Jennifer J. Michal

Obesity gene atlas in mammals.

Obesity in humans has increased at an alarming rate over the past two decades and has become one of the leading public health problems worldwide. Studies have revealed a large number of genes/markers that are associated with obesity and/or obesity-related phenotypes, indicating an urgent need to develop a central database for helping the community understand the genetic complexity of obesity. In the present study, we collected a total of 1,736 obesity associated loci and created a freely available obesity database, including 1,515 protein-coding genes and 221 microRNAs (miRNAs) collected from four mammalian species: human, cattle, rat, and mouse. These loci were integrated as orthologs on comparative genomic views in human, cattle, and mouse. The database and genomic views are freely available online at: http://www.integratomics-time.com/fat_deposition. Bioinformatics analyses of the collected data revealed some potential novel obesity related molecular markers which represent focal points for testing more targeted hypotheses and designing experiments for further studies. We believe that this centralized database on obesity and adipogenesis will facilitate development of comparative systems biology approaches to address this important health issue in human and their potential applications in animals.

Two-Dimensional Liquid Chromatography–Tandem Mass Spectrometry Coupled with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) Labeling Approach Revealed First Proteome Profiles of Pulmonary Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated the pig industry worldwide for almost 25 years, and its virus (PRRSV) preferentially infects and replicates in pulmonary alveolar macrophages (PAMs). To discover cellular protein responses in PRRSV-infected PAMs, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins between the PRRSV-infected groups and the controls. A total of 160 cellular proteins in PAMs that were significantly altered post-infection were identified. These differentially expressed proteins are related to the biological processes of virus binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. This is the first report that analyzed the cellular protein profile of PRRSV-infected PAMs using iTRAQ technology, and this data provides important information to help understand the host response to PRRSV and to define the cellular requirements for the underlying mechanism of PRRSV replication and pathogenesis.

Evaluation of candidate gene effects for beef backfat via Bayesian model selection

Candidate gene approaches provide tools for exploring and localizing causative genes affecting quantitative traits and the underlying variation may be better understood by determining the relative magnitudes of effects of their polymorphisms. Diacyglycerol O-acyltransferase 1 (DGAT1), fatty acid binding protein (heart) 3 (FABP3), growth hormone 1 (GH1), leptin (LEP) and thyroglobulin (TG) have been previously identified as genes contributing to genetic control of subcutaneous fat thickness (SFT) in beef cattle. In the present research, Bayesian model selection was used to evaluate effects of these five candidate genes by comparing competing non-nested models and treating candidate gene effects as either random or fixed. The analyses were implemented in SAS to simplify the programming and computation. Phenotypic data were gathered from a F2 population of Wagyu × Limousin cattle. The five candidate genes had significant but varied effects on SFT in this population. Bayesian model selection identified the DGAT1 model as the one with the greatest model probability, whether candidate gene effects were considered random or fixed, and DGAT1 had the greatest additive effect on SFT. The SAS codes developed in the study are freely available and can be downloaded at: http://www.ansci.wsu.edu/programs/.

Two-dimensional liquid chromatography–tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome, which has spread worldwide. Monocyte/macrophage lineage cells are the major target cells of PCV2. To discover cellular protein responses of pulmonary alveolar macrophages (PAMs) to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PAMs from the PCV2-infected group compared to the uninfected control group. A total of 145 cellular proteins in PAMs that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. The high number of differentially expressed proteins identified should be very useful to elucidate the mechanism of replication and pathogenesis of PCV2 in the future.

Corticotropin releasing hormone is a promising candidate gene for marbling and subcutaneous fat depth in beef cattle

The gene corticotropin releasing hormone (CRH) is mapped on bovine chromosome 14 (BTA14), where more than 30 fat-related quantitative trait loci (QTLs) have been reported in dairy and beef cattle. The gene product regulates secretion of adrenocorticotrophin hormone, the hypothalamic-pituitary-adrenal axis, and multiple hypothalamic functions; therefore, we hypothesized that CRH is a promising candidate gene for beef marbling score (BMS) and subcutaneous fat depth (SFD) in a Wagyu x Limousin F2 population. Two pairs of primers were designed and a total of 5 single nucleotide polymorphisms (SNPs) were identified: g.9657C>T, c.10718G>C, c.10841G>A, c.10893A>C, and c.10936G>C (AAFC03076794.1). Among the 4 cSNPs, c.10718G>C, c.10841G>A, and c.10936G>C are missense mutations leading to amino acid changes from arginine to proline, from serine to asparagine, and from aspartic acid to histidine, respectively. These 5 SNPs were genotyped on ~250 F2 progeny, but only 4 were selected as tagging SNPs for association analysis because no historical recombination was observed between c.10718G>C and c.10893A>C. Statistical analysis showed that g.9657C>T, c.10718G>C, and c.10936G>C and their haplotypes had significant effects on SFD, but only c.10936G>C was significantly associated with BMS. The SNP in the promoter (g.9657C>T) led to gain/loss of a CpG site and 4 potential regulatory binding sites. Different haplotypes among the 4 cSNPs significantly affected mRNA secondary structures but were not associated with phenotypes. Overall, our results provide further evidence that CRH is a promising candidate gene for a concordant QTL related to lipid metabolism in mammals.

Genome-Wide Genetic Diversity and Differentially Selected Regions among Suffolk, Rambouillet, Columbia, Polypay, and Targhee Sheep

Sheep are among the major economically important livestock species worldwide because the animals produce milk, wool, skin, and meat. In the present study, the Illumina OvineSNP50 BeadChip was used to investigate genetic diversity and genome selection among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds from the United States. After quality-control filtering of SNPs (single nucleotide polymorphisms), we used 48,026 SNPs, including 46,850 SNPs on autosomes that were in Hardy-Weinberg equilibrium and 1,176 SNPs on chromosome × for analysis. Phylogenetic analysis based on all 46,850 SNPs clearly separated Suffolk from Rambouillet, Columbia, Polypay, and Targhee, which was not surprising as Rambouillet contributed to the synthesis of the later three breeds. Based on pair-wise estimates of F ST, significant genetic differentiation appeared between Suffolk and Rambouillet (F ST = 0.1621), while Rambouillet and Targhee had the closest relationship (F ST = 0.0681). A scan of the genome revealed 45 and 41 differentially selected regions (DSRs) between Suffolk and Rambouillet and among Rambouillet-related breed populations, respectively. Our data indicated that regions 13 and 24 between Suffolk and Rambouillet might be good candidates for evaluating breed differences. Furthermore, ovine genome v3.1 assembly was used as reference to link functionally known homologous genes to economically important traits covered by these differentially selected regions. In brief, our present study provides a comprehensive genome-wide view on within- and between-breed genetic differentiation, biodiversity, and evolution among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds. These results may provide new guidance for the synthesis of new breeds with different breeding objectives.

Functional UQCRC1 Polymorphisms Affect Promoter Activity and Body Lipid Accumulation

Obesity and type 2 diabetes constitute leading public health problems worldwide. Studies have shown that insulin resistance affiliated with these conditions is associated with skeletal muscle lipid accumulation, while the latter is associated with mitochondrial dysfunctions. However, the initiation and regulation of mitochondrial biogenesis rely heavily on ∼1000 nuclear‐encoded mitochondrial regulatory proteins. In this study, we targeted the ubiquinol‐cytochrome c reductase core protein I gene, a nuclear‐encoded component of mitochondrial complex III, for its association with subcutaneous fat depth (SFD) and skeletal muscle lipid accumulation (SMLA) using cattle as a model. Four promoter polymorphisms were identified and genotyped on ∼250 Wagyu × Limousin F2 progeny. Statistical analysis revealed that two completely linked polymorphic sites, g.13487C>T and g.13709G>C (r2 = 1), were significantly associated with both SFD (p < 0.01) and SMLA (p < 0.0001). The difference between TTCC and CCGG haplotypes was 0.178 cm for SFD and 0.624 scores for SMLA. Interestingly, the former haplotype produced higher promoter activities than the latter by 43% to 49% in three cell lines (p < 0.05). In addition to Rett syndrome and breast/ovarian cancer observed in other studies, we report evidence for the first time, to our knowledge, that overexpression of ubiquinol‐cytochrome c reductase core protein I might affect mitochondrial morphology and/or physiology and lead to development of obesity and related conditions.

Genome Wide Sampling Sequencing for SNP Genotyping: Methods, Challenges and Future Development.

Genetic polymorphisms, particularly single nucleotide polymorphisms (SNPs), have been widely used to advance quantitative, functional and evolutionary genomics. Ideally, all genetic variants among individuals should be discovered when next generation sequencing (NGS) technologies and platforms are used for whole genome sequencing or resequencing. In order to improve the cost-effectiveness of the process, however, the research community has mainly focused on developing genome-wide sampling sequencing (GWSS) methods, a collection of reduced genome complexity sequencing, reduced genome representation sequencing and selective genome target sequencing. Here we review the major steps involved in library preparation, the types of adapters used for ligation and the primers designed for amplification of ligated products for sequencing. Unfortunately, currently available GWSS methods have their drawbacks, such as inconsistency in the number of reads per sample library, the number of sites/targets per individual, and the number of reads per site/target, all of which result in missing data. Suggestions are proposed here to improve library construction, genotype calling accuracy, genome-wide marker density and read mapping rate. In brief, optimized GWSS library preparation should generate a unique set of target sites with dense distribution along chromosomes and even coverage per site across all individuals.

Reactomes of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derived from 0 hour mock-infected and 6, 12, 16 and 24 hours PRRSV-infected porcine alveolar macrophages (PAMs) produced a total 643,255 sequenced tags with 91,807 unique tags. Differentially expressed (DE) tags were then detected using the Bayesian framework followed by gene/mRNA assignment, arbitrary selection and manual annotation, which determined 699 DE genes for reactome analysis. The DAVID, KEGG and REACTOME databases assigned 573 of the DE genes into six biological systems, 60 functional categories and 504 pathways. The six systems are: cellular processes, genetic information processing, environmental information processing, metabolism, organismal systems and human diseases as defined by KEGG with modification. Self-organizing map (SOM) analysis further grouped these 699 DE genes into ten clusters, reflecting their expression trends along these five time points. Based on the number one functional category in each system, cell growth and death, transcription processes, signal transductions, energy metabolism, immune system and infectious diseases formed the major reactomes of PAMs responding to PRRSV infection. Our investigation also focused on dominant pathways that had at least 20 DE genes identified, multi-pathway genes that were involved in 10 or more pathways and exclusively-expressed genes that were included in one system. Overall, our present study reported a large set of DE genes, compiled a comprehensive coverage of pathways, and revealed system-based reactomes of PAMs infected with PRRSV. We believe that our reactome data provides new insight into molecular mechanisms involved in host genetic complexity of antiviral activities against PRRSV and lays a strong foundation for vaccine development to control PRRS incidence in pigs.

Whole transcriptome analysis with sequencing: methods, challenges and potential solutions

Whole transcriptome analysis plays an essential role in deciphering genome structure and function, identifying genetic networks underlying cellular, physiological, biochemical and biological systems and establishing molecular biomarkers that respond to diseases, pathogens and environmental challenges. Here, we review transcriptome analysis methods and technologies that have been used to conduct whole transcriptome shotgun sequencing or whole transcriptome tag/target sequencing analyses. We focus on how adaptors/linkers are added to both 5′ and 3′ ends of mRNA molecules for cloning or PCR amplification before sequencing. Challenges and potential solutions are also discussed. In brief, next generation sequencing platforms have accelerated releases of the large amounts of gene expression data. It is now time for the genome research community to assemble whole transcriptomes of all species and collect signature targets for each gene/transcript, and thus use known genes/transcripts to determine known transcriptomes directly in the near future.