The GTPase activating Rap/RanGAP domain-like 1 gene is associated with chicken reproductive traits.

Abstract

Abundant evidence indicates that chicken reproduction is strictly regulated by the hypothalamic-pituitary-gonad (HPG) axis, and the genes included in the HPG axis have been studied extensively. However, the question remains as to whether any other genes outside of the HPG system are involved in regulating chicken reproduction. The present study was aimed to identify, on a genome-wide level, novel genes associated with chicken reproductive traits.
Suppressive subtractive hybridization (SSH), genome-wide association study (GWAS), and gene-centric GWAS were used to identify novel genes underlying chicken reproduction. Single marker-trait association analysis with a large population and allelic frequency spectrum analysis were used to confirm the effects of candidate genes. Using two full-sib Ningdu Sanhuang (NDH) chickens, GARNL1 was identified as a candidate gene involved in chicken broodiness by SSH analysis. Its expression levels in the hypothalamus and pituitary were significantly higher in brooding chickens than in non-brooding chickens. GWAS analysis with a NDH two tail sample showed that 2802 SNPs were significantly associated with egg number at 300 d of age (EN300). Among the 2802 SNPs, 2 SNPs composed a block overlapping the GARNL1 gene. The gene-centric GWAS analysis with another two tail sample of NDH showed that GARNL1 was strongly associated with EN300 and age at first egg (AFE). Single marker-trait association analysis in 1301 female NDH chickens confirmed that variation in this gene was related to EN300 and AFE. The allelic frequency spectrum of the SNP rs15700989 among 5 different populations supported the above associations. Western blotting, RT-PCR, and qPCR were used to analyze alternative splicing of the GARNL1 gene. RT-PCR detected 5 transcripts and revealed that the transcript, which has a 141 bp insertion, was expressed in a tissue-specific manner.
Our findings demonstrate that the GARNL1 gene contributes to chicken reproductive traits.

Full text

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The GTPase Activating Rap/RanGAP Domain-Like 1 Gene
Is Associated with Chicken Reproductive Traits
Xu Shen1,2, Hua Zeng1, Liang Xie1,3, Jun He1,2, Jian Li1,2, Xiujuan Xie1,2, Chenglong Luo4, Haiping Xu1,2,
Min Zhou5, Qinghua Nie1,2, Xiquan Zhang1,2*
1Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China,
2Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Guangzhou, China, 3Institute of Animal Science and Veterinary, Hainan Academy of
Agricultural Sciences, Haikou, Hainan, China, 4Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong, China,
5Biotechnology Institute, Jiang Xi Education College, Nanchang, Jiangxi, China
Abstract
Background: Abundant evidence indicates that chicken reproduction is strictly regulated by the hypothalamic-pituitary-
gonad (HPG) axis, and the genes included in the HPG axis have been studied extensively. However, the question remains as
to whether any other genes outside of the HPG system are involved in regulating chicken reproduction. The present study
was aimed to identify, on a genome-wide level, novel genes associated with chicken reproductive traits.
Methodology/Principal Finding: Suppressive subtractive hybridization (SSH), genome-wide association study (GWAS), and
gene-centric GWAS were used to identify novel genes underlying chicken reproduction. Single marker-trait association
analysis with a large population and allelic frequency spectrum analysis were used to confirm the effects of candidate
genes. Using two full-sib Ningdu Sanhuang (NDH) chickens, GARNL1 was identified as a candidate gene involved in chicken
broodiness by SSH analysis. Its expression levels in the hypothalamus and pituitary were significantly higher in brooding
chickens than in non-brooding chickens. GWAS analysis with a NDH two tail sample showed that 2802 SNPs were
significantly associated with egg number at 300 d of age (EN300). Among the 2802 SNPs, 2 SNPs composed a block
overlapping the GARNL1 gene. The gene-centric GWAS analysis with another two tail sample of NDH showed that GARNL1
was strongly associated with EN300 and age at first egg (AFE). Single marker-trait association analysis in 1301 female NDH
chickens confirmed that variation in this gene was related to EN300 and AFE. The allelic frequency spectrum of the SNP
rs15700989 among 5 different populations supported the above associations. Western blotting, RT-PCR, and qPCR were
used to analyze alternative splicing of the GARNL1 gene. RT-PCR detected 5 transcripts and revealed that the transcript,
which has a 141 bp insertion, was expressed in a tissue-specific manner.
Conclusions/Significance: Our findings demonstrate that the GARNL1 gene contributes to chicken reproductive traits.
Citation: Shen X, Zeng H, Xie L, He J, Li J, et al. (2012) The GTPase Activating Rap/RanGAP Domain-Like 1 Gene Is Associated with Chicken Reproductive Traits. PLoS
ONE 7(4): e33851. doi:10.1371/journal.pone.0033851
Editor: Patrick Callaerts, VIB & Katholieke Universiteit Leuven, Belgium
Received May 23, 2011; Accepted February 19, 2012; Published April 9, 2012
Copyright: ? 2012 Shen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by China Agriculture Research System (CARS-42-G05) and the National Natural Scientific Foundation of China, project
(No. 31000544). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: xqzhang@scau.edu.cn
Introduction
Egg number at 300 d of age (EN300), age at the first egg (AFE),
and brooding behavior are valuable indices of chicken reproduc-
tive ability. In female chickens, sexual maturity is usually expressed
as AFE. The AFE trait has been under artificial selection to
enhance egg production efficiency [1]. EN300 is another
reproductive trait of economic importance, while incubation
behavior also affects egg production, as it results in the cessation of
egg laying [2]. Chicken reproduction is controlled by photoperiod
[3]. Generally, the process of chicken egg production is strictly
regulated by the hypothalamic-pituitary-gonad (HPG) axis [4].
Gonadotrophin releasing hormone (GnRH) and its receptor
(GnRHR) start the cascade, and neuropeptide Y (NPY) is known
to inhibit GnRH secretion via its receptor (Y1R) and to control
ovulation [5]. Under photo-stimulation, GnRH is synthesized,
secreted by the hypothalamus and binds to its receptor, which
stimulates the pituitary gland to secret gonadotrophins that evoke
steroid synthesis in the gonad, regulating ovarian follicle growth
and ovulation in hens [6,7]. The hypothalamic vasoactive
intestinal peptide (VIP) - pituitary prolactin (PRL) neuroendocrine
pathway also controls reproductive cycles via dopaminergic
neurotransmission in avian HPG system [8–10]. PRL is a key
hormone that is absolutely necessary for egg laying and incubation
behavior in poultry [11,12]. After stimulation by VIP, PRL
inhibits the release of gonadotropins and thereby induces and
maintains chicken incubation behavior [13–15].
The genetic mechanism behind incubation behavior has been
widely studied because of its potential effect on egg production.
This mechanism is a polygenic trait that is controlled by a set of
autosomal genes [16]. Genes in the HPG axis showed high
association with reproductive traits such as broodiness and egg
production [9,17–30], however, this association depends on the
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population used [22,30]. Aside from the genes distributed in HPG
axis, other novel genes have been discovered to affect chicken
reproduction traits [31–33].
Several approaches have been applied to identify the novel
genes involved in chicken reproduction. A genome-wide scan is a
powerful approach to understanding this complex trait. Quanti-
tative trait loci (QTLs) for egg number, egg production rate, AFE
and broodiness were identified through genome-wide scans [34–
42]. Genome-wide association studies based on high density SNPs
can be performed to detect QTLs that could not be detected by
previous studies based on microsatellite genotyping [43–47]. A
genome-wide association study attempts to obtain information on
all variants, but a gene-centric SNP approach would be efficient
enough to capture SNPs associated with particular traits [48,49].
Transcriptome profiling can be also used to identify new genes
associated with chicken reproductive traits. Although many studies
on the genetic effects of candidate chicken reproduction genes
have been reported, few studies have reported transcriptomic and
proteomic changes. In previous studies, transcripts related to high-
egg production were identified by suppressive subtractive hybrid-
ization analysis (SSH), and several of the identified transcripts were
further confirmed to be significantly increased in hens with higher
egg production, though they were not part of the HPG axis [50–
52]. Therefore, it is valuable to identify novel genes related to
chicken reproduction.
The aim of the present study is to identify novel genes involved
in chicken reproductive traits using SSH analysis, an Illumina 60K
chicken Beadchip GWAS, and a gene-centric GWAS, with
confirmation via analysis of single marker-traits, allelic frequency
spectra, and alternative splicing.
Results
GARNL1 identified as a candidate gene underlying
chicken broodiness by suppression subtractive
hybridization
A subtraction library was made by subtracting cDNA from the
pituitary at the egg-laying stage. As shown in Figure 1,
construction of the pituitary-subtracted cDNA libraries was
successful. Genes differentially expressed between brooding and
non-brooding chicken pituitary glands were enriched for and
sequenced, and 57 annotation transcripts and 20 unknown
transcripts were characterized (Table S1). Gene ontology (GO)
analysis was performed to investigate the functions of the
putatively differentially expressed transcripts. Biological process
accounted for the major portion of GO annotations, compared
with cellular component and molecular function. Among the
category of biological process, genes were involved in processes
such as eye photoreceptor cell development, ovarian follicle
development, epinephrine biosynthesis,
GTPase mediated signal transduction, G-protein coupled receptor
protein signaling pathways, and so forth (Table S2). On the basis
of biological process annotations, 10 transcripts were selected to be
validated by qPCR. Among the 10 transcripts, one was identified
as belonging to the chicken GARNL1 gene (Figure S1). The
GARNL1 gene was differentially expressed between tissues
(Figure 2). Low levels of mRNA expression were detected in the
ovary, oviduct, liver, spleen, lung, kidney, muscular stomach,
sebum, abdomen fat, and duodenum; however, higher expression
levels were observed in the cerebrum, cerebellum, hypothalamus,
pituitary, heart, and glandular stomach. Gene expression levels in
the cerebrum, cerebellum, hypothalamus, pituitary, ovary, oviduct
and spleen were significantly higher in broody chickens than in
non-broody chickens (P,0.05), with the levels in tissues from
regulationof small
broody chickens 1.6 times to 4.3 times higher than those of non-
broody chickens. In contrast, GARNL1 expression in leg muscle
was 2-fold higher in non-broody chickens.
GWAS indicates that SNPs associated with chicken
reproductive traits are located in the GARNL1 gene
Before GWAS analysis was carried out, stratification analysis
was conducted in the two-tail sample. The IBS was not
significantly different between two tails sample (Ppermu,0.05). In
all, 2802 SNPs were associated with EN300 in the NDH two tail
sample at the 5% genome-wide level (validated by 10000
permutation tests), and of this total, 470 SNPs were at significant
at the 1% level (Table S3). On chicken chromosome 5, 118 SNPs
were associated with EN300 (Table S3). Among the 118 EN300-
associated SNPs, rs14533299 and GgaluGA282818 composed a
haplotype block. The linkage distance in this block is 1691 kb, and
the GARNL1 gene was observed to be located within this block
(Figure S2).
Gene-centric GWAS reveals an association of several
SNPs in the GARNL1 gene with chicken EN300 and AFE
Six SNPs were highly significantly associated with both EN300
and AFE (Ppermu,0.05 and Ppermu,0.01) (Table 1). A SNP cluster
located on chromosome 5 was associated with both AFE and
EN300 in another NDH two tail sample. Among the SNP cluster,
5 SNPs were located in the GARNL1 gene.
The association of GARNL1 SNPs with chicken EN300 and AFE
was further analyzed in a NDH population comprising 1301
individuals. As showed in Table 2, corrected by SLIDE,
rs15700989 was significantly associated with EN300 (P,0.01)
and rs15701085 was associated with AFE (P,0.05).The block
composed of rs14532787 and rs14532779 was also significantly
associated with EN300 (P=0.0088) (Table 3). In this block, there
are 4 haplotypes, including H1 (TG, 70.1%), H2 (TC, 5.5%), H3
(CG, 23.3%), and H4 (CC, 1.1%). H2H2 and H2H4 had higher
EN300 than the other diplotypes.
Allelic frequency spectrum of the chicken GARNL1 gene
Allelic frequencies of rs15700989 were different among the 5
populations. The frequency of rs15700989 was 1.0 in Leghorn
layers (Table 4), with a highly significant difference between
Leghorn layer and the other 4 native Chinese chickens. The chi-
square test values for the genotype distribution of rs15700989
showed significant difference between Leghorn layer and the other
4 Chinese native chickens (P,0.01) (Table 5), in accordance with
their egg-production performance.
Figure 1. Determination of subtraction efficiency. The chicken
housekeeping gene G3PDH was amplified from the subtracted sample
(showed in lane 1–4) and the unsubtracted sample (showed in lane 5–
8). The number above the lane represents the PCR cycle.
doi:10.1371/journal.pone.0033851.g001
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Alternative splicing of the chicken GARNL1 gene
The chicken GARNL1 gene is predicted to be located on
chromosome 5 and to span positions 38,617,769–38,729,036 on
the reverse strand, with a total gene size of 111,268 bp. Four, six,
and seven isoforms from the pituitary, ovary, and oviduct,
respectively, could be detected by Western blotting. The molecular
weights of these isoforms ranged from 150 KDa to 250 KDa
(Figure 3).
Five alternatively spliced transcripts, GARNL1-w (NCBI acces-
sion number: JF330255), GARNL1-v1 (NCBI accession number:
JF330256), GARNL1-v2 (NCBI accession number: JF330257),
GARNL1-v3 (NCBI accession number: JF330258, and GARNL1-
v4 (NCBI accession number: JF330259) were detected in the
cDNA pool prepared from cerebrum, cerebellum, hypothalamus,
pituitary, ovary, and oviduct tissues. Five transcripts were
generated as a result of exon skipping and intron inclusion
(Table 6 and Figure S3). The wild-type transcript, GARNL1-w,
which is composed of 41 exons and 40 introns, was successfully
cloned. The complete coding sequence of GARNL1-w is 6,108 bp
long and encodes 2,035 amino acids. Chicken GARNL1 shares a
high amino acid sequence identity with those of human (89.3%
with AY596971, 89.4% with AY596970), mouse (87.4% with
AY596972, 87.6% with AY596972), and zebrafish (73.2% with
AB476643, 74.3% with AB476644), and it is predicted to be a
nuclear protein (with 63% probability). Similar to the human
GARNL1 gene and the mouse GARNL1 gene, all 5 transcripts
contain a Rap/Ran-GAP domain (AA 1825–AA 2004), two
transmembrane helices (AA 1203–AA 1225, AA 1385–AA 1407),
and a leucine zipper motif (AA 1068–AA 1089), but have lost the
N-terminal coiled coil domain (shown in figure S4).
The variant GANRL1-v2 (deduced to encode a 2134 AA peptide)
skips exon 40 and includes a 141 bp intron sequence between the
exon 16 and exon 17. RT-PCR showed that the 141 bp intron
inclusion was tissue specific, being observed only in the cerebrum,
cerebellum, hypothalamus, heart, pectoral muscle, and leg muscle.
Its mRNA expression level was higher than the other isoforms
without 141 bp intron inclusion (Figure 4). Similarly, the GARNL1-
v4 transcript contained a 201 bp fragment of intron 19, and a
single amino acid change, N (Asn) to D (Asp), occurs at the new
exon-exon junction. GARNL1-v4 mRNA with the 201 bp intron
fragment was present at very low levels (data not shown). The
mRNA expression levels of transcripts with the 141 bp intron
inclusion sequence (Figure 5) in the cerebrum, cerebellum, and
hypothalamus were almost the same between brooding and non-
brooding chickens. However, its expression levels in heart and
pectoral muscles of the brooding chickens were 1.5 and 2 times
greater than those of the non-brooding chickens, respectively. In
leg muscle, the expression was 8-fold higher in the non-brooding
chickens than in brooding chickens.
Discussion
In this study, data from a SSH analyses, a GWAS, and a gene-
centric GWAS indicate that the GARNL1 gene is involved in
reproduction and that some GARNL1 variants are associated with
chicken reproductive traits.
The SSH analysis indicated that the GARNL1 gene was involved
in chicken brooding behavior. Comparing to the digital gene
expression methods, such as RNA-seq, SSH is not a prevailing
experimental method for detecting differentially expressed genes.
Figure 2. Comparison of GARNL1 mRNA expression level between brooding and non-brooding chickens. qPCR was performed to
validate the mRNA level of the GARNL1 gene between the brooding and egg-laying stages in NDH chickens. The horizontal axis indicates the tissues
used for detection, and the vertical axis indicates the 22DCtvalue (shown as average 6 SEM).
doi:10.1371/journal.pone.0033851.g002
Table 1. The 6 SNPs significantly associated with both EN300
and AFE in a two-tail sample.
Chr1
SNP IDGeneChr: bp Type
EN300AFE
Ppermu
Value
Ppermu
Value
5rs16492011 GARNL1 38621623 intron_390.02699*
0.01099*
5 rs15701085 GARNL138674045 exon_26 0.02849*
0.004998**
5 rs16492034 GARNL138680830 intron_200.008496**
0.04598*
5 rs13585983 GARNL138681662 exon_200.009495**
0.03998*
5 rs14532787 GARNL138699909 exon_150.002999**
0.02899*
24rs16199186 NCAM15972092exon_5 0.0009995**0.02399*
1The chromosome where the associated SNPs located.
* and ** indicate Ppermu,0.05, and Ppermu,0.01, respectively.
doi:10.1371/journal.pone.0033851.t001
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SSH have several limitations, relatively low throughput, highly
false positives, and generally not statistical significance. But the
following qPCR validation would help to get some good results
with the following experiment validation [53–55]. This result was
consistent with previous findings. Chicken GARNL1 was identified
as being potentially related to high egg production in Taiwan
Country chickens [50], and higher GARNL1 expression levels have
been observed in high egg producing strains [51]. Furthermore,
the mRNA level of the GARNL1 gene was specifically associated
with total egg number at 500 d of age or egg rate after the first egg
[52]. The cerebellum was found to have the highest expression
level of human GARNL1 gene among the brain tissues,
corresponding to its influence on 14q13-linked neurological
phenotypes [56]. In zebrafish, GARNL1 was a strong candidate
gene for brain developmental delay [57]. In our study, the
expression of chicken GARNL1 gene varied at different stages. We
found GARNL1 to be predominantly expressed in the brain, and
the levels of the GARNL1 gene were consistently higher in the
hypothalamus, pituitary, ovary, and oviduct of broody hens. The
expression level of transcripts that included the 141 bp intron
sequence suggested that the cerebellum may be an important
action region in chickens and the variants of GARNL1 do not
impair their function on chicken reproductive traits. In conclusion,
the expression levels of the GARNL1 gene could reflect its functions
in chicken reproduction.
Two tail samples were used to detect SNPs associated with
broodiness and EN300 in this study. The first QTLs for broodiness
were recently detected in a region within 95 cM of GGA5 [42],
where the GARNL1 gene is located. In the present study, the
haplotype block between rs14533299 and GgaluGA282818 was
also shown to be related to EN300 (data not showed). The
GARNL1 gene is located in this region. Among all 25 protein-
coding genes located on this region, the GARNL1 gene was the
only one that has been reported to be related to reproductive traits
in mRNA level in chickens [50–52]. Therefore, the GARNL1 gene
may be associated with reproduction in chickens.
Table 2. Association of 17 SNPs with chicken reproductive traits in population.
SNP
InformationEN300 trait AFE trait
Position1
Location2
AllelePointwise-P Corrected-PPointwise-PCorrected-P
rs157009493861798239flankingA/G0.5737 1.0000 0.23360.9854
rs1649201138621623 intron 39G/C 0.3796 0.99940.1031 0.8171
rs14532750 38624284intron39T/C 0.01910.26670.41480.9998
rs1570098938648067intron 37A/G 0.0001 0.0023**
0.094980.7912
rs16492027 38654577 intron 36 T/C0.3907 0.9996 0.05072 0.5644
rs1649203138660349 intron 32A/G 0.6481 1.00000.2189 0.9793
rs1570108538674045exon 26G/A 0.06210.63650.002894 0.0496*
rs13585983 38681662 exon 20A/G0.5698 1.00000.11580.8505
rs1649203438680830intron 20T/C 0.10920.83470.03888 0.4716
rs1453277938697653 intron 15 T/C 0.08760.76140.1711 0.9461
rs14532787 38699909exon 15 T/C0.0316 0.40020.02979 0.3819
rs1649205638708121 intron 8T/C 0.01900.2656 0.1233 0.8695
rs1570111938711935 intron7 TTAAA/- 0.48080.9999 0.39750.9996
rs14532824 3873000159flanking T/C0.4837 0.99990.75531
rs14532808 38724344intron1A/G 0.9870 1.00000.3928 0.9996
rs14532819 38726759intron1T/A 0.26980.99280.60311
rs145328313873184759flankingT/C 0.19010.96260.030880.3935
1The position of the site on chromosome 5 in coordinates from the chicken genome database at UCSC (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start).
2the location of the variants found inside the GARNL1 gene.
Pointwise P indicated the P value gained by PLINK and Corrected-P means the P value corrected by SLIDE,*and**indicate P,0.05, and P,0.01, respectively.
doi:10.1371/journal.pone.0033851.t002
Table 3. The association of haplotypes composed of rs14532787 and rs14532779 with EN300 traits.
Trait P value
H1H11
(647)
H1H21
(102)
H1H31
(408)
H2H21
(5)
H2H31
(53)
H2H41
(2)
H3H31
(76)
H3H41
(1)
EN300 0.0086**
92.79
6
1.09bB
93.07
6
2.64b
94.06
6
1.32bB
132.31
6
11.63aA
96.84
6
3.72a
131.73
6
18.44a
98.49
6
3.05Ab
106.06
6
25.87a
Values were expressed as least-square means 6 standard errors (SE).
The number in brackets was the number of chickens tested for each diplotype.
**indicate P,0.01.
Thea, borA, Bvalues with no common superscripts within a column for each site that differed significantly (P,0.05) or highly significantly (P,0.01).
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As the GARNL1 gene might be involved in chicken reproduc-
tion, its polymorphisms could be related to chicken reproductive
traits. However, no studies on the association between the
mutations of GARNL1 gene and chicken reproductive traits were
carried out. In humans, the GARNL1 gene was an important
candidate gene for human 14q13 deletion phenotypes, and two
mutations in GARNL1 were identified in a family with idiopathic
basal ganglia calcification [56]. Polymorphisms of the GARNL1
gene were associated with both EN300 and AFE in a two tail
sample in our gene-centric association analysis. This result
confirmed our previous SSH findings and was validated in a large
population. An analysis of the allelic frequency spectra of GARNL1
SNPs further supported the association. The frequencies of the
rs15700989 were associated with EN300 associated with divergent
egg production performance, and the frequency of predominant
alleles of rs15700989 was 1.0 in Leghorn layer and was descending
in Leghorn, BEH, NDH, XH, and RJF. The predominant allele of
rs15700989 was related to higher EN300 trait in NDH population.
Thus, the allelic frequency data supports the conclusion that the
chicken GARNL1 gene contributes to chicken reproduction. The
block composed of rs14532787 and rs14532779 was significantly
associated with EN300 traits. Although both of them were not
showed significantly relationship with EN300 after corrected by
SLIDE in single marker association, the CC genotype of
rs14532787 resulted in a higher EN300 and an earlier AFE than
did the other two genotypes (Table S4). However, the genotype
CC can be only observed in the NDH population. Compared to
the variance of total egg number at 40 week between early sexual
mature group and later sexual mature group in Leghorn layer
[58], rs14532787 might undergo artificial selection in NDH
population, aiming at the increase of egg production by early
sexual mature and shortening the interval of oviposition. These
two SNP may contribute to EN300 by interacting each other.
Further analysis of the organization, tissue expression, and
alternative splicing of the chicken GARNL1 gene was conducted.
Using Western blotting, 5 alternatively spliced transcripts of the
GARNL1 gene were isolated from chickens in this study. Note that
none of the alternative splicing isoforms had impaired protein
domains. Chicken GARNL1 is conserved with mammals, but it
has some unique features. A variant of the human GARNL1
lacking exon 40, has been found and corresponded to GARNL1-
v1 in chicken [56]. Chicken GARNL1 has lost the N-terminal
coiled coil domain and subsequently the ability to bind to other
proteins. In mice, GARNL1 plays a crucial role during brain
formation and maintenance. A partial murine GARNL1 product
identified as GRIPE (GAP-related interacting protein to E12)
binds to the helix-loop-helix domain of transcription factor E12
and regulates E12-dependent target gene transcription [59].
Similar to the murine GRIPE, the region responsible for binding
to HLH domains was present in all isoforms of chicken GARNL1.
The Rap/Ran-GAP domain is widely distributed in signaling
proteins [60–62], and two arginine residues in Rap/Ran-GAP
domain are important for the GAP activity of GRIPE in mice
[59]. Two arginine residues were found in the Rap/Ran-GAP
domain of chicken GARNL1.
In conclusion, we reveal that the chicken GARNL1 gene has an
important effect on chicken reproductive traits, as determined
from the data from SSH analyses, GWAS, and gene-centric
GWAS. This effect was validated by analysis of allele frequency
spectra, and further characterization of several aspects of the gene
and its expression.
Materials and Methods
Ethics Statement
The study was approved by the Animal Care Committee of
South China Agricultural University (Guangzhou, People’s
Republic of China) with approval number SCAU#0011. Animals
involved in this study were humanely sacrificed as necessary to
ameliorate their suffering.
Table 4. Allelic frequencies of rs15700989 in the GARNL1 gene in the 5 chicken populations.
SiteAllele
LH
(n=60)
BEH
(n=41)
NDH
(n=82)
XH
(n=50)
RJF
(n=33)
rs15700989G1 0.3520.31 0.090.22
LH=Leghorn layers, BEH=Baier Huang chickens, NDH=Ningdu Huang chicken, XH=Xinghua chicken, RJF=Red Jungle Fowl.
The number in brackets was the number of chickens used.
Hardy-Weinberg equilibrium was set at the 0.01 level.
doi:10.1371/journal.pone.0033851.t004
Table 5. Chi-square test of genotype frequency for rs15700989 in the 5 populations.
Site Populations
x2Value1
RJFXH LHNDH
rs15700989BEH 0.1427.35**
101.00**
5.27
RJF21.22**
93.00**
3.29
XH119.00**
20.64**
LH 120.79**
1x20.05(df=1)=3.841; x20.01(df=1)=6.635; x20.05(df=2)=5.991; x20.01(df=2)=9.21;
*P,0.05;
**P,0.01.
doi:10.1371/journal.pone.0033851.t005
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