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  • RNA-seq analysis for detecting quantitative trait-associated genes

    Scientific Reports | 13 April 2016

    Many recent RNA-seq studies were focused mainly on detecting the differentially expressed genes (DEGs) between two or more conditions. In contrast, only a few attempts have been made to detect genes associated with quantitative traits, such as obesity index and milk yield, on RNA-seq experiment with large number of biological replicates. This study illustrates the linear model application on trait associated genes (TAGs) detection in two real RNA-seq datasets: 89 replicated human obesity related data and 21 replicated Holsteins’ milk production related RNA-seq data. Based on these two datasets, the performance between suggesting methods, such as ordinary regression and robust regression, and existing methods: DESeq2 and Voom, were compared. The results indicate that suggesting methods have much lower false discoveries compared to the precedent two group comparisons based approaches in our simulation study and qRT-PCR experiment. In particular, the robust regression outperforms existing DEG finding method as well as ordinary regression in terms of precision. Given the current trend in RNA-seq pricing, we expect our methods to be successfully applied in various RNA-seq studies with numerous biological replicates that handle continuous response traits.

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  • Comprehensive identification of sexually dimorphic genes in diverse cattle tissues using RNA-seq

    BMC Genomics

    Background
    Molecular mechanisms associated with sexual dimorphism in cattle have not been well elucidated. Furthermore, as recent studies have implied that gene expression patterns are highly tissue specific, it is essential to investigate gene expression in a variety of tissues using RNA-seq. Here, we employed and compared two statistical methods, a simple two group test and Analysis of deviance (ANODEV), in order to investigate bovine sexually dimorphic genes in 40 RNA-seq samples distributed across two factors: sex and tissue.

    Results
    As a result, we detected 752 sexually dimorphic genes across tissues from two statistical approaches and identified strong tissue-specific patterns of gene expression. Additionally, significantly detected sex-related genes shared between two mammal species (cattle and rat) were identified using qRT-PCR.

    Conclusions
    Results of our analyses reveal that sexual dimorphism of metabolic tissues and pituitary gland in cattle involves various biological processes. Several differentially expressed genes between sexes in cattle and rat species are shared, but show tissue-specific patterns. Finally, we concluded that two distinct statistical approaches have their advantages and disadvantages in RNA-seq studies investigating multiple tissues.

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  • Characterizing milk production related genes in Holstein using RNA-seq

    Asian-Australasian Journal of Animal Sciences

    Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents, Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (FDR adjusted P-value < 0.1) detected as milk production related DEGs. Of these genes, five (ALB, NOS3, RNPC3, SECTM1, and SPTSSB) were technically validated using qRT-PCR in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including CAV1, M-SAA3.2, LAP, CATHL4, CATHL6 have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidate for determining milk production.

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  • Comparative analysis of the complete genome of Lactobacillus Plantarum GB-LP2 and potential candidate genes for host immune system enhancement

    Journal of Microbiology and Biotechnology

    Acute respiratory virus infectious diseases are growing health problem, particularly in children and elderly. Much effort has been made to apply probiotics preventing influenza virus infections by enhancing innate immunity in the respiratory tract until available vaccines. Lactobacillus plantarum GB-LP2, isolated from the traditional Korean fermented vegetable, has shown the preventive effect on influenza virus infection in mice. To identify the molecular bases of this strain, we conducted whole genome assembly. The single circular DNA chromosome of 3, 284, 304 bp was completely assembled and 3, 250 protein-encoding genes were predicted. Evolutionarily accelerated genes related with the phenotypic trait of anti-infective activities for influenza virus were determined. These genes encode three integral membrane proteins, a teichoic acid export ATP-binding protein and a glucosamine--fructose-6-phosphate aminotransferase involved in host innate immunity, the non-specific DNA-binding protein Dps protecting bacteria from oxidative damage, and the response regulator of three-component quorum-sensing regulatory system related to the capacity of adhesion to the surface of the respiratory tract and competition with pathogens. This is the first study for identifying the genetic backgrounds of anti-viral activity in L. plantarum strains and it will provide insight about the anti-infective activities of L. plantarum and the development of preventive probiotics.

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  • HGTree: database of horizontally transferred genes determined by tree reconciliation

    Nucleic Acids Research

    The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree (http://hgtree.snu.ac.kr) represents a useful addition to the biological community and enables quick and easy retrieval of information for HGT-acquired genes to better understand microbial taxonomy and evolution. The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information.

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  • Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs

    PLoS ONE

    The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6, 991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

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  • Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon

    BMC Genomics | 9 November 2015

    Background
    Male sterility is an important mechanism for the production of hybrid seeds in watermelon. Although fruit development has been studied extensively in watermelon, there are no reports on gene expression in floral organs. In this study, RNA-sequencing (RNA-seq) was performed in two near-isogenic watermelon lines (genic male sterile [GMS] line, DAH3615-MS and male fertile line, DAH3615) to identify the differentially expressed genes (DEGs) related to male sterility.

    Results
    DEG analysis showed that 1259 genes were significantly associated with male sterility at a FDR P-value of?<?0.01. Most of these genes were only expressed in the male fertile line. In addition, 11 functional clusters were identified using DAVID functional classification analysis. Of detected genes in RNA-seq analysis, 19 were successfully validated by qRT-PCR.

    Conclusions
    In this study, we carried out a comprehensive floral transcriptome sequence comparison of a male fertile line and its near-isogenic male sterile line in watermelon. This analysis revealed essential genes responsible for stamen development, including pollen development and pollen tube elongation, and allowed their functional classification. These results provided new information on global mechanisms related to male sterility in watermelon.

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  • Caloric restriction improves diabetes-induced cognitive deficits by attenuating neurogranin-associated calcium signaling in high-fat diet-fed mice

    Journal of Cerebral Blood Flow & Metabolism | 5 October 2015

    Diabetes-induced cognitive decline has been recognized in human patients of type 2 diabetes mellitus and mouse model of obesity, but the underlying mechanisms or therapeutic targets are not clearly identified. We investigated the effect of caloric restriction on diabetes-induced memory deficits and searched a molecular mechanism of caloric restriction-mediated neuroprotection. C57BL/6 mice were fed a high-fat diet for 40 weeks and RNA-seq analysis was performed in the hippocampus of high-fat diet-fed mice. To investigate caloric restriction effect on differential expression of genes, mice were fed high-fat diet for 20 weeks and continued on high-fat diet or subjected to caloric restriction (2?g/day) for 12 weeks. High-fat diet-fed mice exhibited insulin resistance, glial activation, blood-brain barrier leakage, and memory deficits, in that we identified neurogranin, a down-regulated gene in high-fat diet-fed mice using RNA-seq analysis; neurogranin regulates Ca(2+)/calmodulin-dependent synaptic function. Caloric restriction increased insulin sensitivity, reduced high-fat diet-induced blood-brain barrier leakage and glial activation, and improved memory deficit. Furthermore, caloric restriction reversed high-fat diet-induced expression of neurogranin and the activation of Ca(2+)/calmodulin-dependent protein kinase II and calpain as well as the downstream effectors. Our results suggest that neurogranin is an important factor of high-fat diet-induced memory deficits on which caloric restriction has a therapeutic effect by regulating neurogranin-associated calcium signaling.

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  • Comprehensive Identification of Sexual Dimorphism-Associated Differentially Expressed Genes in Two-Way Factorial Designed RNA-Seq Data on Japanese Quail (Coturnix coturnix japonica)

    PLoS ONE

    Japanese quail (Coturnix coturnix japonica) reach sexual maturity earlier, breed rapidly and successfully, and cost less and require less space than other birds raised commercially. Given the value of this species for food production and experimental use, more studies are necessary to determine chromosomal regions and genes associated with gender and breed-differentiation. This study employed Trinity and edgeR for transcriptome analysis of next-generation RNA-seq data, which included 4 tissues obtained from 3 different breeding lines of Japanese quail (random bred control, heavy weight, low weight). Differentially expressed genes shared between female and male tissue contrast groups were analyzed to identify genes related to sexual dimorphism as well as potential novel candidate genes for molecular sexing. Several of the genes identified in the present study as significant sex-related genes have been previously found in avian gene expression analyses (NIPBL, UBAP2), and other genes found differentially expressed in this study and not previously associated with sex-related differences may be considered potential candidates for molecular sexing (TERA, MYP0, PPR17, CASQ2). Additionally, other genes likely associated with neuronal and brain development (CHKA, NYAP), as well as body development and size differentiation (ANKRD26, GRP87) in quail were identified. Expression of homeobox protein regulating genes (HXC4, ISL1) shared between our two sex-related contrast groups (Female Brain vs. Male Brain and Ovary vs. Testis) indicates that these genes may regulate sex-specific anatomical development. Results reveal genetic features of the quail breed and could allow for more effective molecular sexing as well as selective breeding for traits important in commercial production.

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  • A novel splice variant of the decapentaplegic (dpp) gene in the wild silkworm, Bombyx mandarina

    Biochemical and Biophysical Research Communications

    Decapentaplegic (dpp) is a member of the transforming growth factor-β superfamily. Although the dpp gene and related pathways are known to play important roles in insect development, few studies have examined its function in Bombyx mori and Bombyx mandarina. To date, there have been no previous reports on novel splice variants of dpp in silkworm. In the present study, we conducted RT-PCR to examine dpp expression in the mid-gut tissue of B. mandarina and discovered a novel dpp isoform. The isoform sequence was confirmed using sequencing analysis and found to have 333 bp deletion compared to full-length cDNA encoding dpp. The deleted sequence encodes a region of the latency associated peptide (LAP) region of transforming growth factor-β (TGF-β), which may affect the activity and specificity of TGF-β. Using variant calling analyses, we detected 7 candidate single nucleotide variants (SNVs) for different alternative splicing in dpp. This is the first report of a novel splice variant of the dpp gene in B. mandarina and these results provide insight about the domestication process and distinct phenotypic traits of B. mori and B. mandarina.

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