Christa Kuehn

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

BackgroundDeep 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.ResultsA 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.ConclusionsThe 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.

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

BackgroundSubstantial 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 (DGAT1) 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.ResultsBased on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 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 DGAT1 promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits.ConclusionNon-additive effects like those identified at the bovine DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 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.

A systems biology approach using metabolomic data reveals genes and pathways interacting to modulate divergent growth in cattle

BackgroundSystems biology enables the identification of gene networks that modulate complex traits. Comprehensive metabolomic analyses provide innovative phenotypes that are intermediate between the initiator of genetic variability, the genome, and raw phenotypes that are influenced by a large number of environmental effects. The present study combines two concepts, systems biology and metabolic analyses, in an approach without prior functional hypothesis in order to dissect genes and molecular pathways that modulate differential growth at the onset of puberty in male cattle. Furthermore, this integrative strategy was applied to specifically explore distinctive gene interactions of non-SMC condensin I complex, subunit G (NCAPG) and myostatin (GDF8), known modulators of pre- and postnatal growth that are only partially understood for their molecular pathways affecting differential body weight.ResultsOur study successfully established gene networks and interacting partners affecting growth at the onset of puberty in cattle. We demonstrated the biological relevance of the created networks by comparison to randomly created networks. Our data showed that GnRH (Gonadotropin-releasing hormone) signaling is associated with divergent growth at the onset of puberty and revealed two highly connected hubs, BTC and DGKH, within the network. Both genes are known to directly interact with the GnRH signaling pathway. Furthermore, a gene interaction network for NCAPG containing 14 densely connected genes revealed novel information concerning the functional role of NCAPG in divergent growth.ConclusionsMerging both concepts, systems biology and metabolomic analyses, successfully yielded new insights into gene networks and interacting partners affecting growth at the onset of puberty in cattle. Genetic modulation in GnRH signaling was identified as key modifier of differential cattle growth at the onset of puberty. In addition, the benefit of our innovative concept without prior functional hypothesis was demonstrated by data suggesting that NCAPG might contribute to vascular smooth muscle contraction by indirect effects on the NO pathway via modulation of arginine metabolism. Our study shows for the first time in cattle that integration of genetic, physiological and metabolomics data in a systems biology approach will enable (or contribute to) an improved understanding of metabolic and gene networks and genotype-phenotype relationships.

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

BackgroundA 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.ResultsTherefore, 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.ConclusionThe 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.

Revisiting the quantitative trait loci for milk production traits on BTA6

A parallel association study was performed in two independent cattle populations based on 41 validated, targeted single nucleotide polymorphisms (SNPs) and four microsatellite markers to re-evaluate the multiple quantitative trait loci (QTL) architecture for milk performance on bovine chromosome 6 (BTA6). Two distinct QTL located in the vicinity of the middle region of BTA6, but differing unambiguously regarding their effects on milk composition and yield traits were validated in the German Holstein population. A highly significant association of the protein variant ABCG2 p.Tyr581Ser with milk composition traits reconfirmed the causative molecular relevance of the ABCG2 gene in QTL region 1, whereas in QTL region 2, significant and tentative associations between gene variants RW070 and RW023 (located in the promoter region and exon 9 of the PPARGC1A gene for milk yield traits) were detected. For the German Fleckvieh population, only RW023 showed a tentative association with milk yield traits, whereas those loci with significant effects in German Holsteins (ABCG2 p.Tyr581Ser, RW070) showed fixed alleles. Even though our new data highlight two variants in the PPARGC1A gene (RW023, RW070) in QTL region 2, based on the results of our study, currently no unequivocal conclusion about the causal background of this QTL affecting milk yield traits can be drawn. Notably, the German Holstein and Fleckvieh populations, known for their divergent degree of dairy type, differ substantially in their allele frequencies for the growth-associated NCAPG p.Ile442Met locus.

Tissue-specific mRNA expression patterns reveal a coordinated metabolic response associated with genetic selection for milk production in cows

The molecular mechanisms regulating the physiological adaptation of tissues important for nutrient partitioning and metabolism in lactating cows are still not completely understood. The aim of our study was to identify tissue-specific regulatory mechanisms necessary to accommodate metabolic changes associated with different genetic potential for milk performance. For this purpose, we analyzed mRNA expression of genes involved in energy metabolism of segregating F(2) beef type cows with a combined genetic dairy and beef background (Charolais × German Holstein cross, CH×GH) in contrast to purebred German Holstein (GH) dairy cows. Three groups of cows differing in milk performance were examined using quantitative real-time PCR in liver, mammary gland, and skeletal muscle. Our results describe substantial tissue-specific differences in mRNA transcription profiles between cow groups in relation to their genetic potential for milk performance and highlight genes exhibiting specific, partially yet-unknown functions in dairy and beef type cows, e.g., upregulation of PCK2 transcripts in the mammary gland and FBP2 transcripts in skeletal muscle of dairy cows. Noticeably, PCCA and PPARGC1A mRNA abundance varied significantly across experimental groups in all three tissues, pointing to potential key gene functions in the metabolic adaptation relative to divergent milk production performance. Correlations of mRNA expression levels to milk performance traits indicate that gene transcriptional processes may play a regulatory role in liver, mammary gland, and skeletal muscle to enable cows with different genetic potential for milk performance to cope with metabolic lactation-associated challenges.

Systems Biology Analysis Merging Phenotype, Metabolomic and Genomic Data Identifies Non-SMC Condensin I Complex, Subunit G (NCAPG) and Cellular Maintenance Processes as Major Contributors to Genetic Variability in Bovine Feed Efficiency

Feed efficiency is a paramount factor for livestock economy. Previous studies had indicated a substantial heritability of several feed efficiency traits. In our study, we investigated the genetic background of residual feed intake, a commonly used parameter of feed efficiency, in a cattle resource population generated from crossing dairy and beef cattle. Starting from a whole genome association analysis, we subsequently performed combined phenotype-metabolome-genome analysis taking a systems biology approach by inferring gene networks based on partial correlation and information theory approaches. Our data about biological processes enriched with genes from the feed efficiency network suggest that genetic variation in feed efficiency is driven by genetic modulation of basic processes relevant to general cellular functions. When looking at the predicted upstream regulators from the feed efficiency network, the Tumor Protein P53 (TP53) and Transforming Growth Factor beta 1 (TGFB1) genes stood out regarding significance of overlap and number of target molecules in the data set. These results further support the hypothesis that TP53 is a major upstream regulator for genetic variation of feed efficiency. Furthermore, our data revealed a significant effect of both, the Non-SMC Condensin I Complex, Subunit G (NCAPG) I442M (rs109570900) and the Growth /differentiation factor 8 (GDF8) Q204X (rs110344317) loci, on residual feed intake and feed conversion. For both loci, the growth promoting allele at the onset of puberty was associated with a negative, but favorable effect on residual feed intake. The elevated energy demand for increased growth triggered by the NCAPG 442M allele is obviously not fully compensated for by an increased efficiency in converting feed into body tissue. As a consequence, the individuals carrying the NCAPG 442M allele had an additional demand for energy uptake that is reflected by the association of the allele with increased daily energy intake as observed in our study.

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

BackgroundThe silver homologue(SILV) gene plays a major role in melanosome development. SILV 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 SILV is involved, are still not completely understood. While there are many studies addressing SILV function upon a eumelaneic pigment background, there is a substantial lack of information regarding the further relevance of SILV, e.g. for phaeomelanosome development.ResultsIn contrast to previous results in other species reporting SILV expression exclusively in pigmented tissues, our experiments provide evidence that the bovine SILV gene is expressed in a variety of tissues independent of pigmentation. Our data show that the bovine SILV 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.ConclusionThe expression of the bovine SILV gene independent of pigmentation suggests SILV functions exceeding melanosome development in cattle. This hypothesis is further supported by transcript variants lacking functional key elements of the SILV protein relevant for eumelanosome development. Thus, the bovine SILV gene can serve as a model for the investigation of the putative additional functions of SILV. Furthermore, the splice variants of the bovine SILV 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.

Analysis of structure and gene expression of bovine CCDC3 gene indicates a function in fat metabolism

Our study reports the molecular analysis of the bovine gene encoding the coiled-coil domain-containing protein 3 (CCDC3). Based on comparative sequence analysis and in silico sequence merging of predicted gene models, a new full-length gene model for the bovine CCDC3 gene was predicted and confirmed experimentally. The CCDC3 gene was assigned to bovine chromosome 13. It consists of three exons comprising 2599bp encoding for a respective protein of 274 amino acids. The strong CCDC3 sequence homology on amino acid level between species suggests a conserved universal function of this protein. In mice, the CCDC3 gene had been found to be highly expressed in adipocytes and regulated by hormonal-nutritional alternations and in obesity. The tissue expression pattern of bovine CCDC3 mRNA indicates a ubiquitous physiological function of the gene. Significant differences in CCDC3 mRNA expression in skeletal muscle between individuals characterized by divergent intramuscular fat deposition support the potential function of the gene in fat or energy metabolism, which possibly could also be inferred for other mammalian species. This first report of structural analysis and molecular characterization of the CCDC3 gene in cattle will contribute to a better understanding of the yet unknown physiological role of the respective protein in mammals.

Novel transcripts discovered by mining genomic DNA from defined regions of bovine chromosome 6.

BackgroundLinkage analyses strongly suggest a number of QTL for production, health and conformation traits in the middle part of bovine chromosome 6 (BTA6). The identification of the molecular background underlying the genetic variation at the QTL and subsequent functional studies require a well-annotated gene sequence map of the critical QTL intervals. To complete the sequence map of the defined subchromosomal regions on BTA6 poorly covered with comparative gene information, we focused on targeted isolation of transcribed sequences from bovine bacterial artificial chromosome (BAC) clones mapped to the QTL intervals.ResultsUsing the method of exon trapping, 92 unique exon trapping sequences (ETS) were discovered in a chromosomal region of poor gene coverage. Sequence identity to the current NCBI sequence assembly for BTA6 was detected for 91% of unique ETS. Comparative sequence similarity search revealed that 11% of the isolated ETS displayed high similarity to genomic sequences located on the syntenic chromosomes of the human and mouse reference genome assemblies. Nearly a third of the ETS identified similar equivalent sequences in genomic sequence scaffolds from the alternative Celera-based sequence assembly of the human genome. Screening gene, EST, and protein databases detected 17% of ETS with identity to known transcribed sequences. Expression analysis of a subset of the ETS showed that most ETS (84%) displayed a distinctive expression pattern in a multi-tissue panel of a lactating cow verifying their existence in the bovine transcriptome.ConclusionThe results of our study demonstrate that the exon trapping method based on region-specific BAC clones is very useful for targeted screening for novel transcripts located within a defined chromosomal region being deficiently endowed with annotated gene information. The majority of identified ETS represents unknown noncoding sequences in intergenic regions on BTA6 displaying a distinctive tissue-specific expression profile. However, their definite regulatory function has to be analyzed in further studies. The novel transcripts will add new sequence information to annotate a complete bovine genome sequence assembly, contribute to establish a detailed transcription map for targeted BTA6 regions and will also be helpful to dissect of the molecular and regulatory background of the QTL detected on BTA6.