Multiple splice variants within the bovine silver homologue (SILV) gene affecting coat color in cattle indicate a function additional to fibril formation in melanophores

<p>Abstract</p> <p>Background</p> <p>The silver homologue(<it>SILV</it>) gene plays a major role in melanosome development. <it>SILV </it>is a target for studies concerning melanoma diagnostics and therapy in humans as well as on skin and coat color pigmentation in many species ranging from zebra fish to mammals. However, the precise functional cellular mechanisms, in which <it>SILV </it>is involved, are still not completely understood. While there are many studies addressing <it>SILV </it>function upon a eumelaneic pigment background, there is a substantial lack of information regarding the further relevance of <it>SILV</it>, e.g. for phaeomelanosome development.</p> <p>Results</p> <p>In contrast to previous results in other species reporting <it>SILV </it>expression exclusively in pigmented tissues, our experiments provide evidence that the bovine <it>SILV </it>gene is expressed in a variety of tissues independent of pigmentation. Our data show that the bovine <it>SILV </it>gene generates an unexpectedly large number of different transcripts occurring in skin as well as in non-pigmented tissues, e.g. liver or mammary gland. The alternative splice sites are generated by internal splicing and primarily remove complete exons. Alternative splicing predominantly affects the repeat domain of the protein, which has a functional key role in fibril formation during eumelanosome development.</p> <p>Conclusion</p> <p>The expression of the bovine <it>SILV </it>gene independent of pigmentation suggests <it>SILV </it>functions exceeding melanosome development in cattle. This hypothesis is further supported by transcript variants lacking functional key elements of the <it>SILV </it>protein relevant for eumelanosome development. Thus, the bovine <it>SILV </it>gene can serve as a model for the investigation of the putative additional functions of <it>SILV</it>. Furthermore, the splice variants of the bovine <it>SILV </it>gene represent a comprehensive natural model to refine the knowledge about functional domains in the SILV protein. Our study exemplifies that the extent of alternative splicing is presumably much higher than previously estimated and that alternatively spliced transcripts presumably can generate molecules of deviating function compared to their constitutive counterpart.</p

Epistatic interactions between at least three loci determine the “rat-tail” phenotype in cattle

BACKGROUND: The “rat-tail” syndrome (RTS) is an inherited hypotrichosis in cattle, which is exclusively expressed in diluted coloured hair. The affected animals also suffer from disturbed thermoregulation, which impairs their health and growth performance. Phenotypic features that are similar to RTS are observed in dogs with black hair follicle dysplasia. RESULTS: We used a resource cross population between German Holstein and Charolais cattle breeds to prove that epistatic interactions between at least three independent genetic loci are required for the expression of the RTS phenotype. In this population, the RTS is exclusively expressed in animals with a eumelanic background that is due to the dominant E D allele at the melanocortin 1 receptor gene located on Bos taurus autosome (BTA) 18. In addition, only the individuals that are heterozygous at the dilution locus on BTA5 that corresponds to the premelanosome protein or silver gene variant c.64G&gt;A were classified as displaying a RTS phenotype. Linkage and whole-genome association analyses using different models and different pedigrees allowed us to map a third locus (hereafter referred to as the RTS locus) that is essential for the expression of the RTS phenotype to the chromosomal region between 14 and 22 Mb on BTA5. Our findings clearly demonstrate that the RTS and dilution loci are distinct loci on BTA5. CONCLUSIONS: Our study provides evidence that the RTS locus has effects on hair conformation and coat colour dilution and that the effect on coat colour dilution is clearly independent from that of the dilution locus. Finally, our results excluded several other loci that were previously reported to be associated with or to underlie hair conformation or pigmentation traits as the causal mutations of RTS and also several major functional candidate genes that are associated with hypotrichosis in humans. Our finding on the identification of a three-locus interaction that underlies RTS provides a prime example of epistatic interaction between several independent loci that is required for the expression of a distinct phenotype

Association of an ACSL1 gene variant with polyunsaturated fatty acids in bovine skeletal muscle

<p>Abstract</p> <p>Background</p> <p>The intramuscular fat deposition and the fatty acid profiles of beef affect meat quality. High proportions of unsaturated fatty acids are related to beef flavor and are beneficial for the nutritional value of meat. Moreover, a variety of clinical and epidemiologic studies showed that particularly long-chain omega-3 fatty acids from animal sources have a positive impact on human health and disease.</p> <p>Results</p> <p>To screen for genetic factors affecting fatty acid profiles in beef, we initially performed a microsatellite-based genome scan in a F₂Charolais × German Holstein resource population and identified a quantitative trait locus (QTL) for fatty acid composition in a region on bovine chromosome 27 where previously QTL affecting marbling score had been detected in beef cattle populations. The <it>long-chain acyl-CoA synthetase 1 (ACSL1) </it>gene was identified as the most plausible functional and positional candidate gene in the QTL interval due to its direct impact on fatty acid metabolism and its position in the QTL interval. ACSL1 is necessary for synthesis of long-chain acyl-CoA esters, fatty acid degradation and phospholipid remodeling. We validated the genomic annotation of the bovine <it>ACSL1 </it>gene by <it>in silico </it>comparative sequence analysis and experimental verification. Re-sequencing of the complete coding, exon-flanking intronic sequences, 3' untranslated region (3'UTR) and partial promoter region of the <it>ACSL1 </it>gene revealed three synonymous mutations in exons 6, 7, and 20, six noncoding intronic gene variants, six polymorphisms in the promoter region, and four variants in the 3' UTR region. The association analysis identified the gene variant in intron 5 of the <it>ACSL1 </it>gene (<it>c.481-233A>G</it>) to be significantly associated with the relative content of distinct fractions and ratios of fatty acids (e.g., n-3 fatty acids, polyunsaturated, n-3 long-chain polyunsaturated fatty acids, trans vaccenic acid) in skeletal muscle. A tentative association of the <it>ACSL1 </it>gene variant with intramuscular fat content indicated that an indirect effect on fatty acid composition via modulation of total fat content of skeletal muscle cannot be excluded.</p> <p>Conclusions</p> <p>The initial QTL analysis suggested the <it>ACSL1 </it>gene as a positional and functional candidate gene for fatty acid composition in bovine skeletal muscle. The findings of subsequent association analyses indicate that <it>ACSL1 </it>or a separate gene in close proximity might play a functional role in mediating the lipid composition of beef.</p

A gene-based high-resolution comparative radiation hybrid map as a framework for genome sequence assembly of a bovine chromosome 6 region associated with QTL for growth, body composition, and milk performance traits

BACKGROUND: A number of different quantitative trait loci (QTL) for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of bovine chromosome 6 (BTA6). Dense genetic and physical maps and, ultimately, a fully annotated genome sequence as well as their mutual connections are required to efficiently identify genes and gene variants responsible for genetic variation of phenotypic traits. A comprehensive high-resolution gene-rich map linking densely spaced bovine markers and genes to the annotated human genome sequence is required as a framework to facilitate this approach for the region on BTA6 carrying the QTL. RESULTS: Therefore, we constructed a high-resolution radiation hybrid (RH) map for the QTL containing chromosomal region of BTA6. This new RH map with a total of 234 loci including 115 genes and ESTs displays a substantial increase in loci density compared to existing physical BTA6 maps. Screening the available bovine genome sequence resources, a total of 73 loci could be assigned to sequence contigs, which were already identified as specific for BTA6. For 43 loci, corresponding sequence contigs, which were not yet placed on the bovine genome assembly, were identified. In addition, the improved potential of this high-resolution RH map for BTA6 with respect to comparative mapping was demonstrated. Mapping a large number of genes on BTA6 and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments (human, mouse, rat, dog, chicken) achieved a refined accurate alignment of conserved segments and evolutionary breakpoints across the species included. CONCLUSION: The gene-anchored high-resolution RH map (1 locus/300 kb) for the targeted region of BTA6 presented here will provide a valuable platform to guide high-quality assembling and annotation of the currently existing bovine genome sequence draft to establish the final architecture of BTA6. Hence, a sequence-based map will provide a key resource to facilitate prospective continued efforts for the selection and validation of relevant positional and functional candidates underlying QTL for milk production and growth-related traits mapped on BTA6 and on similar chromosomal regions from evolutionary closely related species like sheep and goat. Furthermore, the high-resolution sequence-referenced BTA6 map will enable precise identification of multi-species conserved chromosome segments and evolutionary breakpoints in mammalian phylogenetic studies

Identification of Regulatory Functions of LncRNAs Associated With T. circumcincta Infection in Adult Sheep

[EN] Several recent studies have demonstrated the role of long non-coding RNAs (lncRNAs) in regulating the defense mechanism against parasite infections, but no studies are available that investigated their relevance for immune response to nematode infection in sheep. Thus, the aim of the current study was to (i) detect putative lncRNAs that are expressed in the abomasal lymph node of adult sheep after an experimental infection with the gastrointestinal nematode (GIN) Teladorsagia circumcincta and (ii) to elucidate their potential functional role associated with the differential host immune response. We hypothesized that putative lncRNAs differentially expressed (DE) between samples from animals that differ in resistance to infection may play a significant regulatory role in response to nematode infection in adult sheep. To obtain further support for our hypothesis, we performed co-expression and functional gene enrichment analyses with the differentially expressed lncRNAs (DE lncRNAs). In a conservative approach, we included for this predictive analysis only those lncRNAs that are confirmed and supported by documentation of expression in gastrointestinal tissues in the current sheep gene atlas. We identified 9,105 putative lncRNA transcripts corresponding to 7,124 gene loci. Of these, 457 were differentially expressed lncRNA loci (DELs) with 683 lncRNA transcripts. Based on a gene co-expression analysis via weighted gene co-expression network analysis, 12 gene network modules (GNMs) were found significantly correlated with at least one of 10 selected target DE lncRNAs. Based on the principle of “guilt-by-association,” the DE genes from each of the three most significantly correlated GNMs were subjected to a gene enrichment analysis. The significant pathways associated with DE lncRNAs included ERK5 Signaling, SAPK/JNK Signaling, RhoGDI Signaling, EIF2 Signaling, Regulation of eIF4 and p70S6K Signaling and Oxidative Phosphorylation pathways. They belong to signaling pathway categories like Cellular Growth, Proliferation and Development, Cellular Stress and Injury, Intracellular and Second Messenger Signaling and Apoptosis. Overall, this lncRNA study conducted in adult sheep after GIN infection provided first insights into the potential functional role of lncRNAs in the differential host response to nematode infectionSIFinancial support for the experimental work of this project was received from the LE248U14 project of the Junta de Castilla and León regional government, whereas the storage and processing of the generated sequencing datasets have been funded by the RTI2018-093535-B-I00 project from the Spanish Ministry of Science and Innovation (MICINN). PC was funded by a Short- Term Scientific Missions of the Functional Annotation of Animal Genomes-European network (FAANG-Europe) COST Action CA15112 to do a short research stay at Leibniz Institute for Farm Animal Biology (FBN). MM-V was also funded by the “Ramón y Cajal” Programme (RYC-2015-18368) from MICIN

eQTL-Detect: nextflow-based pipeline for eQTL detection in modular format with sharable and parallelizable scripts

Bioinformatic pipelines are becoming increasingly complex with the ever-accumulating amount of Next-generation sequencing (NGS) data. Their orchestration is difficult with a simple Bash script, but bioinformatics workflow managers such as Nextflow provide a framework to overcome respective problems. This study used Nextflow to develop a bioinformatic pipeline for detecting expression quantitative trait loci (eQTL) using a DSL2 Nextflow modular syntax, to enable sharing the huge demand for computing power as well as data access limitation across different partners often associated with eQTL studies. Based on the results from a test run with pilot data by measuring the required runtime and computational resources, the new pipeline should be suitable for eQTL studies in large scale analyses

Dominance and parent-of-origin effects of coding and non-coding alleles at the acylCoA-diacylglycerol-acyltransferase (DGAT1) gene on milk production traits in German Holstein cows

<p>Abstract</p> <p>Background</p> <p>Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (<it>DGAT1</it>) gene by using data sets including sires with accumulated phenotypic observations of daughters (breeding values, daughter yield deviations). However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits.</p> <p>Results</p> <p>Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of <it>DGAT1 </it>promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the <it>DGAT1 </it>K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the <it>DGAT1 </it>loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited <it>DGAT1 </it>promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits.</p> <p>Conclusion</p> <p>Non-additive effects like those identified at the bovine <it>DGAT1 </it>locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The <it>DGAT1 </it>alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits.</p

Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing

BACKGROUND: Deep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue. RESULTS: A total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin. CONCLUSIONS: The results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge

Selection signatures in four German warmblood horse breeds: Tracing breeding history in the modern sport horse.

The study of selection signatures helps to find genomic regions that have been under selective pressure and might host genes or variants that modulate important phenotypes. Such knowledge improves our understanding of how breeding programmes have shaped the genomes of livestock. In this study, 942 stallions were included from four, exemplarily chosen, German warmblood breeds with divergent historical and recent selection focus and different crossbreeding policies: Trakehner (N = 44), Holsteiner (N = 358), Hanoverian (N = 319) and Oldenburger (N = 221). Those breeds are nowadays bred for athletic performance and aptitude for show-jumping, dressage or eventing, with a particular focus of Holsteiner on the first discipline. Blood samples were collected during the health exams of the stallion preselections before licensing and were genotyped with the Illumina EquineSNP50 BeadChip. Autosomal markers were used for a multi-method search for signals of positive selection. Analyses within and across breeds were conducted by using the integrated Haplotype Score (iHS), cross-population Extended Haplotype Homozygosity (xpEHH) and Runs of Homozygosity (ROH). Oldenburger and Hanoverian showed very similar iHS signatures, but breed specificities were detected on multiple chromosomes with the xpEHH. The Trakehner clustered as a distinct group in a principal component analysis and also showed the highest number of ROHs, which reflects their historical bottleneck. Beside breed specific differences, we found shared selection signals in an across breed iHS analysis on chromosomes 1, 4 and 7. After investigation of these iHS signals and shared ROH for potential functional candidate genes and affected pathways including enrichment analyses, we suggest that genes affecting muscle functionality (TPM1, TMOD2-3, MYO5A, MYO5C), energy metabolism and growth (AEBP1, RALGAPA2, IGFBP1, IGFBP3-4), embryonic development (HOXB-complex) and fertility (THEGL, ZPBP1-2, TEX14, ZP1, SUN3 and CFAP61) have been targeted by selection in all breeds. Our findings also indicate selection pressure on KITLG, which is well-documented for influencing pigmentation