A novel single-nucleotide polymorphism of the visfatin gene and its associations with performance traits in the chicken.

Abstract

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.

Full text

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Introduction
A variety of adipocytokines and peptides secreted
from adipocytes have been considered to play a
crucial role in obesity, insulin resistance, and
type 2 diabetes. Visfatin has been recently identi-
fied as a novel adipokine, which was known as a
pre-B-cell colony-enhancing factor (PBEF1) and
demonstrated to bind insulin receptor (Fukuhara
et al. 2005; Hug and Lodish 2005). Previous stud-
ies suggested that genetic variations of the visfatin
gene (also called the PBEF1 gene) possibly had a
minor effect on visceral and subcutaneous visfatin
mRNA expression and parameters of glucose and
insulinmetabolism(Böttcheretal.2006).Thebio-
logicalroleofvisfatinremainsunconfirmed,since
subsequent studies failed to repeat some original
observations. Previous studies did not answer the
expectation of the associations between visfatin
and the parameters of insulin sensitivity as well as
between visceral fat mass and plasma visfatin in
humans (Berndt et al. 2005; Pagano et al. 2006).
The genetic variants of the visfatin gene in chil-
dren were not likely to contribute significantly to
the variation in body mass index (BMI) and in the
metabolism of glucose, insulin, and lipids (Körner
et al. 2007). Furthermore, there was no significant
changeofvisfatinmRNA
adipocytes from obese WOKW rats compared
with lean Dark Agouti rats (Kloting N and Kloting
I 2005). On the other hand, the porcine visfatin
gene and protein expression measured in fat tissue
abundancein
J Appl Genet 51(1), 2010, pp. 59–65
Original article
A novel single-nucleotide polymorphism of the visfatin gene and
its associations with performance traits in the chicken
R-L. Han1,2, X-Y. Lan1, L-Z. Zhang1, G. Ren1, Y-J. Jing1, M-J. Li1, B. Zhang1, M. Zhao1, Y-K. Guo1,
X-T. Kang2, H. Chen1,3
1College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for
Agriculture, Yangling, Shaanxi 712100, P. R. China
2College of Livestock Husbandry and Veterinary Engineering, Henan Agricultural University, Henan Research Center of Breeding
Resources for Poultry, Zhengzhou, 450002, P. R. China
3Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou 221116, P. R. China
Abstract.Visfatinisapeptidethatispredominantlyexpressedinvisceraladiposetissueandishypothesizedtobe
related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was
found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymor-
phism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2resource population of Gushi chicken
crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with
chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found be-
tween the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting
length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density
(BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the
polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.
Keywords: association analysis, chicken, forced restriction fragment length polymorphism, single-nucleotide
polymorphism, visfatin.
Received: December 12, 2008. Revised: June 15, 2009. Accepted: October 11, 2009.
Correspondence: H. Chen, College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory
of Molecular Biology for Agriculture, Yangling, Shaanxi 712100, P. R. China; e-mail: chenhong1212@263.net

Page 2

is negatively correlated with subcutaneous, vis-
ceral, and total body fat tissue weights (Palin
2008). It is still unknown whether visfatin expres-
sion and genetic variation are associated with the
development of obesity or glucose and insulin pa-
rameters.
The visfatin gene is composed of 11 exons,
spanning34.7kb.Severalmutationsofthevisfatin
gene have been found in humans (Böttcher et al.
2006). No polymorphism of the visfatin gene has
been reported so far in the chicken. In this study
we report on a novel mutation in the chicken
visfatingeneandanalyzeitseffectonchickenper-
formance traits.
Materials and methods
Resource populations
An F2 resource population was generated from
Gushi (G) chicken (24 hens and 2 roosters), repre-
senting a slow-growing Chinese native chicken
breed, and Anka (A) broilers (12 hens and 4 roost-
ers), representing a fast-growing broiler. The F2
populationconsisted
(A-roosters mated with G-hens) and 3 reciprocal
families (G-roosters mated with A-hens). To build
the F2population, 9 F1females were selected from
eachof7families(6unrelatedroosterfamiliesand
1halfsib).The63F1femaleswerematedwith7F1
males from 7 families. Over 2 hatches, occurring
attwoweekintervals,theresourcepopulationwas
established. It included 42 grandparents, 70 F1
parents, and 860 F2chickens (Figure 1). All chick-
ens were managed in cages, according to the na-
tional standard.
of4crossfamilies
Measurement of phenotypes
Growth traits analyzed in this study included body
weight and body size indices including breast
bone length (BBL) and body slanting length
(BSL). Body weight was individually measured
every 2 weeks from birth to slaughter, while BBL
an BSL were measured every 4 weeks.
In total 836 F2chickens were slaughtered at the
age of 84 days. The following carcass traits were
measured then: carcass weight (CW), semi-evis-
cerationweight(SEW),eviscerationweight(EW),
breast muscle weight (BMW), leg muscle weight
(LMW), fat bandwidth (FBW), and skin fat thick-
ness (SFT).
Additionally, meat quality traits were mea-
sured, including breast muscle water loss rate
(BWLR), leg muscle water loss rate (LWLR),
breast muscle fiber density (BFD), and leg muscle
fiber density (LFD). The final animal numbers
were different for some traits because of failing to
measure or record them.
DNA samples and polymerase chain reaction
(PCR) conditions
Genomic DNA samples were extracted from 836
F2chickens by the phenol-chloroform method. To
amplifythe 372-bpfragmentincludingexon7and
its flanking region of the visfatin gene, one primer
pair (F1: 5’-TCT GTT AGG AGA TGC AGA
ACG TG-3’; and R1: 5’-CAG CCA GTC GTT
ACA AAA CAA AC-3’) was designed according
to a previously reported gene sequence of the
chicken visfatin gene (NC_006088). The 25-?L
volumecontained50ngofgenomicDNA,0.5?M
of each primer, 1 × buffer (including 1.5 mM
MgCl2), 200 ?M dNTPs (dATP, dTTP, dCTP and
dGTP) and 0.625 units of Taq DNA polymerase
(MBI, Vilnius, Lithuania). The cycling protocol
was as follows: 5 min at 95oC, 34 cycles of dena-
turation at 94oC for 35 s, annealing at 67oC for 35
s, extension at 72oC for 40 s, with a final extension
at 72oC for 10 min.
Detection of the novel single-nucleotide
polymorphism (SNP) of the visfatin gene
All PCR amplifications were subjected to sin-
gle-stranded conformation polymorphism (SSCP)
analysis. Aliquots of 5 ?L of PCR products were
mixed with 5 ?L of denaturing solution (95%
formamide, 25 mM EDTA, 0.025% xylene–
cyanole, and 0.025% bromophenol blue), then the
mixturewasheatedfor10minat98°C,andchilled
60R-L. Han et al.
Figure 1. The F2 designed resource population’s
construction. Black = Anka broiler; white = Gushi;
square = male; circle = female.

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on ice. Next, 7 ?L of the mixture was subjected to
polyacrylamide gel electrophoresis (PAGE) pat-
terns (80 mm × 73 mm × 0.75 mm) in 1 × TBE
buffer and constant voltage (200 V) for 2.0 h. The
gel was stained with 0.1% silver nitrate (Lan et al.
2007). In case of existing polymorphism, the PCR
products showed different electrophoresis pat-
terns, which were sequenced by ABI PRIZM 377
DNA sequencer (Perkin-Elmer) and analyzed us-
ing the BioXM software (version 2.6).
Forced restriction fragment length
polymorphism PCR (F-RFLP-PCR)
The F-RFLP-PCR method, described by Murray
(2006),wasusedtogenotypethepolymorphismof
exon 7 (NC_006088:g.17873C>T). Another re-
verse primer (R2: 5’-GCG TTA CCT TTA AAA
CAG TGT CT©~AG-3’) was designed. The re-
verse primer was deliberately introduced by a
point mismatch, which would create an XbaI re-
striction site (T/CTAGA) in PCR products from
visfatin gene carrier chickens, whereas the PCR
products from the noncarriers lacked this site. The
amplification was carried out as described above,
except annealing at 58.4oC. The 10 ?L of PCR
products were digested with 10 ?L of XbaI (MBI,
Vilnius, Lithuania) overnight at 37oC, and the di-
gested products were then separated on 12%
PAGE gel and visualized with ethidium bromide.
Statistical analysis
Statistical analysis was performed by using the
GLM procedure (SPSS 2004) including the ran-
domeffectoffamilyandfixedeffectsofgenotype,
sex, and hatch. For growth and meat quality traits,
model I was used. Considering the effect of body
weight on carcass traits, model II was applied to
carcass traits. In case the effect of genotype was
significant, Benferroni test was used for multiple
comparisons of the genotypes.
Model I: yijklm= ? + Gi+ Sj+ Hk+ fl+ eijklm
Model II: yijklm= ? + Gi+ Sj+ Hk+ fl+ b (Wijklm-
W) + eijklm
where yijklmis an observation of a trait; ? is the
overall population mean; Giis the fixed effect of
genotype (i= 1, 3); Sjis the fixed effect of sex
(j = 1, 2); Hkis the fixed effect of hatch (k = 1, 2);
flis the random effect of family (l = 1, 7); b is the
regression coefficient for slaughter weight; Wijklm
istheindividualslaughterweight;W istheaverage
slaughter weight; and eijklmis the random error.
Least square means (LSMs) were estimated
and compared among 3 visfatin genotypes for
growth, carcass and meat quality traits.
Results
ThePCRproductsofdifferentSSCPvariantswere
sequenced, and the obtained sequences were com-
paredwiththepreviouslyreportedchickenvisfatin
gene NC_006088.
(NC_006088:g.17873C>T) in exon 7 of the
visfatin gene was found (Figure 2). The DNA se-
quence was deposited in the GenBank database
(accession number: FJ384362). The novel SNP at
exon 7 of the visfatin gene was a silent mutation
(p.P318P) (Figure 3).
The 312-bp F-RFLP-PCR products including
exon7anditsflankingregionswereamplified.All
products were digested by XbaI endonuclease and
the results are shown in Figure 4. The allele char-
acterized by the presence of C was named
visfatin-C, and the alternative allele T was called
visfatin-T.TheamplifiedDNAfragmentsweredi-
gested with XbaI, and results showed 2 fragments
(287bpand25bp)forthevisfatin-Talleleandone
fragment (312 bp) for the visfatin-C allele. Corre-
spondingly, genotypes CC, CT, and TT have one
band (312 bp), 3 bands (312, 287 and 25 bp), and
2 bands (287 and 25 bp), respectively. The 25-bp
fragment was too short to be visible in Figure 4.
Theassociationbetweengenotypesandgrowth
traits were shown in Table 1. The silent mutation
was significantly linked with body weight at
4 weeks (BW4), body weight at 6 weeks (BW6),
and slanting length at 4 weeks (BSL4) (P < 0.05).
It was also highly significantly linked with breast
bone length at 4 weeks (BBL4) (P < 0.01). The
17873C>T variant also tended to be associated
with BW10 as well as BBL8 (P < 0.1). Values of
BW4, BW6, BSL4, and BBL4 in individuals with
genotype TT were significantly higher than in
those with genotypes CC and CT. Besides the
growthrate,genotypeTTshowedacontributionto
fat metabolism, as average fat bandwidth (FBW)
in individuals with this genotype was 10.98% and
9.76% higher (P < 0.05) than in those with geno-
types CC and CT, respectively. The polymor-
phism was significantly associated with breast
muscle water loss rate (BWLR) and breast muscle
fiber density (BFD) (P < 0.05). The advantages of
genotype TT were also demonstrated in breast
musclewaterlossrate(BWLR),whichwas8.53%
and 7.43% lower in individuals with genotype TT
than in those with genotypes CT and CC, respec-
tively (Table 1). Breast muscle fiber density
(BFD) in genotype TT was significantly lower
than in genotypes CC and CT (P < 0.05).
AnovelSNP
Polymorphisms of chicken visfatin gene 61

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Discussion
In this study, a novel silent SNP of the chicken
visfatin gene was identified by F-RFLP. Once a
forced restriction site is introduced into one of a
pair of primers, the PCR product will contain a
certain restriction enzyme site. The F-RFLP has
become a rapid, simple, and definitive technique
for SNP detection, and was used previously to ge-
62R-L. Han et al.
Figure2.SNPlocationforexon7anditsflankingregion.Note:NmeansCorT.Thegrayshade:exon7(ACCisthefirst
codon and AAG is the last codon in this coding region). The forward boldface: the forward primer. The backward
boldface: the reverse primer (R2). The frame: XbaI polymorphism enzyme site (T/CTAGA). The boldface and italic: a
point mismatch deliberately introduced into the reverse primer.
Figure
NC_006088:g.17873 (318 amino acids) from different
genotypes of a novel SNP for exon 7 in the chicken
visfatin gene.
3. Sequencingmapsatposition
Figure 4. The 12% PAGE patterns of PCR products
including exon 7 of the chicken visfatin gene digested by
XbaIendonuclease.Lanes1,4,7=genotypeCC(312bp);
lanes 2, 6 = genotype CT (312 and 287 bp); lane 3 =
marker 1 (600, 500, 400, 300, 200, and 100 bp); lane 5 =
genotype TT (287 bp).

Page 5

notype prolific sheep (Davis et al. 2002; Guan
et al. 2006; Hua et al. 2008) and swine (Kim et al.
2003). The results showed that F-RFLP is a robust
and accurate method for genotyping SNPs. To get
clearer results of electrophoresis, PAGE could be
usedinsteadofagarosegelelectrophoresis(AGE).
The SNP in exon 7 of the visfatin gene is a
novel silent mutation, which will not cause any
change at the level of amino acids. Although the
function of synonymous mutation remains un-
known, it is generally accepted that synonymous
mutations play an important role in regulating
geneexpression.Previousstudiesshowedthatnat-
urally occurring silent SNPs possibly lead to the
synthesis of protein products with the same amino
acid sequence but different structural and func-
tional properties (Komar 2007). Although the
physiological role of visfatin remains unclear, its
biological properties are similar to the growth fac-
tor-like properties of some of the cytokines that
have an anti-apoptotic role and promote cell pro-
liferation (Sethi and Vidal-Puig 2005). This may
explainthefactthattheSNPofexon7wassignifi-
cantly linked with body weight and many other
growth traits at early ages (Table 1). Three SNPs
(rs9770242, –948G>T, rs4730153) have been re-
ported in the human visfatin gene, but no associa-
tion of type 2 diabetes mellitus (T2DM) with any
of the genotyped SNPs or their haplotypes was
found(P < 0.05) in
(Böttcher et al. 2006). There was no association of
the SNPs with BMI and BMI-SDS in schoolchil-
dren, whereas there were mild differences in the
waist-to-hip ratio (WHR) (Körner et al. 2007). In
this study, however, the silent SNP was associated
with chicken fat bandwidth. There was no signifi-
cant association between this SNP and the other
carcass traits. With a significant lower breast mus-
cle water loss rate (BWLR), genotype TT had a
potentially better meat quality. Reduced breast
muscle fiber density (BFD) in genotype TT could
be caused by fast growth of TT chickens. Growth
rate also influences muscle fiber properties
(Ruusunen and Puolanne 2004). Advantages of
genotype TT in some growth and meat quality
traits were found in this study. However, it is un-
non-diabeticsubjects
Polymorphisms of chicken visfatin gene63
Table 1. Associations of XbaI polymorphism in exon 7 of the chicken visfatin gene with
growth, carcass, and meat quality traits
Trait Least squares mean ± standard error (N)
CC
122.35±0.98 (347)
319.70±2.28b(345)
559.68±4.29b(358)
813.000±6.45 (355)
1112.26±8.09 (363)
1354.85±9.55 (370)
6.19±0.03B(344)
8.90±0.03 (354)
10.99±0.03 (370)
11.36±0.04b(344)
16.27±0.06 (354)
19.79±0.05 (370)
1188.01±8.34 (373)
1102.96±2.52 (373)
922.18±2.69 (373)
138.61±1.06 (372)
202.07±4.55 (372)
0.73±0.01a(373)
0.46±0.010 (373)
16.72±0.25 (343)
24.22±0.26a(360)
923.74±16.55 (330)
606.14±10.73b(316)
P value
CT
122.42±0.93 (373)
321.08±2.18b(378)
560.47±4.06b(355)
813.555±6.28 (370)
1114.75±7.82 (391)
1349.39±9.12 (403)
6.21±0.03B(378)
8.90±0.03 (371)
10.98±0.03 (403)
11.38±0.04b(378)
16.17±0.06 (371)
19.75±0.05 (403)
1187.68±8.01 (403)
1102.51±2.42 (404)
924.06±2.59 (403)
140.82±1.01 (403)
205.64±4.37 (402)
0.74±0.01b(402)
0.46±0.010 (403)
16.88±0.23 (380)
24.51±0.25a(391)
957.86±16.22 (338)
643.15±10.15a(348)
TT
126.41±3.55 (56)
336.92±5.59a(57)
589.34±10.84a(55)
841.301±16.43 (54)
1167.50±22.81 (58)
1403.34±27.16 (58)
6.42±0.06A(57)
9.09±0.09 (54)
11.13±0.09 (58)
11.63±0.10a(57)
16.27±0.14 (54)
20.00±0.13 (58)
1228.56±23.85 (58)
1101.85±6.37 (58)
919.69±6.80 (58)
143.87±2.66 (58)
194.90±11.57 (57)
0.82±0.03b(58)
0.44±0.03 (58)
17.65±0.90 (55)
22.42±0.65b(58)
951.23±40.56 (54)
598.24±26.49b(51)
BW2 (g)
BW4 (g)
BW6 (g)
BW8 (g)
BW10 (g)
BW12 (g)
BBL4 (cm)
BBL8 (cm)
BBL12 (cm)
BSL4 (cm)
BSL8 (cm)
BSL12 (cm)
CW(g)
SEW(g)
EW(g)
BMW(g)
LMW(g)
FBW (mm)
SFT (mm)
LWLP (%)
BWLR (%)
LFD (root/mm2)
BFD (root/mm2)
Means with different superscripts are significantly different at P<0.05 (small letters) or P<0.01 (capital let-
ters).BW=bodyweightat2,4,6,8,10,and12weeksofage;BBL=breastbonelengthat4,8,and12weeksof
age; BSL = body slanting length at 4, 8, and 12 weeks of age; CW = carcass weight; SEW = semi-evisceration
weight;EW=eviscerationweight;BMW=breastmuscleweight;LMW=legmuscleweight;FBW=fatband-
width; SFT = skin fat thickness; LWLR = leg muscle water loss rate; BWLR = breast muscle water loss rate;
LFD = leg muscle fiber density; BFD = breast muscle fiber density
0.536
0.017
0.034
0.258
0.071
0.168
0.003
0.088
0.224
0.039
0.423
0.193
0.252
0.984
0.888
0.113
0.632
0.036
0.858
0.584
0.011
0.334
0.025