Bovine Genome Research Articles

Transcription initiation mapping in 31 bovine tissues reveals complex promoter activity, pervasive transcription, and tissue-specific promoter usage.

Characterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and contains a low number of transcripts in comparison to human and mouse. By using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein-coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE to produce a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-seq. Coexpression networks revealed considerable use of tissue-specific promoters, especially between brain and testicle, which expressed several genes in common from alternate loci. Furthermore, regions surrounding coexpressed modules were enriched in binding factor motifs representative of each tissue. The comprehensive annotation of promoters in such a large collection of tissues will substantially contribute to our understanding of gene expression in cattle and other mammalian species, shortening the gap between genotypes and phenotypes.

Development of polymorphic markers in the immune gene complex loci of cattle

The addition of cattle health and immunity traits to genomic selection indices holds promise to increase individual animal longevity and productivity, and decrease economic losses from disease. However, highly variable genomic loci that contain multiple immune-related genes were poorly assembled in the first iterations of the cattle reference genome assembly and underrepresented during the development of most commercial genotyping platforms. As a consequence, there is a paucity of genetic markers within these loci that may track haplotypes related to disease susceptibility. By using hierarchical assembly of bacterial artificial chromosome inserts spanning 3 of these immune-related gene regions, we were able to assemble multiple full-length haplotypes of the major histocompatibility complex, the leukocyte receptor complex, and the natural killer cell complex. Using these new assemblies and the recently released ARS-UCD1.2 reference, we aligned whole-genome shotgun reads from 125 sequenced Holstein bulls to discover candidate variants for genetic marker development. We selected 124 SNPs, using heuristic and statistical models to develop a custom genotyping panel. In a proof-of-principle study, we used this custom panel to genotype 1,797 Holstein cows exposed to bovine tuberculosis (bTB) that were the subject of a previous GWAS study using the Illumina BovineHD array. Although we did not identify any significant association of bTB phenotypes with these new genetic markers, 2 markers exhibited substantial effects on bTB phenotypic prediction. The models and parameters trained in this study serve as a guide for future marker discovery surveys particularly in previously unassembled regions of the cattle genome.

Signatures of selection and candidate genes for adaptation to extreme environmental factors in the genomes of Turano-Mongolian cattle breeds

Изменения, происходящие в окружающей среде, заставляют популяции организмов адаптироваться к новым условиям либо за счет фенотипической пластичности, либо за счет генетических или эпигенетических изменений. Следы отбора, такие как специфические изменения частот аллелей и гаплотипов, снижение или повышение генетического разнообразия, помогают выявить изменения генома крупного рогатого скота в ответ на искусственный и естественный отбор, а также локусы и варианты, непосредственно влияющие на адаптивные и экономически важные признаки. Достижения генетики и биотехнологии дают возможность быстрого переноса уникальных генетических вариантов, возникших у местных пород крупного рогатого скота в процессе адаптации к локальной среде обитания, в геномы интернациональных высокопроизводительных пород с целью сохранения их выдающихся свойств в новых условиях обитания. Возможно и использование методов геномной селекции для повышения частот адаптивных аллелей у интернациональных пород. В обзоре рассмотрены недавние работы по истории происхождения и эволюции турано-монгольских пород крупного рогатого скота, адаптации турано-монгольского скота к экстремальным условиям среды. Сделано обобщение имеющихся сведений о потенциальных генах-кандидатах адаптации в геномах турано-монгольских пород, включая гены устойчивости к холоду, гены иммунного ответа и гены адаптации к высокогорью. Авторы приходят к выводу, что имеющиеся данные литературы не позволяют отдать предпочтение одному из двух возможных сценариев происхождения турано-монгольских пород – в результате доместикации дикого тура на территории Восточной Азии или вследствие миграции тауринной протопопуляции из Ближнего Востока. Турано-монгольским породам свойственна высокая адаптация к экстремальным климатическим условиям (холод, жара и недостаток кислорода в горах) и паразитам (гнус, клещи, бактериальные и вирусные инфекции). В результате высокопроизводительного генотипирования и секвенирования геномов и транскриптомов представителей этих пород в последнее время были выявлены перспективные гены-кандидаты и генетические варианты, участвующие в адаптации к факторам внешней среды.

Concordance rate in cattle and sheep between genotypes differing in Illumina GenCall quality score.

Proper quality control of data prior to downstream analyses is fundamental to ensure integrity of results; quality control of genomic data is no exception. While many metrics of quality control of genomic data exist, the objective of the present study was to quantify the genotype and allele concordance rate between called single nucleotide polymorphism (SNP) genotypes differing in GenCall (GC) score; the GC score is a confidence measure assigned to each Illumina genotype call. This objective was achieved using Illumina beadchip genotype data from 771 cattle (12428767 genotypes in total post-editing) and 80 sheep (1557360 SNPs genotypes in total post-editing) each genotyped in duplicate. The called genotype with the lowest associated GC score was compared to the genotype called for the same SNP in the same duplicated animal sample but with a GC score of >0.90 (assumed to represent the true genotype). The mean genotype concordance rate for a GC score of <0.300, 0.300-0.549, and ≥0.550 in the cattle (sheep in parenthesis) was 0.9467 (0.9864), 0.9707 (0.9953), and 0.9994 (0.99997) respectively; the respective allele concordance rate was 0.9730 (0.9930), 0.9849 (0.9976), and 0.9997 (0.99998). Hence, concordance eroded as the GC score of the called genotype reduced, albeit the impact was not dramatic and was not very noticeable until a GC score of <0.55. Moreover, the impact was greater and more consistent in the cattle population than in the sheep population. Furthermore, an impact of GC score on genotype concordance rate existed even for the same SNP GenTrain value; the GenTrain value is a statistical score that depicts the shape of the genotype clusters and the relative distance between the called genotype clusters.

Molecular characterization of coat color gene in Sahiwal versus Karan Fries bovine

BackgroundMelanocortin-1-receptor gene (MC1R) plays a significant role in signaling cascade of melanin production. In cattle, the coat colors, such as red and black, are an outcome of eumelanin and pheomelanin pigments, respectively. The coat colors have become critical factors in the animal selection process. This study is therefore aimed at the molecular characterization of reddish-brown coat-colored Sahiwal cattle in comparison to the black and white-colored Karan Fries. ResultsThe Sequence length of the MC1R gene was 954 base pairs in Sahiwal cattle. The sequences were examined and submitted to GenBank Acc.No. MG373575 to MG373605. Alignment of both (Sahiwal and Karan Fries) protein sequences by applying ClustalO multiple sequence alignment programs revealed 99.8–96.8% sequence similarity within the bovine. MC1R gene phylogenetic studies were analyzed by MEGA X. The gene MC1R tree, protein confines, and hereditary difference of cattle were derived from Ensemble Asia Cow Genome Browser 97. One unique single-nucleotide polymorphism (c.844C>A) (SNP) was distinguished. Single amino acid changes were detected in the seventh transmembrane structural helix region, with SNP at p.281 T>N of MC1R gene in Karan Fries cattle. ConclusionsIn this current research, we first distinguished the genomic sequence of the MC1R gene regions that showed evidence of coat variation between Indian indigenous Sahiwal cattle breed correlated with crossbreed Karan Fries. These variations were found in the Melanocortin 1 receptor coding regions of the diverse SNPs. The conclusions of this research provide new insights into understanding the coat color variation in crossbreed compared to the Indian Sahiwal cattle. Graphical abstract▪

Assessing genomic diversity and signatures of selection in Jiaxian Red cattle using whole-genome sequencing data

BackgroundNative cattle breeds are an important source of genetic variation because they might carry alleles that enable them to adapt to local environment and tough feeding conditions. Jiaxian Red, a Chinese native cattle breed, is reported to have originated from crossbreeding between taurine and indicine cattle; their history as a draft and meat animal dates back at least 30 years. Using whole-genome sequencing (WGS) data of 30 animals from the core breeding farm, we investigated the genetic diversity, population structure and genomic regions under selection of Jiaxian Red cattle. Furthermore, we used 131 published genomes of world-wide cattle to characterize the genomic variation of Jiaxian Red cattle.ResultsThe population structure analysis revealed that Jiaxian Red cattle harboured the ancestry with East Asian taurine (0.493), Chinese indicine (0.379), European taurine (0.095) and Indian indicine (0.033). Three methods (nucleotide diversity, linkage disequilibrium decay and runs of homozygosity) implied the relatively high genomic diversity in Jiaxian Red cattle. We used θπ, CLR, FST and XP-EHH methods to look for the candidate signatures of positive selection in Jiaxian Red cattle. A total number of 171 (θπ and CLR) and 17 (FST and XP-EHH) shared genes were identified using different detection strategies. Functional annotation analysis revealed that these genes are potentially responsible for growth and feed efficiency (CCSER1), meat quality traits (ROCK2, PPP1R12A, CYB5R4, EYA3, PHACTR1), fertility (RFX4, SRD5A2) and immune system response (SLAMF1, CD84 and SLAMF6).ConclusionWe provide a comprehensive overview of sequence variations in Jiaxian Red cattle genomes. Selection signatures were detected in genomic regions that are possibly related to economically important traits in Jiaxian Red cattle. We observed a high level of genomic diversity and low inbreeding in Jiaxian Red cattle. These results provide a basis for further resource protection and breeding improvement of this breed.

94 Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning

Some European cattle breeds, such as Charolais and Maine Anjou, have natural mutations in the myostatin gene (MSTN) that inhibit its expression and result in an increase in muscle mass and protein content. An innovative hallmark would be the invitro introduction of this genotype in South America cattle breeds to improve their commercial value. To achieve this, we aimed to disrupt MSTN gene expression in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate MSTN-edited embryos by somatic cell nuclear transfer (SCNT). BFFs were isolated from a cloned fetus of a Brangus bull with a prized genetic background and nucleofected with the Cas9 ribonucleoprotein-gRNA complex previously assessed to target exon 2 of the bovine MSTN gene. To evaluate MSTN editing, genomic DNA from the wild-type (WT) and nucleofected BFFs were isolated, exon 2 of MSTN was amplified by PCR, and the PCR product was Sanger sequenced. In all cases, the sequencing results were analysed using the indel Synthego software tool. According to indel analysis, MSTN gene editing efficiency of the BFFs was 58.83%±3.2 (n=6). The resulting edit consisted of insertion of thymine in exon 2 of the MSTN gene that shifted the gene reading frame, introducing a premature stop codon and generating a truncated MSTN protein. The nucleofected BFFs were then used to generate embryos by SCNT, and WT BFFs were included as controls. Embryo cleavage and blastocyst development rates were evaluated at Day 2 and 7, respectively (Chi-squared test, P&amp;lt;0.05). Although lower cleavage rates were obtained in the MSTN-edited BFFs group [65.3% (n=273/418) vs. 87.6% (n=169/193)], no differences were observed at the blastocyst stage [19.1% (n=80/418) vs. 25.4% (n=49/193)]. To confirm the efficiency of MSTN editing in the cloned embryos, 10 blastocysts generated with the MSTN-edited BFFs were individually analysed for MSTN exon 2 sequence as described before. The results showed that 30% of the blastocysts (3/10) presented a homozygous biallelic edition, which consisted of a thymine base insertion, as expected. In summary, the strategy we used allowed production of MSTN null cloned Brangus embryos avoiding putative undesired integration of exogenous DNA into the bovine genome, such as plasmid sequence, regardless of off-target occurrences. We conclude that CRISPR/Cas9 is an efficient technique to disrupts MSTN gene expression in bovine embryos; this work represents a step toward improving the production efficiency of South American cattle breeds.

Allele Specific Expression (ASE) analysis between Bos Taurus and Bos Indicus cows using RNA-Seq data at SNP level and gene level.

In the current study, allele specific expression analysis was performed in two subspecies cows (Bos taurus and Bos indicus) at SNP and gene levels. RNA-Seq data of 21,078,477 and 20940063 paired end reads from pooling of whole blood samples (Leukocyte) from 40 US Holstein (Bos Taurus) and 45 Cholistani cows (Bos indicus) obtained from SRA database in NCBI. Quality control and trimming of row RNA-Seq data were processed by FASTQC and Trimmomatic softwares. The transcriptome was assembled by TopHat2 software in two cow's population by aligning and mapping the RNA-Seq reads on bovine reference genome. The SNPs were discovered by Samtools software and ASE analysis was performed by Chi-square test. Results showed that 50183 and 137954 SNPs were discovered on the assembled transcriptome of Holstein and Cholistani cow samples, respectively, and 15308 SNPs were common in both breeds. 10158 SNPs from 50183 (20%) in Holstein and 31523 SNPs from 137954 (23%) in Cholistani cows were identified as ASE-SNPs. Reference allele and alternative allele count in Holstein and Cholistani cows were 3041 and 7155, respectively. Among 131 discovered SNPs in 41 genes with different expression in Holstein and Cholistani cows, 31 ASE-SNPs (5 in Holstein; 26 in Cholistani cows) were discovered.

The impact of reference composition and genome build on the accuracy of genotype imputation in Australian Angus cattle

Context Genotype imputation is an effective method to increase the number of SNP markers available for an animal and thereby increase the overall power of genome-wide associations and accuracy of genomic predictions. It is also the key to achieve a common set of markers for all individuals when the original genotypes are obtained using multiple genotyping platforms. High accuracy of imputed genotypes is crucial to their utility. Aims In this study, we propose a method for the construction of a common set of medium density markers for imputation, which relies on keeping as much information as possible. We also investigated the impact of changing marker coordinates on the basis of the new bovine genome assembly, ARS-UCD 1.2, on imputation accuracy. Methods In total, 49 754 animals with 45 364 single nucleotide polymorphism markers were used in a 10-fold cross-validation to compare four different imputation scenarios. The four scenarios were based on two alternative designs for the reference datasets. (1) A traditional reference panel that was created using the overlapping SNP from five medium density arrays and (2) a composite reference panel created by combining SNPs across the five arrays. Each of the reference datasets was used to test imputation accuracy when the SNPs were aligned on the basis of two genome assemblies (UMD 3.1 and ARS-UCD 1.2). Key results Our results showed that a composite reference panel can achieve higher imputation accuracies than does a traditional overlap reference. Incorporating mapping information on the basis of the recent genome build slightly improved the imputation accuracies, especially for lower density chips. Conclusions Markers with unreliable mapping information and animals with low connectedness to the imputation reference dataset benefited the most from the ARS-UCD 1.2 assembly and composite reference respectively. Implications The presented method is straightforward and can be used to setup an optimal imputation for accurate inference of genotypes in Australian Angus cattle.

Quantitative determination of histone methylation via fluorescence resonance energy transfer (FRET) technology in immortalized bovine mammary alveolar epithelial cells supplemented with methionine.

Methionine (Met) is an essential precursor of S-adenosylmethionine (SAM), which is the primary methyl donor required for biological processes such as DNA and histone methylation, which alter gene expression. In dairy cows, dietary Met has been observed to exert transcriptional alterations with beneficial effects on milk biosynthesis; however, the extent of these effects via SAM remains unknown. Therefore, we evaluated the effect of Met supply on histone methylation in lysine residues K9 and K27 in the histone tail H3 via a fluorescence resonance energy transfer (FRET) system in immortalized bovine mammary alveolar epithelial cells (MACT) incubated varying concentration of Met. The histone methylation data was complemented with global DNA methylation, cellular protein synthesis, and RT-qPCR analysis of genes related to Met cycle, DNA and histone methylation, AA transporters, and protein synthesis. The histone methylation data was performed on MACT cells seeded at 30,000 cells/well in 96-well plates 24 h prior to transfection. The transfections of FRET gene reporter plasmids H3K9 and H3K27 was performed with 0.3 μL/well of Lipofectamine® 3000 and 50 ng of plasmid DNA per well. At 24 h post-transfection, cells were treated with 0, 125, 250, and 500 μM of Met, and quantification of histone methylation was performed at 0, 12, and 24 h post-treatment as well as cell viability at 24 h using CellProfiler software. An inverted microscope for live imagining (EVOS® FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37˚C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification. A more defined response on histone methylation was observed in H3K9 than H3K27 to Met supply, where maximal histone methylation in H3K9 was observed with 125 μM of Met. This greater histone methylation in H3K9 at 125 μM was accompanied by greater cellular protein concentration. The linear increase in Met supply causes a linear decrease in global DNA methylation, while linearly upregulating genes related to the Met cycle (i.e., MAT1A, PEMT, SAHH, and MTR). The histone methylation data suggest that, to some extent, methyl-donors such as Met may affect the methylation sites, H3K9 and H3K27, and consequently causing a different epigenetic alteration. In the context of the dairy cow, further refinement to this FRET assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

Genome-wide characterisation of regions under intense selection based on runs of homozygosity in Charolais cattle

Submitted 2020-06-22 | Accepted 2020-07-20 | Available 2020-12-01 https://doi.org/10.15414/afz.2020.23.mi-fpap.347-352 In this study, 68 genotyped purebred cows and bulls of Charolais cattle were used to determine runs of homozygosity (ROH) for evaluation of selection effect on the genome structure. ROH analysis was performed for 41,153 SNPs, and homozygous segments considering a minimum of 15 consecutive SNPs. The aim was to identify if regions of the genome with a high frequency of SNPs in ROH include signatures of selection. The most significant outlier SNPs were found on BTA2, 5, 7 and 19 (11 regions), with a sum of ROH length of 15.85 Mb. These regions contained genes included in various biological processes associated with the functioning of the immune system, growth, reproduction and metabolism. Various quantitative traits loci (QTLs) which affected the characteristics of meat production and reproduction have been identified in these regions. Overall, obtained results suggest that the Charolais cattle genome includes selection signatures reflecting the improvement of meat production and reproduction in accordance to breeding objecives. Keywords: artificial selection, autozygosity, beef, bovine, candidate genes References Aspatwar, A. et al. (2014). Carbonic anhydrase related proteins: molecular biology and evolution. Subcell. Biochem. 75: 135–156. Biscarini, F. et al. (2019). detectRUNS: an R package to detect runs of homozygosity and heterozygosity in diploid genomes. The R Project. Retrieved June 8, 2019 from https://cran.r-project.org/web/packages/detectRUNS/vignettes/detectRUNS.vignette.html Dixit, S. P. et al. (2020). Genome-Wide Runs of Homozygosity Revealed Selection Signatures in Bos indicus. Front. Genet. https://doi.org/10.3389/fgene.2020.00092 Gaudet, P. et al. (2011). Phylogenetic-based Propagation of Functional Annotations Within the Gene Ontology Consortium. 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Impact of Interbeef on national beef cattle evaluations

International evaluation models for beef cattle allow to compare animals’ estimated breeding values (EBV) across different countries, thanks to sires having offspring in more than one country. In this study we aimed to provide an up-to-date picture of the Interbeef international beef cattle evaluations from a national perspective, considering both large and small populations. Limousin age-adjusted weaning weight (AWW) phenotypes were available for 3,115,598 animals from 10 European countries, born between 1972 and 2017. EBV and reliabilities were obtained using a multi-trait animal model including maternal effects where AWW from different countries are modelled as different traits. We investigated the country of origin of the sires with internationally publishable EBV and, among them, the country of origin of the top 100 sires for each country scale. All countries had 20 to 28,557 domestic sires whose EBV were publishable, according to Interbeef’s rules, on the scale of other countries. All countries, except one, had domestic sires that ranked among the top 100 sires on other country scales. Across countries, inclusion of information from relatives recorded in other countries increased the reliability of EBV for domestic animals on average by 9.6 percentage points for direct EBV, and 8.3 percentage points for maternal EBV. In conclusion, international evaluations provide small countries access to a panel of elite foreign sires with EBV on their country scale and a more accurate estimation of EBV of domestic animals, while large countries obtain EBV for their sires on the scale of different countries which helps to better promote them.

Male recombination map of the autosomal genome in German Holstein

BackgroundRecombination is a process by which chromosomes are broken and recombine to generate new combinations of alleles, therefore playing a major role in shaping genome variation. Recombination frequencies (theta) between markers are used to construct genetic maps, which have important implications in genomic studies. Here, we report a recombination map for 44,696 autosomal single nucleotide polymorphisms (SNPs) according to the coordinates of the most recent bovine reference assembly. The recombination frequencies were estimated across 876 half-sib families with a minimum number of 39 and maximum number of 4236 progeny, comprising over 367 K genotyped German Holstein animals.ResultsGenome-wide, over 8.9 million paternal recombination events were identified by investigating adjacent markers. The recombination map spans 24.43 Morgan (M) for a chromosomal length of 2486 Mbp and an average of ~ 0.98 cM/Mbp, which concords with the available pedigree-based linkage maps. Furthermore, we identified 971 putative recombination hotspot intervals (defined as theta > 2.5 standard deviations greater than the mean). The hotspot regions were non-uniformly distributed as sharp and narrow peaks, corresponding to ~ 5.8% of the recombination that has taken place in only ~ 2.4% of the genome. We verified genetic map length by applying a likelihood-based approach for the estimation of recombination rate between all intra-chromosomal marker pairs. This resulted in a longer autosomal genetic length for male cattle (25.35 cM) and in the localization of 51 putatively misplaced SNPs in the genome assembly.ConclusionsGiven the fact that this map is built on the coordinates of the ARS-UCD1.2 assembly, our results provide the most updated genetic map yet available for the cattle genome.

PSXII-21 Genome-wide search for genomic regions under putative selection in two Russian native cattle breeds using high-density SNP Bead Chip

Abstract National livestock genetic resources are the valuable source of genetic variability, which are necessary to ensure the sustainability of the local animal production systems. The aim of our work was to characterize the genomic architecture and to detect signatures of selection in two oldest Russian native cattle breeds, using high-density DNA arrays. Fifty-six animals of Yaroslavl (n = 31) and Kholmogor (n = 25) were selected for the study. The Illumina Bovine HD BeadChip (777,962 SNPs) was used for genotyping. High-density SNP genotypes for Holsteins (n = 25) were used as the reference. Three methods (Fst, hapFLK, and ROH) were implemented to detect the genomic regions to be under putative selection. The results of the PCA-plot, Neighbor-Net analysis and Admixture clustering showed clear genetic differences between Yaroslavl and Kholmogor breeds as well as between them and Holsteins. Comparison of the results produced by different methods showed presence of twelve regions in genome of Yaroslavl breed and nine regions in genome of Kholmogor breeds, which were identified at least by two methods. We have confirmed nine regions under putative selection in genome of Yaroslavl cattle and six regions in genome of Kholmogor cattle, which were described previously based on medium-density SNP genotypes, herewith the flanking positions of the most of these regions were expanded. Additionally, we detected three new putative genomic regions affected by selection in each studied breed, which were localized on chromosomes 4, 15 and 17 in Yaroslavl breed and on chromosomes 12, 15 and 18 in Kholmogor breed. The functional annotation of genes localized within identified regions was carried out. Our research results can be useful for genetic improvement of the studied cattle breeds and for developing the programs for their conservation. The study was supported by RSF within project No. 19-76-20012.

46 Endometrial transcriptome profiles associated with bovine preimplantation pregnancy outcome and parity

Abstract Pregnancy failure in dairy cattle appears to be attributable to losses during embryogenesis, mainly within the first month. Models have estimated that pregnancies lost after day 30 of fertilization cost producers US $550/cow. The reasons for these losses are not well understood, and the transcriptome profile of the reproductive tract during the establishment of pregnancy may point to mechanisms. Thirty-eight cows in lactations 1 to 3 were artificially inseminated, and endometrial biopsies were collected on day 15 after artificial insemination to investigate the effect of parity on the preimplantation pregnancy outcome. Pregnancy status was determined by the presence of interferon-tau in the uterine flushing, revealing 19 pregnant (P) and 19 non-pregnant (NP) cows. Nineteen biopsy samples [9 cows in lactation 1 (6 P and 3 NP), 4 in lactation 2 (2 P and 2 NP), and 6 in lactation 3 (3P and 3NP)] with an average RNA integrity number of 7 were selected for RNA sequencing with an Illumina HiSeq analyzer. Sequence reads were assembled to the ARS_USD1.2.99 bovine reference genome using the CLC genomics workbench software. On average, the samples generated ~56 million reads and 94.99% were mapped to the reference genome. Differential gene expression analysis between P and NP cows identified 187, 60, and 136 differentially expressed genes (DEG) in lactation 1, 2 and 3, respectively (P &amp;lt; 0.01, FDR &amp;lt; 0.05, FC &amp;gt; ±2). Metabolic pathway enrichment analysis identified several DEG upregulated in lactations 2 and 3 associated with IL-17 signaling pathway, although expression of IL-17 itself was not different. This pathway is part of the protective response against extracellular bacteria and likely participates in pregnancy maintenance through local regulation of immune function. Further functional genomic analyses will be performed to determine additional metabolic pathways, key regulator genes, and functional SNPs associated with the establishment of pregnancy.

24 Structural variants affecting mRNAs isoforms splice sites associated with marbling in Nellore cattle

Abstract The mRNA identification and associated splice variants affecting meat marbling in Nellore cattle could contribute to a better understanding of the genetic architecture of this complex trait. Longissimus thoracis samples from the 20 most extreme bulls (out of 80 bulls set) for marbling [high (n=10) and low (n=10) groups] trait were used to perform transcriptomic analysis using RNA-Sequencing. The CLC Genomics Workbench software was used to align the fragments of each sample to the bovine reference genome (ARS-UCD1.2) and to perform differential expressed transcript analysis (DE). An average of 37,000 transcripts and 14 mRNA DE (P ≤ 0.001; FC &amp;gt; 2) were detected in the muscle transcriptome. Among them, three transcripts were up-regulated (COL4A2-202, GHRH-201 and ENSBTAT00000070459.1) and 11 down-regulated (HBB-201, HBA-201, RPL14-202, CATHL1-201, CATHL4-201, MAPKAPK2-204, PCBD1-201, RPL30-203, NEURL1-202, PGLYRP1-201 and ENSBTAT00000038384.2) in high marbling group in relation to low group. Structural variants, including Single Nucleotide Variant (SNV), insertion or deletion were also found in the mRNA DE (4 in high and 6 in low marbling groups), causing possible splice site disruption. For high marbling group, in the COL4A2-202 transcript, three SNVs [one guanine (G) located on chromosome 12 at 85,104,867 bp, two cytosine (C), at the positions: 85,119,139 and 85,145,937 bp] were found. From these, one is a non-synonyms mutation [SNV (G) 85,145,937bp], causing an amino acid change (ENSBTAP00000005916.5: p.Glu1088Asp) in Collagen alpha-2(IV) chain protein (COL4A2). Interestingly, animals with low marbling presented also deletions (85,100,930 pb) in this same gene, causing an amino acid change (ENSBTAP00000005916.5:p.Pro113fs)in the COL4A2 protein. Functional enrichment analysis, using the DE mRNAs list, identified significant metabolic pathways (FDR &amp;lt; 0.05), such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis and phenylalanine degradation. The results suggest potential key regulatory genes associated with marbling, an economically important trait for the beef industry and for the consumer. Funding FAPESP (#2009/16118-5, #11/21241-0, #2017/02470-3, #2017/10630–2, #2018/ 20026–8, #2018/11154-2) and CAPES (#001).

11 Genome-wide association study using repeated measures model for stillbirth in Holstein dairy cattle

Abstract Reduced fertility is one of the main causes of economic losses in dairy farms. The financial losses are estimated in US$938 per stillbirth case in Holstein herds. The identification of genomic regions associated with stillbirth could help to develop better management and breeding strategies aiming to reduce the frequency of undesirable gestation outcomes. The same cow may show different outcomes in different gestations. Therefore, evaluation of a single time-point may provide a biased indication of the genetic causes of stillbirth. A weighted single-step genomic best linear unbiased prediction (WssGBLUP) was performed using the BLUPF90 software. A total of 14,145 cows (with three or more delivery records) were genotyped for 42,909 SNP markers mapped against the ARS-UCD1.2 bovine reference genome. Three rounds of WssGBLUP were performed using 50,541 stillbirth records (4.8±1.3 records per animal). In total, 5,796 stillbirth cases were observed out of the 50,541 records (~11%). The statistical model included: calf size, calf sex, and dam age class (&amp;lt; 36, 36–50, 50–63, 63–75, ³75 months) as fixed effects; and herd-year-season, cow additive genetic merit, cow permanent environment, and service sire as random effects. Nine windows explaining more than one percent of the total genetic variance were identified, where the top 3 windows explained 7.86% (BTA13:37558814-38550118), 4.92% (BTA18:59897080-60892357) and 2.17% (BTA12:80568320-81508005). The top candidate window, on BTA13, harbors the OVOL2 gene, which codifies a zinc-finger protein crucial for embryo development during the angiogenesis, heart formation, hematopoietic, endothelial, neural tube and placental development. Additionally, OVOL2 is crucial during the spermatogenesis and was already identified as down-regulated between ectopic and eutopic endometrial tissues in woman with endometriosis. These results pinpoint new genomic candidate regions for stillbirth in Holstein, helping to better understand the genetic mechanisms associated with fertility problems.

PSIII-6 Comparisons of high-density genomic data in Holsteins differing in coat type

Abstract Preliminary studies have reported that wild type-haired (WT) Holstein cattle have greater productive potential at a genomic level than their slick-haired (SL) counterpart. The objective of the present study was to evaluate, through a panel of commercial genetic markers (Igenity Elite, Neogen Corp), 49 Holsteins descendants SL [32 SL (n = 11 males and 21 females) and 17 WT (n = 2 males and 15 females)] from the Agricultural Experiment Station of Puerto Rico. These animals received a predictive genomic transmission capacity (PTA) based on DNA tests using more than 100,000 bovine genome markers, which contain information from their parents for a more accurate measure of genetic potential. Traits evaluated included measures of productivity, health, type and other indexes such as: net merit, milk yield, somatic cell score, among others. The data was analyzed using Proc GLIMMIX in SAS and the differences between means were determined by the Tukey test. Various subsets of animals were compared; however, no main differences were found between SL and WT (P &amp;gt;0.05), except for stature (-1.07±0.17 vs -0.39±0.26; P = 0.02, respectively). In a second subset, only lactating cows were evaluated (SL n = 7 and WT n = 7) and differences were found for: hypocalcemia (0.03±0.06 vs -0.21±0.06; P = 0.01), mastitis (-0.11±0.24 vs 0.90±0.24; P = 0.01), PTA type (-0.98±0.26 vs -0.11±0.26; P = 0.03), udder composite (-0.96±0.26 vs -0.07±0.26; P = 0.02), stature (-0.91±0.30 vs 0.04±0.30; P = 0.04), dairy form (-1.64±0.26 vs -0.65±0.26; P = 0.02), fore udder attachment (-0.77±0.30 vs 0.41±0.30; P = 0.02), rear udder height (-1.39±0.34 vs -0.18±0.34; P = 0.03) and width (-1.42±0.29 vs -0.17±0.29; P = 0.01), and udder depth (-0.73±0.27 vs 0.82±0.27; P = 0.002), respectively. These results suggest that SL males and females are genomically similar to WT for the most important economic traits when descending from a SL dam and when compared as a single group. However, differences in type and health traits may occur if the females are compared alone.

PSVII-8 miRNAs explain the variation in muscle and blood transcriptomes of beef calves born from dams with or without energy restriction during late gestation

Abstract Maternal energy restriction during late gestation affects the expression of genes related to energy metabolism in muscle and immune response in blood. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. The aim of this study was to identify potentials miRNA involved in the expression of differentially expressed genes (DEG) in muscle and blood following exposure to maternal gestational energy restriction. Forty days before the expected calving date, cows were assigned to one of two diets: 100% (CTRL) or 70% (REST) of the daily energy requirement. For RNA-seq analysis, muscle samples were collected from 12 heifers and 12 steers, and blood samples were collected from 12 steers. miRNAs were identified from the RNA-seq data based on the bovine genome annotation, with 38 and 10 miRNAs identified in blood and muscle, respectively. The expression of the miRNAs and the previously identified 160 and 450 DEGs in muscle and blood, respectively, was pre-adjusted for fixed effects before final analyses. A stepwise selection (P-value &amp;lt; 0.05) was used to identify miRNAs (dependent variables) explaining variation in DEGs, for each DEG at a time, and analyses performed separately for blood and muscle. The R2 of selected models ranged from 0.88 to 0.99 in muscle and 0.92 to 0.99 in blood. Of the most selected miRNA in muscle, MiR-133a and MiR-1 are known to be related to muscle hypertrophy, and MiR-143 and bta-let-7i promote adipocyte differentiation. Of the most selected miRNA in blood, MiR-21 regulates immune system by different pathways. Using RNA-seq data, we identified miRNAs explaining a large amount of the variation of DEGs, with the identification of important miRNAs related to muscle development and immune system.