Abstract
Bovine mastitis is a common inflammatory disease caused by multiple factors in early lactation or dry period. Genome wide association studies (GWAS) can provide a convenient and effective strategy for understanding the biological basis of mastitis and better prevention. 2b-RADseq is a high-throughput sequencing technique that offers a powerful method for genome-wide genetic marker development and genotyping. In this study, single nucleotide polymorphisms (SNPs) of the immune-regulated gene correlative with mastitis were screened and identified by two stage association analysis via GWAS-2b-RADseq in Chinese Holstein cows. We have screened 10,058 high quality SNPs from 7,957,920 tags and calculated their allele frequencies. Twenty-seven significant SNPs were co-labeled in two GWAS analysis models [Bayesian (P < 0.001) and Logistic regression (P < 0.01)], and only three SNPs (rs75762330, C > T, PIC = 0.2999; rs88640083, A > G, PIC = 0.1676; rs20438858, G > A, PIC = 0.3366) were annotated to immune-regulated genes (PTK2B, SYK, and TNFRSF21). Identified three SNPs are located in non-coding regions with low or moderate genetic polymorphisms. However, independent sample population validation (Case-control study) data showed that three important SNPs (rs75762330, P < 0.025, OR > 1; rs88640083, P < 0.005, OR > 1; rs20438858, P < 0.001, OR < 1) were significantly associated with clinical mastitis trait. Importantly, PTK2B and SYK expression was down-regulated in both peripheral blood leukocytes (PBLs) of clinical mastitis cows and in vitro LPS (E. coli)–stimulated bovine mammary epithelial cells, while TNFRSF21 was up-regulated. Under the same conditions, expression of Toll-like receptor 4 (TLR4), AKT1, and pro-inflammatory factors (IL-1β and IL-8) were also up-regulated. Interestingly, network analysis indicated that PTK2B and SYK are co-expressed in innate immune signaling pathway of Chinese Holstein. Taken together, these results provided strong evidence for the study of SNPs in bovine mastitis, and revealed the role of SYK, PTK2B, and TNFRSF21 in bovine mastitis susceptibility/tolerance.
Highlights
Bovine mastitis is the most complex and costly inflammatory disease with high incidence, which seriously affects developing dairy industry worldwide [1,2,3]
The average distance between adjacent tags was 9,589 bp (Figure 1C), and the unique tag alignment ratio for all samples was 59.69%−72.71%. 10,058 high quality single nucleotide polymorphism (SNP) from 7,957,920 unique tags were selected for RAD typing, and the distribution of SNPs on chromosomes was analyzed by sliding window (Figure S2)
We used two-stage correlation analysis to find 27 significant SNPs associated with risk of mastitis, among which three SNPs were annotated to immune-regulated genes (PTK2B, SYK, and TNFRSF21)
Summary
Bovine mastitis is the most complex and costly inflammatory disease with high incidence, which seriously affects developing dairy industry worldwide [1,2,3]. Previous studies have shown that cow mastitis has complex traits (clinical and subclinical mastitis) and is affected by multiple factors, including genetic features, nutrition, and hygiene standard, pathogen infections, and seasonal changes. Mastitis-related immune response is a complex biological process involving immune cells, mammary epithelial cells, and endothelial cells [8]. It is well-known that mastitis causes a dramatic increase in bovine milk somatic cell counts (SCC), mainly neutrophils [8, 15, 16]. Screening and identifying susceptible or resistant genes associated with SCC or SCS will provide novel strategies to reduce the incidence of mastitis and improve the quality of dairy cows populations [17,18,19]
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