Multi-Gene Marker Assisted Selection And Study In Molecular Biology Of Porcine Lbx Gene Family

With the development of molecular and quantitative genetics and its related subjects, it made a great progress on the research about animal genetic marker assisted selection (MAS). We have known that polymorphism of MC4R (Melanocortin-4 Receptor), H-FABP (Heart FABP), LEP (Leptin), HAL (Halothane), CAST (calpastatin) and MyoG (myogenin) genes associated relation with meat quality in recent research. PCR-RFLP was used to analyze these polymorphisms in a swine breed composite (DIV2) and 7 swine breeds. The implications of the allelic distribution of these genes for meat production, and applicability of molecular markers in pig breeding were discussed. In all animals investigated so far, both the protostome genes and the vertebrate Lbx genes (Ladybird-like genes) were found to play crucial roles in muscle and neural development. As candidate genes of animal growth and meat quality, the porcine Lbx1 and Lbx2 were cloned and identified and genetics effect were analyzed. DNA methylation is an important epigenetic system. It plays many crucial roles in the gene regulation. In this study we investigated whether DNA methylation regulates the expression of the Lbx1 and Lbx2 genes in porcine skeletal muscle. In addition, we analyzed the DNA methylation pattern of four genes (MyoD, Myf5, Pax7 and Lbx1) in myogenic differentiation in C2C12 cells. The results are summarized as follows:1 PCR-RFLP was used to analyze the polymorphisms of MC4R, LEP, H-FABP, HAL, CAST, MyoG genes in a swine breed composite (DIV2) and 7 swine breeds. The association study of these polymorphisms with several economic traits was carried out on a DIV2 population. The results obtained showed that MC4R/TaqI genotype had an effect for average backfat thickness (P<0.05) and lean meat percentage (P<0.05); At locus LEP/HinfI animals of AA genotype had lower test daily gain than that of BB (P<0.01) or AB genotype (P<0.05); At the H-FABP/Haeâ…¢locus lean meat percentage of the individuals with genotype DD were higher than that with genotype dd (P<0.05); At locus CAST/MspI animals of FF genotype had lower test daily gain than that of EF(P<0.01) or EE genotype (P<0.05).2 Linkage disequilibrium analysis among MC4R, LEP and H-FABP revealed that these genes were independent. Therefore, exerting selection pressure on one locus should not influence the allelic frequencies of the other locus. This represented two or more genes that could be combined together within one genotype in order to facilitate breeding for objective traits.3 We obtained the full coding region of the 2 genes with electronic cloning technology. (1) The cDNA sequence of porcine Lbx1 contains an ORF of 846 bp encoding a protein of 281 residues. (2) The cDNA sequence of porcine Lbx2 contains an ORF of 591 bp encoding a protein of 196 residues. Phylogenetic trees were constructed by aligning the amino acid sequences of different species. The porcine protein sequence of Lbx1 has high sequence similarity with Bos taurus, Mus musculus, Homo sapiens, Canis familiaris, Pan troglodytes than with other species, and the porcine protein sequence of Lbx2 has high sequence similarity with Bos taurus, Pongo abelii, Canis familiaris than with other species.4 RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues, Lbx2 was lowly expressed in liver, kidney and spleen. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. The percentage of differentiating satellite cells was highest in 1-week-old pigs and significantly decreased in 7-week-old pigs. It is very interesting that the expression of the porcine Lbx1 gene in skeletal muscle shows the same trend of a higher expression in early postnatal porcine growth. Therefore, we deduce that Lbx1 gene may have a relationship with postnatal hyperplastic mechanisms. Lbx1 gene expressed at higher levels in bicepsfemoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Thus, our study indicates that there is no direct relationship between the Lbx1 transcription level and type of muscle fiber.5 Bisulfite sequencing (BS) technique was used to analyze the specific methylation on promoter and exon 1 regions of Lbx1 and Lbx1 genes in longissimus dorsi muscle. (1) Lbx1, the differentially methylated region was found in a CpG island of the exon 1. (2) Lbx2, the differentially methylated region was outside of the CpG island. These results suggested that the high DNA methylation in CpG island might be involved in the down regulation of Lbx1 in longissimus dorsi muscle.6 Single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.-1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P<0.05) and internal fat rate (P<0.05). The g.752A>G does not affect the encoded protein structure. It means that a direct effect of this polymorphism is unlikely. Our results suggest that the associations are caused by linkage disequilibrium with QTLs for these traits.7 RT-PCR analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. In addition, the C2C12 cells do not express endogeneous Lbxl. In myogenic differentiation in C2C12 cells, the changes of promoter and exon 1 methylation of MyoD, Myf5, Pax7 and Lbxl genes were observed. These results suggested the methylation of these genes is a dymamic process, which play a dominant role in regulating gene expression for development of muscle.