Mariusz Pierzchała

Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid.

Expression patterns of candidate genes with important functions in animal metabolism can help to identify potential molecular markers for cattle production traits. Reverse transcription followed by polymerase chain reaction is a method for rapid and accurate mRNA quantification. However, for exact comparison of mRNA quantity in various samples or tissues, it is important to choose appropriate reference genes. In cattle, little information is available on the expression stability of housekeeping genes (HKGs). The aim of the present study is to develop a set of reference genes that can be used for normalization of concentrations of mRNAs of genes expressed in the bovine liver, kidney, pituitary and thyroid. The study was performed on 6-, 9-, and 12-month-old bulls of dairy and meat cattle breeds. Six HKGs were investigated:ACTB, GAPDH, HPRTI, SDHA, TBP, andYWHAZ. The most stably expressed potential reference HKGs differed among tissues/organs examined:ACTB, TBP, YWHAZ, GAPDH, HPR TI, andSDHA in the liver;GAPDH andYWHAZ in the kidney;GAPDH andSDHA in the pituitary; andTBP andHPRTI in the thyroid. The results showed that the use of a single gene fornormalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to representative samples from specific experimental conditions.

Polymorphisms in coding and regulatory regions of the porcineMYF6 andMYOG genes and expression of theMYF6 gene inm. longissimus dorsi versus productive traits in pigs

MYOG andMYF6 belong to theMyoD gene family. They code for the bHLH transcription factors playing a key role in later stages of myogenesis: differentiation and maturation of myotubes. Three SNPs in porcineMYF6 and two in porcineMYOG were analysed in order to establish associations with chosen carcass quality and growth rate traits in Polish Landrace, Polish Large White and line 990 sows. No statistically significant effect of SNP in the promoter region of theMYF6 gene on its expression measured on mRNA level was found. Associations between the genotype at theMYF6 locus and carcass quality traits appeared to be breed-dependent. The C allele in the case of SNP in the promoter region and GC haplotype in exon 1 were advantageous for right carcass side weight in Polish Landrace sows and disadvantageous for this trait in Polish Large White sows. These gene variants were also the most advantageous for loin and ham weight in sows of line 990. The mutation in exon 1 of theMYOG gene had no statistically significant association with carcass quality traits and the mutation in the 3’-flanking region had the breed-dependent effect as well. These results suggest that SNPs analysed in this study are not causative mutations, but can be considered as markers of some other, still unrevealed genetic polymorphism that influences the physiological processes and phenotypic traits considered in this study.

Identification of proteomic biomarkers in M. Longissimus dorsi as potential predictors of pork quality

Meat quality traits have low heritability and large environmental influences. To predict, improve and manage meat quality, proteomic biomarkers are superior to genetic markers. The objectives of this research were (1) to find associations between proteome profiles of longissimus muscle at slaughter and meat quality accuracies of prediction of traits ranged from 20 up to 80%. Differentially expressed proteins related to drip loss and ultimate pH were identified by NanoLC-FTMSMS. The proteins highlight biological mechanisms that may explain how these traits develop biologically and how they are related to each other.

Sex-related differences of morphometric, densitometric, and geometric parameters of tibia and tarsometatarsal bone in 14-month-old ostriches (Struthio camelus)

The aim of the current study was to investigate basic morphometric, geometric, and densitometric parameters of tibia and tarsometatarsus in 14-mo-old male and female ostriches, and interrelationships between these parameters. The study was conducted on 20 tibiae and 20 tarsometatarsal bones of the left pelvic limb derived from 20 healthy 14-mo-old ostriches (Struthio camelus): 10 males and 10 females. The following parameters were determined using peripheral quantitative computed tomography: bone mineral content (BMC), volumetric bone mineral density (vBMD), cortical content (CRT_CNT), cortical density (CRT_DEN), trabecular content, trabecular density (TRAB_DEN), bone area (TOT_A), trabecular area (TRAB_A), cortical area (CRT_A), cortical thickness (CRT_THK_C), periosteal circumference (PERI_C), endocortical circumference (ENDO_C), and strength-strain index (SSI) in the metaphysis and diaphysis of the bones. Statistical evaluation of the obtained results was performed using Students t-test, and the Pearson correlation coefficient between the investigated parameters was determined. The obtained results have shown significant differences in proximal metaphysis between males and females when evaluating such parameters as CRT_DEN, TOT_A, TRAB_A, PERI_C, SSI in tibia (P < 0.05), and BMC, vBMD, TRAB_DEN, CRT_CNT, TOT_A, TRAB_A, cortical area, PERI_C, ENDO_C, SSI in tarsometatarsus (P < 0.05). Significant differences between the 2 sexes were found in all the investigated parameters of diaphysis of tibia, except for CRT_DEN and ENDO_C (P < 0.05). Significant positive correlations between BW and bone weight were found (P < 0.05). Furthermore, numerous correlations of morphometric, geometric, and densitometric parameters of metaphysis and diaphysis of the tibia and tarsometatarsus were stated (P < 0.05). In conclusion, the present investigation demonstrated sex-related differences in morphometric, densitometric, and geometric properties of tibia and tarsometatarsus in 14-mo-old ostriches. Numerous correleations observed between the investigated parameters have shown that ostrich tibia and tarsometatarsus may present a valuable model for further studies on bone tissue metabolism regulation in breeding birds.

Effect of a diet enriched with omega-6 and omega-3 fatty acids on the pig liver transcriptome.

The optimal ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) is important for keeping the homeostasis of biological processes and metabolism, yet the underlying biological mechanism is poorly understood. The objective of this study was to identify changes in the pig liver transcriptome induced by a diet enriched with omega-6 and omega-3 fatty acids and to characterize the biological mechanisms related to PUFA metabolism.Polish Landrace pigs (n = 12) were fed diet enriched with linoleic acid (LA, omega-6) and α-linolenic acid (ALA, omega-3) or standard diet as a control. The fatty acid profiling was assayed in order to verify how feeding influenced the fatty acid content in the liver, and subsequently next-generation sequencing (NGS) was used to identify differentially expressed genes (DEG) between transcriptomes between dietary groups. The biological mechanisms and pathway interaction networks were identified using DAVID and Cytoscape tools. Fatty acid profile analysis indicated a higher contribution of PUFAs in the liver for LA- and ALA-enriched diet group, particularly for the omega-3 fatty acid family, but not omega-6. Next-generation sequencing identified 3565 DEG, 1484 of which were induced and 2081 were suppressed by PUFA supplementation. A low ratio of omega-6/omega-3 fatty acids resulted in the modulation of fatty acid metabolism pathways and over-representation of genes involved in energy metabolism, signal transduction, and immune response pathways.In conclusion, a diet enriched with omega-6 and omega-3 fatty acids altered the transcriptomic profile of the pig liver and would influence animal health status.

A porcine gluteus medius muscle genome-wide transcriptome analysis: dietary effects of omega-6 and omega-3 fatty acids on biological mechanisms

BackgroundThe level of omega-6 and omega-3 polyunsaturated fatty acids can affect many cellular systems and function via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate changes in the muscle transcriptome and the biological functions regulated by increased consumption of omega-3 and omega-6 fatty acids in the pig gluteus medius muscle.ResultsThe transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database pointing differentially expressed genes (DEG). Comparative expression analyses identified 749 genes significantly differing at least in the twofold of change between two groups of animals fed with divergent level of omega-3 and omega-6 fatty acids. The expression of 219 genes was upregulated, and the expression of 530 genes was downregulated in the group of pigs supplemented with omega-3 and omega-6 fatty acids in relation to control group pigs. Results of RNA-seq indicated a role of fatty acid in the regulation of the expression of genes which are essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including inflammatory response, signaling, lipid metabolism, and homeostasis.ConclusionsSummarizing, obtained results provide strong evidence that omega-6 and omega-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.

Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology

Background RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. Results The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. Conclusions This study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments.

Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology.

Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs/CGs with RNA-seq data identified KCNIP4, CCSER1, DPP6, MAP3K5 and GHR CGs with highest SNPs hit loci in all three breeds and developmental ages. However, CAST CG with more than 100 SNPs hits were observed only in Polish HF and Hereford breeds.These findings are important for identification and construction of novel tissue specific SNP-db and breed specific SNP-db dataset by screening of putative SNPs according to QTL db and candidate genes for bovine growth and reproduction traits, one can develop genomic selection strategies for growth and reproductive traits.

Proteomics biomarkers for pork quality

Proteome expression profiles can be used as biomarkers to predict traits with low heritability and large environmental influences. Porcine meat quality typically is such a trait. Proteome profiles of 150 meat samples were determined and associated with measured technical meat quality traits. Biomarkers tests were developed and their predictive potential determined. Differentially expressed proteins related to variation in drip loss and ultimate pH were determined and the underlying biological mechanism evaluated with bioinformatics. The biological mechanism also explains the connections between these two meat quality traits.

The relationship between the polymorphism of the porcine CAST gene and productive traits in pigs

Urbanski, P., Pierzchala, M., Terman, A., Kamyczek, M., Rózycki, M., Roszczyk, A. and Czarnik, U. 2015. The relationship between the polymorphism of the porcine CAST gene and productive traits in pigs. Can. J. Anim. Sci. 95: 361-367. The aim of the study was to characterize the polymorphism of the calpastatin gene identified with ApaLI, Hpy188I and PvuII restriction enzymes in two pig breeds and one line bred in Poland, and to evaluate the relationship between the CAST genotype and carcass traits. The analysis covered a total of 617 pigs of two breeds, Polish Landrace (185) and Polish Large White (216), and synthetic line L990 (216). All animals studied appeared to be monomorphic at two loci: CAST/ApaLI and CAST/Hpy188I, while three genotypes were observed at CAST/PvuII locus. Statistical analysis was carried out for each breed separately using the least square methods of the GLM procedure. The model included the effect of the CAST genotype, fixed effect of the RYR1 genotype and the effect of the sire. Because the RYR1 genotype could significantly modify the effect of other genes, the effect of the RYR1 genotype was included in the statistical model. The relationship between the polymorphism and several productive traits was identified in each of the study groups of pigs. Animals carrying the heterozygous genotype at this locus showed most extreme values for some of the traits tested. Our results suggest that the CAST /PvuII genotype might be utilized in the selection of valuable pig carcass traits, particularly weight and size of the loin.