Identification and application of pig CD4 gene function mutation site molecular breeding marker

[0124] Genome-wide association analysis (GWAS) of porcine 60K gene chip and T cell subtypes

[0125] The 60K gene chip (Illumina, USA) of the Duroc × Erhualian F2 population was used to analyze the relationship between each SNP and the T cell subtype using a mixed model. The model can be expressed as Y=Xb+Sα+Zμ+e, where Y is the table type vector, b is the estimated value of fixed effect gender and batch, μ is the additive effect of gene (μ-N(0,Gσ α 2 ), G is the genome similarity matrix, σ α 2 is the polygenic additive variance), X and Z are the correlation matrices of b and μ, S is the correlation vector of α, and e is the residual matrix (in the form of N(0,Iσ e 2 ) distribution, where I is the identity matrix, σ e 2 is the residual variance). For specific analysis, the software package in R was used, referring to the research of Aulchenko et al. (Aulchenko et al., 2007a,b), and the thresholds for genome-level significance and suggested significance were set after Bonfe

[0127] Determination of two haplotypes of porcine CD4 gene

[0128] The experimental method is as follows:

[0129] (1) Using TRIZOL lysis method to extract total white blood cell RNA from Duroc × Erhualian F2 population

[0130] 1) Take out the white blood cell lysate added with 1ml TRIZOL from -80°C, dissolve it on ice, shake vigorously to lyse the cells;

[0131] 2) Add 0.2ml of chloroform, shake vigorously for 15s, let stand at room temperature for 2-3min, RNA is dissolved in the supernatant water sample layer, DNA and protein are dissolved in the organic layer below;

[0132] 3) Place the centrifuge tube in a refrigerated centrifuge, centrifuge at 12000rpm at 4°C for 15min, carefully absorb the water sample layer and transfer it to a new 1.5ml centrifuge tube, add an equal volume of isopropanol, mix well and let stand at room temperature for 15min to precipitate total RNA;

[0133] 4) Place the centrifuge tube in a refrigerated centrifuge, centrifuge at 12,000 rpm at 4°C

[0169]Mutation vector construction and identification of key mutation sites

[0170] The experimental method is as follows:

[0171] (1) Mutation vector construction

[0172] Using the haplotype A and haplotype B overexpression vectors as templates, design mutant vector primers with primer sequences such as SEQ ID NO: 5-8, and carry out the amplified SNPs region containing type A and the SNPs region containing type B To reassemble the connection, the specific steps are as follows:

[0173] 1) PCR amplification recovery. Using the constructed haplotype A and haplotype B vectors as templates, PCR amplification was performed with primer pairs of SEQ ID NO: 5-8, and the amplification system was as follows (50 μL):

[0174]

[0175] After mixing and centrifuging, perform PCR amplification. The reaction procedure is as follows:

[0176]

[0177] The PCR product was recovered by gel, and the recovered product was detected by electrophoresis.

[0178] 2) Recombination connecti