A novel SNP of Lysozyme Gene and Its Association with Mastitis Trait in Chinese Holstein

Mastitis is the most frequent and important disease in the dairy industry worldwide, which leads to great economic losses in the dairy industry (Nash et al. 2003). Direct selection for resistance to clinical mastitis may be very difficult and indirect selection has been practiced widely. The recommended measure is to record herd milk somatic cell scores (SCS) based on the positive correlation between clinical mastitis and milk SCS (Rupp et al. 1999). Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymor pho nuclear leukocytes and macrophages. The lysozyme level is an index of macrophage functional status (Di Luzio 1979). Seyfert et al. (1996) suggested the Lyz gene as a candidate gene for improvement of mastitis resistance. The activity of lysozyme may be the result of single nucleotide change. The Lyz gene is located on autosome 5, containing 4 exons and 3 introns. The purposes of this study were to reveal single nucleotide polymorphisms (SNPs) in the encoding region of Lyz gene and to evaluate the possible association of SNPs with SCS in Chinese Holstein populations.


Background
Mastitis is the most frequent and important disease in the dairy industry worldwide, which leads to great economic losses in the dairy industry (Nash et al. 2003).Direct selection for resistance to clinical mastitis may be very difficult and indirect selection has been practiced widely.The recommended measure is to record herd milk somatic cell scores (SCS) based on the positive correlation between clinical mastitis and milk SCS (Rupp et al. 1999).
Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymor pho nuclear leukocytes and macrophages.The lysozyme level is an index of macrophage functional status (Di Luzio 1979).Seyfert et al. (1996) suggested the Lyz gene as a candidate gene for improvement of mastitis resistance.The activity of lysozyme may be the result of single nucleotide change.The Lyz gene is located on autosome 5, containing 4 exons and 3 introns.The purposes of this study were to reveal single nucleotide polymorphisms (SNPs) in the encoding region of Lyz gene and to evaluate the possible association of SNPs with SCS in Chinese Holstein populations.

PCR amplification condition
Deoxyribonucleic acid was isolated from blood samples of 610 Chinese Holstein, the offspring of 30 bulls, using a phenol-chloroform extraction protocol followed by an ethanol precipitation step.Polymerase chain reaction (PCR) was carried out in a typical 20 μl system containing 100 ng template, 1 μl primer (10 pmol/μl), 0.4 μl dNTPs (10 mmol/μl), 1.0 μl MgCl2 (25 mmol), 1.5 U Taq DNA polymerase and 2 μl 10× buffer.Polymerase chain reaction ampli fi ca tion conditions were used as follows: pre-denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s and a final extension at 72 °C for 10 min.The PCR products were detected on a 1.5 % agarose gel.

Detection of Lyz gene polymorphisms
The polymorphism of Lyz gene was detected by the method of PCR-Single Strand Confor ma tion Polymorphism (PCR-SSCP).A total of 2.0 μl PCR product was mixed with 8 μl of the denaturation solution (50 mmol/l NaOH, 1 mmol/l EDTA) and 1 μl of the loading buffer, containing 0.25 % bromophenol blue and 0.25 % xylene cyannol, denatured for 10 min at 98 °C and rapidly chilled in −20 °C.The samples were then electrophoresed in 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).A thermostatically controlled refrigerated circulator was used to maintain a constant temperature (4 °C) of the gels.The gels were run under the following conditions: 250 V, 40 mA, 10 min (pre-electrophoresis) and 150 V, 24 mA, for 8 h.The gels were then stained with Silver Stain (Kucharczyk Techniki Elektroforetyczne).The patterns of DNA bands were observed and photographed with the GDS7500 System (UVP Inc., Upland, CA, USA).After the polymorphism was detected, 4 samples of each type of band were sequenced and analysed.

Detection of Lysozyme concentration in milk
Twenty-four milk samples from the offspring were collected for the ELISA test (8 offspring from each genotype).

Statistical analysis
The frequencies of alleles and genotypes were analysed with POPGENE software (ver.1.31).The χ 2 -test was to test linkage disequilibrium.All the production data were analysed with the GLM procedure of SAS 9.0 software (SAS Institute Inc., Cary, NC, USA).The relation of the Lyz gene polymorphism with 305-d milk yield and somatic SCS were analysed with a general linear model: Where y is the individual phenotypic value, μ is the overall mean, b is the fixed effect for sire (30), ys is the fixed effect of year and season, p is the fixed effect of parity (1-4), mm is the fixed effect of milking month (day in milk, 1-12), cs is the fixed effect of calving season (3-5: spring, 6-8: summer, 9-11: autumn, 12-2: winter), g is the fixed effect of genotype and e is the random residual effect.The somatic cell count (SCC) was converted into the SCS (Wiggans et al. 1987) (SCS=log2 (SCC/100 000) +3) and rectified to eliminate the effect of lactation days and period of sampling on SCS (Houng et al. 2000).
Statistical significance of the parameter values among the different genotype was tested by ANOVA procedure.The treatment means were separated by Duncan's multiple range test and accepted if P<0.05.

SNP identification and distributions of alleles and genotype
Results for PCR-SSCP showed that three types of SSCP bands were detected in primer 1. Direct DNA sequencing of the PCR productions of Lys gene revealed a single base nucleotide transversion from T to G at c. 115T>G site in the exon 1, resulting in replacement of Arg by Leu.The TT, TG and GG genotypes were designated, respectively.The genotypic and allelic frequencies of the mutations are shown in Table 1.The predominant allele was T (0.524) with TG presented at a high frequency (0.508) and GG presented at a low frequency (0.222).The χ 2 value was 288.11, indicating deviation from Hardy-Weinberg equilibrium in Chinese Holstein.

Table 1
The frequencies of genotype and allele and least squares mean and standard errors for SCS, 305 d milk yield and lysozyme concentration of different genotypic groups in Chinese Hostein Small letters means values in the same column with different letters significantly differ at P<0.05; capital letters means values in the same column with different letters highly significant differ at P<0.01