Isolation, Identification Of Four Porcine Candidate Genes And The Association Analysis Of Molecular Markers With Heterosis

With the development and application of DNA marker technology, the molecular breeding techniques such as marker-assisted selection (MAS) and marker-assisted introgression (MAI) combining with traditional breeding methods have greatly promoted the process of genetic improvement in pigs. The basis of MAS is to seek the major genes or markers which are closely connected with the significant economic traits. Skeletal muscle is the most abundant tissue in animals, and its related growth and development traits are the most important ones to the livestock. So in this research, four genes which are pertinent to the growth and metabolism of the muscle were chosen as the candidate genes for pig production traits and were studied on. With the integration of 21 reported microsatellites and the 4 SNPs markers discovered in this study, the relationship between molecular markers and heterosis was discussed. The results are as follows:1. GSTM2: (1) The full coding sequence, 5'-RACE sequence and partial genomic sequences of GSTM2 gene from Large White, Landrace, Meishan were isolated. (2) With the help of CLUSTALW,CDD,Signal P3.0,Prosite software, the structure and function of the coded protein were analyzed and predicted, the phylogenetic trees were constructed as well. (3) 32 potential SNPs were identified, thereinto, the C27T mutation the exon 5 would lead to nonsense mutation and thus produce a PTC. (4) PCR-Taq I-RFLP was developed to detect C27T substitution in exon 5. In the different seven pig populations, TT genotype was not found. In all these populations, the frequency of allele C was higher than that of allele T. Especially in Large White×Landrace pigs, the frequency of allele C amounted to 1 while that of allele T was 0. (5) Association analysis of markers and production traits found that the relation between GSTM2 gene and FMP, RLF, BP, all fat thickness traits, CL1 and CL2 was highly significant. Pigs with genotype CC had lower all fat thickness traits, FMP and higher RLF, BP, CL than that of pigs with genotype with TT. (6) The possibility analysis of the nonsense-mediated mRNA decay of the exon 5 nonsense mutation discovered that allele T was absent in pig GSTM2 cDNA when it existed in genomic DNA, which indicated that that because of the existence of PTC, the mRNA of allele T might be degraded by NMD path. (7) The tissue expression profile analysis showed that the GSTM2 gene was expressed at a high level in liver and testis, a medium in longissimus dorsi muscle, adipose tissue, spleen and lung, a low in kidney and a lower in heart and embryo. (8) Real-time RT-PCR analysis showed that in the skeletal muscle of Large White and Meishan pigs, the expression level of GSTM2 gene decreased with the increasing of days, while it made no apparent difference between Large White and Meishan pigs.2. TCAP: (1) The full coding sequence and intron sequences of TCAP gene from Large White, Landrace, Meishan were isolated. (2) With the help of CLUSTALW,CDD,Signal P3.0,Prosite software, the structure and function of the coded protein were analyzed and predicted, the phylogenetic trees were constructed as well. (3) 4 potential SNPs were identified by sequence alignment. ASP was developed to detect G334A substitution in exon 2. In the different seven pig populations except Meishan, the frequency of allele G was higher than that of allele A. Particularly in Large White×Landrace pigs, the frequency of allele G reached 1. (4) Association analysis of markers and production traits found that the relation between TCAP gene and DP, LMP, RFT, TFT, BFT, AFT, LEH, LEW, CL1, CL2, FP, SFT, RLF, DLR, WHC, MCV1, WM are significant or highly significant. (5) The tissue expression profile analysis showed that the TCAP gene was expressed at a high level in muscle, heart, kidney and embryo, a decreased in adipose tissues and lung, a lower in spleen and liver, and had no expression in uterus, ovary and stomach. (6) Real-time RT-PCR analysis showed that in the skeletal muscle of Large White and Meishan pigs, the expression level of TCAP gene decreased with the increasing of days, and it was extremely high at newly- born period. At 60 days old it decreased sharply until 120 days when the decrease was no so apparent. During the two periods of 60 and 120 days, the expression level of TCAP gene in Large White pigs was higher than that in Meishan pigs.3. ACTA2: (1) The full coding sequence and partial genomic sequences of ACTA2 gene from Large White, Landrace, Meishan were isolated. (2) With the help of CLUSTALW,CDD,Signal P3.0,Prosite software, the structure and function of the coded protein were analyzed and predicted, the phylogenetic trees were constructed as well. (3) 27 potential SNPs were identified by sequence alignment. PCR-Hinf I-RFLP was developed to detect C1554T substitution in exon 2. In the different seven pig populations, the frequency of allele C in Large White was higher than that of allele T. The frequencies of allele C and T were the same in Meishan×Large White, while in all the other populations the frequency of allele C is higher than that of allele T. (4) Association analysis of markers and production traits found that the relation between ACTA2 gene and SP, SFT, FP, LMP, BFT, LEH, pH(BF), IMF are significant or highly significant. Compared with CC genotype, TT had a higher LMP, a lower FP and backfat thickness. (5) The tissue expression profile analysis showed that the expression level of ACTA2 gene was the highest in muscles, uterus and kidney, decreased in embryo, spleen, ovary, stomach and lung, lower in heart, and lowest in adipose tissues, liver. (6) Real-time RT-PCR analysis showed that in the skeletal muscle of Large White and Meishan pigs, the expression level of ACTA2 gene decreased with the increasing of days. And during each period, the expression level was higher in Meishan pigs than in Large White pigs.4. DUSP13: (1) Part of the eDNA sequence and partial genomic sequences of DUSP13 gene from Large White, Landrace, Meishan were isolated. (2) With the help of CLUSTALW,CDD,Signal P3.0,Prosite software, the structure and function of the coded protein were analyzed and predicted. (3) 16 potential SNPs were identified by sequence alignment. PCR-Hinf I-RFLP was developed to detect T302C substitution in intron 1. In all the different seven pig populations, the frequency of allele T was higher than that of allele C. (4) Association analysis of marker and production traits found that the relation between DUSP13gene and DP, LEH, LEW, RLF, FP, LMP, MM1, MM2, IMF are significant or highly significant. (5) The tissue expression profile analysis showed that the expression level of DUSP13 gene was the highest in muscles and ovary, decreased in kidney and lung, lower in embryo, uterus, heart, spleen, adipose tissues, liver, and lowest in stomach. (6) Real-time RT-PCR analysis was made in the skeletal muscle of different populations at different growing period. The results showed that in Large White pigs, the expression level ofDUSP13 gene was the highest at 1 day old, dropped sharply at 60 days old, and rose slightly at 120 days old. While in Meishan pigs, the expression level of DUSP13 gene decreased gradually with the increasing of days. Additionally, at 60 days old, the expression level of DUSP13 gene in Meishan pigs was higher than in Large White pigs and at 120 days old, it was higher in Large White pigs than in Meishan pigs.5. Single-marker analysis between markers and heterosis of traits: single-marker analysis of the heterosis of ten traits in LY＋YL and YM＋MY discovered the marker loci which were of great significance in both populations: SWR2516 (LEA), SW2408 and S0165 (SP), SW2185, SW2021 and SW1443 (LMP), SW18 79 (BWT), SW2185 (FMR), SW1879 (ABF) .6. Association analysis between Individual Heterozygosity (Hi) and heterosis of traits: in LY＋YL and YM＋MY, the results indicated that individual heterozygosity and BWT heterosis were significantly positively related. Moreover, a significant positive association between individual heterozygosity and ABF heterosis was also showed in LY＋YL. No significant association was demonstrated between individual heterozygosity and heterosis of other traits in these two populations, and these significant associations were both positive and negative.7. Heterosis diversity among different Hi gradations: analysis of the diversity among different Hi gradations was made in LY＋YL and YM＋MY. The results showed that in both populations, SP heterosis demonstrated significant diversity in different gradations of Hi. Additionally, BP, BWT, and DG heterosis were significantly diverse among different Hi gradations in YM＋MY. Most studied heterosis of traits in YM＋MY had the tendency to augment with the increase of Hi gradations.