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Background: Runs of homozygosity (ROH) islands are stretches of homozygous sequence in the genome of a large proportion of individuals in a population. Algorithms for the detection of ROH depend on the similarity of haplotypes. Coverage gaps and copy number variants (CNV) may result in incorrect identification of such similarity, leading to the detection of ROH islands where none exists. Misidentified hemizygous regions will also appear as homozygous based on sequence variation alone. Our aim was to identify ROH islands influenced by marker coverage gaps or CNV, using Illumina BovineHD BeadChip (777 K) single nucleotide polymorphism (SNP) data for Austrian Brown Swiss, Tyrol Grey and Pinzgauer cattle., Methods: ROH were detected using clustering, and ROH islands were determined from population inbreeding levels for each marker. CNV were detected using a multivariate copy number analysis method and a hidden Markov model. SNP coverage gaps were defined as genomic regions with intermarker distances on average longer than 9.24 kb. ROH islands that overlapped CNV regions (CNVR) or SNP coverage gaps were considered as potential artefacts. Permutation tests were used to determine if overlaps between CNVR with copy losses and ROH islands were due to chance. Diversity of the haplotypes in the ROH islands was assessed by haplotype analyses., Results: In Brown Swiss, Tyrol Grey and Pinzgauer, we identified 13, 22, and 24 ROH islands covering 26.6, 389.0 and 35.8 Mb, respectively, and we detected 30, 50 and 71 CNVR derived from CNV by using both algorithms, respectively. Overlaps between ROH islands, CNVR or coverage gaps occurred for 7, 14 and 16 ROH islands, respectively. About 37, 44 and 52% of the ROH islands coverage in Brown Swiss, Tyrol Grey and Pinzgauer, respectively, were affected by copy loss. Intersections between ROH islands and CNVR were small, but significantly larger compared to ROH islands at random locations across the genome, implying an association between ROH islands and CNVR. Haplotype diversity for reliable ROH islands was lower than for ROH islands that intersected with copy loss CNVR., Conclusions: Our findings show that a significant proportion of the ROH islands in the bovine genome are artefacts due to CNV or SNP coverage gaps.

The Slovak Pinzgau breed faces the bottleneck effect and the loss of diversity due to unequal use of founders and a significant population decline. Further population size reduction can lead to serious problems. Information obtained here and in other studies from high-throughput genotyping of 179 individuals was used to characterise genetic diversity and differentiation of Slovak Pinzgau, Austrian Pinzgau, Cika and Piedmontese cattle by Bayesian clustering algorithm. A gene flow network for the clusters estimated from admixture results was produced. The low estimate of genetic differentiation (F ST ) in Pinzgau cattle populations indicated that differentiation among these populations is low, particularly owing to a common historical origin and high gene flow. Changes in the log marginal likelihood indicated Austrian Pinzgau as the most similar breed to Slovak Pinzgau. All populations except the Piedmontese one displayed two ways of gene flow among populations, indicating that Piedmontese cattle was involved in producing of the analysed breeds while these breeds were not involved in creation of Piedmontese. Genetic evaluation represents an important tool in breeding and cattle selection. It is more strategically important than ever to preserve as much of the livestock diversity as possible, to ensure a prompt and proper response to the needs of future generations. Information provided by the fine-scale genetic characterization of this study clearly shows that there is a difference in genetic composition of Slovak and Austrian populations, as well as the Cika and Piedmontese cattle. Despite its population size, the Slovak Pinzgau cattle have a potential to serve as a basic gene reserve of this breed, with European and world-wide importance.

Background: Runs of homozygosity are long, uninterrupted stretches of homozygous genotypes that enable reliable estimation of levels of inbreeding (i.e., autozygosity) based on high-throughput, chip-based single nucleotide polymorphism (SNP) genotypes. While the theoretical definition of runs of homozygosity is straightforward, their empirical identification depends on the type of SNP chip used to obtain the data and on a number of factors, including the number of heterozygous calls allowed to account for genotyping errors. We analyzed how SNP chip density and genotyping errors affect estimates of autozygosity based on runs of homozygosity in three cattle populations, using genotype data from an SNP chip with 777,972 SNPs and a 50 k chip., Results: Data from the 50 k chip led to overestimation of the number of runs of homozygosity that are shorter than 4 Mb, since the analysis could not identify heterozygous SNPs that were present on the denser chip. Conversely, data from the denser chip led to underestimation of the number of runs of homozygosity that were longer than 8 Mb, unless the presence of a small number of heterozygous SNP genotypes was allowed within a run of homozygosity., Conclusions: We have shown that SNP chip density and genotyping errors introduce patterns of bias in the estimation of autozygosity based on runs of homozygosity. SNP chips with 50,000 to 60,000 markers are frequently available for livestock species and their information leads to a conservative prediction of autozygosity from runs of homozygosity longer than 4 Mb. Not allowing heterozygous SNP genotypes to be present in a homozygosity run, as has been advocated for human populations, is not adequate for livestock populations because they have much higher levels of autozygosity and therefore longer runs of homozygosity. When allowing a small number of heterozygous calls, current software does not differentiate between situations where these calls are adjacent and therefore indicative of an actual break of the run versus those where they are scattered across the length of the homozygous segment. Simple graphical tests that are used in this paper are a current, yet tedious solution.

While mitogenome mutations leading to pathological manifestations are rare, more than 200 such mutations have been described in humans. In contrast, pathogenic mitogenome mutations are rare in domestic animals and have not been described at all in cattle. In the small local Slovenian cattle breed Cika, we identified (next-generation sequencing) two cows with the T10432C mitogenome mutation in the ND4L gene, which corresponds to the human T10663C mutation known to cause Leber's hereditary optic neuropathy (LHON). Pedigree analysis revealed that the cows in which the mutation was identified belong to two different maternal lineages with 217 individual cows born between 1997 and 2020. The identified mutation and its maternal inheritance were confirmed by Sanger sequencing across multiple generations, whereas no single analysis revealed evidence of heteroplasmy. A closer clinical examination of one cow with the T10432C mutation revealed exophthalmos, whereas histopathological examination revealed retinal ablations, subretinal oedema, and haemorrhage. The results of these analyses confirm the presence of mitochondrial mutation T10432C with homoplasmic maternal inheritance as well as clinical and histopathological signs similar to LHON in humans. Live animals with the mutation could be used as a suitable animal model that can improve our understanding of the pathogenesis of LHON and other mitochondriopathies. [ABSTRACT FROM AUTHOR]

While mitochondriopathies, mitochondrial diseases, caused by mutations in mitochondrial DNA (mtDNA), are well documented in humans, single pathogenic mtDNA mutation or disorders are unknown in livestock populations. In a survey of 799 complete cattle mtDNAs belonging to more than 120 breeds two mutations, one in ND1 (C4171T) and the other in ND4L (T10663C) gene were identified, that are confirmed to be pathogenic in humans causing Leber's hereditary optic neuropathy (LHON). In one Cika cow with T10663C mutation, which was in humans reported to cause an acute onset of visual loss or/and many other LHON associated clinical manifestations, an exophthalmia of the right eye that might fit to the pathogenesis of LHON was observed. This work supports the existence of potentially detrimental mtDNA mutations in cattle, while aetiology and pathogenesis need to be further documented. [ABSTRACT FROM AUTHOR]

The extraction of amplifiable DNA is a crucial step for genetic analysis of farm animals. The aim of present study was to determine the quality and the quantity of DNA extracted from blood, milk, hair and tissue in cattle. It has been shown that milk and hair samples can be used as a reliable source for obtaining good quality and quantity of DNA ready for the next generation sequencing (NGS). Commercially available kit, with several modifications, was included in the evaluation. Raw milk or milk somatic cell pellet storage under -80 °C or -20 °C has shown to be a reliable procedure for preservation when large number of milk samples were collected at once. The quality (purity) and the quantity of extracted DNA were measured using a nanophotometer. The adequacy of the DNA extract was assessed by estimating average read depth of all captured bases of whole NGS mitogenome sequences. Although DNA extracted from different types of samples was suitable for NGS analysis, hair samples showed the largest variation of DNA quantity, low purity and DNA integrity. Non-invasive sampling methods such as milk and hair collection can be used for dairy cattle while sampling from free-range requires handling, capturing and caution. Milk and hair can be used for obtaining optimal DNA concentrations for NGS analysis. [ABSTRACT FROM AUTHOR]

The use of relatively low numbers of sires in cattle breeding programs, particularly on those for carcass and weight traits in Nellore beef cattle (Bos indicus) in Brazil, has always raised concerns about inbreeding, which affects conservation of genetic resources and sustainability of this breed. Here, we investigated the distribution of autozygosity levels based on runs of homozygosity (ROH) in a sample of 1,278 Nellore cows, genotyped for over 777,000 SNPs. We found ROH segments larger than 10 Mb in over 70% of the samples, representing signatures most likely related to the recent massive use of few sires. However, the average genome coverage by ROH (>1Mb) was lower than previously reported for other cattle breeds (4.58%). In spite of 99.98% of the SNPs being included within a ROH in at least one individual, only 19.37% of the markers were encompassed by common ROH, suggesting that the ongoing selection for weight, carcass and reproductive traits in this population is too recent to have produced selection signatures in the form of ROH. Three short-range highly prevalent ROH autosomal hotspots (occurring in over 50% of the samples) were observed, indicating candidate regions most likely under selection since before the foundation of Brazilian Nellore cattle. The putative signatures of selection on chromosomes 4, 7, and 12 may be involved in resistance to infectious diseases and fertility, and should be subject of future investigation. [ABSTRACT FROM AUTHOR]

Inbreeding depression is known to affect quantitative traits such as male fertility and sperm quality, but the genetic basis for these associations is poorly understood. Most studies have been limited to examining how pedigree- or marker-derived genome-wide autozygosity is associated with quantitative phenotypes. In this study, we analyzed possible associations of genetic features of inbreeding depression with percentage of live spermatozoa and total number of spermatozoa in 19,720 ejaculates obtained from 554 Austrian Fleckvieh bulls during routine artificial insemination programs. Genome-wide inbreeding depression was estimated and genomic regions contributing to inbreeding depression were mapped. Inbreeding depression did affect total number of spermatozoa, and such depression was predicted by pedigree-based inbreeding levels and genomewide inbreeding levels based on runs of homozygosity (ROH). Genome-wide inbreeding depression did not seem to affect percentage of live spermatozoa. A model incorporating genetic effects of the bull, environmental factors, and additive genetic and ROH status effects of individual single-nucleotide polymorphisms revealed genomic regions significantly associated with ROH status for total number of spermatozoa (4 regions) or percentage of live spermatozoa (5 regions). All but one region contains genes related to spermatogenesis and sperm morphology. These genomic regions contain genes affecting sperm morphogenesis and efficacy. The results highlight that next-generation sequencing may help explain some of the genetic factors contributing to inbreeding depression of sperm quality traits in Fleckvieh bulls. [ABSTRACT FROM AUTHOR]