Kamil Oleński

Genome-wide association study for poor sperm motility in Holstein-Friesian bulls

The aim of the study was to screen the whole bull genome to identify markers and candidate genes underlying poor sperm motility. The analyzed data set originates from the Polish Holstein-Friesian bull population and consists of 41 Case and 279 Control bulls (selected from 1581 bulls). The most distinguishing trait of case group was very poor sperm motility (average 25.61%) when compared to control samples (average 72.95%). Each bull was genotyped using the Illumina BovineSNP50 BeadChip. Genome-wide association analysis was performed with the use of GoldenHelix SVS7 software. An additive model with a Cohran-Armitage test, Correlation/Trend adjusted by Bonferroni test were used to estimate the effect of Single Nucleotide Polymorphism (SNP) marker for poor sperm motility. Markers (n=34) reached genome-wide significance. The most significant SNP were located on chromosome 24 (rs110876480), 5 (rs110827324 and rs29011704), and 1 (rs110596818), in the close vicinity of melanocortin 4 receptor (MC4R), PDZ domain containing ring finger 4 (PDZRN4) and ethanolamine kinase 1 (ETNK1), olfactory receptor 5K3-like (LOC785875) genes, respectively. For five other candidate genes located close to significant markers (in distance of ca. 1 Mb), namely alkaline phosphatase, liver/bone/kidney (ALPL), tripartite motif containing 36 (TRIM36), 3-hydroxyisobutyrate dehygrogenase (HIBADH), kelch-like 1 (KLHL1), protein kinase C, beta (PRKCB), their potential role in sperm motility was confirmed in the earlier studies. Five additional candidate genes, cystic fibrosis transmembrane conductance regulator (CFTR), insulin-like growth factor 1 receptor (IGF1R), steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2), cation channel, sperm associated 1 (CATSPER1) calpain 1 (mu/I) large subunit (CAPN1) were suggested to be significantly associated with sperm motility or semen biochemistry. Results of the present study indicate there is a genetic complexity of poor sperm motility but also indicate there might be a causal polymorphism useful in marker-assisted selection. Identifying genomic regions associated with poor sperm motility may be very important for early recognition of a young sire as unsuitable for effective semen production in artificial insemination centers.

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.

Genome-wide association study for semen volume and total number of sperm in Holstein-Friesian bulls

In artificial insemination industry bulls producing high volume of semen with relatively high concentration of sperm are very desirable since they ensure stable production of commercial straws especially in case of top bulls. The aim of the study was to screen the entire bull genome to identify markers and candidate genes underlying semen volume (SV) and total number of sperm (TNS) in ejaculate produced by Holstein-Friesian bulls. Data on semen production were retrieved from records of AI center and included a population of 877 Holstein-Friesian bulls. Each bull was genotyped using the Illumina BovineSNP50 BeadChip. Genome-wide association analysis was performed with the use of GoldenHelix SVS7 software. An additive model for Linear Regression Analysis was used to estimate the effect of SNP marker for SV and TNS. After Bonferroni correction, 3 markers located on chromosome 22 reached the highest significance (rs41625599, rs41584616, rs42012507) for both traits. In the vicinity of these significant markers 3 genes are located (DCP1A, SFMBT1, TMEM110). Moreover, marker rs110109069 located on chromosome 25 was significantly associated with TNS and marker rs42438348 located on chromosome 10 has been found to be associated with SV. Some additional candidate genes were suggested to be potentially involved in analyzed traits (GALC, PRKCD, PHF7, TLR9, SPATA7). Identifying SNPs associated with the lower total number of sperm may be very useful for early recognition of a young sire as less suitable for effective semen production in artificial insemination centers.

Milk from cows of different β-casein genotypes as a source of β-casomorphin-7.

The aim of this study was to quantify β-casomorphin-7 in raw, hydrolyzed and processed milk in different stages of the cow lactation. The obtained results lead to the following conclusion: the highest amount of β-casomorphin-7 released from the hydrolyzed and processed milk is related to the β-casein A1 allele, irrespective of a lactation period. Some traces of β-casomorphin-7 in milk from cows with the β-casein A2 variant are probably a result of the acid hydrolysis of β-casein during its digestion with pepsin. It has been shown that processing of raw milk at high temperatures affects, in a slight degree, the differences between β-casomorphins-7 originating from different β-casein genotypes. The obtained results suggest a possibility to provide a new nutritional factor for milk quality based on the content of β-casomorphin-7 liberated in vivo from milk digested by a mixture of the gastrointestinal enzymes.

On the origin of mongrels: evolutionary history of free-breeding dogs in Eurasia

Although a large part of the global domestic dog population is free-ranging and free-breeding, knowledge of genetic diversity in these free-breeding dogs (FBDs) and their ancestry relations to pure-breed dogs is limited, and the indigenous status of FBDs in Asia is still uncertain. We analyse genome-wide SNP variability of FBDs across Eurasia, and show that they display weak genetic structure and are genetically distinct from pure-breed dogs rather than constituting an admixture of breeds. Our results suggest that modern European breeds originated locally from European FBDs. East Asian and Arctic breeds show closest affinity to East Asian FBDs, and they both represent the earliest branching lineages in the phylogeny of extant Eurasian dogs. Our biogeographic reconstruction of ancestral distributions indicates a gradual westward expansion of East Asian indigenous dogs to the Middle East and Europe through Central and West Asia, providing evidence for a major expansion that shaped the patterns of genetic differentiation in modern dogs. This expansion was probably secondary and could have led to the replacement of earlier resident populations in Western Eurasia. This could explain why earlier studies based on modern DNA suggest East Asia as the region of dog origin, while ancient DNA and archaeological data point to Western Eurasia.

Towards an integrated approach to study SNPs and expression of candidate genes associated with milk protein biosynthesis

MilkProtChip is oligonucleotide microarray allowing bovine genotyping based on single nucleotide polymorphisms (SNPs) in genes influencing milk protein biosynthesis. A total of 71 SNPs in 42 genes were selected as associated with milk protein biosynthesis. Genotyping of about 300 animals of Polish Black-and-White cattle showed that SNPs in acyl-CoA:1,2-diacylglycerol O-transferase (DGAT1), lactoferrin (LTF), casein kappa (CSN3) and growth hormone receptor (GHR) genes were associated with several milk performance traits. Analysis of correlations between SNPs and milk production traits showed that SNPs in single genes rarely affect the investigated traits. Only 4 of 42 investigated single SNPs had impact on milk production traits while 22 combinations of paired SNPs in these genes had impact. Positive effect SNP combinations in two genes can be a result of additive effect on these SNPs on the same traits or effect of genes interaction. The MilkBovExp chip representing 90 genes encoding transcription factors expressed in the bovine mammary gland and/or involved in mammary gland signaling pathways was designed for further investigation of impact of gene expression and/or its encoded products on milk traits performance.

Diversifying selection between pure-breed and free-breeding dogs inferred from genome-wide SNP analysis

Domesticated species are often composed of distinct populations differing in the character and strength of artificial and natural selection pressures, providing a valuable model to study adaptation. In contrast to pure-breed dogs that constitute artificially maintained inbred lines, free-ranging dogs are typically free-breeding, i.e., unrestrained in mate choice. Many traits in free-breeding dogs (FBDs) may be under similar natural and sexual selection conditions to wild canids, while relaxation of sexual selection is expected in pure-breed dogs. We used a Bayesian approach with strict false-positive control criteria to identify FST-outlier SNPs between FBDs and either European or East Asian breeds, based on 167,989 autosomal SNPs. By identifying outlier SNPs located within coding genes, we found four candidate genes under diversifying selection shared by these two comparisons. Three of them are associated with the Hedgehog (HH) signaling pathway regulating vertebrate morphogenesis. A comparison between FBDs and East Asian breeds also revealed diversifying selection on the BBS6 gene, which was earlier shown to cause snout shortening and dental crowding via disrupted HH signaling. Our results suggest that relaxation of natural and sexual selection in pure-breed dogs as opposed to FBDs could have led to mild changes in regulation of the HH signaling pathway. HH inhibits adhesion and the migration of neural crest cells from the neural tube, and minor deficits of these cells during embryonic development have been proposed as the underlying cause of “domestication syndrome.” This suggests that the process of breed formation involved the same genetic and developmental pathways as the process of domestication.

Genome-wide association study for sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls

The aim of the study was to screen the entire bull genome to identify SNP markers and propose candidate genes potentially involved in the variation of sperm membrane integrity in Holstein-Friesian bulls. Two hundred eighty eight bulls kept in one AI center were included in the study. Each bull was genotyped for 54.001 Single Nucleotide Polymorpisms (SNP) by the Illumina BovineSNP50 BeadChip. Commercial straws of frozen-thawed semen were used for the evaluation of sperm plasma membrane integrity (SYBR-14/PI staining) and sperm mitochondrial function (JC1/PI staining). An additive model for Linear Regression Analysis was applied to estimate the effect of SNP marker for sperm membrane integrity (by the use of GoldenHelix SVS7 software). Five significant markers (encompassing 2,2 MB region located on chromosome 6) for SYBR-14/PI were found. Among them one marker-rs41570391 passed Bonferroni correction test. Within approximately 3 Mb genomic region including significant markers three candidate genes: SGMS2 (Sphingomyelin Synthase 2), TET2 (Methylcytosine dioxygenase 2) and GSTCD genes (Gluthatione S-transferase C terminal domain) were proposed as potentially involved in sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls.

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.

Genomic variability in the extinct steppe bison (Bison priscus) compared to the European bison (Bison bonasus)

In 2009, a frozen mummy of the steppe bison (SB) (Bison priscus) was discovered between the lower Kolyma River and the Alazeya River in northeast Siberia, Russia. The specimen was dated with 14C and estimated to have lived more than 48,000 14C years before present (BP). The relationship between SB and the European Bison (EB) (Bison bonasus), also known as wisent or European wood bison, is unresolved and it is unclear whether the SB and EB overlapped in space and time. The aim of our study was to compare genetic variability between the SB specimen and modern EB. We expected higher SB variability due to substantial bottlenecks in the EB approximately one century ago when it became extinct in the wild. The EB (n = 167) and the SB specimen were genotyped with the Illumina BovineHD BeadChip with 777,962 single-nucleotide polymorphism (SNP) markers. Steppe bison DNA was extracted and genotyped six times to account for genotyping errors due to low-quality DNA. We obtained a final set of 7786 SNPs. The mean number of private alleles in EB was 0.027 (± 0.0002) and in SB, it was 0.288 (± 0.0006). This could be explained by factors including differences between species, spatiotemporal divergence, and bottleneck effects. Investigation of historic EB samples could help resolve phylogenetic relationships, the role of the recent bottleneck, and provide information for conservation management to reduce the incidence of disease in the population and maintain its evolutionary potential.