Distribution of runs of homozygosity ROH on ECA3

Overlapping runs of homozygosity (ROH islands) shared by the majority of a population are hypothesized to be the result of selection around a target locus. In this study we investigated the impact of selection for coat color within the Noriker horse on autozygosity and ROH patterns. We analyzed overlapping homozygous regions (ROH islands) for gene content in fragments shared by more than 50% of horses. Long‐term assortative mating of chestnut horses and the small effective population size of leopard spotted and tobiano horses resulted in higher mean genome‐wide ROH coverage (SROH) within the range of 237.4–284.2 Mb, whereas for bay, black and roan horses, where rotation mating is commonly applied, lower autozygosity (SROH from 176.4–180.0 Mb) was determined. We identified seven common ROH islands considering all Noriker horses from our dataset. Specific islands were documented for chestnut, leopard spotted, roan and bay horses. The ROH islands contained, among others, genes associated with body size (ZFAT, LASP1 and LCORL/NCAPG), coat color (MC1R in chestnut and the factor PATN1 in leopard spotted horses) and morphogenesis (HOXB cluster in all color strains except leopard spotted horses). This study demonstrates that within a closed population sharing the same founders and ancestors, selection on a single phenotypic trait, in this case coat color, can result in genetic fragmentation affecting levels of autozygosity and distribution of ROH islands and enclosed gene content.

Article: Grilz-Seger G., Neuditschko M., Mesaric M. et al. (2019): Analysis of ROH patterns in the Noriker horse breed reveals signatures of selection for coat color and body size, Animal genetics 59(2). DOI: 10.1111/age.12797


Intensive artificial and natural selection have shaped substantial variation among European horse breeds. Whereas most equine selection signature studies employ divergent genetic population structures in order to derive specific inter-breed targets of selection, we screened a total of 1476 horses originating from 12 breeds for the loss of genetic diversity by runs of homozygosity (ROH) utilizing a 670,000 single nucleotide polymorphism (SNP) genotyping array. Overlapping homozygous regions (ROH islands) indicating signatures of selection were identified by breed and similarities/dissimilarities between populations were evaluated. In the entire dataset, 180 ROH islands were identified, whilst 100 islands were breed specific, all other overlapped in 36 genomic regions with at least one ROH island of another breed.

chr11 Copyright grilz

Furthermore, two ROH hot spots were determined at horse chromosome 3 (ECA3) and ECA11. Besides the confirmation of previously documented target genes involved in selection for coat color (MC1R, STX17, ASIP), body size (LCORL/NCAPG, ZFAT, LASP1, HMGA2), racing ability (PPARGC1A), behavioral traits (GRIN2B, NTM/OPCML) and gait patterns (DMRT3), several putative target genes related to embryonic morphogenesis (HOXB), energy metabolism (IGFBP-1, IGFBP-3), hair follicle morphogenesis (KRT25, KRT27, INTU) and autophagy (RALB) were highlighted. Furthermore, genes were pinpointed which might be involved in environmental adaptation of specific habitats (UVSSA, STXBP4, COX11, HLF, MMD).

Article: Grilz-Seger G, Neuditschko M, Ricard A, Velie B, Lindgren G, Mesarič M, Cotman M, Horna M, Dobretsberger M, Brem G, Druml T. Genome-Wide Homozygosity Patterns and Evidence for Selection in a Set of European and Near Eastern Horse Breeds. Genes. 2019; 10(7):491.


The sample ascertainment bias due to complex population structures remains a major challenge in genome-wide investigations of complex traits. In this study we derived the high-resolution population structure and levels of autozygosity of 377 Lipizzan horses originating from five different European stud farms utilizing the SNP genotype information of the high density 700k Affymetrix Axiom™ Equine genotyping array. Scanning the genome for overlapping runs of homozygosity (ROH) shared by more than 50% of horses, we identified homozygous regions (ROH islands) in order to investigate the gene content of those candidate regions by gene ontology and enrichment analyses.


The high-resolution population network approach revealed well-defined substructures according to the origin of the horses (Austria, Slovakia, Croatia and Hungary). The highest mean genome coverage of ROH (SROH) was identified in the Austrian (SROH=342.9), followed by Croatian (SROH=214.7), Slovakian (SROH=205.1) and Hungarian (SROH=171.5) subpopulations. ROH island analysis revealed five common islands on ECA11 and ECA14, hereby confirming a closer genetic relationship between the Hungarian and Croatian as well as between the Austrian and Slovakian samples.







 Private islands were detected for the Hungarian and the Austrian Lipizzan subpopulations. All subpopulations shared a homozygous region on ECA11, nearly identical in position and length containing among other genes the homeobox-B cluster, which was also significantly (p<0.001) highlighted by enrichment analysis. Gene ontology terms were mostly related to biological processes involved in embryonic morphogenesis and anterior/posterior specification. Around the STX17 gene (causative for greying), we identified a ROH island harbouring the genes NR4A3, STX17, ERP44 and INVS. Within further islands on ECA14, ECA16 and ECA20 we detected the genes SPRY4, NDFIP1, IMPDH2, HSP90AB1, whereas SPRY4 and HSP90AB1 are involved in melanoma metastasis and survival rate of melanoma patients in humans.


We demonstrated that the assessment of high-resolution population structures within one single breed supports the downstream genetic analyses (e.g. the identification of ROH islands). By means of ROH island analyses, we identified the genes SPRY4, NDFIP1, IMPDH2, HSP90AB1, which might play an important role for further studies on equine melanoma. Furthermore, our results highlighted the impact of the homeobox-A and B cluster involved in morphogenesis of Lipizzan horses.


Article: G. Grilz-Seger, T. Druml, M. Neuditschko, M. Dobretsberger, M. Horna and G. Brem (2019): High-resolution population structure and runs of homozygosity reveal the genetic architecture of complex traits in the Lipizzan horse, BMC Genomics 20/1. DOI: 10.1186/s12864-019-5564-x.




Long consecutive homozygous genotype segments, runs of homozygosity (ROH), are a result of parents transmitting identical haplotypes, which can be used to estimate autozygosity. Based on 612K singlenucleotide polymorphisms, we computed three ROH parameters (genome length covered by ROH, SROH; number of ROH, NROH; and autozygosity, FROH) to investigate different scenarios in contemporary horse breeding: limited census (Bosnian mountain horse), conservation breeding (Posavje horse), and selection within closed studbook (Haflinger). The ROH parameters revealed well-defined differences between breeds. SROH was highest in the Bosnian mountain horse with 296.32 Mb, followed by the Haflinger sample (SROH 270.35 Mb) and the Posavje sample with 192.68 Mb. The highest number of ROH segments (ROHs) was observed within the Haflinger sample followed by the Posavje sample. FROH ranged at a population level from 8.59% in Posavje, over the Haflinger (mean FROH 12.05%) to 13.21% in the Bosnian mountain horse breed. Bottlenecks were detected for Bosnian mountain horse and Haflinger, whereas for the Posavje, a positive effect of the conservation breeding program was documented.
Investigating the distribution of ROHs across the genome, we detected four common ROH islands on equine chromosomes ECA 6, ECA 11, and ECA 17, which were present in all breeds. On breed level, the Bosnian mountain horses contained 10, the Posavje, four, and the Haflinger, 11 distinct ROH islands (containing the MC1R locus on ECA 3). With this analysis, we were able to compare genomic levels of inbreeding between breeds differing in management, pedigree completeness, and genes under selection.

Authors: Grilz-Seger G., Mesaric M., Cotman M., Neuditschko M., Druml T.and Brem G. (2018): Runs of Homozygosity and Population History of Three Horse Breeds With Small Population Size. Journal of Equine Verterinary science 71, 24-34.

Within the framework of genome‐wide analyses using the novel Axiom® genotyping array, we investigated the distribution of two previously described coat color patterns, namely sabino1 (SBI), associated with the KIT gene (KI16+1037A), and splashed white, associated with the PAX3 gene (ECA6:g.11429753C>T; PAX3C70Y), including a total of 899 horses originating from eight different breeds (Achal Theke, Purebred Arabian, Partbred Arabian, Anglo‐Arabian, Shagya Arabian, Haflinger, Lipizzan and Noriker). Based on the data we collected we were able to demonstrate that, besides Quarter horses, the PAX3C70Y allele is also present in Noriker (seven out of 189) and Lipizzan (three out of 329) horses. The SB1 allele was present in three breeds (Haflinger, 14 out of 98; Noriker, four out of 189; Lipizzan one out of 329). Furthermore, we examined the phenotypes of SB1‐ and PAX3C70Y‐carrier horses for their characteristic white spotting patterns. None of the SB1/sb1‐carrier horses met the criteria defining the Sabino1 pattern according to current applied protocols. From 10 heterozygous PAX3C70Y‐carrier horses, two had nearly a splashed white phenotype. The results of this large‐scale experiment on the genetic association of white spotting patterns in horses underline the influence of gene interactions and population differences on complex traits such as Sabino1 and splashed white.

Article: Druml et al. (2018), Novel insights into Sabino1 and splashed white coat color patterns in horses, Anim Genet., doi: 10.1111/age.12657




Shape model for phenotyping the horses On the Lipizzan stallion a 31 single landmarks

Crossbreeding between individuals of different breeds and introgression, the transfer of genes between breeds and/or populations mediated primarily by backcrossing, have been characteristic tools used in the refinement or optimisation of practical horse breeding. In this study we analysed the genetic contribution of the Arabian horse to the gene pool of the Lipizzan horse and its association with the overall type via shape regression analysis in 158 Lipizzan horses from the Austrian federal stud farm of Piber and the Spanish Riding School. Although crossbreeding with Arabian horses took place between 1776 and 1945, we found a significant association between Lipizzan body shape (p < 0.003) and individual coefficients of Arabian gene proportion, which varied from 21 to 29 %. In order to compare and interpret the estimated Lipizzan shape transitions from Iberian type towards the oriental type, we included a sample of 32 Shagya Arabians from the Slovak National stud farm Topol'ćianky. The estimated shape transitions in Lipizzans due to an increasing proportion of Arabian genes are similar to those we observed in the population comparison study of Lipizzan and Shagya Arabian horses. The main morphometric differences due to increasing Arabian genetic contributions in Lipizzans were found in the conformation of head, neck, withers, and legs. Although selection in the Austrian Lipizzan breed favours the Iberian type, Arabian shape characteristics are still present, indicating the segregation of Arabian founder haplotypes in the population. We also demonstrated that techniques of shape analysis are able to differentiate phenotypes associated with the gene pool and can be applied for phenotypic evaluation and prediction in crossbreeding programs.

Article: Thomas Druml, Michaela Horna, Gertrud Grilz-Seger, Maximilian Dobretsberger und G.
Brem (2018):Association of body shape with amount of Arabian genetic contribution in the Lipizzan horse,
Archives of Animal Breeding, 61, 79-83

Within the scope of current genetic diversity analyses, population structure and homozygosity measures are independently analysed and interpreted. To enhance analytical power, we combined the visualization of recently described high-resolution population networks with runs of homozygosity (ROH). In this study, we demonstrate that this approach enabled us to reveal important aspects of the breeding history of the Haflinger horse. We collected high-density genotype information of 531 horses originating from seven populations which were involved in the formation of the Haflinger, namely 32 Italian Haflingers, 78 Austrian Haflingers, 190 Noriker, 23 Bosnian Mountain Horses, 20 Gidran, 33 Shagya Arabians, and 155 Purebred Arabians. Model-based cluster analysis identified substructures within Purebred Arabian, Haflinger and Noriker that reflected distinct genealogy (Purebred Arabian), geographic origin (Haflinger) and coat colour patterns (Noriker). Analysis of ROH revealed that the two Arabian populations (Purebred and Shagya Arabians), Gidran and the Bosnian Mountain Horse had the highest genome proportion covered by ROH segments (306Mb - 397Mb). The Noriker and the Austrian Haflinger showed the lowest ROH coverage (228Mb, 282Mb). Our combined visualization approach made it feasible to clearly identify outbred (admixture) and inbred (ROH segments) horses. Genomic inbreeding coefficients (FROH) ranged from 10.1% (Noriker) to 17.7% (Purebred Arabian). Finally it could be demonstrated, that the Austrian Haflinger sample has a lack of longer ROH segments and a deviating ROH spectrum, which is associated with past bottleneck events and the recent mating strategy favouring out-crosses within the breed.


Druml T., Neuditschko M., Grilz-Seger G., Horna M., Ricard A., Mesarič M., Cotman M., Pausch H.and Brem, G. (2017): Population networks associated with runs of homozygosity reveal new insights into the breeding history of the Haflinger horse, Journal of Heredityhttps://doi.org/10.1093/jhered/esx114



tiger grilz
Die Farbzucht, insbesondere die Tigerzucht, spielt beim österreichischen Noriker eine zentrale Rolle und ist im Zuchtprogramm explizit verankert. Die Methode der Tiger-zucht ist durch die 350 Jahre nachweisbare Kontinuität spezifisch und unterscheidet sich sowohl im Zuchtziel als auch in der Nomenklatur vom amerikanischen System. In dieser Arbeit wurden beide Ansätze gegenübergestellt und diskutiert. In weiterer Folge wurde das Klassifikationsschema beim Noriker anhand von 111 Tieren (ent-spricht 43% der aktuellen Tigerzuchtpopulation) mittels Bildanalyse-methoden hinsichtlich ihres Weißpixelanteiles (äquivalent zu den unpig-mentierten Fellbereichen) analysiert. Die vier Klassifikationskatego­rien Schimmel, Volltiger, Schabracken- und Lendentiger und Grieseltiger unterschieden sich sig­nifikant voneinander. Im Longitudinalvergleich von 24 Tigern, die auf fortschreitende alters­bedingte Depigmentierung untersucht wurden, ergaben sich zwei Gruppen: veränderliche und unver-änderliche Tiger. Um die zugrunde liegende genetische Komponente zu bestim­men, wurden die bislang bekannten Tiger assoziierten Allele LP (ECA1 g. 108 297 929_108 297 930 ins1378) und PATN1 (SNP ECA3:23 658 447T > G in 3) genotypisiert. Von den 24 Tieren aus den drei Hauptkategorien Volltiger, Schabracken- und Lendentiger sowie Griesel-tiger, deren Weißpixelanteile zwischen 1288% variierten, hatten alle den Genotyp LP/lp PATN1/-. Damit konnte die phänotypische Varianz zwischen den betreffenden Kategorien nicht anhand des Genotyps erklärt werden. In dieser Arbeit konnte gezeigt werden, dass das Klassifikationssystem der Tigerzucht beim Noriker gemeinsam mit der hohen Selektionsintensität zugunsten des Schwarzflecktigers und den Rotationspaarungen von Volltigern an einfarbige Pferde aus Tigerlinien, zielführende Mittel sind, den Anteil an heterozygoten LP/lp Tieren und die Frequenz des PATN1 Allels in der Population zu erhö­hen. Gleichzeitig reflektieren die unterschiedlichen Klassifikationschemata auch die spezifischen Phänotypenfrequenzen in den Rassen Noriker und Appaloosa.


Artikel: Gertrud Grilz-Seger, ,T. Druml, Barbara Neuhauser , G. Brem (2017): Die züchterische Relevanz der Klassifikation und Nomenklatur der Tigerung beim Noriker Pferd, Züchtungskunde 89 (5), 359-374.



Figure3Classical dressage and the schools above the ground as performed in the Spanish Riding School Vienna, require special psychological and physical properties from riding horses. To document the training and performing level of the Lipizzan riding stallions from the Spanish Riding School Vienna we analysed the horses’ performance traits retrieved from chief riders’ evaluations in relation to training levels and age classes and we studied the interplay of performing status with the horses’ body shape. In total the mean age of all 80 riding stallions was 11.9 years (min 4 years, max. 26 years). Completely trained stallions (competition level S and higher) were on average 15.6 years old (min. 10 years, max. 26 years). From ten recorded performance traits (five physical traits and five psychological traits) walk, trot and collection ratings showed significant differences for levadeurs, caprioleurs and courbetteurs, the psychological traits reactibility, diligence and sensibility showed significant differences between age class (3-4 years, 5-8 years, 9-16 years, >16 years) and number of flying gallop changes. Further we found, that 80% of the chief riders’ ratings of physical performance traits reached significant levels in the shape regressions, indicating an association of their ratings with body shape variation. The resulting mean body shapes from the significant regressions, illustrated the requirements of the school above the ground and the classical dressage on the horses’ conformation. We showed that the evaluation of subjective ratings on valuating scales applying shape regressions can help to optimize the quality of scoring data in equine performance traits.


Article: Druml T., Dobretsberger M. and Brem G. (2017) The interplay of performing level and conformation – a characterisation study of the Lipizzan riding stallions from the Spanish Riding School Vienna, Journal of Veterinary Equine Science, DOI: 10.1016/j.jevs.2017.06.003


Genetic analyses of coat colors are frequently restricted to subjectively categorized phenotype information. The aim of this study was to develop a method to numerically quantify the variability of leopard complex (LP) spotting phenotypes introducing tools from image analysis. Generalized Procrustes analysis eliminates systematic errors due to imaging process. The binarization of normalized images and the application of principal component analysis (PCA) on the derived pixel matrices, transform pixel information into numerical data space. We applied these methods on 90 images to ascertain the specific leopard patterns within the Noriker breed. Furthermore, we genotyped a representative sample of 191 Noriker horses for the known LP spotting associated loci. Ninety-seven percentage of the genotyped leopard spotted horses were heterozygous for LP and had at least one copy of the PATN1 allele. However, the remaining pattern variation was great, indicating other genetic factors influencing the expression of LP spotting. Based upon this data, we estimated effect sizes of the modifier PATN1, and additional factors including sex, age, base color, and spotting phenotype of parents. The PCA of the pixel matrix resulted in 2 significant components accounting for 51% of the variation. Applying a linear model, we identified significant effects for age groups and base color on the first and second components, while for sex and parents’ LP phenotype significant effects were found on 4 additional component.

Article: Druml T., Grilz-Seger G., Neuhauser B., Neuditsko M., Gottfried Brem; Phenotypic and Genetic Analysis of the Leopard Complex Spotting in Noriker Horses. J Hered 2017. https://doi.org/10.1093/jhered/esx039