Czech J. Anim. Sci., 62, 2017 (4): 157–167 Original Paper doi: 10.17221/97/2015-CJAS Genetic Variations of the Bovine MX1 and Their Association with Mastitis Ningbo Chen, Fengqiao Wang, Nongqi Yu, Yuan Gao, Jieping Huang, Yongzhen Huang, Xianyon Lan, Chuzhao Lei, Hong Chen, Ruihua Dang* College of Animal Science and Technology, Northwest A&F University, Yangling, P.R. China *Corresponding author: [email protected] ABSTRACT Chen N., Wang F., Yu N., Gao Y., Huang J., Huang Y., Lan X., Lei C., Chen H., Dang R. (2017): Genetic variations of the bovine MX1 and their association with mastitis. Czech J. Anim. Sci., 62, 157–167. The primary agent of mastitis is a wide spectrum of bacterial strains; however, viral-related mastitis has also been reported. The MX dynamin-like GTPase 1 (MX1) gene has been demonstrated to confer positive antiviral responses to many viruses, and may be a suitable candidate gene for the study of disease resistance in dairy cattle. The present study was conducted to investigate the genetic diversity of theMX1 gene in Chinese cattle breeds and its effects on mastitis in Holstein cows. First, polymorphisms were identified in the complete coding region of the bovine MX1 gene in 14 Chinese cattle breeds. An association study was then carried out, utilizing polymorphisms detected in Holstein cows to determine the associations of these single nucleotide polymor- phisms (SNPs) with mastitis. We identified 13 previously reported SNPs in Chinese domestic cattle and four of them in Holstein cattle. A novel 12 bp indel was also discovered in Holstein cattle. In addition, haplotype frequencies and linkage disequilibrium of four SNPs detected in Holstein cows were investigated. Analysis of these four SNPs in Chinese Holstein cows revealed two SNPs (g.143181370 T>C and g.143182088 C>T) significantly P( < 0.05) associated with somatic cell score (SCS). The results indicated that SNPs in the MX1 gene might contribute to the variations observed in the SCS of dairy cattle. Therefore, implementation of these two mutations in selection indexes of the dairy industry might be beneficial by favouring the selection individuals with lower SCS. Keywords: MX dynamin-like GTPase 1; polymorphism; marker assisted selection; disease resistance; viral infection; somatic cell score The Mx dynamin-like GTPase (MX) 1 gene plays essential for the regulation of GTPase activity and an important role in the immune response, induc- the C-terminal portion contains a leucine zipper tion of apoptosis, and signal transduction (Haller et motif that is involved in forming a homooligomer al. 2015). MX1 possesses a N-terminal GTPase do- (Melen et al. 1992). The MX1 protein has been main, a bundle signalling element, and a C-terminal demonstrated to have potent activity against several stalk domain. The N-terminal region of MX1 is RNA viruses, such as the influenza virus, vesicular Supported by the National Natural Science Foundation of China (No. 81270439), the Research Foundation for Advanced Talents (No. Z111021202), the China Postdoctoral Science Foundation (2015M572607), and the Program of the National Beef Cattle Industrial Technology System (CARS-38). N. Chen and F. Wang contributed equally to this paper. 157 Original Paper Czech J. Anim. Sci., 62, 2017 (4): 157–167 doi: 10.17221/97/2015-CJAS stomatitis virus (VSV), and Thogoto virus (THOV) In addition, most studies that identify quantita- (Verhelst et al. 2013; Haller et al. 2015). Loss of tive trait loci (QTL) for mastitis resistance use MX1 function in human or mice enhances infection the somatic cell score (SCS) as an indicator trait by multiple viruses, including orthomyxoviruses, for both subclinical and clinical mastitis (CM). paramyxoviruses, and hepadnaviruses (Verhelst et Breeding schemes often use the SCS because it al. 2015). The MX1 gene increases fitness through shows a moderate to high genetic correlation to antiviral activity in mice, human, cows (Gerardin CM and it is easier to record (Lund et al. 2007). et al. 2004; Nakatsu et al. 2004), chickens (Ko et Taking the functions of the MX1 protein into con- al. 2002), and pigs (Asano et al. 2002). In cattle, sideration, we hypothesized that MX1 might also overexpression of the MX1 protein inhibits rep- have an indirect impact on mastitis in dairy cattle. lication of foot-and-mouth disease virus (FMD) Therefore the aims of the present study were to (Diaz-San Segundo et al. 2013). The porcine MX1 detect polymorphisms of the bovine MX1 gene in gene has been proposed as a candidate gene for 13 Chinese cattle breeds (including Yellow cattle immunity traits (Wang et al. 2012). Furthermore, a and Holstein cows), and to determine the associa- polymorphism in the promoter region of the MX1 tion of these SNPs with milk SCS (considered a gene is associated with porcine reproductive traits mastitis indicator) in 297 Chinese Holstein cows. and respiratory syndrome virus infection (Li et We expect to discover candidate markers related al. 2015). These findings suggest that MX1 is an to CM in Chinese dairy cattle. interesting candidate gene for disease resistance in farm animals. Bovine MX1 cDNA (GenBank Acc. No. AF047692) MATERIAL AND METHODS was originally cloned from an Angus-Gelbvieh cross-bred cow by Ellinwood et al. (1998). The Animals. We investigated genetic variation in bovine MX1 gene is mapped to chromosome 1 the bovine MX1 gene using 466 individuals from and is made up of 13 exons (Gerardin et al. 2004) 13 cattle breeds in China. All experimental proce- that encode a 654 amino acid protein (Ellinwood dures were performed according to authorization et al. 1999). A total of 23 polymorphisms have granted by the Chinese Ministry of Agriculture. A been identified in the bovine MX1 gene so far, total of 120 animals from 12 yellow cattle breeds including 10 synonymous mutations, one missense (ten animals per each breed) were selected from substitution, and a 13 bp indel mutation in the 3' different Chinese farms: Mongolian cattle, Anxi untranslated regions (UTR) (Nakatsu et al. 2004). cattle, Qinchuan cattle, Jinnan cattle, Luxi cattle, In addition, the MX1 gene of other domestic ani- Jiaxian cattle, Zaosheng cattle, Nanyang cattle, mals has been identified, however, its impact on Xuanhan cattle, Wannan cattle, Leizhou cattle, and disease susceptibility remains unclear (Ellinwood Hainan cattle. In addition, a total of 346 Chinese et al. 1999; Asano et al. 2002; Ko et al. 2002). In Holstein cattle were selected for the study. For light of this potential application, the present the association analysis, 297 Chinese Holstein study was designed to detect polymorphisms of cows were randomly selected from the Agricul- the MX1 gene in native Chinese cattle. tural Machinery Corporation of Xi’an city in the Mastitis is the most significant health prob- province of Shaanxi. Blood samples were collected lem affecting dairy cattle. The primary agents and genomic DNA was extracted from leukocytes of mastitis include a wide spectrum of bacterial using the standard phenol-chloroform method strains; however, the incidence of viral-related (Green and Sambrook 2012). Genomic DNA con- mastitis has also been reported. Viruses can either centrations were measured and working solutions be directly or indirectly involved in the etiology were prepared and adjusted to concentrations of bovine mastitis. In natural cases of mastitis, of 50 ng/µl. Ten DNA samples from each breed bovine herpesvirus 1 (BHV1), BHV4, FMD, and were selected to construct genomic DNA pools PI3 viruses have all been isolated from milk. It is for yellow cattle. DNA samples from 50 Holstein likely that some viruses (BHV2, cowpox, pseudo- cows were randomly selected for a DNA pool. cowpox, FMD, VSV, and papillomaviruses) cause The genomic DNA pool provided the templates teat lesions, thereby damaging the integrity of the for PCR amplification to explore genetic variation bovine udder, and indirectly contribute to mastitis. in the MX1 gene. 158 Czech J. Anim. Sci., 62, 2017 (4): 157–167 Original Paper doi: 10.17221/97/2015-CJAS Phenotypic data. All of the dairy cows were PCR-restricted fragment length polymorphisms 4.0–7.0 years old with the same lactation period. (PCR-RFLP) (Table 1). Three different genotypes Milk samples were taken from each cow once each of each SNP were confirmed by DNA sequencing month for the entire lactation period. The test- again. Aliquots (6 µl) of the PCR products were day milk yield was recorded, SCS, fat, and protein digested with 10 U AvaI for g.143181451 A>G, ScrFI percentages were collected from the laboratory of for g. 143182088C>T, and HaeIII for g.143189365 a dairy herd improvement (DHI) center (Shaanxi, T>C at 37°C for 10 h. The locus g.143181370 T>C China) using a milk composition analyzer (Foss has no natural restriction endonuclease cleavage Milk Scan FT 6000, Denmark) (Supplementary site, so an artificially created restriction site-PCR Table S1). In the cow, SCS is calculated according (ACRS-PCR) primers were designed, forward to Shook (1993): primer MX1-g.143181370 T>C with G instead of A at the 3' end was designed to introduce a HincII SCS = log2(SCC/100) + 3 recognition site. In addition, a 12 bp indel could be genotyped by different length of PCR products. where: The products were separated by 12% polyacryla- SCC = cells/µl mide gel (PAGE) in 1× Tris-borate-EDTA (TBE) The mean and standard error of test day record buffer under constant voltage (200 V) for 2 h. The of average milk yield, fat percentage (%), protein gels were stained with ethidium bromide and the percentage (%), and SCS were 20.779 ± 0.208 kg, genotypes were determined based on difference 4.309 ± 0.019%, 3.161 ± 0.009%, and 4.962 ± 0.075, in electrophoretic patterns. respectively, in the tested animals (Supplementary Statistical analysis.