Gnetic Analysis Of Three Laboratory Miniature Pig Breeds Using Microsatellite Loci And SNPs Of RBPs Gene Family

The laboratory miniature pig populations of Guangxi Bama, Wuzhishan, Guizhou A strain and Guizhou B strain are bred for experimental and medical research for many years. Laiwu Black pig is a Chinese indigenous pig breed which inhabits in Laiwu county of Shandong province. The samples of Laiwu Black pig used in this study were collected in the Conservation Farm of Laiwu Black pig. Using 19 microsatellite loci and SNPs of porcine cellular retinol binding protein gene familys(RBPs), the population genetic structure within and differentiation between the Laiwu Black pig, Guangxi Bama, Wuzhishan, Guizhou A strain and Guizhou B strain were assayed. The results are presented as follows:1. The average observed allele numbers over 19 microsatellite loci of Laiwu Black pig population and Guangxi Bama, Guizhou A strain, Guizhou B strain and Wuzhishan miniature pig populations are 4.37±1.46, 3.00±0.82, 4.47±1.47, 3.68±1.00 and 3.47±1.22(mean±SD), respectively; the observed heterozygosity of the five populations are 0.629±0.188, 0.449±0.261, 0.551±0.177, 0.536±0.215 and 0.451±0.226 (mean±SD), respectively; and the expected heterozygosity 0.615±0.162, 0.406±0.218, 0.636±0.169, 0.534±0.189 and 0.440±0.194(mean±SD), respectively.2. Three distinct exact tests(probability-test, heterozygote deficiency, and heterozygote excess) were performed for testing of deviations from Hardy-Weinberg equilibrium(HWE) proportions under the same null hypothesis of a random union of gametes. There are five(SW24, S0355, S0101, SW72, S0218), three(SW951, S0386, S0218), five(SW951, SW240, S0355, S010, S0218), two (SW951, S0218) and two(SW911, SW902) loci deviated from HWE in Laiwu Black pig population and Guangxi Bama, Guizhou A strain, Guizhou B strain and Wuzhishan miniature pig populations, respectively. There is no single population at all loci or single locus in all populations deviated from HWE.3. The null alleles were tested for every population at each locus using the computer program Micro-Checker version 2. 2. 3. Only one null allele at S0218 locus in both Laiwu Black population and Guizhou B strain miniature pig population.4. The genetic differentiation among Laiwu Black pig and Guangxi Bama, Guizhou A strain, Guizhou B strain and Wuzhishan miniature pig populations is considerable, as the value of coancestry coefficient (F_ST; Weir and Cockerham, 1984) and the relative magnitude of gene differentiation(Gst; Nei, 1987) are 0.402 and 0.327, respectively.5. The principal component analysis(PCA) was performed for five swine populations using the computer program Multivariate Statistical Package version 3.13g. The first, the second and the third principal components (PC1, PC2 and PC3) account for 31.56ï¼…, 28.99ï¼…and 24.15ï¼…of the total genetic variation estimated from the allele frequencies at 19 microsatellite loci, respectively. The two strains of Guizhou miniature pig and Laiwu Black pig are separated from Bama and Wuzhishan miniature pigs by the PC1, the two strains of Guizhou miniature pig are separated further from Laiwu Black pig by the PC2, and the Bama miniature pig is separated from Wuzhishan miniature pig by the PC3.6. Population structure is deduced using computer software STRUCTURE version 2.2 and the results demonstrated that the allele components are homogenous in Guangxi Bama miniature and Wuzhishan miniature pig populations and also in Laiwu Black pig population. But the allele components of the two strains of Guizhou miniature pig are a little complex.7. The genetic distance between the two strains of Guizhou miniature pig is the least among all population pairs of the five populations, and the largest genetic distance is found between Wuzhishan miniature pig population and Laiwu Black pig population.8. The porcin cellular retinol binding protein gene 1, 2, 5, 7(RBP1, RBP2, RPB5 and RBP7), plasma retinol binding protein gene(RBP4), cellular retinoic acid binding protein genel and 2(CRABP1 and CRABP2) are assigned to SSC 13q31, 13q31, 5q21-q24, 6q12-q21, 14q25-q26, 7q11-q23 and 4q22-q23, respectively.9. Using specific primer pair for each gene, 728 bp, 558 bp, 545bp and 585bp long cDNA fragments which contain the complete CDS of porcine RBP5, RBP7, CRABP1 and CRABP2 genes were amplified by RT-PCR. The CDS sequences of porcine RBP5, RBP7, CRABP1 and CRABP2 genes were deposited in the GenBank(GenBank accession nos. EF-208120, EF-208119, EF-397416 and EF-397415, respectively).10. Expression patterns of porcine RBP1, RBP2, RBP4, RBP5, RBP7, CRABP1 and CRABP2 genes in 12 tissues(lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph code, small intestine, liver, brain, kidney and fat) of adult Wuzhishan pig and the expression patterns in three stage of fetal development (33, 65, 90 dpc) of Tongcheng pig were detected by RT-PCR.11. The SNP loci of RBP2-A/G⁹³, -A/G¹³⁸, -C/T¹¹⁷, RBP4-A/G¹⁵⁶, RBP5-A/G⁶³, -A/G³⁹, -A/G⁴², -A/G^intronl-91, -T/C^intronl-90, -A/G⁵¹⁷, -A/G⁶⁴⁹, -T/C⁶⁵³, RBP7-T/C⁶ and CRABP2-G/C⁵⁰⁰ were confirmed by digestion with enzymes or direct sequencing of PCR products in a set of samples. With the SNP loci RBP2-C/T¹¹⁷, RBP5-A/G⁶³, -T/C^introl-90 and -A/G⁵¹⁷, the PCR-RFLP method was employed to analyse all samples of Wuzhishan, Laiwu, Guangxi Bama, Guizhou A and B strains, Tongcheng, Yorkshire and Landrace breeds, and the gene frequences and the genotype frequences of each locus in each population were calculated.12. Association analysis of genotypes of these SNP loci with economic traits was done in our experimental populations. Significant associations of different genotypes of RBP2-C/T¹¹⁷, RBP4-A/G¹⁵⁶, RBP5-A/G⁶³, RBP5-A/G⁵¹⁷ and RPB5-T/C^intronl-90 loci with traits including days to slaughter weight(Age), maximum carcass length (LM), minimum carcass length(LN), marbling score(MS), back fat thickness at shoulder(SBF), muscle shear force(MSF), meat color score(MCS), hematocrit (HCT), hemoglobin(HGB) and mean corpuscular volume hemoglobin(MCH) were detected.13. At most loci, one allele at RBP2-C/T¹¹⁷, RBP4-A/G¹⁵⁶, RBP5-A/G⁶³, RBP5-A/G⁵¹⁷ and RPB5-T/C^intronl-90 SNP loci in Wuzhishan, Guizhou A and B strains and Guangxi Bama miniature pig populations was dominant. The results indicated that these four laboratory miniature pig populations have a certain degree of gene homozygosity.