Genetic variation at the porcine MYF-5 gene locus. Lack of association
with meat production traits
M.F.W. te Pas, F.L. Harders, A. Soumillion, L. Born, W. Buist, T.H.E. Meuwissen
DLO-Institute for Animal Science and Health (ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands
Received: 9 July 1998 / Accepted: 22 September 1998
Abstract. The number of muscle fibers at birth appears to deter-
mine the maximal lean meat growth capacity in pigs and in cattle.
Development of muscle fibers is regulated by the MyoD gene
family consisting of MyoD1, myf-5, myf-6, and myogenin. Myf-5 is
expressed in proliferating myoblasts. Here we report the genomic
sequence of the porcine myf-5 gene with three microsatellites and
two RFLPs located close to the coding sequences. Two of the
microsatellites are located in the promoter region. The allelic dis-
tribution differs between breeds and selection lines. In two GY
selection lines, 1216 pigs of two-generation families were geno-
typed for the HinfI RFLP, which was segregating in the GY breed.
The other polymorphic loci are physically linked to this RFLP
locus, and therefore the results can be extrapolated to these loci.
Statistical analysis revealed no association with birth weight,
growth rate, weight at slaughter age, carcass meat weight, and
backfat thickness. Thus, in this study myf-5 did not explain genetic
variation in meat (muscle) development in pigs.
Breeding in meat-producing animals focuses on growth rate and
lean meat deposition. In pigs, the number of muscle fibers appears
to determine the maximal lean meat growth capacity (Handel and
Stickland 1984, 1988). Meat production capacity in cattle seems
also to be determined by the number of myocytes, since double-
muscled cattle show a higher number of myofibers than other
cattle (Swatland and Kiefer 1974; Hanset et al. 1982). Mammalian
myofiber formation is a strictly embryonic process, regulated by
the MyoD gene family. The MyoD gene family consists of four
structurally related genes, MyoD1, myogenin, myf-5, and myf-6.
These genes encode basic helix-loop-helix (bHLH) proteins that
are involved in muscle cell determination and differentiation by regu-
lating the expression of muscle differentiation-stage-specific genes
(for extensive reviews see Olson 1990; Weintraub et al. 1991; Lyons
and Buckingham 1992; Rudnicki and Jaenisch 1995; Ordahl and
Williams, 1998). Myf-5 and MyoD1 are expressed during prolifera-
tion of myoblasts, myogenin is expressed during terminal differentia-
tion, and myf-6 is mainly expressed during postnatal life.
Both myoblast proliferation rate and length of time, and timing
of the onset of differentiation potentially influence the number of
muscle fibers formed, and thus maximal lean meat growth capacity
(Te Pas and Visscher 1994). Variation in myoblast proliferation
rate has been observed in QL mice (Penney et al. 1983), and
MyoD1 polymorphisms have been associated with muscle repair
processes (Kay et al. 1993), while delayed onset of differentiation
related to myofiber hyperplasia has been observed in quail
(Coutinho et al. 1993). Thus, we studied the MyoD genes to un-
derstand their role in porcine meat development. Previously, we
reported the chromosomal localization of the myf-5 gene (Soumil-
lion et al. 1997a). Here we report the porcine myf-5 genomic
sequence, locus-specific polymorphisms, and association with
muscle development-related meat production traits.
Materials and methods
Cloning and sequencing of the porcine myf-5 gene.
The screening of
a porcine genomic DNA library (Soumillion et al. 1997b) yielded four myf-
5-positive-phage clones. The clones were identified by PCR (Soumillion et al.
1997a). Phage insert DNA was amplified with Expand-PCR (Boehringer
Mannheim, Mannheim, Germany) and standard PCR technology (Sambrook
et al. 1989). PCR products were digested with PvuII and EcoRI according to
the manufacturer’s recommendations, and myf-5-containing fragments were
identified by use of previously reported PCR and partial porcine myf-5 se-
quence information (Soumillion et al. 1997a, X95702), and subcloned. Sub-
cloning was performed in pUC18 and pT7blue plasmids. Clones were se-
quenced twice by standard molecular biology technology (Sambrook et al.
1989) and an automated sequencer, ABI373A (Applied Biosystems, Foster
City, Calif., USA). Also, several genomic DNA PCR reaction products were
cloned and sequenced. BLAST searches were done to investigate the sequence
identity to the homologous gene of other species.
Southern blotting and RFLP analysis of porcine genomic
Southern blotting and RFLP analysis was performed as described
before (Soumillion et al. 1997b). Genomic DNA was digested with 100 U
PvuII (Boehringer Mannheim) for 16 h at 37°C.
Detection of PCR-RFLP and microsatellite polymorphisms.
PCR-RFLP test for the polymorphic HinfI site amplified a 322-bp product
with the primers 5Ј-CTCCGAATTAGTGTGGCTTC-3Ј (forward) and 5Ј-
GTTCTTTCGGGACCAGACAGG-GCTG-3Ј (reverse), anneal temperature
60°C. Ten microliter of the PCR product was digested with 10 U HinfI
according to the manufacturer’s recommendations and analyzed by agarose
gel electrophoresis. The PCR-RFLP is bi-allelic showing either undigested
PCR product (322 bp, allele A), or 170 bp and 143 bp digestion products
Microsatellite (MS) sequences were identified by sequencing and am-
plified as follows: MS-promoter (closest to the coding sequences) 5Ј-
CACAGAGCCTACAAAACGGCAGGC-3Ј (forward primer); 5Ј-
GGGTGTCACAGGCCAAAGGAACG-3Ј (reverse primer), anneal tem-
perature 56°C; MS-intron2: 5Ј-TACCGAGGGTGCTTTTCCACTCC-3Ј
(forward primer); 5Ј-GCAATCCAGGCTGGATAAGGAGC-3Ј (reverse
primer), anneal temperature 58°C. The use of Amplitaq Gold (Perkin
Elmer Cetus, Norwalk, Conn.) is needed for the amplification of the second
microsatellite PCR. The amplification products from 332 to 344 bp and 247
to 259 bp length, respectively, were analyzed on the ABI373A sequencer
with the genescan and genotyper (Applied Biosystems) software.
Animals and data collection.
Yorkshire pigs from two commercial sire
selection lines were used in this association study. Parents were genotyped
for the HinfI polymorphism, and crosses between heterozygous and be-
tween heterozygous and homozygous animals were made, of which the
Correspondence to: M.F.W. te Pas
The nucleotide sequence data reported in this paper have been submitted to
the EMBL database and have been assigned the accession number Y17154.
Mammalian Genome 10, 123–127 (1999).
© Springer-Verlag New York Inc. 1999