Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series.

Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

Widespread horse-based mobility arose around 2200 BCE in Eurasia.

Horses revolutionized human history with fast mobility1. However, the timeline between their domestication and their widespread integration as a means of transport remains contentious2-4. Here we assemble a collection of 475 ancient horse genomes to assess the period when these animals were first reshaped by human agency in Eurasia. We find that reproductive control of the modern domestic lineage emerged around 2200 BCE, through close-kin mating and shortened generation times. Reproductive control emerged following a severe domestication bottleneck starting no earlier than approximately 2700 BCE, and coincided with a sudden expansion across Eurasia that ultimately resulted in the replacement of nearly every local horse lineage. This expansion marked the rise of widespread horse-based mobility in human history, which refutes the commonly held narrative of large horse herds accompanying the massive migration of steppe peoples across Europe around 3000 BCE and earlier3,5. Finally, we detect significantly shortened generation times at Botai around 3500 BCE, a settlement from central Asia associated with corrals and a subsistence economy centred on horses6,7. This supports local horse husbandry before the rise of modern domestic bloodlines.

Historical Introgression from Wild Relatives Enhanced Climatic Adaptation and Resistance to Pneumonia in Sheep.

How animals, particularly livestock, adapt to various climates and environments over short evolutionary time is of fundamental biological interest. Further, understanding the genetic mechanisms of adaptation in indigenous livestock populations is important for designing appropriate breeding programs to cope with the impacts of changing climate. Here, we conducted a comprehensive genomic analysis of diversity, interspecies introgression, and climate-mediated selective signatures in a global sample of sheep and their wild relatives. By examining 600K and 50K genome-wide single nucleotide polymorphism data from 3,447 samples representing 111 domestic sheep populations and 403 samples from all their seven wild relatives (argali, Asiatic mouflon, European mouflon, urial, snow sheep, bighorn, and thinhorn sheep), coupled with 88 whole-genome sequences, we detected clear signals of common introgression from wild relatives into sympatric domestic populations, thereby increasing their genomic diversities. The introgressions provided beneficial genetic variants in native populations, which were significantly associated with local climatic adaptation. We observed common introgression signals of alleles in olfactory-related genes (e.g., ADCY3 and TRPV1) and the PADI gene family including in particular PADI2, which is associated with antibacterial innate immunity. Further analyses of whole-genome sequences showed that the introgressed alleles in a specific region of PADI2 (chr2: 248,302,667-248,306,614) correlate with resistance to pneumonia. We conclude that wild introgression enhanced climatic adaptation and resistance to pneumonia in sheep. This has enabled them to adapt to varying climatic and environmental conditions after domestication.

Whole genome sequencing reveals a complex introgression history and the basis of adaptation to subarctic climate in wild sheep.

To predict species responses to anthropogenic disturbances and climate change, it is reasonable to use species with high sensitivity to such factors. Snow sheep (Ovis nivicola) could represent a good candidate for this; as the only large herbivore species adapted to the cold and alpine habitats of northeastern Siberia, it plays a crucial role in its ecosystem. Despite having an extensive geographical distribution among all ovine species, it is one of the least studied. In this study, we sequenced and analysed six genomes of snow sheep in combination with all other wild sheep species to infer key aspects of their evolutionary history and unveil the genetic basis of their adaptation to subarctic environments. Despite their large census population size, snow sheep genomes showed remarkably low heterozygosity, which could reflect the effect of isolation and historical bottlenecks that we inferred using the pairwise sequential Markovian coalescent and runs of homozygosity. F4 -statistics indicated instances of introgression involving snow sheep with argali (Ovis ammon) and Dall (Ovis dalli) sheep, suggesting that these species might have been more widespread during the Pleistocene. Furthermore, the introgressed segments, which were identified using mainly minimum relative node depth, covered genes associated with immunity, adipogenesis and morphology-related traits, representing potential targets of adaptive introgression. Genes related to mitochondrial functions and thermogenesis associated with adipose tissue were identified to be under selection. Overall, our data suggest introgression as a mechanism facilitating adaptation in wild sheep species and provide insights into the genetic mechanisms underlying cold adaptation in snow sheep.

Equine vitiligo-like depigmentation in grey horses is related to genes involved in immune response and tumor metastasis.

BACKGROUND: In horses, the autoimmune disease vitiligo is characterized by the loss of melanocytes and results in patchy depigmentation of the skin around the eyes, muzzle and the perianal region. Vitiligo-like depigmentation occurs predominantly in horses displaying the grey coat colour and is observed at a prevalence level of 26.0-67.0% in grey horses compared with only 0.8-3.5% in non-grey horses. While the polygenetic background of this complex disease is well documented in humans, the underlying candidate genes for this skin disorder in horses remain unknown. In this study we aim to perform a genome-wide association study (GWAS) for identifying putative candidate loci for vitiligo-like depigmentation in horses. METHODS: In the current study, we performed a GWAS analysis using high-density 670 k single nucleotide polymorphism (SNP) data from 152 Lipizzan and 104 Noriker horses, which were phenotyped for vitiligo-like depigmentation by visual inspection. After quality control 376,219 SNPs remained for analyses, the genome-wide Bonferroni corrected significance level was p < 1.33e-7. RESULTS: We identified seven candidate genes on four chromosomes (ECA1, ECA13, ECA17, ECA20) putatively involved in vitiligo pathogenesis in grey horses. The highlighted genes PHF11, SETDB2, CARHSP1 and LITAFD, are associated with the innate immune system, while the genes RCBTB1, LITAFD, NUBPL, PTP4A1, play a role in tumor suppression and metastasis. The antagonistic pathogenesis of vitiligo in relation to cancer specific enhanced cell motility and/or metastasis on typical melanoma predilection sites underlines a plausible involvement of RCBTB1, LITAFD, NUBPL, and PTP4A1. CONCLUSIONS: The proposed candidate genes for equine vitiligo-like depigmentation, indicate an antagonistic relation between vitiligo and tumor metastasis in a horse population with higher incidence of melanoma. Further replication and expression studies should lead to a better understanding of this skin disorder in horses.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

High-resolution population structure and runs of homozygosity reveal the genetic architecture of complex traits in the Lipizzan horse.

BACKGROUND: 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 700 k 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. RESULTS: 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. CONCLUSIONS: 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.

High-density genotyping reveals signatures of selection related to acclimation and economically important traits in 15 local sheep breeds from Russia.

BACKGROUND: Domestication and centuries of selective breeding have changed genomes of sheep breeds to respond to environmental challenges and human needs. The genomes of local breeds, therefore, are valuable sources of genomic variants to be used to understand mechanisms of response to adaptation and artificial selection. As a step toward this we performed a high-density genotyping and comprehensive scans for signatures of selection in the genomes from 15 local sheep breeds reared across Russia. RESULTS: Results demonstrated that the genomes of Russian sheep breeds contain multiple regions under putative selection. More than 50% of these regions matched with intervals identified in previous scans for selective sweeps in sheep genomes. These regions contain well-known candidate genes related to morphology, adaptation, and domestication (e.g., KITLG, KIT, MITF, and MC1R), wool quality and quantity (e.g., DSG@, DSC@, and KRT@), growth and feed intake (e.g., HOXA@, HOXC@, LCORL, NCAPG, LAP3, and CCSER1), reproduction (e.g., CMTM6, HTRA1, GNAQ, UBQLN1, and IFT88), and milk-related traits (e.g., ABCG2, SPP1, ACSS1, and ACSS2). In addition, multiple genes that are putatively related to environmental adaptations were top-ranked in selected intervals (e.g., EGFR, HSPH1, NMUR1, EDNRB, PRL, TSHR, and ADAMTS5). Moreover, we observed that multiple key genes involved in human hereditary sensory and autonomic neuropathies, and genetic disorders accompanied with an inability to feel pain and environmental temperatures, were top-ranked in multiple or individual sheep breeds from Russia pointing to a possible mechanism of adaptation to harsh climatic conditions. CONCLUSIONS: Our work represents the first comprehensive scan for signatures of selection in genomes of local sheep breeds from the Russian Federation of both European and Asian origins. We confirmed that the genomes of Russian sheep contain previously identified signatures of selection, demonstrating the robustness of our integrative approach. Multiple novel signatures of selection were found near genes which could be related to adaptation to the harsh environments of Russia. Our study forms a basis for future work on using Russian sheep genomes to spot specific genetic variants or haplotypes to be used in efforts on developing next-generation highly productive breeds, better suited to diverse Eurasian environments.

Population structure and genetic diversity of 25 Russian sheep breeds based on whole-genome genotyping.

BACKGROUND: Russia has a diverse variety of native and locally developed sheep breeds with coarse, fine, and semi-fine wool, which inhabit different climate zones and landscapes that range from hot deserts to harsh northern areas. To date, no genome-wide information has been used to investigate the history and genetic characteristics of the extant local Russian sheep populations. To infer the population structure and genome-wide diversity of Russian sheep, 25 local breeds were genotyped with the OvineSNP50 BeadChip. Furthermore, to evaluate admixture contributions from foreign breeds in Russian sheep, a set of 58 worldwide breeds from publicly available genotypes was added to our data. RESULTS: We recorded similar observed heterozygosity (0.354-0.395) and allelic richness (1.890-1.955) levels across the analyzed breeds and they are comparable with those observed in the worldwide breeds. Recent effective population sizes estimated from linkage disequilibrium five generations ago ranged from 65 to 543. Multi-dimensional scaling, admixture, and neighbor-net analyses consistently identified a two-step subdivision of the Russian local sheep breeds. A first split clustered the Russian sheep populations according to their wool type (fine wool, semi-fine wool and coarse wool). The Dagestan Mountain and Baikal fine-fleeced breeds differ from the other Merino-derived local breeds. The semi-fine wool cluster combined a breed of Romanian origin, Tsigai, with its derivative Altai Mountain, the two Romney-introgressed breeds Kuibyshev and North Caucasian, and the Lincoln-introgressed Russian longhaired breed. The coarse-wool group comprised the Nordic short-tailed Romanov, the long-fat-tailed outlier Kuchugur and two clusters of fat-tailed sheep: the Caucasian Mountain breeds and the Buubei, Karakul, Edilbai, Kalmyk and Tuva breeds. The Russian fat-tailed breeds shared co-ancestry with sheep from China and Southwestern Asia (Iran). CONCLUSIONS: In this study, we derived the genetic characteristics of the major Russian local sheep breeds, which are moderately diverse and have a strong population structure. Pooling our data with a worldwide genotyping set gave deeper insight into the history and origin of the Russian sheep populations.

Whole-genome SNP analysis elucidates the genetic structure of Russian cattle and its relationship with Eurasian taurine breeds.

BACKGROUND: The origin of native and locally developed Russian cattle breeds is linked to the historical, social, cultural, and climatic features of the diverse geographical regions of Russia. In the present study, we investigated the population structure of nine Russian cattle breeds and their relations to the cattle breeds from around the world to elucidate their origin. Genotyping of single nucleotide polymorphisms (SNPs) in Bestuzhev (n = 26), Russian Black-and-White (n = 21), Kalmyk (n = 14), Kholmogor (n = 25), Kostromsky (n = 20), Red Gorbatov (n = 23), Suksun (n = 20), Yakut (n = 25), and Yaroslavl cattle breeds (n = 21) was done using the Bovine SNP50 BeadChip. SNP profiles from an additional 70 breeds were included in the analysis as references. RESULTS: The observed heterozygosity levels were quite similar in eight of the nine studied breeds (HO = 0.337-0.363) except for Yakut (Ho = 0.279). The inbreeding coefficients FIS ranged from -0.028 for Kalmyk to 0.036 for Russian Black-and-White and were comparable to those of the European breeds. The nine studied Russian breeds exhibited taurine ancestry along the C1 axis of the multidimensional scaling (MDS)-plot, but Yakut was clearly separated from the European taurine breeds on the C2 axis. Neighbor-Net and admixture analyses, discriminated three groups among the studied Russian breeds. Yakut and Kalmyk were assigned to a separate group because of their Turano-Mongolian origin. Russian Black-and-White, Kostromsky and Suksun showed transboundary European ancestry, which originated from the Holstein, Brown Swiss, and Danish Red breeds, respectively. The lowest level of introgression of transboundary breeds was recorded for the Kholmogor, Yaroslavl, Red Gorbatov and Bestuzhev breeds, which can be considered as an authentic genetic resource. CONCLUSIONS: Whole-genome SNP analysis revealed that Russian native and locally developed breeds have conserved authentic genetic patterns in spite of the considerable influence of Eurasian taurine cattle. In this paper, we provide fundamental genomic information that will contribute to the development of more accurate breed conservation programs and genetic improvement strategies.

Population Networks Associated with Runs of Homozygosity Reveal New Insights into the Breeding History of the Haflinger Horse.

Within the scope of current genetic diversity analyses, population structure and homozygosity measures are independently analyzed 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 7 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 color patterns (Noriker). Analysis of ROH revealed that the 2 Arabian populations (Purebred and Shagya Arabians), Gidran and the Bosnian Mountain Horse had the highest genome proportion covered by ROH segments (306-397 Mb). The Noriker and the Austrian Haflinger showed the lowest ROH coverage (228, 282 Mb). 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 favoring out-crosses within the breed.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

A missense mutation in damage-specific DNA binding protein 2 is a genetic risk factor for limbal squamous cell carcinoma in horses.

Squamous cell carcinoma (SCC) is the most common cancer of the equine eye, frequently originating at the limbus, with the potential to invade the cornea, cause visual impairment, and result in loss of the eye. Several breeds of horses have a high occurrence of limbal SCC implicating a genetic basis for limbal SCC predisposition. Pedigree analysis in the Haflinger breed supports a simple recessive mode of inheritance and a genome-wide association study (N = 23) identified a 1.5 Mb locus on ECA12 significantly associated with limbal SCC (Pcorrected = 0.04). Sequencing the most physiologically relevant gene from this locus, damage specific DNA binding protein 2 (DDB2), identified a missense mutation (c.1013 C > T p.Thr338Met) that was strongly associated with limbal SCC (P = 3.41 × 10-10 ). Genotyping 42 polymorphisms narrowed the ECA12 candidate interval to 483 kb but did not identify another variant that was more strongly associated. DDB2 binds to ultraviolet light damaged DNA and recruits other proteins to perform global genome nucleotide excision repair. Computational modeling predicts this mutation to be deleterious by altering conformation of the ß loop involved in photolesion recognition. This DDB2 variant was also detected in two other closely related breeds with reported cases of ocular SCC, the Belgian and the Percheron, suggesting it may also be a SCC risk factor in these breeds. Furthermore, in humans xeroderma pigmentosum complementation group E, a disease characterized by sun sensitivity and increased risk of cutaneous SCC and melanomas, is explained by mutations in DDB2. Cross-species comparison remains to be further evaluated.

Detection of two non-synonymous SNPs in SLC45A2 on BTA20 as candidate causal mutations for oculocutaneous albinism in Braunvieh cattle.

BACKGROUND: Cases of albinism have been reported in several species including cattle. So far, research has identified many genes that are involved in this eye-catching phenotype. Thus, when two paternal Braunvieh half-sibs with oculocutaneous albinism were detected on a private farm, we were interested in knowing whether their phenotype was caused by an already known gene/mutation. RESULTS: Analysis of genotyping data (50K) of the two albino individuals, their mothers and five other relatives identified a 47.61-Mb candidate haplotype on Bos taurus chromosome BTA20. Subsequent comparisons of the sequence of this haplotype with sequence data from four Braunvieh sires and the Aurochs genome identified two possible candidate causal mutations at positions 39,829,806 bp (G/A; R45Q) and 39,864,148 bp (C/T; T444I) that were absent in 1682 animals from various bovine breeds included in the 1000 bull genomes project. Both polymorphisms represent coding variants in the SLC45A2 gene, for which the human equivalent harbors numerous variants associated with oculocutaneous albinism type 4. We demonstrate an association of R45Q and T444I with the albino phenotype by targeted genotyping. CONCLUSIONS: Although the candidate gene SLC45A2 is known to be involved in albinism in different species, to date in cattle only mutations in the TYR and MITF genes were reported to be associated with albinism or albinism-like phenotypes. Thus, our study extends the list of genes that are associated with bovine albinism. However, further research and more samples from related animals are needed to elucidate if only one of these two single nucleotide polymorphisms or the combination of both is the actual causal variant.

Phenotypic and Genetic Analysis of the Leopard Complex Spotting in Noriker Horses.

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 components.

Aging of Cloned Animals: A Mini-Review.

The number of species for which somatic cell nuclear transfer (SCNT) protocols are established is still increasing. Due to the high number of cloned farm, companion, and sport animals, the topic of animal cloning never ceases to be of public interest. Numerous studies cover the health status of SCNT-derived animals, but very few cover the effects of SCNT on aging. However, only cloned animals that reach the full extent of the species-specific lifespan, doing so with only the normal age-related afflictions and diseases, would prove that SCNT can produce completely healthy offspring. Here, we review the available literature and own data to answer the question whether the aging process of cloned animals is qualitatively different from normal animals. We focus on 4 main factors that were proposed to influence aging in these animals: epigenetic (dys)regulation, accumulation of damaged macromolecules, shortened telomeres, and (nuclear donor-derived) age-related DNA damage. We find that at least some cloned animals can reach the species-specific maximum age with a performance that matches that of normal animals. However, for most species, only anecdotal evidence of cloned animals reaching high age is available. We therefore encourage reports on the aging of cloned animals to make further analysis on the performance of SCNT possible.

Exploring the oviductal fluid proteome by a lectin-based affinity approach.

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin-bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin-bound fraction were known glycoproteins (p-value [FDR] = 1.45E-31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.

Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots.

Due to post-translational modifications such as phosphorylation, proteins exist as distinct charge variants. Two-dimensional (2D) gel electrophoresis followed by immunoblotting enables the detection of these isoforms. For their accurate relative quantitation in different samples, a loading control is necessary to compensate for technical errors such as imprecise sample loading or transfer. The study reveals that the combinatory approach of SYPRO Ruby and chemiluminescence-based 2D Western blot analysis exhibits high linearity and excellent reproducibility and is applicable for limited sample amounts.

A Study of Applicability of SNP Chips Developed for Bovine and Ovine Species to Whole-Genome Analysis of Reindeer Rangifer tarandus.

Two sets of commercially available single nucleotide polymorphisms (SNPs) developed for cattle (BovineSNP50 BeadChip) and sheep (OvineSNP50 BeadChip) have been trialed for whole-genome analysis of 4 female samples of Rangifer tarandus inhabiting Russia. We found out that 43.0% of bovine and 47.0% of Ovine SNPs could be genotyped, while only 5.3% and 2.03% of them were respectively polymorphic. The scored and the polymorphic SNPs were identified on each bovine and each ovine chromosome, but their distribution was not unique. The maximal value of runs of homozygosity (ROH) was 30.93Mb (for SNPs corresponding to bovine chromosome 8) and 80.32Mb (for SNPs corresponding to ovine chromosome 7). Thus, the SNP chips developed for bovine and ovine species can be used as a powerful tool for genome analysis in reindeer R. tarandus.

Tracing the Dynamical Genetic Diversity Changes of Russian Livni Pigs during the Last 50 Years with the Museum, Old, and Modern Samples.

The pig industry is usually considered an intensive livestock industry, mainly supported by hybrid breeding between commercial pig breeds. However, people's pursuit of a more natural environment and higher meat quality has led to an increasing demand for eco-friendly and diverse pig feeding systems. Therefore, the importance of rearing and conserving local pig breeds is increasing. The Livni pig is a local breed with good adaptability to the environmental and fodder conditions in central Russia. In this study, we aimed to analyze the genetic diversity and population structure of Livni pigs using whole-genome single nucleotide polymorphism (SNP) data. We utilized the Porcine GGP HD BeadChip on genotype samples from old (n = 32, 2004) and modern (n = 32, 2019) populations of Livni pigs. For the museum samples of Livni pigs (n = 3), we extracted DNA from their teeth, performed genomic sequencing, and obtained SNP genotypes from the whole-genome sequences. SNP genotypes of Landrace (n = 32) and Large White (n = 32) pigs were included for comparative analysis. We observed that the allelic richness of Livni pigs was higher than those of Landrace and Large White pigs (AR = 1.775-1.798 vs. 1.703 and 1.668, respectively). The effective population size estimates (NE5 = 108 for Livni pigs, NE5 = 59 for Landrace and Large White pigs) confirmed their genetic diversity tendency. This was further supported by the length and number of runs of homozygosity, as well as the genomic inbreeding coefficient (almost twofold lower in Livni pigs compared to Landrace and Large White pigs). These findings suggest that the Livni pig population exhibits higher genetic diversity and experiences lower selection pressure compared to commercial pig populations. Furthermore, both principal component and network tree analyses demonstrated a clear differentiation between Livni pigs and transboundary commercial pigs. The TreeMix results indicated gene flow from Landrace ancestors to Livni pigs (2019) and from Large White ancestors to Livni pigs (2004), which was consistent with their respective historical breeding backgrounds. The comparative analysis of museum, old, and modern Livni pigs indicated that the modern Livni pig populations have preserved their historical genomic components, suggesting their potential suitability for future design selection programs.