Isolation, Imprinting Identification And Association With Production Traits Of Seven Candidate Imprinted Genes In Pigs

Genomic imprinting indicates that alleles from one parent are expressed, but allelesfrom the other parent are not expressed or express little in some tissues or cell types ofdiploid organism. Imprinted genes are genes that preferentially expressed from either thematernally inherited allele or the paternally inherited allele. The researches on genomicimprinting in mammals show that imprinted genes have important roles in the regulationof fetal growth, development, function of the placenta and postnatal behavior. At present,most studies on genomic imprinting are in human and mouse but little in livestock. Inpigs, there is no report on the identification of imprinted genes at the molecular level.Therefore, it is of interest to identify more imprinted genes in pigs for analyzing theconservation of genomic imprinting among different species. Most of the imprinted genesthat have been studied are regulatory: transcription factors, alternative splicer, tumorsuppressors, growth factors, or are involved in complex signal pathways. Because of theimportant regulatory function of imprinted genes on growth and development, we couldpredict that imprinted genes may affect growth and development largely in pigs. Whetherthe imprinted genes may be used as molecular mark in pigs' breeding is not known. Basedon above, our research includes two main aspects: first, analyzing the imprinted status of7 candidate imprinted genes with RT-PCR-RFLP or sequencing directly in the F₁ hybridsof Large White boars×Meishan sows and Meishan boars×Large White sows; second,PCR-RFLP analysis of 4 genes is used to detect the association of the polymorphism inimprinted genes and the carcass traits in the pigs of Large White×Meishan F₂ hybrids.Our results are as follows:1. We chose PLAGL1,PEG10,PPP1R9A,XIST,MEST,NAP1L5 and PEG3 genesas candidate imprinted genes in pigs according to the imprinted status of the genes inhuman and mouse and their biological functions. We obtained some sequences includesome complete ORF of the genes with electronic cloning and comparative genomictechnology. (1) PLAGL1, obtained cDNA sequence 2238bp, ORF (Open reading frame)1392bp, GenBank accession number DQ28899; (2) PEG10, obtained cDNA sequence6379bp, ORF 1212bp, GenBank accession number DQ779285; (3) PPP1R9A, obtainedcDNA sequence 1946bp, GenBank accession number EF619476; (4) XIST, obtainedcDNA sequence 1039b, GenBank accession number EF619477; (5) MEST, obtainedcDNA sequence 2167bp, ORF 981bp, GenBank accession number DQ EF619473; (6)NAP1L5, obtained cDNA sequence, GenBank accession number EF619474; (7) PEG3,obtained cDNA sequence 2052bp, GenBank accession number EF619475. Comparing thesimilarity between the cDNA sequences of the 7 genes and the corresponding homology in human and mouse show that the sequences similarities between human and pigs are allhigher than the sequences similarities between mouse and pigs.2. Aligning the cDNA sequences of the 7 genes between 3 Large White pigs and 3Meishan pigs revealed that: (1) PLAGL1, one SNP existed in coding region and one SNPexisted in the 3' UTR region, both of them are transition mutation. (2) PEG10, weidentified 4 SNPs in the 3' UTR region. Of 3 are transition mutation, 1 isinsertion/deletion mutation. (3) PPP1R9A, we identified 8 SNPs in the 3' UTR region. Of4 are transition mutation, 3 are transversion mutation, 1 is insertion/deletion mutation. (4)XIST, we identified 4 SNPs in the 3' UTR region. Of 3 are transition mutation, 1 isinsertion/deletion mutation. (5) MEST, two SNPs existed in the 3' UTR region, both ofthem are transition mutation. (6) NAP1L5, two SNPs existed in the 3' UTR region, one istransversion mutation and the other is insertion/deletion mutation. (7) PEG3, weidentified 3 SNPs in the 3' UTR region. Of 1 is transition mutation, 2 areinsertion/deletion mutation.3. Using the model of F₁ hybrids of Large White boar×Meishan sow and Meishanboar×Large White sow, we identified the imprinted status of the 7 genes in 14 differenttissues of 12 two-month pigs in the model through RT-PCR-RFLP analysis andsequencing directly. (1) PLAGL1gene is paternally expressed in skeletal muscle, fat, heart,liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary and testicle. (2) PEG10gene is paternally expressed in skeletal muscle, fat, heart, liver, spleen, lung, kidney,stomach, small intestine, uterus and ovary. (3) PPP1R9A gene escapes genomicimprinting in skeletal muscle, fat, heart, liver, spleen, lung, kidney, stomach, smallintestine, uterus, ovary and pituitary. (4) Xist gene is biallelically expressed in skeletalmuscle, fat, heart, liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary andpituitary. (5) MEST gene is biallelically expressed in liver, spleen and ovary, butpaternally expressed in skeletal muscle, fat, heart, lung, kidney, stomach, uterus, ovaryand pituitary. (6)NAP1L5 gene is paternally expressed in skeletal muscle, fat, liver,spleen, lung, kidney, stomach, small intestine, uterus, ovary and pituitary. (7) PEG3 geneis biallelically expressed in fat, heart, lung, stomach, small intestine and ovary, butpaternally expressed in skeletal muscle, liver, spleen, kidney and uterus. Although theimprinted status of the 7 genes is different in various tissues, they are relativelyconservative among pigs, human and mouse as a whole.4. Using PCR-RFLP, we detected 4 SNPs of 4 genes in different pig populations andobserved associations with traits in Large White and Meishan F₂ hybrids. The resultsshowed: (1) PLAGL1, C1428T-TaqI-RFLP is significant association with shoulder backfat thickness, internal fat ratio and biceps femoris pH (p＜0.05). (2) PEG10,C5274T-TaqI-RFLP is significant association with lion eye height and biceps femoris pH(p＜0.05), higher significant association with lion eye width (p＜0.01). (3) PPP1R9A,A1688C-Eco47I-RFLP is significant association with shoulder backfat thickness, buttockfat thickness and intramuscular fat (p＜0.05), higher significant association with averagebackfat thickness, carcass length, lion eye area and bone percentage (p＜0.01). (4) Xist,598-TCCATGG-Eco47I-RFLP is significant association with buttock fat thickness,average backfat thickness, intramuscular fat and water moisture (p＜0.05), highersignificant association with fat percentage, lean meat percentage, 6～7 rib fat thickness andthorax-waist fat thickness (p＜0.01) in the population of male pigs. But in the populationof female pigs, 598-TCCATGG-Eco47I-RFLP is significant association with fatpercentage, average backfat thickness and lion eye height (p＜0.05), higher significantassociation with buttock fat thickness and 6～7 rib fat thickness (p＜0.01). Three of the 4candidate genes are associated with fat deposition, which showed that imprinted genes inparticular Xist gene located on sexual chromosome are fit for the molecular breeding.