Expression of myogenes in <i>longissimus dorsi</i> muscle during prenatal development in commercial and local Piau pigs

Reis, Evelyze Pinheiro dos; Paixão, Débora Martins; Brustolini, Otávio José Bernardes; Silva, Fabyano Fonseca e; Silva, Walmir; Araújo, Flávio Marcos Gomes de; Salim, Anna Christina de Matos; Oliveira, Guilherme; Guimarães, Simone Eliza Facioni

doi:10.1590/1678-4685-GMB-2015-0295

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Animal Genetics • Genet. Mol. Biol. 39 (4) • Oct-Dec 2016 • https://doi.org/10.1590/1678-4685-GMB-2015-0295 copy

Expression of myogenes in longissimus dorsi muscle during prenatal development in commercial and local Piau pigs

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

This study used qRT-PCR to examine variation in the expression of 13 myogenes during muscle development in four prenatal periods (21, 40, 70 and 90 days post-insemination) in commercial (the three-way Duroc, Landrace and Large-White cross) and local Piau pig breeds that differ in muscle mass. There was no variation in the expression of the CHD8, EID2B, HIF1AN, IKBKB, RSPO3, SOX7 and SUFU genes at the various prenatal ages or between breeds. The MAP2K1 and RBM24 genes showed similar expression between commercial and Piau pigs but greater expression (p < 0.05) in at least one prenatal period. Pair-wise comparisons of prenatal periods in each breed showed that only the CSRP3, LEF1, MRAS and MYOG genes had higher expression (p < 0.05) in at least one prenatal period in commercial and Piau pigs. Overall, these results identified the LEF1 gene as a primary candidate to account for differences in muscle mass between the pig breeds since activation of this gene may lead to greater myoblast fusion in the commercial breed compared to Piau pigs. Such fusion could explain the different muscularity between breeds in the postnatal periods.

Keywords
commercial line; gene expression; myogenesis; pig; Piau breed

Gene	Accession number	Primer sequences (5’ → 3’)	Amplicon size¹ 1 Amplicon size in nucleotide number,
CHD8	XM_003482263.1	F: AGTGAGGACGAGAAGGAAGA	104
		R: GGGAATCCATCTTGGGACATAG
CSRP3	NM_001172368.1	F: CAGCAACCCTTCCAAGTTCA	91
		R: CATCACCTTCTCAGCAGCATAG
EID2B	XM_003127131.1	F: CGCCACTATCTGGAACACTAC	122
		R: CGCTGATATTCGGCATCAAAC
HIF1AN	XM_003359328.1	F: GTACTGGTGGCATCACATAGAG	118
		R: CTGATGGGCTTTGAGAGGATATT
IKBKB	NM_001099935.1	F: GATGGCGACAGTCAGGAAAT	107
		R: TTGCAAACCACCGTCTTACT
LEF1	NM_001129967.1	F: CTATTGTAACGCCTCAGGTCAA	99
		R: TTGGCTCTTGCTCCTTTCTC
MAP2K1	NM_001143716.1	F: GGAGCTGGAGCTGATGTTT	110
		R: GTCGGCTGTCCATTCCATAA
MRAS	XM_003358570.2	F: GGTCGATTTGATGCATTTGAGG	96
		R: TCCTTGGCACTGGTTTCTATG
MYOG	NM_001012406.1	F: CAGGCTCAAGAAGGTGAATGA	118
		R: GCACTCGATGTACTGGATGG
RBM24	XM_001925447.3	F: TACCTGCCCACTATGTCTATCC	118
		R: GCAGCTCCCGTGTAATCAAT
RSPO3	NM_032784.4	F: GAAACACGGGTCCGAGAAATA	110
		R: CCCTTCTGACACTTCTTCCTTT
SOX7	XM_003359052.1	F: TCTCCACTCCAACCTCCA	120
		R: TCATTGCGATCCATGTCCTC
SUFU	XM_001928912.4	F: GGAGCCCTCATTCCTCTTTG	83
		R: GCCATGTCACCTGTGATACTT
ACTB² 2 Reference gene. CHD8 – chromodomain helicase DNA binding protein 8, CSRP3 – cysteine and glycine-rich protein 3, EID2B – EP300 interacting inhibitor of differentiation 2B, HIF1AN – hypoxia inducible factor 1, α subunit inhibitor, IKBKB - inhibitor of κ light polypeptide gene enhancer in B-cells, kinase β, LEF1 – lymphoid enhancer-binding factor 1, MAP2K1 – mitogen-activated protein kinase kinase 1, MRAS – muscle RAS oncogene homolog, MYOG – myogenin (myogenic factor 4), RBM24 – RNA binding motif protein 24, RSPO3 – R-spondin 3, SOX7 – SRY (sex determining region Y)-box 7, SUFU – suppressor of fused homolog, ACTB – β-actin, GAPDH – glyceraldehyde-3-phosphate dehydrogenase, HPRT1 – hypoxanthine phosphoribosyltransferase 1.	XM_003124280.3	F: AAGATCAAGATCATCGCGCCTCCA	108
		R: ACTCCTGCTTGCTGATCCACATCT
GAPDH² 2 Reference gene. CHD8 – chromodomain helicase DNA binding protein 8, CSRP3 – cysteine and glycine-rich protein 3, EID2B – EP300 interacting inhibitor of differentiation 2B, HIF1AN – hypoxia inducible factor 1, α subunit inhibitor, IKBKB - inhibitor of κ light polypeptide gene enhancer in B-cells, kinase β, LEF1 – lymphoid enhancer-binding factor 1, MAP2K1 – mitogen-activated protein kinase kinase 1, MRAS – muscle RAS oncogene homolog, MYOG – myogenin (myogenic factor 4), RBM24 – RNA binding motif protein 24, RSPO3 – R-spondin 3, SOX7 – SRY (sex determining region Y)-box 7, SUFU – suppressor of fused homolog, ACTB – β-actin, GAPDH – glyceraldehyde-3-phosphate dehydrogenase, HPRT1 – hypoxanthine phosphoribosyltransferase 1.	NM_001206359.1	F: ACAGTCTTCTGGGTGGCAGTGAT	176
		R: CATGTTTGTGATGGGCGTGAACAA
HPRT1² 2 Reference gene. CHD8 – chromodomain helicase DNA binding protein 8, CSRP3 – cysteine and glycine-rich protein 3, EID2B – EP300 interacting inhibitor of differentiation 2B, HIF1AN – hypoxia inducible factor 1, α subunit inhibitor, IKBKB - inhibitor of κ light polypeptide gene enhancer in B-cells, kinase β, LEF1 – lymphoid enhancer-binding factor 1, MAP2K1 – mitogen-activated protein kinase kinase 1, MRAS – muscle RAS oncogene homolog, MYOG – myogenin (myogenic factor 4), RBM24 – RNA binding motif protein 24, RSPO3 – R-spondin 3, SOX7 – SRY (sex determining region Y)-box 7, SUFU – suppressor of fused homolog, ACTB – β-actin, GAPDH – glyceraldehyde-3-phosphate dehydrogenase, HPRT1 – hypoxanthine phosphoribosyltransferase 1.	NM_001032376.2	F: GCTGACCTGCTGGATTACAT	101
		R: CTGGTCATTACAGTAGCTCTTCAG

		Gene ontologies
Gene	Metabolic pathway	Cellular component	Molecular function	Biological process
CHD8	WNT signaling pathway	Nuclear lumen	Histone and DNA binding	Embryo development
CSRP3	–	Cytoskeleton	Contractile fiber	Muscle organ development/Muscle structure development/Muscle system process
EID2B	–	Nucleus	Muscle differentiation	Muscle organ development
HIF1AN	–	Nucleus/cytosol	Protein binding	Muscle organ development/Muscle structure development
IKBKB	MAPK signaling pathway	Nucleus/cytosol	Protein binding	Muscle system process/Musculoskeletal movement/Skeletal muscle contraction
LEF1	WNT signaling pathway	Nucleus	Chromatin and DNA binding	Embryo development
MAP2K1	MAPK signaling pathway	Cytoskeleton/cytosol/nucleus	Protein kinase activity	Muscle system process
MRAS	MAPK signaling pathway	Intracellular	GTPase activity/nucleotide binding	Muscle organ development/Organ development
MYOG	–	Nucleus	Chromatin and DNA binding	Muscle organ development/Muscle structure development
RBM24	–	Nucleus/cytoplasm	Nucleotide binding	Muscle organ development/Organ development
RSPO3	–	Extracellular region	Receptor binding	Embryo development
SOX7	–	Nucleus/cytoplasm	Nucleic acid binding	Embryo development
SUFU	Hedgehog signaling pathway	Nucleus/cytoplasm	Transcription corepressor activity	Embryo development/Organ development

Genes	Factors
Genes	Breed	Period	Breed x Period
CHD8	0.9764	0.3615	0.6094
CSRP3	0.8615	< 0.0001	< 0.0001
EID2B	0.9072	0.1615	0.4284
HIF1AN	0.5757	0.2535	0.5533
IKBKB	0.7473	0.6656	0.6948
LEF1	0.9772	< 0.0001	0.0004
MAP2K1	0.4445	0.0125	0.0712
MRAS	0.6557	0.0019	0.0205
MYOG	0.7314	< 0.0001	< 0.0001
RBM24	0.9866	0.0284	0.1756
RSPO3	0.7198	0.0885	0.2230
SOX7	0.3451	0.4870	0.6915
SUFU	0.6504	0.2974	0.4175

Genes	Comparisons
Genes	21d x 40d^a a 21d, 40d, 70d and 90d indicate the prenatal ages. Values in bold were statistically significant (p < 0.05).	21d x 70d	21d x 90d	40d x 70d	40d x 90d	70d x 90d
MAP2K1	0.0027	0.0772	0.1882	0.1177	0.0456	0.6143
RBM24	0.0192	0.0165	0.0188	0.9412	0.9917	0.9495

Genes	Breed	Comparisons
Genes	Breed	21d x 40d^a a 21d, 40d, 70d and 90d indicate the prenatal ages. Values in bold were statistically significant (p < 0.05) by Student's t-test.	21d x 70d	21d x 90d	40d x 70d	40d x 90d	70d x 90d
CSRP3	Commercial	0.0032	0.0003	0.0003	0.2524	0.3070	0.8964
	Piau	0.0309	0.0029	0.0035	0.2719	0.3062	0.9365
LEF1	Commercial	0.5880	0.0049	0.0058	0.0155	0.0183	0.9356
	Piau	0.0257	0.0045	0.0007	0.4091	0.1052	0.3973
MRAS	Commercial	0.0575	0.8639	0.3663	0.0412	0.0089	0.4609
	Piau	0.1992	0.3261	0.1414	0.0318	0.0107	0.6007
MYOG	Commercial	0.0026	0.0002	0.0012	0.2565	0.7099	0.4366
	Piau	0.0001	0.0006	0.0004	0.4341	0.5396	0.8629

Sociedade Brasileira de Genética Rua Cap. Adelmio Norberto da Silva, 736, 14025-670 Ribeirão Preto SP Brazil, Tel.: (55 16) 3911-4130 / Fax.: (55 16) 3621-3552 - Ribeirão Preto - SP - Brazil
E-mail: editor@gmb.org.br

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[1] Send correspondence to Evelyze Pinheiro dos Reis. Departamento de Zootecnia, Universidade Federal de Viçosa (UFV), Avenida Peter Henry Rolfs, Viçosa, 36570-000, MG, Brazil. E-mail: evelyzepinheiro@yahoo.com.br