Paweł Brym

Single nucleotide polymorphism in the promoter region of the lactoferrin gene and its associations with milk performance traits in Polish Holstein-Friesian cows

Bovine lactoferrin (LTF) is a multifunctional small glycoprotein found in milk acting mainly as a defense factor in the mammary gland. Many polymorphisms have been found in the bovine LTF gene but almost none were considered as genetic markers of production traits in dairy cattle. In this study, the promoter fragment of LTF gene containing mutation (G/C) in position +32 has been amplified by PCR followed by genotyping by the SSCP and RFLP method. Three hundred fifty-eight Polish Holstein-Friesian cows were screened, giving the following frequency of genotypes: 0.628, 0.313 and 0.059 for GG, GC and CC, respectively. GLM (General Linear Model) analysis was applied to evaluate the associations of lactoferrin with milk performance traits, including SCC (somatic cell count). It was found that CC cows show significantly higher (P ≤ 0.01) protein content in milk in comparison with GG cows. The values of other milk performance traits were also higher but at nonsignificant levels. SCC in milk was the lowest in CC cows, but also at a nonsignificant level.

Associations Between Milk Performance Traits in Holstein Cows and 16 Candidate SNPs Identified by Arrayed Primer Extension (APEX) Microarray

An oligonucleotide microarray—which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis—was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples. The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.

Evaluation of reference genes for qRT-PCR gene expression studies in whole blood samples from healthy and leukemia-virus infected cattle.

Real-time quantitative reverse transcription PCR (qRT-PCR) is the method of choice to investigate the alterations in gene expression involved with BLV pathogenesis. However, the reliability of qRT-PCR data critically depends on proper normalization to a set of stably expressed reference genes. The aim of the study was to validate the expression stability of ten candidate reference genes in RNA isolated from whole blood cells of BLV-negative and BLV-positive cows with hematological abnormalities. The rankings of the candidate genes according to their expression stability were calculated using BestKeeper, NormFinder and qBase(PLUS) software with implemented geNorm(PLUS) algorithm. The results showed that two genes are sufficient for normalization of qRT-PCR studies in whole blood RNA isolated from cows infected with BLV. According to geNorm, UCHL5 and RPLP0 were the best choice, but taking into account possible intergroup variation, NormFinder recommended RPLP0 and B2M as a most suitable pair. The overall ranking based on the geometric mean of the ranking numbers from each method separately showed UCHL5, RPLP0 and TBP as the most stable candidate reference genes. In addition, all three methods unanimously pointed at the commonly used ACTB and GAPDH as the least stable genes. These results further emphasize the need to accurately validate candidate reference genes before use in gene expression qRT-PCR studies.

SNiPORK – A Microarray of SNPs in Candidate Genes Potentially Associated with Pork Yield and Quality – Development and Validation in Commercial Breeds

SNiPORK is an oligonucleotide microarray based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for pork yield and quality traits. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5′ end to a glass slide and terminating one nucleotide before the polymorphic site. Extension with one fluorescently labeled dideoxynucleotide complementary to the template reveals the polymorphism. Ninety SNPs were selected from those associated directly or potentially with pork traits. Of the 90 SNPs, 5 did not produce a positive signal. For 85 SNPs, 100% repeatiblity was proved by double genotyping of 13 randomly chosen boars. In addition, the accuracy of genotyping was verified in 2 sib-families by a Mendelian inheritance of 49–50 homozygous genotypes from sire to sons. Three genotype discrepancies were found (97% accuracy rate). All inaccurities were confirmed by an alternative method (sequencing and PCR-RFLP assays). Moreover, the exclusion power of the chip was evalueted by an SNP inheritance analysis of unrelated boars within each sib-family. In the validation step, 88 boars (13 Pietrain, 31 Landrace, 16 Large White, 8 Duroc, 7 Hampshire x Pietrain crosses, and 13 other hybrid lines) were screened to validate SNPs. Among the 85 selected SNPs, 12 were found to be monoallelic, the rest showing at least two genotypes for the entire population under study. The primary application of the SNiPORK chip is the simultaneous genotyping of dozens of SNPs to study gene interaction and consequently better understand the genetic background of pork yield and quality. The chip may prospectively be used for evolutionary studies, evaluation of genetic distances between wild and domestic pig breeds, traceability tests, as well as the starting point for developing a platform for identification and paternity analysis.

Genome-wide association study for host response to bovine leukemia virus in Holstein cows.

The mechanisms of leukemogenesis induced by bovine leukemia virus (BLV) and the processes underlying the phenomenon of differential host response to BLV infection still remain poorly understood. The aim of the study was to screen the entire cattle genome to identify markers and candidate genes that might be involved in host response to bovine leukemia virus infection. A genome-wide association study was performed using Holstein cows naturally infected by BLV. A data set included 43 cows (BLV positive) and 30 cows (BLV negative) genotyped for 54,609 SNP markers (Illumina Bovine SNP50 BeadChip). The BLV status of cows was determined by serum ELISA, nested-PCR and hematological counts. Linear Regression Analysis with a False Discovery Rate and kinship matrix (computed on the autosomal SNPs) was calculated to find out which SNP markers significantly differentiate BLV-positive and BLV-negative cows. Nine markers reached genome-wide significance. The most significant SNPs were located on chromosomes 23 (rs41583098), 3 (rs109405425, rs110785500) and 8 (rs43564499) in close vicinity of a patatin-like phospholipase domain containing 1 (PNPLA1); adaptor-related protein complex 4, beta 1 subunit (AP4B1); tripartite motif-containing 45 (TRIM45) and cell division cycle associated 2 (CDCA2) genes, respectively. Furthermore, a list of 41 candidate genes was composed based on their proximity to significant markers (within a distance of ca. 1 Mb) and functional involvement in processes potentially underlying BLV-induced pathogenesis. In conclusion, it was demonstrated that host response to BLV infection involves nine sub-regions of the cattle genome (represented by 9 SNP markers), containing many genes which, based on the literature, could be involved to enzootic bovine leukemia progression. New group of promising candidate genes associated with the host response to BLV infection were identified and could therefore be a target for future studies. The functions of candidate genes surrounding significant SNP markers imply that there is no single regulatory process that is solely targeted by BLV infection, but rather the network of interrelated pathways is deregulated, leading to the disruption of the control of B-cell proliferation and programmed cell death.

Polymorphism and expression of the tumour necrosis factor-alpha (TNF-alpha) gene in non-infected cows and in cows naturally infected with the bovine leukaemia virus (BLV).

The objective of this study was to determine the influence of TNF-alpha gene polymorphism at position -824 A > G on TNF-alpha gene expression in BLV-positive animals with aleukaemic (AL) and persistent lymphocytosis (PL) forms of enzootic bovine leukosis (EBL) and in BLV-negative cows. Polymorphism of the TNF- alpha gene at position -824 A > G had a complex influence on TNF-alpha gene expression in cows infected with BLV. In animals with various TNF-alpha genotypes, mRNA levels were stable and similar, but significant differ - ences were noted in the percentages of PBMCs expressing membrane TNF-alpha (mTNF-alpha) protein (PBMCs that were positive for mTNF-alpha). In healthy cows, significant differences in TNF-alpha gene expression were not noted at any of the analysed levels. BLV infection and EBL progression resulted in differential TNF-alpha gene expression at both mRNA and protein levels, but the differences in the amount of the transcript and the percentages of mTNF-alpha+ cells exhibited a reverse trend. The lowest mRNA levels and the highest percentage of PBMCs expressing mTNF-alpha protein were determined in BLV-positive cows. These complex results regard- ing TNF-alpha gene expression in BLV-positive cows could be attributed to the presence of virus or viral protein/ proteins modifying the expression of the TNF-alpha gene.

Polymorphism and expression of the tumor necrosis factor receptor II gene in cows infected with the bovine leukemia virus.

A single T>C nucleotide polymorphism (rs42686850) of bovine tumor necrosis factor receptor type II gene (TNF-RII) is located within a sequence with allele-specific affinity to bind E2F transcription factors, considered pivotal in the regulation of cell cycle and cell proliferation. The objective of the study was to determine the effect of this SNP and BLV infection on the TNF-RII gene expression at the mRNA and protein levels in peripheral blood mononuclear cells (PBMC). We noted that analyzed TNF-RII gene polymorphism influenced the expression of the TNF-RII gene at the mRNA level but only in BLV-positive cows. Concurrently, no statistically significant association was found between gene polymorphism and TNF-RII expression at the protein level. However, we found a significant effect of BLV infection status on the amount of TNF-RII mRNA and the percentage of PBMC expressing TNF-RII. These results show an unclear effect of considered T>C polymorphism on TNF-RII gene expression in bovine leukocytes and they suggest the involvement of BLV in modifying the TNF-RII expression in BLV-infected cows potentially implying the EBL (Enzootic Bovine Leukosis) associated pathogenesis.

Associations between 60 SNPs identified by APEX microarray and growth rate, meatiness and selection index in boars

A total of 312 boars (201 Landrace and 111 Large White) were genotyped with a custom-made low throughput genotyping microarray (called SNiPORK) based on array primer extension (APEX) technology. The results were used to association studies between genotyped SNP markers and daily gains, meat content and selection index. Among the 60 SNP markers analyzed, 14 of them showed statistically significant associations between the genotype and the level of at least one trait. In order to find extremely beneficial or unfavorable intergenic diplotype combinations, 5 SNP markers were selected: CAST A499C, MYF6 T255C, PKLR C384T, SFRS1 C1146T and TNNT3 T153C, which showed statistically significant associations at P ≤ 0.01 within one of the traits and the frequency of homozygotes with a minor allele of at least 0.1. Among 10 possible permutations, statistically significant associations were found only for a combination of SNP markers in TNNT3 × SFRS1 genes and, interestingly, for all combinations with SNP located within the calpastatin (CAST) gene commonly known as a gene influencing pork quality traits. This study also found that CAST allele A (which is beneficial for pork tenderness) is also favorable for growth rate. This effect is clearly increased with additive alleles C of myogenic factor MYF6 and troponin T3 (TNNT3) and is decreased with each allele T from serine rich splicing factor 1 (SFRS1) gene. For meat content, the most favorable genotype of calpastatin gene was AC, the effect of which was generally increased with each C allele of MYF6 and TNNT3 and decreased with each allele T from SFRS1 and PKLR (puruvate kinase) genes, respectively. The calpastatin AC genotype seemed to be beneficial for selection index, although in combination with the CC genotype of troponin T3, calpastatin genotypes AA and CC reached higher values. In the case of the combination of genotypes TNNT3 × SFRS1, the most preferable for all analyzed traits is the CC genotype of TNNT3, especially in combination with CT or CC genotypes in the SFRS1 gene. We conclude that searching for interaction effects between candidate SNPs (even of moderate influence) may lead to interesting and valuable findings enabled better understanding and applications of SNPs in pork yield and quality improvement programmes.