Investigation of candidate genes for metabolic disorders expressed in liver and pituitary gland by comparing the RNA-seq data of Polish-HF and Polish-Red cattle

Background: Metabolic disorder is a major health problem in dairy cattle, particularly to high milk producing dairy cattle. It is worthily emphasized that metabolic diseases have a very complex etiology and pathogenesis, and the impact of these diseases on hepatic and pituitary gland gene expression and organism oxidative balance is not fully described. The presented study was aimed to determine and predict the hepatic and pituitary gland expression of potential candidate genes in context to maintenance of oxidative balance, negative nitrogen balance, as well as ketosis in Polish HF and Polish Red cattle.Methods: Based on the RNA-seq experimental data, we investigated the candidate genes (SOD1, SOD2, SOD3, GPx2, GPx3, GPx5, GPx6, GPx7, GPx8, BDH1, FN1, ACSL3, HMGCL, HMGCS2, BDH2, ACSL6, ACAT2, IDH3B, ACAT1, HMGCS1, ACSL4, ACSL1, PC, CPT1A, OXCT1 and ACSL5 respectively) expressions in liver and pituitary gland tissues of Polish HF and Polish Red cattle. The RNA-seq experimental design comprised of young bulls aged between 6 to 12 months were investigated. For each breed, six liver and six pituitary gland tissues were sequenced using Next-seq 500 illumina platform. The RNA-seq expression data were normalized by the reads per kilobase of exon per million reads mapped (RPKM) method.Results: By comparing the RNA-seq data of liver and pituitary gland tissues, the investigated candidate genes were highly expressed in the hepatic tissues than to pituitary gland in investigated cattle breeds. However, by comparing the Polish HF and Polish Red cattle breeds, results revealed a similar trend of gene expression profiling of all investigated candidate genes for both metabolic tissues. In case of hepatic gene expression profiling, the SOD1, FN1, HMGCL, HMGCS2, ACAT2, ACAT1, HMGCS1, ACSL1 and ACSL5 were highly expressed (FPKM values of >40), followed by SOD2, GPX3, IDH3B, PC and BDH2 as moderately expressed (FPKM values: >10 to <40), and averagely expressed SOD3, GPX5, GPX6, GPX7, GPX2, GPX8, BDH1, ACSL3, ACSL6, ACSL4, CPT1A and OXCT1 respectively, in Polish HF and Polish Red breeds. In case of pituitary gland gene expression profiling, the SOD1 and GPx3 were highly expressed (FPKM values of >40), followed by SOD2, GPX8, IDH3B, ACAT1, ACSL4 and PC as moderately expressed (FPKM values: >10 to <40), and averagely expressed SOD3, GPX3,GPX5, GPX6, GPX7, GPX2, BDH1, BDH2, ACSL3, ACSL6, CPT1A, OXCT1, FN1, HMGCL, HMGCS2, ACAT2, ACAT1, HMGCS1, ACSL1 and ACSL5 respectively, in Polish HF and Polish Red breeds.Conclusions: Based on this presented results on hepatic and pituitary gland gene expression, a further research plan is an essential pre-requisite to validate the identified candidate genes. Study indicated the understanding the genetic factors that predispose metabolic disorders in cattle would benefit the dairy industry as a whole by providing producers, breeding services, and veterinarians a tool to forecast a cow’s susceptibility to metabolic disorders

Identification of CXCL8c.105A>G and CXCL8c.210C>T polymorphism in Polish HF cattle

Background: Bovine chemokine C-X-C motif legend 8 (CXCL8) also known as interleukin 8 (IL8) is a chemotactic factor that attracts neutrophils, basophils, and T-cells, in response to an inflammatory stimulus. The aim of this study was to investigate the novel single nucleotide polymorphisms (SNPs) located at the promoter region of CXCL8 gene in Polish Holstein Friesian (HF) bulls.Methods: Genotypic profiling of CXCL8c.105A>G and CXCL8c.210C>T SNP polymorphism were carried out by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) methods using HpyCH4VI and RsaI restriction enzymes. Polymorphism study was conducted on 151 randomly selected Polish HF bulls.Results: The genotype frequencies of CXCL8c.105A>G SNPs polymorphism in the investigated Polish HF bulls were observed as 0.24%, 0.5% and 0.26% respectively, whereas, the genotype frequencies of CXCL8c.210C>T SNPs polymorphism were observed as 0.27%, 0.5% and 0.23% respectively. Overall, the allele frequencies were recorded higher for “G’ allele for CXCL8c.105A>G and “C” allele and CXCL8c.210C>T SNP polymorphism. In both investigated SNP polymorphism, the state of genetic equilibrium was maintained in Polish HF bulls. Overall, the obtained results identified four haplotypes with the highest frequencies of CG (0.493%) and TA (0.476%) haplotypes, and the lowest frequencies of CA (0.02%) and TG (0.01%) haplotypes, respectively.Conclusions: Study concludes that both polymorphism could be further investigated for the trait-associated studies in the breeding population of Polish HF cattle

Association of bovine CXCL8 polymorphism with clinical mastitis and fertility trait in Polish HF cattle

Background: The aim of this study was to investigate the relationship between CXCL8 (CXCL8c.105A>G and CXCL8c.210C>T) SNP polymorphism and the clinical mastitis and production trait in Polish HF cattle.Methods: The trait-associated CXCL8 SNPs polymorphism study was carried out in the Polish HF bulls (n=151) by PCR-RFLP methods as described in the previous issue of this journal. The phenotypic records (assessment year 2017) of clinical mastitis, functional and fertility trait were collected from the Research Institute of Animal Production, Balice, Poland (http://www.izookrakow.pl).Results: Overall, the trait-associated study revealed no relationship between the CXCL8c.210C>T SNP polymorphism and the breeding value of selected clinical mastitis and fertility trait of breeding bulls. However, TT genotypes showed low levels of significance of differences for the breeding values of somatic cells count (p = 0.27) and stillborn calving of heifers (p=0.27). Similarly, the trait-associated study showed no correlation between the CXCL8c.105A>G polymorphism and the breeding value of selected clinical mastitis and fertility trait of breeding bulls. However, AA genotypes showed low levels of significance of differences for the somatic cell count (p = 0.12), ease calving of heifers (p = 0.14), the difficulty calving of heifers (p = 0.17), live-born calves of heifers (p = 0.21), and AG genotypes showed the low levels of significance of differences for the stillborn calving in heifers (p = 0.21).Conclusions: Study concludes that trait-associated studies of CXCL8 polymorphism did not identify highly significant effects on clinical mastitis and fertility trait in the investigated breeding bulls of Polish HF cattle

Data set for transcriptome analysis of pituitary galnd in cattle breeds

Transcriptome data presented in this article is associated with the research article entitled “Single nucleotide polymorphism discovery in bovine pituitary gland using RNA-seq technology” published in PLOS One [1]. Herein, we provide raw and analysed RNA-seq data of pituitary gland tissues from three cattle breeds, viz., Polish-HF, Polish Red and Hereford cattle breeds. Bioinformatics pipelines of high-quality RNA-seq data includes the FastQC tools for quality controls, Trimmomatic cutadapt tools for trimming RNA-seq data, and BWA version 0.7.5-r404 for mapping and alignment to the Bos taurus reference genome, SAMtools for SNPs identifications in bovine pituitary gland transcriptome. Raw FASTq files for the RNA-seq libraries of bovine pituitary gland were deposited in the NCBI Sequence Read Archive (SRA) and have been assigned BioProject accession PRJNA312148

Data set for transcriptome analysis of liver in cattle breeds

Transcriptome analysis using high-throughput next-generation sequencing (HT-NGS) technology provides the capability to understand global gene expression variations through a wide range of tissue samples in domesticated animals. We provide raw and analysed data for transcriptomic analysis of liver tissues from Polish-HF, Polish Red and Hereford cattle breeds, obtained by RNA-seq. High-quality sequencing data have been analysed using our bioinformatics pipeline which consists of FastQC for quality controls, Trimmomatic for trimming, and BWA version 0.7.5-r404 for alignment to the Bos taurus reference genome, SAMtools for SNPs identifications, and differentially expressed genes (DEGs) identification using DEseq and edgeR pipelines after adjustment for false-discovery rate (FDR) with adjusted two-sided p values <0.01 and the trimmed mean of M values (TMM) normalisation method. The data accompanying the published manuscript describing the SNPs and DEGs identification in the bovine liver transcriptome of cattle breeds. Raw FASTq files for the RNA-seq libraries are deposited in the NCBI Sequence Read Archive (SRA) and have been assigned BioProject accession PRJNA312148. Raw and processed RNA-seq data were deposited and made publicly available on the Gene Expression Omnibus (GEO; GSE114233)

Quality control assessment of the RNA-Seq data generated from liver and pituitary transcriptome of Hereford bulls using StrandNGS software

Background: Quality control (QC) assessment is the most critical step in the high-throughput RNA-seq data analysis to characterize the in-depth understanding of genome and transcriptome assembling to a given reference genome. It provides not only a quick insight into the RNA-seq data quality to allow early identification of good or bad RNA-seq data samples, but also to verify the alignment QC checks for further essential high-throughput bioinformatics analysis such as, identification of novel genetic variants, differentially expressed genes (DEGs), gene network and metabolic pathways.Method: After isolation of total RNA from liver (n=15) and pituitary gland (n=15) tissues of young Hereford bulls, the pooled total RNA (n=30) were fragmented using GeneRead rRNA depletion kit (Qiagen, Hilden, Germany) and cDNA library preparation were preformed using ScriptSeqTM v2 RNA-Seq library preparation kit (Epicentre, illumina, USA), followed by high-throughput sequencing of combined liver and pituitary transcriptome using MiSeq reagent kit v2 (illumina, USA) to obtain high quality of paired-end RNA-seq reads of 251 base-pairs (bps). In this paper, the QC assessment of obtained RNA-seq raw data as well as post-alignment QC of processed RNA-seq data of combined liver and pituitary transcriptome (n=30) of Hereford bulls were performed using the strand NGS software v1.3 (Agilent; http://www.strand-ngs.com/) data analysis package. The reads were aligned with Bowtie using default settings against both Bull and Cow genome assembly.Results: Using two runs of MiSeq platform, a total of over 60 million paired-end RNA-seq reads were successfully obtained and submitted to NCBI SRA resources (https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=312148). Library complexity plot results revealed 72.02% of duplicate reads with a low library complexity value of 0.28. The pre-alignment QC analysis of raw RNA-seq data revealed the sequence read lengths ranged from 35-251 bp size with more than 50% of all reads with length over 200bp and 10% of reads below 100bp.Conclusion: By testing the RNA-seq methodology on Illumina platform, two MiSeq sequencing runs yielded significantly high quality of 30 million sequencing reads per single MiSeq run. Our initial pre-alignment and post-alignment analysis of RNA-seq data analysis revealed that mapping of the Hereford liver and pituitary gland transcriptome to reference Bos taurus genome was successfully performed, however, more than 50% of all reads with length over 200bp were recovered. Therefore, obtained results concludes that liver and pituitary transcriptome sequencing with rRNA depletion method is less effective than mRNA RNA-seq method

RNA-seq based SNP discovery in gluteus medius muscle of Polish Landrace pigs

BackgroundSingle nucleotide polymorphisms (SNPs) are the well-known molecular markers in genetics and breeding studies applied to veterinary sciences and livestock production. Advancement of next generation sequencing (NGS) provides a high-throughput means of potential putative SNP discovery. The aim of the study was to identify the putative genetic variants in gluteus medius muscle transcriptome of Polish Landrace pigs.MethodsRNA-seq based NGS experiment was performed on Polish Landrace pigs fed with omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) and normal diets. Isolation of total RNA from gluteus medius muscle was performed on low PUFAs (n=6) and High PUFAs dietary group of Polish Landrace pigs. The RNA-seq libraries were constructed by mRNA enrichment, mRNA fragmentation, second strand cDNA synthesis, adaptor ligation, size selection and PCR amplification using the illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego CA, USA), followed by NGS sequencing on MiSeq illumina platform. The quality control of raw RNA-seq data was performed using the Trimmomatic and FastQC tools. High QC paired-end RNA-seq data of gluteus medius muscle transcriptome were mapped to the reference genome Sus scrofa v.10.2. Finally, the SNPs discovery was performed using GATK and SAMtools bioinformatics SNPs caller tools.ResultsThe Fastq RNA-seq data generated from two pooled paired-end libraries (151bp) of gluteus medius muscle tissue of Polish Landrace pigs were submitted to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra). Study identified a total of 50.5 million paired-end reads (32.5 million low PUFAs dietary group and 18 million reads high PUFAs dietary group) of gluteus medius muscle transcriptome of Polish Landrace pigs. SNP discovery identified a total of 35436 homozygous and 28644 heterozygous cSNPs in gluteus medius muscle transcriptomes representing both dietary groups of Polish Landrace pig. Moreover, a total of 25187 and 5488 cSNP were identified as synonymous SNPs, and 18005 and 4780 cSNP were identified as nonsynonymous SNPs. Finally, single nucleotide variation (SNV) representing substitutions of all four possibilities (A,T,G,C) were identified ranging 2935 to 3227 SNVs (high PUFAs) and 3528 to 3882 SNVs (low PUFAs) for the heterozygous cSNPs and 2712 to 4058 (high PUFAs) and 4169 to 5692 SNVs (low PUFAs) for the heterozygous SNPs in gluteus medius muscle transcriptomes of Polish Landrace pigs.ConclusionsStudy concluded that identification of cSNPs dataset representing the gluteus medius muscle transcriptome of Polish Landrace pigs fed with a control diet (low) and pigs fed with a PUFAs diet (high) may be helpful to develop a new set of genetic markers specific to Polish Landrace pig breed. Such cSNP markers eventually can be utilized in genome-wide association studies (GWAS) and to finally implement on marker assisted selection (MAS) and genomics selection (GS) program in active breeding population of Polish Landrace pigs in Poland

RNA-seq based SNP discovery in liver transcriptome of Polish Landrace pigs

Background: RNA-seq technology is most commonly used in quantitative measurement of gene expression levels and identification of non-annotated transcripts. It is also used for the coding SNPs (cSNPs) discoveries in an efficient and cost-effective way. The aim of this study was to identify the putative genetic cSNPs variants in liver transcriptome of Polish Landrace pigs fed with high and low (normal) omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) diets.Methods: RNA-seq based NGS experiment was performed on Polish Landrace pigs fed with high and low PUFAs diets. Total RNA were isolated from liver tissues of low PUFAs (n=6) and high PUFAs dietary group (n=6) of Polish Landrace pigs. The RNA-seq libraries preparations were performed by mRNA enrichment, mRNA fragmentation, second strand cDNA synthesis, adaptor ligation, size selection and PCR amplification using the illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego CA, USA), followed by NGS sequencing on MiSeq illumina platform. The quality control (QC) of raw RNA-seq data of liver transcriptome was performed using the Trimmomatic and FastQC tools. The paired-end mapping of the liver transcriptome RNA-seq data (n=12) was performed on the reference genome Sus scrofa v.10.2, followed by cSNPs discovery using GATK and SAMtools bioinformatics SNPs caller tools.Results: Two pooled paired-end libraries of 151bp liver transcriptome of Polish Landrace pigs were generated from MiSeq instrument and subsequent Fastq RNA-seq data were submitted to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra). Our study identified 25.3 million paired-end reads: representing 13,509,248 paired-end reads of high PUFAs dietary group and 11,815,696 paired-end reads of low PUFAs dietary group of Polish Landrace pigs liver transcriptome. The SNP discovery results revealed identification of 25909 homozygous and 23290 heterozygous cSNPs in the liver transcriptome of both dietary groups of Polish Landrace pigs. With regards to same or alternative SNPs alleles encoding amino acids regions, a total of 27141 synonymous cSNP and 5989 non-synonymous cSNPs were identified in liver transcriptome representing high PUFAs dietary group. However, a total of 15128 synonymous cSNPs and 3900 non-synonymous cSNPs were identified in liver transcriptome representing low PUFAs dietary groups of Polish Landrace pigs. The identification of single nucleotide variations (SNVs) representing substitutions of all four possibilities (A,T,G,C) were ranged 2872 to 6868 SNVs (high PUFAs) and 2574 to 3654 SNVs (low PUFAs) in the homozygous cSNPs and 2452 to 2678 SNVs (high PUFAs) and 2094 to 2230 SNVs (low PUFAs) in the heterozygous cSNPs of liver transcriptomes of Polish Landrace pigs, respectively.Conclusions: Study concluded that identification of cSNPs dataset representing the liver transcriptome of Polish Landrace pigs fed with a control diet (low) and pigs fed with a PUFAs diet (high) may be helpful to develop a new set of genetic markers for trait-associated studies (viz., growth and metabolic traits) specific to Polish Landrace pig breed. Such cSNP markers eventually can be utilized in the genetic improvement of the pig production traits using the genome-wide association studies (GWAS) and to finally implement on marker assisted selection (MAS) and genomics selection (GS) program in active breeding population of Polish Landrace pigs in Poland

Comparative Analysis of the Liver Transcriptome among Cattle Breeds Using RNA-seq

Global gene expression in liver transcriptome varies among cattle breeds. The present investigation was aimed to identify the differentially expressed genes (DEGs), metabolic gene networks and metabolic pathways in bovine liver transcriptome of young bulls. In this study, we comparatively analyzed the bovine liver transcriptome of dairy (Polish Holstein Friesian (HF); n = 6), beef (Hereford; n = 6), and dual purpose (Polish-Red; n = 6) cattle breeds. This study identified 895, 338, and 571 significant (p < 0.01) differentially expressed (DE) gene-transcripts represented as 745, 265, and 498 hepatic DE genes through the Polish-Red versus Hereford, Polish-HF versus Hereford, and Polish-HF versus Polish-Red breeds comparisons, respectively. By combining all breeds comparisons, 75 hepatic DE genes (p < 0.01) were identified as commonly shared among all the three breed comparisons; 70, 160, and 38 hepatic DE genes were commonly shared between the following comparisons: (i) Polish-Red versus Hereford and Polish-HF versus Hereford; (ii) Polish-Red versus Hereford and Polish-HF versus Polish-Red; and (iii) Polish-HF versus Hereford and Polish-HF versus Polish-Red, respectively. A total of 440, 82, and 225 hepatic DE genes were uniquely observed for the Polish-Red versus Hereford, Polish-HF versus Hereford, and Polish-Red versus Polish-HF comparisons, respectively. Gene ontology (GO) analysis identified top-ranked enriched GO terms (p < 0.01) including 17, 16, and 31 functional groups and 151, 61, and 140 gene functions that were DE in all three breed liver transcriptome comparisons. Gene network analysis identified several potential metabolic pathways involved in glutamine family amino-acid, triglyceride synthesis, gluconeogenesis, p38MAPK cascade regulation, cholesterol biosynthesis (Polish-Red versus Hereford); IGF-receptor signaling, catecholamine transport, lipoprotein lipase, tyrosine kinase binding receptor (Polish-HF versus Hereford), and PGF-receptor binding, (Polish-HF versus Polish-Red). Validation results showed that the relative expression values were consistent to those obtained by RNA-seq, and significantly correlated between the quantitative reverse transcription PCR (RT-qPCR) and RNA-seq (Pearson’s r  > 0.90). Our results provide new insights on bovine liver gene expressions among dairy versus dual versus beef breeds by identifying the large numbers of DEGs markers submitted to NCBI gene expression omnibus (GEO) accession number GSE114233, which can serve as useful genetic tools to develop the gene assays for trait-associated studies as well as, to effectively implement in genomics selection (GS) cattle breeding programs in Poland

Identification of Differentially Expressed Gene Transcripts in Porcine Endometrium during Early Stages of Pregnancy

During the early stages of pregnancy, the uterine endometrium undergoes dramatic morphologic and functional changes accompanied with dynamic variation in gene expression. Pregnancy-stage specific differentially expressed gene (DEG)-transcript-probes were investigated and identified by comparing endometrium transcriptome at 9th day (9D), 12th day (12D) and 16th day (16D) of early pregnancy in Polish large-white (PLW) gilts. Endometrium comparisons between 9D-vs-12D, 9D-vs-16D and 12D-vs-16D of early pregnancy identified 6049, 374 and 6034 highly significant DEG-transcript-probes (p 2 FC). GO term enrichment analysis identified commonly shared upregulated endometrial DEG-transcript-probes (p 2 FC), that were regulating the gene functions of anatomic structure development and transport (TG), DNA-binding and methyltransferase activity (ZBTB2), ion-binding and kinase activity (CKM), cell proliferation and apoptosis activity (IL1B). Downregulated DEG-transcript-probes (p 2 FC) were involved in regulating the gene functions of phosphatase activity (PTPN11), TC616413 gene-transcript and Susscrofa LOC100525539. Moreover, blastn comparison of microarray-probes sequences against susscrofa11 assembly identified commonly shared upregulated endometrial DEG-transcript-probes (E 2 FC), that were regulating the gene functions of reproduction and growth (SELENOP), cytoskeleton organization and kinase activity (CDC42BPA), phosphatase activity (MINPP1), enzymebinding and cell-population proliferation (VAV3), cancer-susceptibility candidate gene (CASC4), cytoskeletal protein-binding (COBLL1), ion-binding, enzyme regulator activity (ACAP2) Downregulated endometrial DEG-transcript-probes (E 2FC) were involved in regulating the gene functions of signal-transduction (TMEM33), catabolic and metabolic processes (KLHL15). Microarray validation experiment on selected candidate genes showed complementarity to significant endometrial DEG-transcript-probes responsible for the regulation of immune response (IL1B, S100A11), lipid metabolism (FABP3, PPARG), cell-adhesion (ITGAV), angiogenesis (IL1B), intercellular transmission (NMB), cell-adhesion (OPN) and response to stimuli (RBP4) was confirmed by RT-PCR. This study provides a clue that identified pregnancy-stage specific microarray transcript probes could be considered as candidate genes for recognition and establishment of early pregnancy in the pig.</p