Analysis Of Imprinted Genes In Porcine Skeletal Muscle

Genomic imprinting is one of the most common epigenetic modification in mammals.That alleles from parents has a specificity modification during development, and as a resultonly one allele is expressed. Studies have shown that genomic imprinting plays a veryimportant role in the placental development, fetal growth, human genetic disease and cancer.Until now, most studies about genomic imprinting have been focused on human and mice,in pig, although more imprinted genes are identified but still relatively less. Thus,identification of novel imprinted genes in pigs is required for analyzing the conservationbetween species, the function of imprinted genes as well as livestock improvement andbreeding.In this study,“Laiwu-Landrace reciprocal cross model” is used for imprintingidentification. The method is called “expressed SNP identification (cSNP)”, in whichRT-PCR, RFLP analysis and PCR direct sequencing will be used. The purpose of this study isto identify candidate imprinted genes in piglet skeletal muscle and with the hope of exploringthe characteristics and conservation between species, and illustrating the skeletal musclerelated mechanism in epigenetic perspective. Results are listed as follows:(1) Porcine SNRPN cDNA sequence of1304bp is cloned, which includes the completeCDS of a total of723bp encoding240amino acids; and porcine UBE3A cDNA sequence of2907bp is cloned, which includes the complete CDS of a total of2628bp encoding875aminoacids. For the NECD, MKRN3, SNRPN, UBE3A and OSBPL5genes phylogenetic tree wereconstructed based on amino acid sequences acquired from NCBI and Enseml data base. Theresult shows that all the five genes are reasonably conservative, and the highest homology isbetween pig and cattle, followed by pig and primates, birds, reptiles and fish are lessconservative.(2) cSNP discovery: Name, location, and mutations are as follows: the SNRPN geneg.402T>C; NECD genes g.263C>G; UBE3A gene g.340A>G; MKRN3gene g.293G>C, g.361A>G, g.678C>T, g.1376T>C and g.1407A>T; OSBPL5gene g.1873>A. Imprintinganalysis of SNRPN and NECD genes in piglet skeletal muscle shows that both the two genesare maternal imprinted, while the OSBPL5, UBE3A and MKRN3genes are not imprinted. For OSBPL5gene, maternal allele expression is approximately three times of the paternalexpression. For MKRN3gene, the allele from Laiwu pig is always expressed, which requiresfuther study.(3) A CpG island is found in MKRN3gene upstream, and in OSBPL5gene distalpromoter there is also a CpG island. Methylation analysis of the two CpG islands illustratesthat both of the two regions showing high DNA methylation which may be associated withlow expression levels of these genes and theMKRN3gene5’-CpG methylation is associatedwith its expression, but not completely.(4) Analysis of the distribution of OSBPL5gene g.1873G>A mutation in pig populationsshowed that GG type was the dominant genotype in Duroc and Junmu No.1populations andAG heterozygous type is the dominant genotype in Laiwu pigs. However, the result showsLaiwu pig population is not in Hardy-Weinberg equilibrium. Traits correlation analysis showsg.1873is significantly associated with litter size and circumference of cannon bone (P<0.01).In summary, we analyzed imprinting status of five genes (SNRPN, NECD, MKRN3,UBE3A and OSBPL5) by using Laiwu-Landrace reciprocal cross F1piglets. The result showsboth SNRPN and NECD gene are maternally imprinted in piglet skeletal muscle, whileUBE3A and OSBPL5are not, and for MKRN3the allele from Laiwu pig is always expressed.This study also showed that g.1873G>A mutation of OSBPL5gene is related to pig litter sizeand circumference of cannon bone.