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A recent comprehensive genomic analysis based on 50K SNP profiles has shown that the regional Balkan sheep populations have considerable genetic overlap but are distinctly different from surrounding breeds. All eight Croatian sheep breeds were represented by a small number of individuals per breed. Here, we genotyped 220 individuals representing the native Croatian sheep breeds (Istrian Sheep, Krk Island Sheep, Cres Island Sheep, Rab Island Sheep, Lika Pramenka, Pag Island Sheep, Dalmatian Pramenka, Dubrovnik Sheep) and mouflon using the Ovine Infinium® HD SNP BeadChip (606,006 SNPs). In addition, we included publicly available Balkan Pramenka and other Mediterranean sheep breeds. Our analyses revealed the complex population structure of Croatian sheep breeds and their origin and geographic barriers (island versus mainland). Migration patterns confirmed the historical establishment of breeds and the pathways of gene flow. Inbreeding coefficients (FROH>2 Mb) between sheep populations ranged from 0.025 to 0.070, with lower inbreeding coefficients observed in Dalmatian Pramenka and Pag Island Sheep and higher inbreeding in Dubrovnik sheep. The estimated effective population size ranged from 61 to 1039 for Krk Island Sheep and Dalmatian Pramenka, respectively. Higher inbreeding levels and lower effective population size indicate the need for improved conservation management to maintain genetic diversity in some breeds. Our results will contribute to breeding and conservation strategies of native Croatian sheep breeds.

Estimation of inbreeding is important for the survival of genetically small populations. Therefore, the objective of this study was to i) estimate genomic inbreeding coefficients on the X chromosome in three native Croatian cattle breeds, ii) compare estimates of different genomic inbreeding coefficients on the X chromosome based on identity by descent (IBD) with those based on identity by state (IBS), and iii) analyse segmental variation in inbreeding on the X chromosome. The analysis was based on three Croatian cattle breeds (112 individuals) genotyped with the Illumina BovineHD SNP chip (777, 000 SNPs). Our analyses revealed peculiarities in the estimation of genomic X chromosome inbreeding compared to autosomal inbreeding, e.g. higher variation and higher level of inbreeding, which should not be neglected. The obtained results can be used to estimate inbreeding depression and to identify deleterious mutations related to the X chromosome. However, to obtain more general knowledge about the genomic inbreeding level of the X chromosome, more cattle breeds should be analysed.

The aim of this study was to determine the position of two Croatian goats in the global context using genome-wide SNP data, while assessing population structure, admixture, and conservation status. All analyses were performed on the genotypes of 11 Istrian and 33 Croatian coloured goats and a sample of 2255 goats from 66 global goat breeds. Neighbor net and admixture results show that two breeds have high genetic diversity, with much of the historical admixture due to an extensive produc tion system. Principal component analysis (PCA) showed clear clustering between the two breeds, while some Istrian goats showed clear clustering with the Saanen breed. The Croatian Coloured goat was most similar to the Greek goats. The results obtained with the Treemix software confirmed these findings and showed gene flow from the Croatian Coloured goat to the Istrian goat. Despite the low Ne for the Istrian goat, both breeds showed a low degree of inbreeding (FROH~0.05). Consider ing these results, the Istrian goat is at high risk of extinction and should be carefully monitored.

Goat rearing is an important sector of livestock production and managing the genetic diversity of local goat breeds is an important component of genetic improvement and environmental adaptation. The aim of this study was to analyse the ge netic diversity based on 46 862 autosomal SNPs of the Czech and Slovak local goat breeds (Czech White Shorthair – CWS, Slovak White Shorthair – SWS, Czech Brown Shorthair – CBS and Czech Landrace – CLA). This analysis was based on 140 individuals representing Czech and Slovak local breeds and sixteen Austrian and Swiss goat breeds (639 genotypes in total). We demonstrated that the Czech and Slovak local goat breeds are genetically similar populations. All Czech and one Slovak local breeds were closely related to other Austrian and Swiss goats. We also estimated genomic inbreeding level (FROH) and effective population size (NeLD) for each breed. Estimated effective population size (NeLD) for Czech and Slovak local breeds ranged from 56 (CLA) to 267 (CWS). We demonstrated that Czech and Slovak local breeds are affected by a loss of genetic diversity, while they are genetically related. The present results show that appropriate management of genetic diversity in local goat breeds is urgently needed to support the conservation of the specific biocultural system of grazing.

Mitochondria, whose mitochondrial DNA (~16.5 Kb) or mitogenome is passed through maternal inheritance, play a critical role in the general functional aspects of mammalian cells, and their dysfunction has been associated with various metabolic phenotypes related to bioenergy. Therefore, it is surprising that information about the mitogenome has been little used in animal breeding and diversity management. In the following, we first provide an overview of the biological aspects of mito chondrial inheritance and its implications for shaping phenotypic variation. Using cattle as an example, we will show how mitogenome information can help to i) estimate the impact on quantitative traits important for cattle production, ii) identify deleterious mutations that cause disease, iii) identify ancient introgression and upgrading, iv) estimate genetic diversity and relatedness of populations, v) screen and/or identify pedigree errors, and vi) estimate mitochondrial effectiveness and its link to productive traits. Finally, we will present some recent developments where mitogenome information has been implemented in the commercial SNP chip.

Sheep rearing is one of the most important types of extensive livestock in Croatia, occurring mainly in coastal and mountainous areas along the eastern Adriatic. We retrieved mitogenome from the whole genome (12× and 30×) of 23 individuals representing eight indigenous sheep breeds (Cres Island Sheep, Dalmatian Pramenka, Dubrovnik Ruda, Istria Sheep, Krk Island Sheep, Lika Sheep, Pag Island Sheep and Rab Island Sheep) and two mouflons. The average coverage of the mitogenome from the 12-fold sequenced genome of 17 samples was 1, 627 (min 322 ; max 9, 588) and from the 30- fold sequenced genome of eight samples was 1, 851 (min 865 ; max 2, 841). Our 16, 624 bp fasta set consisted of 112 parsim-info sites and 247 singleton sites while obtained 24 haplotypes with a haplotype diversity of 0.997. When compared with the mitogenome variability of 347 sheep (86 breeds) and three mouflon (Ovis aries musimon) available in GenBank, the median-joining network showed that 22 haplotypes belonged to haplogroup B and, interestingly, one belonged to haplogroup A and one to haplogroup C. Bayesian analyses also confirmed a high frequency of haplogroup A in the breeds from Central and East Asia, and a high frequency of haplogroup B in Europe and Anatolia. The rare haplogroups C occur in the Iberian Peninsula area, the Middle East, the Caspian Sea region, China, India, and Nepal, while haplogroups D and E may be found in the Middle East. The haplotypes of our mouflons were assigned to haplogroup B along with those of the other European mouflons. The presence of two haplotypes assigned to haplogroups A and C suggests migratory contacts between the Balkans and the Middle East that need to be further explored. In order to clarify the timing of the appearance of A and C haplotypes in the eastern Adriatic, our future analyses will refer to chronological analyses of ancient sheep bones.

Due to that Czech indigenous goat breeds, together with Slovak indigenous goat breeds, are closed population, face this population loss of genetic diversity, because these populations are closed. The aim of this study was to perform a genome-wide diversity characterization using Illumina GoatSNP50 BeadChip of indigenous Czech Braun Shorthair and White Shorthair goat breeds together with international Alpine goat from Czech Republic and indigenous White Shorthair goat breed from Slovak Republic. For comparison was in this analysis included indigenous alpine goat breed from Austria and Switzerland. To determine key conservation risk parameters, we estimated inbreeding (FROH) and current and historical effective population size (Ne). The observed estimates for Czech and Slovak indigenous breed show similar values as other alpine goat breeds. In addition, by comparing genetic differences by Neighbour-net, no differences were recorded between the Czech-Slovak WSG. The study provides data and information utilizable in the management of conservation programs planned to reduce inbreeding and to minimize loss of genetic variability.

This study used genome-wide SNP data to characterise the Istrian and Croatian Coloured goats genetically. Population structure and conservation status were analysed using 11 Istrian and 33 Croatian Coloured goats and 506 additional animals belonging to 19 European goat breeds. Neighbour-net and admixture results show that the two breeds are unique and retain a great proportion of the ancestral genetic structure. Despite low Ne for Istrian goats, both breeds showed low levels of inbreeding (FROH~0.05). Considering these results, the Istrian goat is at high risk of extinction and should be carefully monitored.

Most of the milk produced in Italy is processed into various types of cheese. Therefore, the insight into the milk processing properties is highly important. Caseins are milk proteins that are considered the most important in the context of cheese production. 54 SNP polymorphisms have been detected in the casein gene cluster on chromosome 6 in Italian Holsteins. With the increasing availability of SNP arrays in livestock species, a lot of genomic information has been generated, enabling a range of genomic analyses, including breeding values, inbreeding, population structure, genome-wide association studies, mapping of positive selection, and others. Runs of Homozygosity and Extended Haplotype Homozygosity are approaches that reveal precise patterns of homozygosity and provide valuable insights into past or ongoing selection events. In this study, 160, 000 Italian Holstein cattle genotyped with a 50k SNP array were used to analyse the casein gene cluster on chromosome 6 for extreme Runs of Homozygosity islands and integrated Haplotype Scores. The results showed that a certain number of animals have high levels of autozygosity on chromosome 6, which is also confirmed for homozygosity at the haplotype level based on high iHS scores. These analyses confirm the effects of selection on certain casein gene variants, although they were not included in the selection scheme. Since the casein gene cluster is important for the Italian Holstein population, the results of the selection signature analysis should be evaluated and hopefully contribute to the selection plans.

The Silesian Norik and the Czech Norik are Czech local draft horse breeds with a common history. These two Czech Norik breeds originated from the Austrian Norik breed. Although the Silesian Norik has been included in the Czech genetic reserve since 1990, genetic differentiation between these historically related Czech draft horse populations is considered to be low due to their shared history. The aim of this study was to analyse the genetic diversity based on 36 981 autosomal SNPs of the two Czech local Norik breeds (Silesian Norik and Czech Norik) together with the one Slovak local Norik breed (Muran Norik) and the original Austrian Norik breed. Other horse breeds kept in the Czech and Slovak Republics (Old Kladruber horse, Hucul and Shagya Arabian) were also included to study population structure, admixture and genetic relationships between Norik populations. Relatedness and population structure were analysed using Wright's FST coefficient, unsupervised Bayesian clustering, and multivariate analyses (principal component and discriminant analysis). We also estimated genomic inbreeding rate (FROH) and effective population size (NeLD) for each breed. We demonstrated that the Czech local Norik breeds are strongly genetically linked. Therefore, in further population management, an appropriate breeding strategy should be applied to preserve the Silesian Norik as a unique Czech genetic reserve, distinct from other Norik breeds.

Breeding programmes for dairy cows focus on improving milk production traits and are characterised by intensive use of a relatively small number of elite bulls, resulting in higher levels of inbreeding and inbreeding depression. The aim of this study was to evaluate the effect of inbreeding (per 1% increase) on milk (MY), protein (PY) and fat (FY) yield in the Czech population of Holstein cattle. Our Bayesian approach to statistical animal models included 4749 genotyped cows (effectively providing information on > 40, 000 SNPs) with records of milk production in the first lactation. Several different models, depending on pedigree (FPED related to pedigree with complete generation equivalent of 12.5) or ROH-based genomic (ROH>4Mb or ROH>8MB) inbreeding coefficients (covariates), were applied. All values in the high posterior density interval (99%HDI) were negative for the MY estimates, with mean genomic inbreeding depression estimates (expressed per 1% increase in inbreeding coefficient) higher than pedigree estimates, bFROH>4Mb=-30.0 (99%HDI from -36.3 to -23.3) and bFROH>8Mb=-34.4 (99%HDI from -40.5 to -28.2) versus bFPED=-13.2 (99%HDI from -16.8 to -9.6). Negative mean estimates of inbreeding depression were obtained in all models, with only one exception (MY estimates for FROH > 8Mb) with respect to PY (bFPED=-0.8, bFROH>4Mb=-0.9, bFROH>8Mb=-0.9) and FY (bFPED=-0.7, bFROH>4Mb=-0.1, bFROH>8Mb=0.0), although positive estimates were observed even at an HDI of 95%, which could be the consequence of the low statistical power of the analyses performed. Therefore, the presented results should be confirmed using a larger and more informative data set.

Mating among related animals is an inevitable consequence of a closed population structure especially when it is coincided with small census size. As a result inbreeding depression may be encountered especially for fitness traits. However, under certain circumstaces the joint effects of inbreeding and selection may at least partly purge the detrimental genes from the population. In the course of this study our objective was to determine the status of purging and to quantify the magnitude of the eliminated genetic load for the survival at birth of Pannon White rabbit kits maintained in a closed nucleus population. The evolution of the survival at birth was evaluated applying the PurgeR software based on the inbreeding-purging model. 22718 kindling records was analyzed for the period of 1992-2017. Concerning the estimated parameters purging coefficient reached the possible maximum value of 0.5 between 1992-1997. Based on the expected fitness and on the expressed opportunity of purging the beneficial effects of purging could be expected after 10 generations where the proportion of the purged genetic load could be between 20 and 70%. These results suggest that large fraction of the inbreeding load may be caused by limited number of genes that could possibly be purged.

Estimates of the inbreeding coefficient based on runs of homozygosity (FROH) are widely used in population genetics and livestock studies. The accuracy of FROH estimates is determined in part by the density of the genotyping chip. Compared with high density (HD) chips, low density (LD) chips tend to overestimate FROH in shorter homozygous segments. It is unclear whether imputation of genotypes from LD to HD would reduce or eliminate this overestimation. Our goal was to determine whether FROH calculations based on imputed LD genotypes are accurate. To this end, FROH estimates were determined for Nelore cows using HD or LD imputed genotypes (FROH_HD and FROH_LDimp). Our results showed that LD imputed genotypes reduced but did not eliminate differences between estimates. Differences were observed in both directions, with under- and overestimates of FROH. This suggests that imputation errors can either disrupt or generate homozygous segments.

Under the pressure of natural and artificial selection, the pattern of genomic landscape of domestic animals is constantly changing, leaving visible signs of selection. Here, we propose a new approach called haplotype richness drop (HRiD), which is able to identify selection signals from genomic information contained in male haplotypes. This applies to males genotyped on the X- chromosome, although extension to diploid genotypes that are accurately phased is also possible. HRiD is based on the assumption that genomic regions subject to positive selection exhibit a sudden decrease (drop) in haplotype richness. Thus, the main task of HRiD is to identify outlying genomic regions that exhibit a sudden drop in haplotype richness. Using empirical datasets, we have shown that HRiD complements other within-population approaches, such as extreme Runs of Homozygosity islands, integration Haplotype Scores, and the number of Segregating Sites by Length. In addition, we demonstrated that HRiD results can be readily combined with phylogenetic analyses of selection signals (haplotypes). This study was supported by the Croatian Science Foundation under the project ANAGRAMS, IP-2018-01-8708, and by the Operational Programme for Competitiveness and Cohesion from 2014 to 2020 under the project SirjeIN, KK.01.1.1.04.0058, while the participation of Luboš Vostry was supported by the Ministry of Agriculture of the Czech Republic (project no. QK1910156).

Ancient records testify the presence of Arabian horse on Croatian grounds for hundreds of years. Its population size varied depending on the given circumstances of a given time and social events. Today’s Croatian Arabian horse originates from the stud farm Borike founded in 1895 which unfortunately ended its existence in 2019. There were four main sources from which today’s Croatian Arabian horses derive their ancestry: purebred Arabian horses from Syria, Saudi Arabia, Yemen, Iraq ; the horses from the stud farm in Babolna (Hungary), the horses from the stud farm in Slawuta (Poland) and one stallion from the stud farm Radautz (Austria). Nowadays, the Arabian horse is bred at a stud farm Višnjica and by the private owners in Croatia. Due to all these circumstances, we assumed that a special type of Arabian horse was created, adapted to the needs and characteristics of Croatian breeding requirements. For that reason, 70 samples of pure Arabian and Shagya-Arab horses were genotyped using GeneSeek® Genomic Profiler™ Equine SNP BeadChip 70K (73860 SNPs). In order to identify the selection signatures, two approaches were used: 1) detection of extreme Runs of Homozygosity islands (eROHi) and 2) across populations identification of SNPs with extremely high FST values (ehFST). To analyse the FST values, we compared the Croatian Arabian horse population with other international and related breeds populations across the world using data available from other studies. The results from this study will certainly contribute to better understanding of the genomic structure of Croatian Arabian horses as well as a good starting point to implement the genomic information in the existing breeding programme.

Livestock Guardian Dogs (LGDs, Canis familiaris) are large dogs adapted to harsh mountain environments and selected to protect grazing livestock from predators in free-ranging grazing systems. Signals of positive selection in a meta- population represented by nine LGD breeds (116 individuals) were analysed based on information from 104, 618 SNP genotypes using the extreme Runs of Homozygosity islands (eROHi) and the integrated Haplotype Score (iHS) approach. We found that 28 genomic regions (11 identified using the eROHi approach and 17 using the iHS approach) had highly significant signals for positive selection. Some of the most pronounced selection signals are associated with genes such as LRIG3, TRIP11, CNTNAP2 and EFNA5. The presented results will contribute to the understanding of the synergistic behavioural interaction of LGDs with humans and grazing animals, especially sheep and goats, and the mammals' adaptation to harsh mountain conditions, as well as to their breeding management.

Native Croatian goats are local breeds kept in an extensive system with low production. Local breeds are part of the genetic and socio-cultural heritage of the local community. For this reason, it is very important to preserve the genetic diversity of the remaining breeds, most of which are trapped in unselected autochthonous breeds. This study aimed to analyse the genetic diversity and conservation status of two indigenous Croatian goat breeds, the Istrian goat and the Croatian Coloured goat, using genome-wide SNP data. Population structure was analysed using 11 Istrian goats, 33 Croatian Coloured goats, and 506 additional animals belonging to 19 European goat breeds and 7 Iranian Bezoar. We used a graphical approach to illustrate the relationship between the analysed breeds in terms of their population structure. In addition, we determined the migration patterns of all goat breeds presented in this study. Our analyses revealed a fine population structure of Croatian goat breeds and their dependence on geographical origin and barriers. The mean observed heterozygosity for the Istrian goat and Croatian coloured goat was 0.39 and 0.40, respectively. The mean fixation index (FST) between the Istrian and Croatian coloured goats was 0.046, indicating an intermediate level of genetic differentiation between these two populations. Principal component analysis (PCA) showed distinct clustering of the two breeds, while some Istrian goats showed a clear clustering with the Saanen breed. The Croatian coloured goat was closest to the Carpathian goat. The results obtained with Treemix software confirmed these results and showed gene flow from Croatian coloured goat to Istrian goat. Our results will contribute to the breeding and conservation strategies for the native Croatian sheep breeds.

Inbreeding is the mating of related individuals that share common ancestors, resulting in increased homozygosity. Inbreeding depression and increased prevalence of genetic defects are known negative consequences of inbreeding. Therefore, the estimation of inbreeding coefficients is important for the survival of genetically small populations. The main objective of this study was to estimate the extent of genomic X-chromosome inbreeding (FXsex), a rarely estimated parameter, and its relationship with autosomal inbreeding (FX) in seven Croatian sheep breeds (99 individuals) genotyped with a high-density SNP array (606, 006 SNPs). All genomic inbreeding coefficients were calculated with respect to the ROH>4 Mb. In all 99 individuals (East Adriatic metapopulation), the mean FXsex (0.054) was 2.26 times higher than FX(0.024) and a similar trend was observed in variation, as the standard deviation of FXsex (0.116) was 2.54 times higher than that of FX (0.046). The highest observed FXsex was 0.572, while 10% of animals had an FXsex>0.250. In contrast, the highest observed FX was 0.301, with only one individual having an FX>0.2500. The highest mean X-chromosome and autosomal inbreeding coefficient (FXsex=0.079 and FX=0.042) was observed in Rab Island Sheep, while the smallest inbreeding coefficients were found in Pag Island Sheep (FXsex=0.024 and FX=0.012). Our results are preliminary because both the number of breeds (7) and the number of individuals representing each breed (13 to 20) were small. Nevertheless, the higher inbreeding estimates observed for the X-chromosome in sheep demonstrate the importance of analysing possible negative inbreeding effects of the X-chromosome.

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (similar to 16.10x) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, similar to 121.2 million single nucleotide polymorphisms, similar to 61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3 '-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.

Intramuscular fat (IMF) plays an important role in determination of meat quality and directly contributes to the positive technological and sensory characteristics such as taste, texture and juiciness. In Black Slavonian pig (BS), numerous studies reported much higher IMF content compared to other local breeds, therefore, understanding the genetic mechanisms of IMF in BS becomes one of the main challenges in scientific research. Transcriptome and gene expression analyses using RNA-seq technology is approach which enables the identification of genes that are differentially expressed in distinct phenotypes. In the last few years, the majority of gene expression studies has been carried out on modern pig breeds, therefore such analyses on local breeds would significantly contribute to the pig breeding of local breeds, but also to the commercial breeding programs. For the analysis of gene expression for IMF in this study, out of the total of 80 pigs in full-sib design, 10 sib pairs with extremely high and low IMF phenotypes were selected, and also analyzed for performance and meat quality traits. Whole transcriptome analysis was performed using RNA-seq technology, while candidate genes were further verified using quantitative real-time PCR (qPCR) analysis. The results from this study will certainly contribute to the understanding of genetic mechanisms of IMF in BS, and will also provide new possibilities for improvement of breeding program.