Isolation, Characterization And Physical Mapping Of Four New Genes On SSC12

Pig is one of the predominant meat producing around the world and so the pig industry is of economic importance to both developed and developing countries. The rediscovery of the law of Mendel and the establishment of quantitative genetics caused a sea change in animal breeding. In order to make the genetic improvement in important economic traits of pigs a rapid progress by using the molecular breeding approach, further effort should be made to establish high-density gene map and have full understand about the gene structure, function and their association with the economic traits. The objective of this thesis was to isolate, characterize and physically map the novel genes on pig chromosome 12 by use of information derived from comparative mapping. The main results are as follows:1. The CATS primers were designed from well conserved-sequence regions of homologous genes between the human chromosome 17 and the mouse chromosome 11. Using the CATS primers, four novel porcine gene fragments were isolated and identified from Erhualian genomic DNA. The four novel porcine genes were as follows: glial fibrillary acidic protein (GFAP) gene ( GeneBank accession number: AF250778), ferredoxin reductase (FDXR) gene ( GeneBank accession number: AF317683), rod cGMP-phosphodiesterase gamma-subunit (PDEG) gene (GeneBank accession number: AF317684), and proteasome activator subunit 3 (PSME3) gene ( GeneBank accession number: AF317685).2. The pig ×rodent somatic cell hybrid panel (SCHP) was used for regional assignment of the four genes on the pig chromosome. SCHP analysis mapped all the four genes to porcine chromosome 12p (SSC12p). The GFAP and PSME3 were localized to SSC12pll- (2/3) p13 with error risk <0.1%. The probability of regional localization and concordance for the two genes was 0.8897, 0.9177 and 0.8901, 1.0, respectively. The FDXR and PDEG genes were physically mapped to SSC12 (1/3 p13)-p15 with error risk <0.1%. The probability of regional localization and concordance for the two genes was 0.8989, 0.9250 and 0.8181, 0.9220, respectively.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 GFAP and PSME3 genes were located closely to GH already placed on chromosome 12, with LOD score threshold 9.17 and 6.52, respectively. The FDXR and PDEG genes, which were mapped on the other linkage group, were closely linked to SO 143 and SW2490, which both had been localized on chromosome 12, withLOD score threshold 12.36 and 7.78, respectively.4. An improved RH comparative map of human chromosome 17 (HSA17) and pig chromosome 12 (SSC12) 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 12 given by the two-point distance was PDEG-FDXR-PSME3-GFAP.Comparing the SSC12 framework map with HSA17 GB4 map, an inverted gene order between PSME3 and GFAP was observed.5. A search for mutation in the amplified fragment of the four genes was performed by PCR-RFLP. The polymorphism of FDXR Hhal-RFLP and PDEG MspI-RFLP were detected and allele frequencies among different pig populations were also determined. RFLP screening with the restriction digestion enzymes Hhal and Alul revealed two polymorphic sites within PSME3 gene .The Hhal site is located in intron 6,whereas the'Alu! site is placed within exon 8.But the GFAP fragment obtained with GFAP CATS primer did not show any polymorphism.6. Codominant inheritance of the FDXR Hhal-RFLP, PDEG MypI-RFLP and PSME3 Hhal-RFLP were demonstrated in the Landrace x Tongcheng and Large White x Tongcheng two-generation pedigrees.7. Within intron 5 of PSME3 gene, a length variation caused by a 147bp fragment deletion was observed. Genotype and allele frequencies among several pig populations were identified.8. Using the information available in the EST database, th...