds, except the Romanian Simmental which displayed the lowest percentage of polymorphic markers (24,66 and 32,48%) from higher MAF category (0.3 to <0.4 and 0.4 to <0.5) and the highest percentage (3.82 and 12.00%) for SNPs from low and intermediate MAF categories (0.05 to <0.1 and 0.1 to <0.2). In the current study, the SNP assay was successfully used to analyze the level of SNP sites of Romanian cattle breeds, however, a higher number of samples and production data are needed for future applications of the results in genomic selection, genome-wide association studies and genetic diversity analysis.

Large-scale mitochondrial DNA analysis reveals new light on the phylogeography of Central and Eastern-European Brown hare (Lepus europaeus Pallas, 1778).

European brown hare, Lepus europaeus, from Central and Eastern European countries (Hungary, Poland, Serbia, Lithuania, Romania, Georgia and Italy) were sampled, and phylogenetic analyses were carried out on two datasets: 1.) 137 sequences (358 bp) of control region mtDNA; and 2.) 105 sequences of a concatenated fragment (916 bp), including the cytochrome b, tRNA-Thr, tRNA-Pro and control region mitochondrial DNA. Our sequences were aligned with additional brown hare sequences from GenBank. A total of 52 and 51 haplotypes were detected within the two datasets, respectively, and assigned to two previously described major lineages: Anatolian/Middle Eastern (AME) and European (EUR). Furthermore, the European lineage was divided into two subclades including South Eastern European (SEE) and Central European (CE). Sympatric distribution of the lineages of the brown hare in South-Eastern and Eastern Europe revealed contact zones there. BAPS analysis assigned sequences from L. europaeus to five genetic clusters, whereas CE individuals were assigned to only one cluster, and AME and SEE sequences were each assigned to two clusters. Our findings uncover numerous novel haplotypes of Anatolian/Middle Eastern brown hare outside their main range, as evidence for the combined influence of Late Pleistocene climatic fluctuations and anthropogenic activities in shaping the phylogeographic structure of the species. Our results support the hypothesis of a postglacial brown hare expansion from Anatolia and the Balkan Peninsula to Central and Eastern Europe, and suggest some slight introgression of individual haplotypes from L. timidus to L. europaeus.

Genetic characterization of indigenous goat breeds in Romania and Hungary with a special focus on genetic resistance to mastitis and gastrointestinal parasitism based on 40 SNPs.

Goat breeding has become an important sector in Eastern Europe, with Romania and Hungary being among the major producer countries. Given the limited number of research done up-to-date concerning genetic studies of indigenous goat breeds reared in Romania and Hungary, the current preliminary study aimed to analyze the variability of genes related to mastitis and gastrointestinal parasitism by using Kompetitive Allele Specific PCR (KASP™). We studied 52 single nucleotide polymorphisms (SNPs) belonging to 19 genes in indigenous breeds from both countries, namely Banat's White (n = 36), Carpatina (n = 35) from Romania and Hungarian Milking (n = 79) and identified 16 polymorphic SNPs among 10 genes (PTX3, IL6, CLEC4E, IL8, IL1RN, IL15RA, TNFSF13, SOCS3, TNF and TLR3) in 150 animals. Furthermore, the diversity of the studied breeds was investigated. The PIC values ranged from 0.042 to 0.691. The mean values of observed and expected heterozygosity were 0.235 and 0.246 respectively. The highest observed heterozygosity was obtained for IL15RA g.10343904C>T in Banat's White (0.464), IL15RA g.10354813C>T in Carpatina (0.577) and SOCS3 g.52626440T>G in Hungarian Milking (0.588). Pairwise FST values between the Romanian breeds and Romanian and Hungarian breeds were small (0.009 and 0.015), indicating the close relationship among the studied goat populations. From all the polymorphic SNPs identified, the Hungarian Milking breed showed the highest proportion of polymorphisms (100%), whereas the Carpatina breed had the lowest percentage (87.5%). The highest value of MAF was obtained for SOCS3 g.52626440T>G (0.46), IL15RA g.10343904C>T (0.47), IL15RA g.10344025C>T (0.45), and IL15RA g.10354813C>T (0.42). The 16 polymorphic SNPs identified in a panel of 150 unrelated individuals belonging to three Romanian and Hungarian indigenous goat breeds could be used in future genomic based breeding schemes as markers for genetic resistance to mastitis and gastrointestinal parasitism in goat breeds found in Eastern and Central Europe.

Kompetitive Allele Specific PCR (KASP™) genotyping of 48 polymorphisms at different caprine loci in French Alpine and Saanen goat breeds and their association with milk composition.

Using a novel and fast genotyping method called Kompetitive Allele Specific PCR (KASP™), we carried out a pilot study on 48 single nucleotide polymorphisms (SNPs) belonging to 40 genes in French Alpine (n = 24) and Saanen (n = 25) goats reared in Romania. Furthermore, the associations of the 13 polymorphic genetic variants with milk production and composition were investigated. Thirty-five SNPs did not show polymorphism in the studied populations. Polymorphic SNPs were detected in the following genes: CAST, CLEC4E, DES, GHRHR, HSP90AA1, IL15RA, IL1RN, IL8, MITF, PPRC1, SOCS3, TNF and TNFSF13. The studied Alpine population was in Hardy-Weinberg disequilibrium at the g.62894878A>G locus (rs671391101) (P < 0.05). The results showed that four SNPs rs671391101 (GHRHR), rs640582069 (IL1RN) rs635583012 (SOCS3) and rs635969404 (IL15RA) out of the 13 polymorphic markers were significantly associated with milk production, protein, fat and lactose content in the Alpine breed. However, no significant effect was recorded in the Saanen population regarding milk yield or milk chemical composition. The current results provide new insights for the development of SNP marker-assisted selection technology in the goat industry and confirm the potential of using SNPs for the GHRHR, IL1RN, SOCS3, and IL15RA genes as candidate genes for selection, highlighting the direct implications of such genes for farm production outputs. The results from this study are relevant for future goat genomic studies and the inclusion of the associated traits into up-to-date selection schemes.

The influence of CSN3 and LGB polymorphisms on milk production and chemical composition in Romanian Simmental cattle.

The aim of this study was to assess the influence of genotypes from the CSN3 and LGB loci on milk production and chemical composition in Romanian Simmental cattle (n=114). For the CSN3 locus a significantly higher frequency (P≤0.001) was observed for the allele A (0.785) compared to allele B (0.215) and for AA (0.631) compared to AB (0.307) and BB (0.062) genotypes, respectively. The AA genotype was associated with a higher milk production (5887.76±115.7 kg) compared to the yields associated with the BB genotype (5619±86.34 kg, P≤0.003), as well as a higher fat percentage (4.19±0.05% vs. 4.01±0.02%, P≤0.001). A higher milk protein percentage was associated with BB (3.4±0.02%) compared to AA (3.27±0.03) and AB (3.29±0.03) genotypes, respectively (P≤0.05). For the LGB locus, the frequency of allele A was significantly higher (0.606, P≤0.008) than allele B (0.394). The AB genotype was the most prevalent in the herd (0.579) compared to AA (0.316) and BB genotypes (0.105). The AB genotype was associated with a higher milk production (5906.54±166.76 kg) compared with the other two genotypes (P≤0.05). The BB genotype was associated with a higher fat percentage in milk (4.23±0.06%) compared with AA (4.17±0.06) and AB (4.2±0.08) genotypes, respectively (P≤0.01). For the LGB locus, no significant differences (P>0.05) were observed for milk protein percentage. In order to increase the quantity and quality of milk, the outcomes obtained in this study encourage improving genetic structure in cattle based on marker assisted selection for genes with economic values.

Preliminary report on ß-casein gene Met183QVal183 polymorphism in Romanian indigenous Zackel sheep breeds.

Genetic polymorphisms of the milk protein genes are important because of their effects on quantitative traits and technological properties of milk manufacturing. In the present study we identified the polymorphism of the beta-casein gene in two local sheep breeds (Racka n=98 and Turcana n=111) in Romania. The most studied variants at the ovine beta-casein (CSN2) locus are: A and G variants. Genomic DNA was extracted from hair follicles and beta-casein genotypes were determined by the rapid TaqMan (Applied Biosystems, USA) genotyping assay. Homozygote genotypes GG were not detected in any of the studied breeds. In both, the Racka and Turcana breeds, the A variant had a much higher frequency, 0.98% and 0.97%, respectively. In the current study, the fast DNA tests for genotyping ovine CSN2 were successfully optimized, however, further samples and correlations of genomic results with milk characteristics and production data are needed for the development of future selection schemes of the Romanian indigenous sheep breeds, with the ultimate purpose to produce low allergen level sheep milk and derived dairy products.

Comparative study on production, reproduction and functional traits between Fleckvieh and Braunvieh cattle.

OBJECTIVE: Aim of the current comparative study was to evaluate production outputs, reproduction efficiency and functional traits in dual-purpose Fleckvieh and Braunvieh cows, reared under temperate European conditions. METHODS: A data-set from 414 Fleckvieh and 42 Braunvieh cows and 799 lactations was analysed. ID tag number, milk yield per milking session, number of steps/interval and milk conductivity were recorded and collected daily using AfiMilk 3.076 A-DU software (Afimilk Ltd., Kibbutz, Israel). Production and milk quality data were taken from the results of the official performance recordings and the reproductive outputs of cows were recorded by the research stations veterinarians. Comparisons between the two genotypes were carried out using the one way analysis of variance protocol, with categorical factor being considered the breed of cows. All the statistical inferences were carried out using Statistica software (StatSoft Inc., Tulsa, OK, USA). RESULTS: Fleckvieh cows significantly outperformed (p≤0.05) the Braunvieh herd, with average milk yields of 5,252.1±35.79 kg and 4,897.6±128.94 kg, respectively. Age at first calving was significantly (p≤0.01) influenced by the breed, with Fleckvieh heifers being more precocious (32.8±0.29 mo) compared to those of Braunvieh breed (35.7±0.84 mo). Reproduction efficiency as defined by the number of inseminations per gestation, calving interval, dystocia, days dry and days open, was not influenced by genotype (p>0.05). Incidences of sub-clinical mastitis, clinical mastitis, lameness and abortions were not influenced by the breed factor (p>0.05). Stayability of cows was significantly (p≤0.001) influenced by genotype, with Braunvieh cows having an average age at culling of 117.88±11.78 months compared to 90.88±2.89 months in Fleckvieh. CONCLUSION: Overall, results have shown that genotype significantly influenced milk yield, age at first calving and longevity.

Genetic polymorphism of CSN2 gene in Banat White and Carpatina goats.

The main objective of the current study was to obtain preliminary results on genetic polymorhism of A and C variants at the CSN2 locus in indigenous Romanian goat (Banat White, Carpatina) breeds using a rapid and efficient genotyping method, TaqMan assay (Applied Biosystems, USA). Hair follicle samples were taken from 73 Banat White and 82 Carpatina purebred goats from Arad, Caras-Severin and Timis counties. After the optimization of the genotyping assay it was found that the most frequent allele at the CSN2 locus was C in Banat White breed, while CSN2*A and CSN2*C showed similar frequencies in Carpatina breed (0.51 and 0.49, respectively). All three genotypes were detected in the two studied breeds, however, AA was the least frequent, especially in Banat White. The studied polymorphisms are potential markers for milk production in the studied breeds and the results will be useful in future works aimed at identifying possible associations with milk production traits, in order to test the feasibility of producing hypoallergenic organic goat milk.

Microsatellite and Mitochondrial DNA Study of Native Eastern European Cattle Populations: The Case of the Romanian Grey.

The Eastern European Grey cattle are regarded as the direct descendants of the aurochs (Bos taurus primigenius). Nowadays in Romania, less than 100 Grey animals are being reared and included in the national gene reserve. We examined the genetic diversity among Romanian Grey, Brown, Spotted and Black and White cattle breeds, with a particular focus on Romanian Grey through the use of (i) 11 bovine specific microsatellite markers on 83 animals and (ii) 638 bp length of mitochondrial DNA (mtDNA) D-loop region sequence data from a total of 81 animals. Both microsatellite and mtDNA analysis revealed a high level of genetic variation in the studied breeds. In Romanian Grey a total of 100 alleles were found, the mean number of observed alleles per locus was 9.091; the average observed heterozygosity was 0.940; the Wright's fixation index (FIS) was negative (-0.189) and indicates that there is no inbreeding and no selection pressure. MtDNA analysis revealed 52 haplotypes with 67 variable sites among the Romanian cattle breeds without any insertion or deletion. Haplotype diversity was 0.980 ± 0.007 and ranged from 0.883 ± 0.056 (Brown) to 0.990 ± 0.028 (Spotted and Black and White). The highest genetic variability of the mtDNA was recorded in the Grey breed, where 18 haplotypes were identified. The most frequent mtDNA D-loop region belonged to T3 haplogroup (80.247%), which was found across all studied breeds, while T2 haplotypes (16.049%) was only found in Grey, Spotted and Black and White genotypes. The T1 haplotypes (3.704%) were found in the Grey and Spotted. The current results contribute to the general knowledge on genetic diversity found in Eastern European cattle breeds and could prove a valuable tool for the conservation efforts of animal genetic resources (FAnGR).