Urszula Czarnik

Deletion/insertion polymorphism of the prion protein gene (PRNP) in Polish Holstein-Friesian cattle

The aim of the present study was to identify the deletion/insertion polymorphism of the bovine prion protein gene (PRNP) within the promoter sequence (23 bp), intron 1 (12 bp) and 3’ untranslated region (14 bp). DNA was isolated from blood of 234 randomly tested Polish Holstein-Friesian cows and from semen of 47 sires used for artificial insemination (AI) in 2004. No statistically significant differences were found in the frequency of genotypes and alleles between cows and breeding bulls in the 3 analysed polymorphic sites within thePRNP gene. Only 3 haplotypes were identified in sires and 4 haplotypes in cows.

Deletion/insertion polymorphism of the prion protein gene (PRNP) in Polish Red cattle, Polish White-backed cattle and European bison (Bison bonasus L., 1758)

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3′ end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle.

Effectiveness of a program aimed at the elimination of BLAD-carrier bulls from Polish Holstein-Friesian cattle

The molecular basis of BLAD is the D128G mutation of the gene coding for the CD 18 subunit of beta—2 integrin. This mutation is lethal, since homozygous (BL/BL) animals die before they reach sexual maturity. In the 1990s, BLAD was the most widespread genetic disease in HF cattle worldwide. The aim of the present study was to determine the frequency of BLAD carriers among 4645 young breeding bulls in Poland in 1995–2006. The frequency of carriers of the mutated allele showed a clear decreasing trend. The highest frequency (7.9%) was recorded while implementing the BLAD control program (1995–1997). Regular monitoring has enabled a great reduction of this threat to the tested population. Today only sporadic cases of BL/TL heterozygotes are reported (ca. 0.8% in 2004–2006).

A novel AAT-deletion mutation in the coding sequence of the BCO2 gene in yellow-fat rabbits

The carcasses of yellow-fat rabbits may be attractive to modern consumers, because they have a relatively high content of biologically active compounds. One of the main candidate genes associated with the yellow-fat trait is β-carotene 9′,10′-oxygenase (BCO2). This study is the first report of the novel AAT-deletion mutation at codon 248 of the BCO2 gene, which has been found in homozygous yellow-fat rabbits. The deletion mutation, located at the beginning of exon 6, results in the absence of asparagine in protein. We also developed a PCR-RFLP test that supports intravital genotyping of indel polymorphism based on genomic DNA.

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.

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.

Polymorphism in the region of bovine prion protein gene and selected haematological indices in cows naturally infected with bovine leukaemia virus

Abstract The objective of this study was to determine whether insertion-deletion (indel) 23 bp polymorphism of the bovine prion protein (PRNP) gene differentiates the total number of leukocytes and lymphocytes and the number of virus-infected lymphocytes. The experimental materials comprised 119 Black-and-White Polish Holstein-Friesian cows. Bovine leukosis was diagnosed by an indirect immunofluorescence based on the detection of viral protein p24 in bovine lymphocytes infected with leukaemia virus (BLV). Indel23 polymorphism was determined by PCR. Blood haematological parameters (total leukocyte counts, total lymphocyte counts, and their percentages) were determined at a specialist haematological laboratory. The examined indices were analysed in three replications, at one-month intervals. It was found that indel23 polymorphism significantly differentiated blood leukocyte counts and the total number and percentage of lymphocytes. Cows with the 23 bp del/del genotype showed significantly higher leukocyte and lymphocyte counts than animals with the remaining two genotypes. Higher values of the analysed haematological parameters noted in homozygotes with 23 bp deletion are similar to the values reported in cows affected by persistent lymphocytosis, thus pointing to an adverse effect of this genotype on the haemopoiesis process. The variations between indel23 genotypes and the number and percentage of BLV-infected lymphocytes are less obvious and more difficult to interpret.