No replicated association of the c.-312A > G in<i> EDG</i>1 with marbling in Niigata population of Japanese Black beef cattle

doi:10.4236/ojas.2013.34040

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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: [email protected]

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

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.

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