Three Novel Single-nucleotide Polymorphisms Of MBL1 Gene In Chinese Native Cattle And Their Associations With Milk Performance Traits

Infectious disease has major adverse effects both on the economics of livestock production and on animal welfare. The costs of disease are estimated as 17% of turnover in the developed world (￡1.7 billion in the UK each year) and 35-50% in the developing world. Cow mastitis is a common disease in dairy industry worldwide, and has caused tremendous loss to the dairy production. A number of therapeutic, prophylactic and management strategies have been proposed to minimize this complex disease. Although these treatments have very good clinical outcome, emerge of drug-resistant strains and remaining antibiotics in milk have brought great concerns. Thus, an approach based on improving the host genetics in resistance to infectious diseases through molecular marker selective breeding is becoming widely accepted.Innate immune responses in animals are mediated by specific pattern recognition receptors or proteins that recognize microbe-specific pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide of Gram-negative bacteria, lipoteichoic acid of Gram-positive bacteria, and 1,3-β-D-glucan of eukaryotic fungi. This process also occurs in mammals by which secreted pattern recognition proteins such as mannose-binding lectin (MBL) and ficolins bind to microbial cells. Mannan-binding lectins (MBL) are collagenous C-type lectins involved in the innate immune response to various microbial pathogens. MBLs in plasma, like other collagenous lectins, are composed of multiple trimers, which further oligomerize toward the N-terminus by disulfide bridges and triple-helical association of the collagen-like domain to form higher order multimers. Antimicrobial functions of MBL, including opsonization, neutralization, and complement activation, depend on oligomeric assembly and clustered orientation of multiple carbohydrate-recognition domains which recognize repeating PAMPs on microbial surfaces. MBL selectively targets invading microorganisms for neutralization by its recognition domain's binding to cell surface mannose and N-acetylglucosamine residues, then activating MBL-associated serine proteases (MASPs). In humans, the single-nucleotide polymorphisms (SNPs) in MBL gene were found to cause various innate immune dysfunctions.Chinese Holstein Cattle is derived from grading cross breeding and selection between the Chinese native cow and pure-bred bull of Holstein introduced to China. The frequency of mastitis in the breed is about 38%～50%. The Luxi yellow cattle and Bohai black cattle are two of the representative indigenous bovine (Bos taurus) breeds in China, which have been bred as beef and draft dual-purpose cattle for thousands of years because of the lower disease and enduring unfavourable feeding condition.In the present study we detected 3 single-nucleotide polymorphisms of MBL1 gene in Chinese native cattle and analyzed their associations with milk traits. By screening the genetic variation of MBL1 in 1053 individuals of three Chinese native cattle breeds including China Holstein, Luxi Yellow and Bohai Black using Created Restriction Site-Polymerase chain reaction (CRS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing techniques, three new SNPs were found. One SNP (g.855G>A) of MBL1 gene was located in intronⅠand the other two SNPs (g.2651G>A, g.2686T>C) were located in exonⅡ, which showed one non-synonymous mutation GTT (Val)>ATT (Ile) and one synonymous mutation GCT (Ala) >GCC (Ala). The SNPs were submitted to the National Centre for Biotechnology Information (submitter SNP numbers: ss172800380, ss172800389, ss172800394) In Holstein cattle and Luxi Yellow cattle and Bohai Black cattle, predominan allele were identical at g. 855G>A, g.2651G>A, and g.2686T>C, which were G, G, C and the allelic frequencies were 0.87/0.58/0.57, 1/0.75/0.74, 1/0.76/0.63, respectivly. Chi-square test indicated that g.855G>A of Chinese Holstein cattle, g.855G>A and g.2651G>A of Luxi Yellow cattle and all the sites of Bohai Black cattle were in accordance with the Hardy-Weinberg equilibrium (P>0.05). The value of polymorphism information content indicated that g.855G>A was slight polymorphism; g.2651G>A and g.2686T>C were moderate polymorphism in the three breed(s0.25A and g.2686T>C and somatic cell score (SCS), however significant associations were found between g.2651G>A and SCS, suggesting a possible role of this SNP in the host response against mastitis. Our data also suggested that combined genotype GGC/AAC with the lowest SCS, AAT/AAT with the highest protein rate and AGC/AGC with the highest 305-d milk yield were favorable for the mastitis resistance and milk production traits. Thus, GGC/AAC, AAT/AAT and AGC/AGC can be used as a possible candidate for marker-assisted selection in dairy cattle breeding program.Optimum conditions for the total complement hemolytic activity assay in Luxi yellow cattle serum were also standardized.