Introduction
Perilipin, a PAT
(perilipin/adipophilin/tail-interacting protein of 47 kDa)
family protein and encoded by the PLIN gene, is a lipid
droplet-associated phosphor-protein that functions as a key regulator
during lipid metabolism and fat deposition that plays an important
role in the formation and maintenance of lipid droplet (Londos et al.,
2005; Liu and Xu, 2006). Un-phosphorylated perilipin molecules coat
the surface of lipid droplets in adipocytes to form a barrier that
prevents lipase from accessing triacylglycerol, thereby inhibiting
lipolysis (Wolins et al., 2005; Xu et al., 2006). Upon activation of
protein kinase A, hormone-sensitive lipase (HSL) and perilipin are
phosphorylated. Phosphorylation of perilipin is essential for the
translocation of HSL from the cytoplasm to lipid droplets, thus
perilipin phosphorylation is an important molecular switch for the
activation of lipolytic reactions (Londos et al., 1999; Xu et al.,
2004).
Primer sequences of PCR amplification of chicken PLIN gene.
Primer
Sequences (5′–3′)
Length of products (bp)
Annealing temp. (∘C)
PLIN Primer 1
F: CTCTGTGCTGTTCTGCCTTTA
242
58.9
R: ATCCAGACGACCAGTTCCTG
PLIN Primer 2
F: AAAGCCAAAGGGCAGGAA
114
56.3
R: TGCAGAGGGAACAGAAACC
Studies indicate that the PLIN gene affects body weight (BW) and fat
deposition in animals (Londos et al., 2005; Bickel et al., 2009). Mutations
to PLIN have been associated with carcass traits and adiposity in
humans (Lü et al., 2015), pigs (Gandolfi et al., 2011; Gol et al., 2015),
cattle (Fan et al., 2010), sheep (Gao et al., 2012), and ducks (Zhang et al.,
2013). To date, only three reports have investigated the association of
PLIN polymorphisms with a limited number of production traits in
chickens. In the first report, three single-nucleotide polymorphisms (SNPs)
of PLIN (g.2272C>T, g.2319C>T, and g.2476G>A) were significantly associated
with living body weight, carcass weight, and leg muscle weight, and strongly
correlated to abdominal fat weight and abdominal fat percentage (Zhou et al.,
2014). In the second study, a SNP located at rs315831750 on chromosome 10 was
found to be associated with the percentages of breast muscle, leg muscle, and
abdominal fat (Zhang et al., 2015). Certainly, not all genetic variations
exert distinct effects on carcass and adipose traits, as concluded by the
third study, which found no significant correlation of a single mutation
(g.2224G>T) of the PLIN gene with carcass and
abdominal traits (Lei et al., 2011).
Hence, most studies on PLIN gene polymorphisms in chickens
have focused on carcass and abdominal traits, while few have
investigated the effect on BW traits. Therefore, the aim of the
present study was to further investigate the contribution of the
PLIN gene to a wider range of performance in chickens,
especially to confirm whether PLIN polymorphisms exert
a significant effect on BW at different ages.
Materials and methods
Ethics statement
The study protocol was approved by the Animal Care Committee of the
Department of Animal Science and Technology of Yangzhou University and
conducted in accordance with the guidelines of the Animal Use
Committee of the Chinese Ministry of Agriculture. All efforts were
made to minimize animal suffering.
Population and samples collection
A total of 322 female Jinmao Hua chickens were selected randomly from
the same feeding batch at Jiangsu Sanditi Animal Husbandry Co.,
Ltd. All birds were hatched on the same day and reared on the ground
under the same nutritional and environmental conditions. Six growth
traits, the BW of chickens at day 0 (BW0) and weeks 2 (BW2), 4 (BW4), 6
(BW6), 8 (BW8) and 10 (BW10), were recorded. Genomic DNA was extracted
by the phenol-chloroform extraction method, dissolved in
Tris-ethylenediaminetetraacetic acid (EDTA) buffer, then quantified by
spectrophotometry and then stored at -20∘C until analyzed.
Polyacrylamide gel electrophoresis results of SSCP analysis
of PCR products in Jinmao Hua chickens.
SSCP analysis of PCR amplification using primer 2 in Jinmao
Hua chickens.
Primer design and variability analysis
Sequence alignment of AA, BB, JJ and JL genotypes of
primer 1.
According to the gallinaceous PLIN gene sequences (GenBank
Accession no. NC_006097.3), Primer 5.0 software was used to design two
pairs of primers to amplify fragments that encompassed exon 5 and
a partial intron of the PLIN gene (Table 1). Primers were
synthesized by Sangon Biotech (Shanghai) Co., Ltd. Polymerase chain
reaction (PCR) was performed in a 20 µL reaction volume
containing 1 µL of genomic DNA, 0.8 µL of each
primer, 7.4 µL of dH2O, and 10 µL of
2× Taq Master Mix for polyacrylamide gel electrophoresis
(Vazyme Biotech Co., Ltd., Nanjing, China). The PCR thermal profile
consisted of pre-denaturation at 95∘C for 5 min,
followed by 30 cycles of denaturation at 95∘C for
30 s, annealing at X∘C for 30 s and
elongation at 72∘C for 30 s and final extension at
72∘C for 10 min (X is the annealing temperature
specific for the primer pairs; see Table 1).
For single-strand conformation polymorphism (SSCP) analysis,
2 µL of each amplification product was mixed with
7 µL of denaturing buffer (98 % formamide, 0.025 %
bromophenol blue, 0.025 % xylene cyanol FF,
10 mmol/LEDTA (at pH 8.0) and 2 % glycerol), heated
for 10 min at 98 ∘C and then cooled on ice for
10 min. Denatured PCR products were subjected to 10 %
non-denaturing polyacrylamide (29:1) gel electrophoresis at 250 V
for the first 5 min and then 120 Vcm-1 for 10 to
12 h. SSCP patterns on the gels were visualized by silver
staining. For each genotype, Sangon Biotech sequenced the PCR products
of four samples.
Parameter estimates of different genotypes on body weight traits for
primer 1 (means±SD).
Traits
Genotype
AA (N=209)
BB (N=9)
AB (N=33)
JJ (N=41)
JL (N=30)
BW0
25.10±2.01ab
23.07±2.54b
24.73±2.39ab
25.60±2.36a
24.49±2.08b
BW2
108.21±16.67
105.68±14.86
111.00±15.68
112.04±17.25
108.17±20.67
BW4
230.03±39.86
232.38±43.84
226.88±36.87
235.21±39.44
219.37±42.57
BW6
400.54±64.02ab
408.32±70.04ab
392.88±60.31ab
415.07±58.14a
379.24±62.97b
BW8
635.18±87.08ab
633.68±98.51ab
610.97±96.45b
653.83±83.60a
608.19±96.16b
BW10
849.93±111.91
849.81±106.61
824.93±123.39
864.07±97.45
827.52±108.84
Note: different letters in the rows indicate significantly different mean
value at P<0.05 and the same letters indicate no significant
difference (P>0.05).
Sequence alignment of CC and DD genotypes of primer 2.
Statistical analysis
PHASE2.1 software
(http://stephenslab.uchicago.edu/phase/download.html) was used
to analyze the types and frequency of haplotypes. The association
between genotypes and growth traits were analyzed using the general
liner model (GLM) procedures of SPSS ver. 19.0 software (SPSS, Inc.,
Chicago, IL, USA). The following statistical model was used:
Y=μ+Gi+e,
where Y is the phenotypic value of traits, μ the population
mean, Gi fixed effects of genotype or diplotype, and e random residual error. Multiple comparisons were performed with least
squares means.
Results
SSCP and sequence analysis
SSCP analysis revealed that the products of the two primer pairs
displayed polymorphisms in the PLIN gene. Five genotypes were
detected in Jinmao Hua chickens with primer pair 1 (AA, AB, BB, JJ and
JL; Fig. 1) and three with primer pair 2 (CC, CD and DD; Fig. 2).
The PCR products of different genotypes were cloned and sequenced.
Sequencing revealed two nucleotide mutations (g.1889C>T and g.1904T>C) in the AA genotype, as
compared with BB genotypes, and two nucleotide mutations
(g.1904T>C and g.1922C>T)
between the JJ and JL genotypes. Individuals with the JJ genotype had
one nucleotide mutation (g.1922C>T), as compared
with the JL genotype (Fig. 3). Sequencing analysis results of the
different genotypes of primer pair 2 are depicted in Fig. 4. Two
mutations were detected by sequencing (g.2014A>G
and g.2020C>T). None of these mutations resulted
in an amino acid change.
Association of genotypes with BW traits
One-way analysis of variance was used to analyze the effects of
different genotypes. The use of primer pair 1 revealed that the
polymorphism was significantly associated with growth traits in of
Jinmao Hua chickens on BW0, BW6 and BW8 (Table 2). Genotype JJ had
a significantly higher hatch weight than that of the BB and
JL genotypes (P<0.05). The JJ genotype was predominant, as
compared with the JL genotype (P<0.05), at 6 weeks of age. At BW8,
chickens with the JJ genotype were significantly heavier than the
JL chickens. There was no distinct difference in the other traits
among the five genotypes (P>0.05).
Parameter estimates of different genotypes on growth traits for
primer 2 (means±SD).
Genotype
Traits
BW0
BW2
BW4
BW6
BW8
BW10
CC (239)
24.99±2.18
109.48±17.32
230.20±9.54
399.65±63.15
633.12±86.11ab
845.21±106.37ab
CD (64)
25.24±2.12
107.94±16.60
231.00±38.42
407.27±57.14
641.57±84.33a
868.29±99.99a
DD (19)
24.53±1.70
105.02±15.00
214.64±42.03
377.18±64.46
594.45±94.94b
799.16±129.68b
Note: different letters in the rows indicate significantly different mean
value at P<0.05 and the same letters indicate no significant
difference (P>0.05).
Haplotypes inferred on the five single-nucleotide polymorphisms.
Haplotype
g.1889C>T
g.1904T>C
g.1922C>T
g.2014A>G
g.2020C>T
Frequency (%)
H1
C
T
C
G
T
58.6957
H2
C
C
T
G
T
15.5280
H3
C
T
C
A
C
11.3354
H4
T
C
C
G
T
5.4348
H5
C
C
C
G
T
4.5031
H6
T
C
C
A
C
2.4845
H7
C
C
T
A
C
1.8634
H8
C
C
C
A
C
0.1553
Diplotypes and frequencies of five SNPs.
Diplotype
Frequency (%)
Diplotype
Frequency (%)
H1H1
47.205
H3H6
2.795
H1H3
15.528
H4H4
1.8634
H1H4
6.8323
H1H6
0.6211
H2H2
10.2484
H4H6
0.3106
H2H5
8.0745
H5H7
0.9317
H2H7
2.4845
H6H6
0.6211
H3H3
2.1739
H7H8
0.3106
The results of multiple comparison analysis of growth traits between
the genotypes for primer pair 2 among all 322 female Jinmao Hua
chickens are presented in Table 3. Significant differences among the
three genotypes were found at BW8 and BW10 (P<0.05). Chickens of
genotype CD had the highest BW and those with the DD genotype had the
lowest. No difference was detected among three genotypes at BW0, BW2,
BW4 and BW6.
Construction of haplotype and association analysis
The parameters of the haplotypes based on the five SNPs are shown in
Table 4. A total of eight haplotypes were detected, with C-T-C-G-T as
the main haplotype, accounting for 58.6957 % of the
observations. Based on the eight haplotypes, 14 diplotypes in Jinmao
Hua chickens were obtained. To ensure that the analysis was accurate,
haplotypes and diplotypes with a frequency of less than 1 % were
excluded from further association analysis. The frequencies of
haplotypes and displotypes are listed in Tables 4 and 5.
The least squares mean multiple comparisons of diplotypes are
displayed in Table 6. Diplotypes were found to be strongly
significantly associated with BW6 (P=0.027) and BW8 (P=0.013). For
BW0 and BW4, there was no significant association although a trend was
observed (P=0.093 and 0.067, respectively). The BW of chickens with
the H2H2 was greater than the other eight diplotypes at each
week. Meanwhile, the BW0, BW2, BW4, BW6, BW8 and BW10 values of
chickens with the H3H3 diplotype were the lowest.
Correlation analysis between different diplotypes and body
weight traits (means±SD).
Traits
BW0
BW2
BW4
BW6
BW8
BW10
P value
0.093
0.430
0.067
0.027*
0.013*
0.238
H1H1 (152)
25.02±2.00
108.72±16.83
230.75±40.30
397.37±65.82
634.82±86.18
844.96±113.09
H1H3 (50)
25.34±2.13
106.71±16.58
228.94±37.13
407.42±58.08
632.19±90.12
851.07±113.26
H3H3 (7)
24.22 ± 1.292
96.71 ± 9.162
202.31 ± 45.052
363.03 ± 58.092
574.29 ± 74.382
801.20 ± 71.222
H1H4 (22)
24.41±2.44
110.77±16.00
228.17±34.77
394.31±56.48
603.62±93.07
821.07±107.89
H3H6 (9)
25.47±1.16
112.37±17.39
223.53±46.79
390.88±78.25
618.08±119.25
813.56±112.60
H4H4 (6)
24.77±2.69
105.68±18.04
226.17±49.21
405.41±83.61
625.43±114.26
836.22±123.81
H2H2 (33)
25.87 ± 2.511
112.54 ± 17.471
238.80 ± 38.951
423.64 ± 58.641
661.51 ± 83.081
869.97 ± 96.611
H2H7 (8)
24.45±1.06
109.96±17.33
217.80±39.33
379.75±42.66
606.60±74.89
834.75±103.69
H2H5 (26)
24.44±2.07
106.77±20.66
212.12±37.70
370.32±56.65
592.35±83.86
813.10±94.25
Note: 1 or 2 italic values are the
highest or lowest least squares means,
respectively; * P≤0.05
Discussion
Polymorphisms of the PLIN gene
According to the dbSNP database of the National Center for Biotechnology
Information, the human PLIN gene has 555 SNPs, including some
that may be closely associated to obesity, lipid metabolism, etc. In
animals, the PLIN gene also shows abundant polymorphism. Fan
et al. (2010) found two SNPs located in the region of exon 3 and 4 in
Qinchuan cattle. Gao et al. (2012) detected mutations in exon 3, 4, 5,
6 and 8 in sheep. Fan et al. (2011) found one SNP in exon 2 and in
intron 1 of the PLIN gene in Peking as well as Cherry Valley
ducks. Vykoukalová et al. (2009) used PCR-restriction fragment
length polymorphism to scan the overlapping PCR fragments covering the
porcine PLIN gene from exon 1 to 9 and detected eight SNPs in
the coding sequence of the porcine PLIN gene. Zhang
et al. (2015) designed primers that covered all eight exons to detect the
PLIN polymorphisms in six broiler populations, and discovered
only one SNP (rs313726543) in exon 1. Additionally, the study on Luqin
chickens revealed three SNPs, two in exon 6 and one in intron 6 (Zhou
et al., 2014).
We scanned the SNPs in exon 5 and partial intron 5 regions of the
PLIN gene of Jinmao Hua chickens. The products amplified by
PLIN primer pair 1 and 2 displayed polymorphism and five SNPs
were identified by sequencing of the two PCR fragments, including
three (g.1889C>T, g.1904T>C
and g.1922C>T) located in exon 5 that formed five
genotypes (AA, AB, BB, JJ and JL), while others (g.2014A>G and g.2020C>T) located in intron 5
formed three genotypes (CC, CD and DD). These results combined with of
those previous reports (Fan et al., 2010, 2011; Gao et al., 2012;
Vykoukalová et al., 2009) indicated that the PLIN gene
had an abundance of polymorphism in different animal breeds that
conform to the comprehensive functions of the PLIN
gene. Moreover, compared with the previous studies (Zhang et al.,
2015; Zhou et al., 2014), the distribution of SNPs of PLIN
gene in chicken breeds is evidently different.
Relationship of the PLIN gene with some economic
traits
The PLIN gene has been shown to be crucial for some economic
traits of animals. Gol et al. (2015) detected a mutation (g.173G > A) of intron 2 in Duroc pigs, which showed some evidence of negative
additive and dominant effects on BW at 120 and 180 days. In sheep,
three genetic mutations with major effects on fat-tail weight and tail
width have been identified in PLIN (Gao et al., 2012). Fan
et al. (2010) found one SNP in the PLIN gene associated with
a significantly greater slaughter weight, carcass weight, back fat
thickness, and loin muscle area of the AB genotype, as compared with
the AA and BB genotypes in Qinchuan cattle (P<0.05). In chickens,
Zhou et al. (2014) detected three SNPs of PLIN gene and found
that the CTA TCA diplotype had a significant effect on living body
weight and breast intramuscular fat content of chickens, while the TCA
TCA diplotype produced the lowest carcass weight and fatness
traits. These findings showed that the PLIN gene had
significant effects on carcass and fat traits, similar to its function
in different animals. However, research on PLIN gene
polymorphisms in chickens has been more focused on slaughter traits
and fat deposition, rather than BW, at different ages. Hence, the
genetic polymorphisms in exon 5 and partial intron 5 of PLIN
gene in Jinmao Hua chickens were screened to discern potential
associations between the nucleotide polymorphisms and BW traits.
The findings from the least squares means of different genotypes and
diplotypes were interesting. The BW of chickens with the JJ genotype was the
heaviest among all the genotypes at each week. Meanwhile, chickens with the
CD genotype had the highest mean BW with the exception at week 2. On the
whole, chickens with the JJ and CD genotypes performed best in this group. To
gain further insight, 322 chickens were divided into eight haplotypes groups
using the five SNPs. The haplotypes were found to be significantly associated
with BW. Notably, the combination between different haplotypes increased the
BW of chickens and the H2H2 diplotype was found to be the best combination.
For instance, the BW of 1-day-old H2H2 chickens was 25.87 g, which
was 0.27 g greater than chicks at the same age with the JJ genotype.
The BW6 of chickens with the H2H2 diplotype was 423.64, which was 8.57 and
16.37 g greater than JJ and CD genotype, respectively. At BW8, the BW
of the H2H2 diplotype was 661.51 g, which was 7.68 and
19.94 g greater than chickens with the JJ and CD genotypes,
respectively. So, the H2H2 diplotype is regarded as the most advantageous for
chicken BW traits. In addition, the combination of haplotypes also reduced
the BW. The H3H3 chickens had the lowest BW in comparison with other
diplotypes, even among all genotypes. As a result, the H3H3 diplotype was
regarded as detrimental to chicken BW traits, suggesting that it should be
deleted during cultivation. Haplotype or haplotype block analysis provide
a practical solution to resolve the innate problems of the single-marker
analysis, such as noisy, unsatisfied, and obscured important localization
information (Daly et al., 2001). Both haplotype diversity and the method of
SNP selection based on maximum haplotype diversity are always preferred
(Huang et al., 2003). According to the data analysis, the SNPs examined in
this study may be functionally linked with BW of chickens.