Vol.3, No.4, 269-272 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34040
No replicated association of the c.-312A > G in
EDG1 with marbling in Niigata population of
Japanese Black beef cattle
Bin Tong1, Narumi Fuke1, Yui Himizu1, Hiroyuki Katou2, Masato Hatano2, Takeshi Ohta3,
Hiroyuki Kose4, Takahisa Yamada1*
1Laboratory of Animal Genetics, Graduate School of Science and Technology, Niigata University, Niigata, Japan;
*Corresponding Author:
2Niigata Prefectural Headquarters, National Federation of Agricultural Cooperative Association, Niigata, Japan
3Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Takatsuki, Japan
4Department of Life Science, Division of Natural Sciences, International Christian University, Mitaka, Japan
Received 5 July 2013; revised 15 August 2013; accepted 1 September 2013
Copyright © 2013 Bin Tong et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Marbling, defined by the amount and the distri-
bution of intramuscular fat and measured as
beef marbling score (BMS), is an economically
important trait of beef cattle in Japan. We re-
cently rep orted that a single nucl eotide poly mor-
phism (SNP), namely, c.-312A > G, in the endo-
thelial differentiation sphingolipid G-protein-
coupled recepto r, 1 (EDG1) gene was associa ted
with the BMS level in the Japanese Black beef
cattle population of Oita prefecture, with the G
allele being associated with a high level of the
BMS. Thus, the c.-312A > G SNP seems to be a
candidate marker for marker-assisted selection.
In this study, we investigated whether this as-
sociation could be replicated in the Japanese
Black beef cattle population of Niigata prefec-
ture and analyzed the effect of the SNP geno-
types on the carcass traits other than the BMS.
No significant differences in the BMS level w ere
detected among the genotypes of the c.-312A >
G SNP in the Niigata Japanese Black beef cattle
population. The SNP genotype had no signifi-
cant effects on the carcass weight, rib eye area
and rib thickness of the cattle population. These
findings suggested that the association of the
c.-312A > G SNP with the BMS level in the Japa-
nese Black beef cattle population was not rep-
licated in the Niigata population, and revealed
no effects of the SNP genotype on the beef
productivity in the Niigata population. Thus, we
concluded that th e c.-312A > G SNP is not use ful
for effective marker-assisted selection to in-
crease meat quality and, additionally, meat pro-
ductivity in Japanese Black beef cattle of Niigata
prefecture.
Keywords: Association; EDG1; Japanese Black
Breed; Marbling; Replication Study; Single
Nucleotide Polymorphism
1. INTRODUCTION
Generally, marbling means the amount of intramuscu-
lar fat in musculus longissimus muscle [1]. In Japan,
marbling is characterized as the amount and distribution
of intramuscular fat in a cross section of musculus long-
issimus muscle, and called Shimofuri [1]. High levels of
such marbling improve the palatability and acceptability
of beef by affecting the taste and tenderness of the meat
[2-4]. Because of the impor tance of the presence of mar-
bling on the economics of beef production, there is great
interest in gaining a better understanding of the molecu-
lar architecture of marbling and in generating new op-
portunities for more effective marker-assisted breeding.
The endothelial d ifferentiation, sphingolipid G -protein
coupled receptor, 1 (EDG1) gene, involved in blood ves-
sel formation [5], has been previously shown to possess
higher expression levels in a high-marbled steer group
than in a low-marbled steer group in musculus longis-
simus muscle across all ages [6,7]. EDG1 is located
within the genomic region of a quantitative trait locu s for
marbling on chromosome 3 [7,8], and thus has been re-
garded as a positional functional candidate for the gene
responsible for marbling [9]. We have recently reported

B. Tong et al. / Open Journal of Animal Sciences 3 (2013) 269-272
270
that a single nucleotide polymorphism (SNP), c.-312A >
G in th e EDG1 was detected between the 2 steer groups,
and associated with marbling in the Japanese Black beef
cattle population in Oita prefecture, with the G allele of
the SNP being associated with a high level of the mar-
bling [9].
Thus, we have now investigated whether this associa-
tion could be replicated in the Japane se Black beef cattle
population of the Niigata prefecture and analyzed the
effects of the SNP genotypes on the carcass traits (car-
cass weight (CWT), rib eye area (REA), and rib thick-
ness (RT)) other than the beef marbling score (BMS), in
order to confirm the application of the c.-312A > G SNP
to effective marker-assisted selection in the Niigata Japa-
nese Black cattle population.
2. MATERIALS AND METHODS
2.1. Samples and Data
Japanese Black cattle population from the Niigata
prefecture were used, and we studied the association of
the c.-312A > G SNP with BMS, CWT, REA, and RT. In
this study, 130 paternal half-sib progeny steers (1 to 16
steers per sire) from 39 sires were used. Hair root speci-
mens of the progeny steers were collected for genotyping
the SNP. DNA samples were prepared from the materials
using DNeasy Blood & Tissue Kit (QIAGEN, Hilden,
Germany).
BMS, CWT, REA, and RT were measured according
to the Japanese meat grading system by certified graders
from the Japan Meat Grading Association (Tokyo, Japan)
[1]. The predicted breeding values of the BMS, CWT,
REA, and RT for the steers were used as the phenotypic
values in this study [10]. The breeding values were pre-
dicted using carcass records of Japanese Black steers and
heifers fattened in the Niigata prefecture, and obtained
from the Niigata Pre-fectural Headquarters, National Fe-
deration of Agricultural Cooperative Association (Niigata,
Japan). The animals slaughtered were part of the popula-
tion used in this study.
This study conformed to the guidelines for animal ex-
perimentation of the Graduate School of Science and Te-
chnology, Niigata University (Niigata, Japan).
2.2. SNP Genotyping
The c.-312 A > G SNP was genotyped by the PCR-re-
striction fragment length polymorphism method as de-
scribed previously [9]. Using this method, 378-bp PCR
fragments containing the SNP site were amplified and
Msc I-digested into 163- and 215-bp fragments at the A
allele, but not the G allele: the GG homozygotes, the AA
homozygotes and the AG heterozygotes yielded 1 band
(378 bp), 2 bands (163 and 215 bp) and 3 bands (163,
215, and 378 bp), respectively.
2.3. Statistical Analyses
Departures from the Hardy-Weinberg equilibrium
were tested for the SNP by chi-square test. Statistical
comparisons between the allele frequencies at the SNP in
the half-sib progeny steers or the frequencies estimated
from maternal alleles possessed by the steers in the Nii-
gata prefecture population and data from the Japanese
Black cattle population of the Oita prefecture, as well as
Japanese Brown, Japanese Short Horn, Holstein, or
Brown Swiss cattle populations [9,11] were also per-
formed by chi-square test. The effect of genotypes at the
SNP on the predicted breeding values for the BMS, CWT,
REA, and RT was analyzed with a model that included
the SNP genotype as a fixed effect and the sire as a ran-
dom effect. Statistical analysis was performed by the
MIXED procedures of the SAS program version 9 (SAS
Institute, Inc., Cary, NC).
3. RESULTS AND DISCUSSION
Genotyping the 130 paternal half-sib progeny steers
for the c.-312A > G SNP revealed 46 animals homozy-
gous for the A allele, 57 animals heterozygous for the A
allele and the G allele and 27 animals homozygous for
the G allele for the c.-312A > G SNP (Tab le 1). The ob-
served heterozygosity value at the SNP was in agreement
with the expected heterozygosity value, indicating that
the Niigata prefecture population conformed to the Har-
dy-Weinberg equilibrium. The frequency of the G allele
of c.-312A > G SNP in the Niigata prefecture population
(0.427) was higher than the frequencies of this allele in
Japanese Brown, Japanese Short Horn, Holstein, and
Brown Swiss cattle populations that have not been
strongly selected for high marbling [11], but lower than
the frequency in Japanese Black cattle population of the
Oita prefecture [9]. However, no statistically significant
difference was detected between the allele frequencies
estimated from maternal alleles possessed by the half-sib
progeny steers in the Niigata prefecture population and
those obtain ed in the Japanese Black cattle populatio n of
the Oita prefecture [9]. These frequencies were signifi-
cantly higher than those of Japanese Brown, Japanese
Short Horn, Holstein, and Brown Swiss cattle popula-
tions [11] (data not shown).
No statistically significant differences in BMS level
were detected among the genotypes (Table 2). These
Tab le 1. Frequencies of c.-312A > G SNP genotypes in Japa-
nese black cattle population of the Niigata prefecture.
Genotype No. of animals Frequency
AA 46 0.354
AG 57 0.438
GG 27 0.208

B. Tong et al. / Open Journal of Animal Sciences 3 (2013) 269-272 271
Tabl e 2. Effects of c.-312A > G SNP genotype on BMS, CWT,
REA, and RT in Japanese Black cattle population of the Niigata
prefecture.
Genotype2
Trait1 P-value AA AG GG
BMS 0.186 1.35 ± 0.06 1.37 ± 0.05 1.53 ± 0.08
CWT 0.157 40.60 ± 4.34 42.08 ± 3.90 53.65 ± 5.70
REA 0.546 6.45 ± 0.59 6.85 ± 0.53 7.52 ± 0.77
RT 0.133 0.53 ± 0.05 0.63 ± 0.04 0.67 ± 0.06
1BMS, Beef marbling score (unit); CWT, Carcass weight (kg), REA, Rib
eye area (cm2); RT, Rib thickness (cm); 2The breeding values are given as
least squares means ± SE.
results were not consistent with the data obtained in our
previous study in the Oita population [9]. These results
did not show replication for the association of the c.-
312A > G SNP with BMS. Based on no replicated asso-
ciation, we propose that the c.-312A > G SNP is in link-
age disequilibrium with not yet identified causative mu-
tation for BMS level in the EDG1 gene in the Oita pre-
fecture population, but not in the Niigata prefecture
population. There may be differences between the history
of these populations. The causative mutation may corre-
spond to a novel SNP, g.1471620G > T, in 5’ flanking
region of the EDG1 gene [12]. Alternatively, a tendency
towards significance for BMS was observed (P = 0.186)
and genotypic profiles of the predicted breeding value
for the BMS showed trends similar to data obtained in
our previous study in the Oita prefecture population [9]
(Ta bl e 2). Thus, our present study in the Niigata prefec-
ture population might not have enough power to detect
an association with the BMS.
The effect of the c.-312A > G SNP genotype was not
statistically significant for the CWT, REA, and RT (Ta-
ble 2). Thus, it is likely that the c.-312A > G SNP is not
associated with the CWT, REA, and RT in the Japanese
Black beef cattle population of the Niigata prefecture.
The marbling quantitative trait locus corresponding to
the genomic position of EDG1 on bovine chromosome 3
has shown a statistically significant effect on RT as well
as on BMS [13]. Based on no association, the marbling
quantitative trait locus was supposed to be distinct from
the quantitative trait locus responsible for RT level. Oth-
erwise, lack of linkage disequilibrium or insufficient de-
tection power might lead to no association of the c.-
312A > G SNP with the RT in the present study.
4. CONCLUSION
We concluded that the c.-312A > G SNP is not useful
for effective marker-assisted selection to increase meat
quality and, additionally, meat productivity in Japanese
Black beef cattle of Niigata prefecture. Thus, this study
will provide information on differences of populatio ns in
marker-assisted selection for Japanese Black beef cat-
tle.
5. ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research
(B) (no. 14360166) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and by the research funds of Japa-
nese Livestock Technology Association.
REFERENCES
[1] JMGA (1988) New beef carcass grading standards. Japan
Meat Grading Association, Tokyo.
[2] Busboom, J.R., Jeremiah, L.E., Gibson, L.L., Johnson,
K.A., Gaskins, C.T., Reeves, J.J. and Wright, R.W.Jr.
(1993) Effects of biological source on cooking and palat-
ability attributes of beef produced for the Japanese market.
Meat Scie nce, 35, 241-258.
http://dx.doi.org/10.1016/0309-1740(93)90054-L
[3] Boylston, T.D., Morgan, S.A., Johnson, K.A., Busboom,
J.R., Wright, R.W.Jr. and Reeves, J.J. (1995) Lipid con-
tent and composition of Wagyu and domestic breeds of
beef. Journal of Agricultural and Food Chemistry, 43,
1202-1207. http://dx.doi.org/10.1021/jf00053a015
[4] Matsuishi, M., Fujimori, M. and Okitani, A. (2001)
Wagyu beef aroma in Wagyu (Japanese Black cattle) beef
preferred by the Japanese over imported beef. Animal
Science Journal, 72, 498-504. https://www.jstage.jst.go.jp
[5] Liu, Y., Wada, R., Yamashita, T., Mi, Y., Deng, C.X.,
Hobson, J.P., Rosenfeldt, H.M., Nava, V.E., Chae, S.S.,
Lee, M.J., Liu, C.H., Hla, T., Spiegel, S. and Proia, R.L.
(2000) Edg-1, the G protein-coupled receptor for sphin-
gosine-1-phosphate, is essential for vascular maturation.
Journal of Clinical Investigation, 106, 951-961.
http://dx.doi.org/10.1172/JCI10905
[6] Sasaki, Y., Nagai, K., Nagata, Y., Doronbekov, K., Ni-
shimura, S., Yoshioka, S., Fujita, T., Shiga, K., Miyake, T.,
Taniguchi, Y. and Yamada, T. (2006) Exploration of genes
showing intramuscular fat deposition-associated changes
in musculus longissimus muscle. Animal Genetics, 37,
40-46.
http://dx. doi.o rg/10.1111/j .1365-2052.2005.01380.x
[7] Sasaki, Y., Yamada, T. and Miyake, T. (2006) Quantitative
and molecular genetic approaches for the improvement of
carcass traits in the Wagyu cattle. 8th World Congress on
Genetics Applied to Livestock Production, Session no.
13-01. Minas Centro Convention Center, Belo Horizonte,
Minas Gerais.
[8] Yamada, T., Taniguchi, Y., Nishimura, S., Yoshioka, S.,
Takasuga, A., Sugimoto, Y. and Sasaki, Y. (2006) Radia-
tion hybrid mapping of genes showing intramuscular fat
deposition-associated expression changes in bovine mus-
culus longissimus muscle. Animal Genetics, 37, 184-185.
http://dx. doi.o rg/10.1111/j .1365-2052.2006.01426.x
[9] Yamada, T., Itoh, M., Nishimura, S., Taniguchi, Y., Mi-
yake, T., Sasaki, S., Yoshioka, S., Fujita, T., Shiga, K.,
Morita, M. and Sasaki, Y. (2009) Association of single
nucleotide polymorphisms in the endothelial differentia-

B. Tong et al. / Open Journal of Animal Sciences 3 (2013) 269-272
272
tion sphingolipid G-protein coupled receptor 1 gene with
marbling in Japanese Black beef cattle. Animal Genetics,
40, 209-216.
http://dx. doi.o rg/10.1111/j .1365-2052.2008.01822.x
[10] Sasaki, Y., Miyake, T., Gaillard, C., Oguni, T., Matsumoto,
M., Ito, M., Kurahara, T., Sasae, Y., Fujinaka, K., Ohta-
gaki, S. and Dougo, T. (2006) Comparison of genetic
gains per year for carcass traits among breeding programs
in the Japanese Brown and the Japanese Black cattle.
Journal of Animal Science, 84, 317-323.
http://www.journalofanimalscience.org
[11] Watanabe, N., Yoshioka, S., Itoh, M., Satoh, Y., Furuta,
M., Komatsu, S., Sumio, Y., Fujita, Y., Yamada, T. and
Sasaki, Y. (2009) The G allele at the c.-312A>G SNP in
the EDG1 gene associated with high marbling in Japanese
Black cattle is at a low frequency in breeds not selected
for marbling. Animal Genetics, 40, 575-582.
http://dx. doi.o rg/10.1111/j .1365-2052.2009.01871.x
[12] Yamada, T., Sasaki, S., Sukegawa, S., Miyake, T., Fujita,
T., Kose, H., Morita, M., Takahagi, Y., Murakami, H.,
Morimatsu, F. and Sasaki, Y. (2009) Novel SNP in 5’
flanking region of EDG1 associated with marbling in
Japanese Black beef cattle. Animal Science Journal, 80,
486-489.
http://dx. doi.o rg/10.1111/j .1740-0929.2009.00665.x
[13] Takasuga, A., Watanabe, T., Mizoguchi, Y., Hirano, T.,
Ihara, N., Takano, A., Yokouchi, K., Fujikawa, A., Chiba,
K., Kobayashi, N., Tatsuda, K., Oe, T., Furukawa-Kuroi-
wa, M., Nishimura-Abe, A., Fujita, T., Inoue, K., Mizo-
shita, K., Ogino, A. and Sugimoto, Y. (2007) Identifica-
tion of bovine QTL for growth and carcass traits in Japa-
nese Black cattle by replication and identical-by-descent
mapping. Mammalian Genome, 18, 125-136.
http://dx.doi.org/10.1007/s00335-006-0096-5