Daniela Elena Ilie

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).

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.

Demographic Study on the Total Sura de Stepă Breed Population in Romania

Abstract The research was conducted from August until November 2012 in the districts of Moldavia (Botoşani, Suceava, Iaşi, Vaslui, Bacău, Neamţ, Galaţi and Vrancea), in the West and the Central part of the country (Cluj and Covasna districts) and in the South-East of the country (Tulcea and Brăila districts). The choice of the districts was established based on the informations received from Animal Improvement and Breeding Offices in Romania. The purpose of this research was to identify the effective of Sura de stepă cattle breed in Romania and the evaluation of the phenotypic characters in steppe animals, which belong to the variety of Moldavian breed. The results showed that the Sura de stepă breed consists of a minor part, being raised in just two districts of Moldavia, Iaşi and Neamţ, respectively, as a pure breed with an average of 0,03 % (83 cows), at the Research and Development Station for Cattle Breeding (R.D.S.C.B.) Dancu-Iaşi, with a core of preservation of 59 cows (0,19 %) and at the Holding TCE 3 Brazi Society, Neamţ district, with a core of 24 cows (0,06 %), and under half-breed form with an average of 0,33 % (592 cows). In Harghita, Covasna and Cluj districts, 295 cows from the Sura de stepă breed, Hungarian variety, were identified, excepting being the animals belonging to University of Agricultural Sciences and Veterinary Medicine Cluj, which come from R.D.S.C.B. Dancu-Iaşi, which are Romanian Sura de stepă, Moldavian variety. In Tulcea district, 20 cows of half-breed Sura de stepă and 50 cows of pure breed cattle, Moldavian variety, were identified, from which 24 cows at a private landlord in Pardina locality, Tulcea district, and 26 cows belonging to nine owners from different localities (C.A. Rosetti, Pardina, Chilia Veche, Sfântu Gheorghe, Crişan), each owning 2-3 cows. The analysis of the main body indices showed that the Sura de stepă cows from Pardina, Tulcea county, are of small size and weight, having smaller values in all analysed parameters, compared to the Sura de stepă cows from R.D.S.C.B. Dancu-Iaşi from the North-East part of the country, which however fitted the specific limits of the breed, Moldavian variety. Rezumat Studiu demografic asupra populaţiei totale din rasa Sura de stepădin România. Studiul a fost efectuat în perioada august-noiembrie 2012 în judeţele din Moldova (Botoşani, Suceava, Iaşi, Vaslui, Bacău, Neamţ, Galaţi, şi Vrancea), în vestul şi centrul ţării (Cluj, Covasna) şi în sud-estul ţării (Tulcea şi Brăila). Alegerea judeţelor s-a facut pe baza informaţiilor primite de la Oficiile de Ameliorare şi Reproducţie în Zootehnie. Scopul acestei lucrări a fost de a identifica efectivele de vaci din rasa Sura de stepă din Romania şi de a evalua caracterele fenotipice de rasă la animalele care aparţin varietăţii moldoveneşti a rasei. Rezultatele au evidenţiat că rasa Sura de stepă are o pondere foarte mică, fiind crescută doar în două judeţe din Moldova, respectiv Iaşi şi Neamţ, ca rasă curată, în medie 0,03 % (83 capete), la Staţiunea de Cercetare- Dezvoltare pentru Creşterea Bovinelor (S.C.D.C.B.) Dancu-Iaşi, cu un nucleu în conservare de 59 capete (0,19%) şi la S.C. Holding TCE 3 Brazi S.R.L., Neamţ county, cu un nucleu de 24 capete (0,06%), iar sub formă de metişi, în medie 0,33 % (592 capete). În judeţele Harghita, Covasna şi Cluj au fost identificate un număr de 295 animale din rasa Sura de stepă, varietatea maghiară, cu exceptia animalelor care aparţin U.Ş.A.M.V. Cluj, provenite de la S.C.D.C.B. Dancu-Iaşi, care sunt Sura de stepă românească, varietatea moldovenească. În judeţul Tulcea a fost identificat un numar total de 2011 capete metişi din rasa Sura de stepă, 50 capete taurine de rasă curată, varietatea moldovenească, din care 24 capete la un proprietar particular în localitatea Pardina, judeţul Tulcea, iar 26 de capete au fost identificate la nouă proprietari din diverse localităţi (C.A. Rosetti, Pardina, Chilia Veche, Sfântu Gheorghe, Crişan ), câte 2-3 capete/gospodărie. Analiza principalilor indici corporali a evidenţiat faptul că vacile din rasa Sura de stepă de la Pardina, judeţul Tulcea, sunt de talie şi greutate mai mici, cu valori medii mai mici la toţi parametri analizaţi, comparativ cu vacile Sura de stepă de la S.C.D.C.B. Dancu-Iaşi, din zona de nord-est a ţării, dar care s-au încadrat în limitele specifice rasei, varietatea moldovenească.

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.

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.

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.

Preliminary report on β-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.

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.