The Separation Of The Four Genes Of The Pig, The Physical Location And Myog Gene Snp Study

In order to make the genetic improvement in important economic traits in pigs by molecular breeding approach, further efforts should be made to establish high-density gene map and fully understood the gene structure, function and their association with the economic traits. The objective of this thesis was to isolate, characterize and physically map the four novel genes on pig chromosome 11 by use of information derived from comparative mapping of Homo sapiens chromosome 13 (HSA13). This was of great importance for the study of the structure and function of four genes. It also founded the basis for the insight into the evolution mechanism of Sus scrofa chromosome 11 (SSC11).Several primers were designed from well conserved-sequence regions of homologous genes between the human chromosome 13 and the mouse chromosome 8. Using the method of comparative genome between human and swine, six fragments of four genes were isolated and identified from genomic DNA in Daweizi and Ningxiang pig. Also, the typing of SNP (G/C) of MyoG gene in the 3'-untranslated region were studied. The results of this thesis were as followed:1. Six fragments of four genes were deposited into GenBank. These genes were growth arrested-special 6 (GAS6) gene (GeneBank accession number: AY880668), osteoblast specific factor (POSTN) gene (GeneBank accession number: AY880669), cell devision cyclin homology 16 (CDC16) gene (GeneBank accession number: AY880670), ephrin-B2 (EFNB2) gene (GeneBank accession number: AY880671), the intron 8 of CDC16 gene (GeneBank accession number: DQ206823) and the intron 3 of EFNB2 gene (GeneBank accession number: DQ206822).2. The pig x rodent somatic cell hybrid panel (SCHP) was used for regional assignment of the four genes. SCHP analysis mapped all four genes to porcine chromosome 11. The GAS6 was localized to SSC 11 q11^ql7 with error risk <0.1%. The probability of regional localization and concordance was 0.9976 and 0.7906 respectively. The POSTN was localized to SSC 11p11 15 with error risk <0.1%. The probability of regional localization and concordance was 0.9992 and 0.7031. The CDC16 gene of pig was localized to SSC11q11- ql7 with error risk <0.1%. The probability of regional localization and concordance was 0.9998 and 0.8563. The EFNB2 was localized to SSC 11 q11^q17 with error risk <0.1%. The probability of regional localization and concordance was 1.0000 and 0.7802.3. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of the four genes. Statistical analysis revealed that both the GAS6 and CDC16 genes were linkaged closely to the microsatellite SW1452 anchored on SSC11 with the rate of retension of 0.22, LOD score threshold were 16.88 and 16.08 -espectively and the distance in map of the radiation hybrid (RH) were 56 cR and 62 cR espectively. The POSTN gene was linkaged closely to the microsatellite SW1486 of SSC11 with0.35 of the rate of retension and the value of LOD of 15.87 and the location in the RH map was 67 cR. The EFNB2 gene was linkaged closely to the microsatellite SW903 of SSCll with 0.29 of the rate of retension, the value of LOD of 6.86 and the location in the RH map was 56 cR.4. An improved RH comparative map of human chromosome 13 (HSA13) and pig chromosome 11 (SSCll) was established by integrating the four genes with the recently published first-generation porcine whole-genome radiation hybrid map. The most likely order of the four genes on chromosome 11 given by the two-point distance was POSTN —GAS6— CDC 16 —EFNB2 while the order of these four genes in the HSA13 was POSTN — EFNB2 — GAS6— CDC16.5. Three single nucleotide polymorphism (SNP) (A/G) were found in the position of 20 bp, 172 bp, 246 bp in the intron9 of GAS6 by the direct sequencing for the products of the intron9 of GAS6 gene. Four point mutations were also existed in the position of 28 bp, 136 bp, 257 bp and 263 bp in that fragment. The mutation type was A/Q G/C, A/G and A/G, respectively. The individual included the native breed Daweizi pig in Hunan province and the exotic breed pig Landrace pig and Large Yorkshine because their genetic constitution is significantly different.6. By constructing the contigs for the express sequence tagged (EST) which were highly homologous to the human CDC16 gene (GenBank accession number. NM₀04661), the entire coding region of CDC16 gene was obtained through in silico cloning. This EST were BP436622, AJ656153, CJ022837 and BP161802. The length of the cloned cDNA was 2057 bp and 5'-untranslated region was 119 bp. The length of 3'-untranslated region was 75 bp. The length of the open reading frame (ORF) was 1863 bp. The deduced amino acid sequence was composed of 617 amino acid. It showed 97.6% and 95.3% similarity to corresponding human and mouse respectively. By alignment for the four pig EST, which were BP436622, AJ656153, CJ022837 and BP161802, four candidate SNP and candidate deletion mutation of 14 bp were found. The deletion sequence was GTT TGG AM CCC AG.7. The genetic variations of 3'-UTR of MyoG gene were detected with PCR-RFLP-Msp I in 630 pigs including Large Yorkshire, Landrace, Duroc, Ningxiang pig, Shaziling pig, Daweizi pig and Taoyuan black pig. The results showed that the genotypes of AA, AB, BB existed in the exotic breeds and the genotype of BB was dominant, and the frequency of allele B was 0.74. Three genotypes also existed in the Chinese native breeds, and the genotype of AA was dominant, and the frequency of allele A was 0.95. The frequencies of allele A and B were remarkably different between the exotic breeds and Chinese native breeds. The y2 value of genotypes indicated that every exotic pig breed had highly significant differences with four native pig breeds'. Taoyuan pig had highly significant differences with Ningxiang pig (p<0.01), Shaziling pig and Daweizi pig. Daweizi pig had significant differences with Ningxiang pig and Shaziling pig. Large Yorkshire had highly significant differences with Landrace pig.