Isolation, Characterization, Polymorphism And Association With Production Traits Of Eight Candidate Genes In Pigs

The use of the marker-assisted selection (MAS) combining with traditional breeding methods promoted the progress of genetic improvement in pigs. The basis of MAS was to seek the major gene or marker which contributed genetically much to the economic trait. With the development of pig genetic map, QTL mapping and functional genome research of human or mouse, it is practice that determining candidate gene associated with economic trait in the chromosome region which homologous to human or mouse based on the results of QTL mapping. In this study, eight candidate genes associated with muscle metabolism were selected by comparative positional genomics to research in pigs. The main results are as follows:1,HK2(Hexokinase II): (1) The full coding sequence and partial genomic sequences of Large White, Landrace, Meishan and Duroc breeds were isolated and deposited to GenBank under accession number DQ432056,DQ432057,AY818385,AY821788. (2) 13 cSNPs (single nucleotide polymorphism in coding sequence), two SNPs within 3' untranslated region and 38 SNPs within intron sequences were identified. (3) Two PCR-Msp I -RFLP were developed to detect A/G substitution in intron 10 and T/G substitution in exon 17. Allele frequencies for Msp I PCR-RFLP in intron 10 were studied in unrelated pigs from six breeds and allele A was found only in Large White and Landrace pigs, while allele A and B were both detected in Chinese pigs. For Msp I PCR-RFLP in exon 17 in unrelated pigs from six breeds, allele A was found only in heterozygocytous pig and the frequencies of two alleles were similar in all studied pig breeds. (4) In the 309 F₂ animals in LargeWhite×Meishan resource family were genotyped by Msp I PCR-RFLP in intron 10 for association analysis. For carcass traits, significant effects were observed for this locus on LEH and AST, including their additive effects; For meat quality traits, this locus was significantly associated with MM(BF). Msp I PCR-RFLP in exon 17 was typed in 186 pigs (Landrace pigs, LargeWhite×Landrace and Landrace×LargeWhite pigs) to study the association between carriers of different genotype and the trait values, statistically significant associations with CL, RNS, BFT1, LEH, LEW, LEA, BFT2, ABF, FMP, LMP and IFR were found. (5) The expression of HK2 gene was specially in skeleton muscle of pigs.2,LMCD1 (LIM and cysteine-rich domains 1) : (1) The full coding sequence of Large White, Landrace and Meishan breeds were isolated and deposited to GenBank under accession number NM₀01008692. (2) 4 cSNPs were identified , of which one was predicted to change amino acid. (3) PCR-SSCP (polymerase chain reaction-Single strand conformational polymorphism) typing was developed to detect G/A substitution in intron 3. The frequencies of allele G were 1 or near to in Large White, Landrace, Erxihei and Wannanhua, while Meishan and Qingping breed were 0.2889 and 0 separately. (4) In the 178 F₂ animals were genotyped for association analysis. For carcass traits, significant effects were observed for this locus on BFT1 and BFT2; For meat quality traits, this locus was near to significantly associated with pH(LD) and pH(SC) and seemed to be dominant in action. (5) LMCD1 gene was highly expressed in heart and muscle while it had a broad expression spectrum across all tissues examined.3,NRAP (Nebulin-related anchoring protein): (1) The full coding sequence of LargeWhite, Landrace and Meishan breeds were isolated. The nucleotide sequence of 5'RACE (rapid amplification of cDNA end) and intron 39 and 40 have been deposited to GenBank under accession number DQ157553 and DQ480149. (2) 35 SNPs were identified by comparing sequences, three of which resulted in amino acid change. (3) PCR-Eco721-RFLP was developed to detect A/G substitution in intron 39. In unrelated pigs from eight breeds, both allele A and B were found in Chinese pig breeds, while only B was in LargeWhite and Landrace. (4) Association analysis in 302 F₂ animals showed significant associations between Eco721-RFLP genotypes with BWT, CFW, LEH and BFT1 for carcass traits and pH(LD), pH(BF), MCV1, MCV2, DLR and WHC for meat quality traits. This locus was predominant in dominance for carcass trait, but in addition for meat quality. (5) NRAP mRNA was transcribed especially in heart and skeleton muscle.4,ECH1 (Enoyl CoA hydratase 1): (1) The full coding sequence of LargeWhite, Landrace and Meishan breeds, 5'RACE and all introns except intron 3 were isolated and deposited to GenBank under accession number DQ157552 and DQ480146DQ157552. (2) 17 SNPs were identified, of which one was cSNP. (3) Two A/G substitution were detected by PCR-BamHâ… -RFLP in intron 1 and PCR- Pstâ… -RFLP in intron 5 separately, and T/C mutation in exon 4 was detected by PCR-SSCP. Among seven different pig breeds examined, results of BamHâ… -RFLP in intron 1 showed that the frequency of allele B was near to 1 in LargeWhite and Landrace; both allele A and B were found in Chinese pig breeds, and allele B was predominant in other breeds except Meishan and Qingping. For PCR- Pstâ… -RFLP in intron 5, in seven commercial pig breeds, both alleles C and D (allele D was dominant) were detected in all other examined breeds except Jianli pig which only had allele D.(4) Association analysis in 296 F₂ animals showed significant associations between BamHâ… -RFLP genotypes and CFW, LEH and WM. And this locus seemed to be dominant in action. For PCR- Pstâ… -RFLP in intron 5 in 306 F₂ animals, for carcass traits, significant effects were observed for this locus on DP and AST; For meat quality traits, this locus was significantly associated with MCV2. (5) Expression of ECH1 gene can be detected in all tissues examined: heart, liver, lung, kidney, spleen, stomach, small intestine, muscle and fat.5,CKMT2 (creatine kinase, mitochondrial 2): (1) The full coding sequence of LargeWhite, Landrace and Meishan breeds were isolated and deposited GenBank under accession number DQ363337. In addition, the sequence of intron 4 can be seen in appendix 14. (2) Of nine SNPs identified within these sequences, three of which resulted in amino acid change. (3) An PCR- Mspâ… -RFLP was developed to detect the A/G polymorphism in intron 4. Among nine pig breeds examined, allele B was 1 or near to 1 in Meishan and Erhualian, while in other seven breeds all were 1 of allele A. (4) 302 F₂ animals were typed for association analysis. For carcass traits, significant effects were observed for this locus on BWT, IFR, FMP, CL, BFT1, BFT2 and BFT3, and their additive effects were predominant; For meat quality traits, this locus was significantly associated with WM. (5) Expression of CKMT2 gene was predominant in heart and skeleton muscle and moderate in lung, kidney and fat, although a weak signal was observed for liver, ovary and testis, and no expression in spleen, stomach, small intestine and uterus.6,TTID (Titin Immunoglobulin Domain Protein): (1) The full coding sequence of LargeWhite, Landrace and Meishan breeds and sequences of all introns except intron 2 and 7 were isolated and deposited to GenBank under accession number DQ157551 and DQ480148. (2) Total 51 SNPs within these intron sequences and no mutation or deletion was found in cDNA sequences. (3) An PCR- Hinfâ… -RFLP was developed to detect T/C substitution in intron 6. Among ten pig breeds which were typed by Hinfâ… -RFLP, results showed that there were significantly different in allele frequencies between Chinese indigenous pig breeds and foreign pig breeds: the frequencies were 1 or near to1 in LargeWhite and Landrace, while allele A was predominant in other five breeds although allele B were only a bit dominant in Erxihei, Qinsping and Bamei. (4) 279 F₂ animals were typed for association analysis. For carcass Waits, significant effects were observed for this locus on SP, LEA and AST, and their additive effects were significant; For meat quality traits, this locus was significantly associated with pH(BF), MCV1 and WM, and their additive effects were significant. (5) Expression of TTID gene was highest in skeleton muscle and heart, with weak level in fat and lung, and no signal in other tissues.7,PFKM (Phosphofructokinase Muscle Type): (1) The full cDNA sequence of LargeWhite, Landrace and Meishan breeds and sequences of 2-21 introns were isolated and deposited to GenBank under accession number DQ363336 and DQ480147. And the length of sequence of intron 1 was identified. (2) Of the 111 SNPs identified within all sequences, four of which resulted in amino acid change. (3) PCR-Taqâ… -RFLP were developed to detect T/C substitution in exon 13 and exon 17. In unrelated individuals from seven different pig breeds, allele frequencies of the Taqâ… -RFLP in exon 13 was compared: frequencies of allele B in LargeWhite, Landrace, Erxihei were 1 or near to 1, and a little dominant of allele B in other four Chinese pig breeds. For the same polymorphism in exon 17, the trend of allele frequencies distribution was the similar to that of exon 13. (4) For association analysis in the 214 F₂ animals, significant effects were observed for different genetype of exon 13 on BWT, IFR, FMP, LMP, RLF, LEH, BFT1, BFI2, BFT4 and ABF of carcass traits. It is notable that both additive effects and dominant effects in BWT, LMP, RLF and LEH were very significant; For meat quality traits, this locus was significantly associated with MCV2, MM1, WM and IMF. Association analysis in 241 F2 animals showed there were significant effects on BFI3 and ABF of carcass traits and on MM1, MM2, DLR, WHC, IMF and WM of meat quality traits with Taqâ… -RFLP in exon 17. (5) Expression of PFKM gene had different levels in each examined tissue except stomach and small intestine, with highest in skeleton muscle, then in heart.8,MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle): (1) The full genomic sequence of of LargeWhite, Landrace and Meishan breeds were isolated and deposited to GenBank under accession number DQ533994. (2)43 SNPs, including 5 cSNPs, were found by comparing sequences of three pig breeds, three of which resulted in amino acid change. (3) PCR-Taqâ… -RFLP and PCR-Mspâ… -RFLP were developed to detect G/A substitution and T/C substitution in intron 1 separately. For Mspâ… -RFLP among seven different pig breeds examined, the frequency of allele B was two times of that of allele A in Landrace, while allele B were predominant in other breeds. For Taqâ… -RFLP, the frequency of allele B was two times of that of allele A in LargeWhite and Landrace. (4) The locus of Mspâ… -RFLP was typed in 140 pigs (Landrace, LargeWhite, Meishan, LargeWhite×Landrace and Landrace×Large White pigs) to study the association between carders of different genotype and the trait values. For carcass traits, significant effects were observed for this locus on LMP, SP, LEW and LEA, with approximative significant on BFT1 and LEH(P＜0.06). This locus appeared to be additive effects with different action direction; For meat quality traits, this locus was significantly associated with MM1, MM2 and WM and IMF, which appeared to be predominant on addictive effects. (5) RT-PCR analysis of MYLPF showed its expression in all tissues examined, with highest levels in heart and skeleton muscle.The results described above provide some valuable basis for the further research of the molecular mechanism of these genes with the production and metabolism of muscle in pig and the application of molecular breeding with MAS. All these works would facilitate the progress of genetic improvement in pig, and establish the basis for the research of functional genome in our country.