2b-RADSeq Screening,Validation And Regulation Of Molecular Mechanisms For Mastitis Resistance Gene Marker In Dairy Cows

Bovine mastitis,including clinical mastitis and subclinical mastitis,is a productive and inflammatory disease caused by pathogenic bacteria invading and colonizing in mammary gland tissues,with an incidence of more than 20%,causing direct or indirect economic losses in many fields,and has seriously affected and restricted the development of dairy farming and dairy products processing industry.Since the modern dairy cows large-scale breeding,mastitis has been plagued researchers and ranchers.Previous studies have found that the mastitis clinical traits of dairy cows have high heritability,multi-gene micro-effect characteristics,and the candidate genes associated with mastitis traits（resistance or susceptibility）are regionally specific,which undoubtedly brings great difficulties to label and molecular mechanism research of mastitis traits candidate genes in dairy cows.Recently,Genome wide trait analysis（GWAS）has been successfully applied to marker single nucleotide polymorphism sites（SNPs）and candidate genes for bovine mastitis traits,bringing new strategies for the prevention and treatment of mastitis in dairy cows.But so far,the key genes or gene clusters for mastitis resistance or susceptibility in dairy cows（including Chinese Holstein）have not been identified.Therefore,how to screen and determine the mastitis resistance or susceptibility SNPs and genes in Chinese Holstein dairy cows is still a huge challenge.In order to screen SNPs that are significantly associated with mastitis,and mark mastitis resistance or susceptibility candidate genes,and further understand the molecular genetics and biological pathways of mastitis trait in Chinese Holstein dairy cows.In this study,for the first time,two-stage trait association analysis was used to mark and verify important SNPs that were significantly associated with mastitis traits in Chinese Holstein dairy cows,and to verify the function of candidate genes through in vivo and in vitro experiments.At I stage,the whole-genome sequencing of 40 Chinese Holstein dairy cows（peripheral blood）in the third lactation period via 2b-RADseq technology,and 10,058 high-quality SNPs were obtained from 7,957,920 tags.Then the Bayesian（P<0.001）and Logistic regression（P<0.01）analysis models were performed to analyze genome-wide association and alignment analysis,and 27 important and significant differences SNPs were gained,of which only 3 significant SNPs（rs75762330,C>T,PIC=0.2999;rs88640083,A>G,PIC=0.1676;rs20438858,G>A,PIC=0.3366）were annotated to immune regulatory genes,respectively,and were located in non-coding regions with low or moderate genetic polymorphisms.The candidate genes（PTK2B,SYK,and TNFRSF21）for mastitis traits in Chinese Holstein dairy cows corresponding the above three SNPs were determined by KEGG pathway,GO classification and enrichment analysis,and Fine Map analysis.At II stage,the population genetic verification of above 3 significant SNPs was performed in another independent sample（383）of Chinese Holstein dairy cows via case-control study,and it’s finally determined that the 3 important SNPs were significantly associated with mastitis traits in Chinese Holstein cows,among them,SNPs rs75762330（P<0.025,OR>1）and rs88640083（P<0.005,OR>1）was significantly correlated with mastitis resistance traits and SNPs rs20438858（P<0.001,OR<1）was significantly correlated with mastitis susceptibility traits.Bovine mastitis candidate genes for resistance traits（PTK2B and SYK）and susceptibility traits（TNFRSF21）were verified（in vivo and in vitro）in Chinese Holstein dairy cows.In vivo,qRT-PCR analysis showed that the mRNA expressions of PTK2B and SYK were down-regulated in peripheral blood lymphocytes（P<0.001,and P<0.001）and mammary gland tissues microenvironment（P<0.01,and P<0.01）in clinical mastitis cows,while TNFRSF21 was significantly up-regulated（P<0.05,and P<0.05）.In vitro,LPS（E.coli）stimulated or S.agalactiae infected bMECs,mRNA expression of the three candidate genes had LPS（E.coli）stimulated or S.agalactiae infection concentration and time-programmed effects;PTK2B and SYK mRNA expression were down-regulated,while TNFRSF21 was also significantly up-regulated.Validation data（in vivo and in vitro）indicated that PTK2B and SYK genes were important candidate genes for mastitis resistance traits,and TNFRSF21 gene was important candidate gene for mastitis susceptibility traits in Chinese Holstein dairy cows.The TLR4-SYK signaling pathway is involved in the immune response of the mammary gland tissue microenvironment in mastitis dairy cows.In vivo,the data showed that the expression of SYK mRNA in peripheral blood lymphocytes was significantly down-regulated,and the protein levels of Syk and its phosphorylated p-Syk were also significantly down-regulated in mastitis cows;Tlr4 and Syk dual-fluorescence co-localization data showed that they had a medium or high correlation（0.4<Pearson Correlation Values PVC=0.5187 or 0.8857<1.0）in mammary gland microenvironment;Co-immunoprecipitation data also confirmed the interaction between Tlr4 and Syk in the microenvironment of dairy cow mammary glands.In vitro,LPS（E.coli）stimulated or S.agalactiae infected the bMECs and bMECs ^SYK-,respectively;under stimulation conditions of the same pathogenic factor,the expressions of TLR4mRNA and protein levels were hardly affected by SYK siRNA in both bMECs,indicated that SYK is an important downstream element of the TLR4 cascade.Research data（in vitro and in vivo）supported the presence of the TLR4-SYK signaling pathway to regulate the mastitis immune response in the mammary gland microenvironment of dairy cows.The SYK-NLRP3 signaling pathway is involved in the inflammatory response of mammary gland microenvironment in dairy cows with mastitis.In vivo,the protein expression levels of Nlrp3 inflammasome and pro-inflammatory factor IL-1βwere significantly up-regulated in in the mammary gland microenvironment;Syk and Nlrp3dual fluorescence co-localization data showed that there had a high-medium or high correlation（0.6<PVC=0.6103 or 0.8533<1.0）in mammary gland microenvironment;Co-immunoprecipitation data also confirmed the interaction between Syk and Nlrp3 in the microenvironment of dairy cow mammary glands..In vitro,mRNA and protein expression levels of NLRP3 inflammasome were significantly up-regulated in LPS（E.coli）stimulated or S.agalactiae infected bMECs,including bMECs ^SYK-（P<0.001）;moreover,under the same conditions,the expression level of Nlrp3 protein in bMECs ^SYK-was significantly higher than that of bMECs,reaching P<0.01（LPS（E.coli））and P<0.05（S.agalactiae）level,suggesting that Syk had negatively regulated the expression of NLRP3 inflammasome in bMECs of dariy cows.Data showed that there was SYK-NLRP3 signaling pathway regulated the inflammatory response in mammary gland tissue microenvironment of the dairy cows with mastitis.AKT1 mRNA was significantly up-regulated in mastitis mammary gland tissue,LPS（E.coli）stimulated or S.agalactiae infected bMECs and the up-regulation trends were completely opposite to the mRNA expression levels of SYK and PTK2B gene.Gene function network analysis indicated that PTK2B and SYK are co-expressed in the Chinese Holstein natural immune signaling pathway,suggested that PTK2B,SYK and AKT1 genes were involved in the immune cascade of bMECs.In addition,the mRNA expressions of IL-1β,IL-8 and IL-10 were significantly up-regulated in mastitis mammary gland tissue,LPS（E.coli）stimulated or S.agalactiae infected bMECs and bMECs ^SYK-,indicated that they played an important role in response to innate immunity caused by pathogenic factors in mammary gland environment of the Chinese Holstein dairy cows.NF-κB mRNA expressions were up-regulated in the mammary gland tissue microenvironment,LPS（E.coli）stimulated or S.agalactiae infected bMECs and bMECs^SYK-,suggesting that TLR2/4-SYK-NF-κB or TNFRSF21-NF-κB signaling pathways may exist to regulate the immune response in bMECs.Taken together,firstly,this study provided strong evidence for the study of significant SNPs（resistance traits SNPs:rs75762330,rs88640083;and susceptibility traits SNPs rs20438858）related to mastitis via 2b-RADseq and GWAS,and determined mastitis candidate genes PTK2B,SYK,and TNFRSF21 through KEGG and GO functional analysis in Chinese Holstein dairy cows.Then the authenticity and reliability of SNPs rs75762330,rs88640083,and rs20438858 were verified by case-control study.Secondly,the Tlr4-Syk and Syk-Nlrp3 signaling pathways were preliminarily determined to participate in immune response and inflammatory response of mammary gland microenvironment in Chinese Holstein cows with mastitis,in vivo and in vitro,via qRT-PCR,WB,Dual Immunofluorescence co-location,Co-immunoprecipitation and siRNA technologies.Finally,new strategies for the prevention and treatment of dairy cow mastitis and related inflammatory diseases can be formed,which can also provide theoretical basis and technical support for the prevention and treatment of mastitis and related inflammatory diseases in other ruminants（such as dairy goats）.