Analysis on DNA sequence of goat RFRP gene and its possibleassociation with average daily sunshine duration

D. W. Huang • R. Di • J. X. Wang •

M. X. Chu • J. N. He • G. L. Cao •

L. Fang • T. Feng • N. Li

Received: 14 November 2011 / Accepted: 9 June 2012 / Published online: 26 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Goat RFRP gene was cloned and its mutations

were detected in thirteen goat breeds whose reproductive

seasonality and litter size were different. Then sequence

characteristics were analyzed and association analyses

were performed to reveal the relationships between muta-

tions of RFRP gene and average daily sunshine duration,

reproductive seasonality as well as litter size in goats.

A 4,862 bp DNA fragment of goat RFRP gene was

obtained and the complete CDS of 591 bp encodes 196

amino acids, having high homology with that of other

mammals. The protein was predicted to be a secreted

protein with a signal peptide of 21 amino acids. Moreover,

two mutations (A712G, T1493C) in 50 regulatory region

and one mutation (A3438T) in exon 2 were detected. The

test of genotype distribution in six selective goat breeds

showed that there was no uniform significant association

between the three polymorphisms and seasonal reproduc-

tion. The association just existed in some goat breeds for

each locus. Interestingly, however, there was a strong

positive correlation (r = 0.830, P = 0.003) between the G

allele frequency of the A712G locus and average daily

sunshine duration in ten local goat breeds, suggesting that

RFRP gene has undergone a selective pressure in sunshine

duration and may have indirect relationship with repro-

ductive seasonality in goats. Additionally, no significant

difference was found in litter size between genotypes in

prolific Jining Grey goats.

Keywords RFRP � Goat � Polymorphism � Photoperiod �Seasonal reproduction � Litter size

Introduction

RFamide-related peptides (RFRPs), the mammalian homo-

logues of avian gonadotropin inhibitory hormone (GnIH),

are characterized by a LPXRF-amide (X = L or Q) motif at

their C-terminus and they belong to RFamide peptides

superfamily. RFRPs are the neuropeptides/neurotransm-

itters synthesized and secreted by the hypothalamic neurons

which were predominantly localized to the dorsomedial

hypothalamic nucleus (DMH), paraventricular nucleus

(PVN), and to neuronal projections from the PVN to the

neurosecretory zone of the median eminence of pituitary [1–

4]. There are three forms of RFRPs in mammals, RFRP-1 to

3 but they are only encoded by a single gene (RFRP gene) [3,

5, 6]. Among them, RFRP1/3 showed gonadotropin inhibi-

tory activity as avian GnIH while RFRP2 was of no bio-

logical activity [3, 5–9].

RFRPs may potently be involved in mammal repro-

duction as an inhibitory regulator firstly because of its

neuron projections to the preoptic area of hypothalamus, in

which GnRH neurons are located [3, 8–13]. And RFRP

immunoreactive terminals have also been found in the

neurosecretory zone of the median eminence in mammals

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-012-1789-3) contains supplementarymaterial, which is available to authorized users.

D. W. Huang � R. Di � J. X. Wang � M. X. Chu (&) �J. N. He � G. L. Cao � L. Fang � T. Feng

Key Laboratory of Farm Animal Genetic Resources and

Germplasm Innovation of Ministry of Agriculture, Institute of

Animal Science, Chinese Academy of Agricultural Sciences,

Beijing 100193, People’s Republic of China

e-mail: mxchu@263.net

N. Li

State Key Laboratory of Agricultural Biotechnology, China

Agricultural University, Beijing 100193,

People’s Republic of China

123

Mol Biol Rep (2012) 39:9167–9177

DOI 10.1007/s11033-012-1789-3

[1–3, 8, 10]. They can provide a functional neuroana-

tomical infrastructure for RFRPs to regulate GnRH neurons

activity in hapothalamus and synthesis and release of

gonadotropins in anterior pituitary [14]. Additionally,

RFRP could decrease LH and/or FSH levels in rat [15–18],

mouse [19], sheep [1, 7] and cattle [20] by direct sup-

pressive action on GnRH neurons [8, 19] or GnRH-stim-

ulated gonadotropins synthesis and secretion. And the

inhibitory effect on gonadotropins may be mediated by a

reduction in the GnRH-stimulated second messenger

phospho-ERK-1/2 in sheep [7] and RFRP potently inhib-

ited GnRH-stimulated mobilization of intracellular calcium

in gonadotropes [1]. Furthermore, there was an inverse

correlation of RFRP3 and gonadal activity or development

in mice [21, 22] and rats [18], further suggesting that RFRP

is a negative regulator of reproduction.

Notably, RFRP may be involved in photoperiodic con-

trol of seasonal breeding in mammals including mice [22],

Syrian and Siberian hamsters [9, 23–25], rats [26] and

sheep [8, 12]. In sexually quiescent Syrian and Siberian

hamsters, the level of RFRP mRNA and the number of

RFRP-immunoreactive cell bodies were reduced under

short-day photoperiod compared with sexually active ani-

mals maintained under long-day photoperiod [9, 25]. And

the photoperiodic variation of RFRP expression was

probably dependent on melatonin [9, 25, 26], which regu-

lates seasonal reproduction as the known internal photo-

period signal of animals [27]. Additionally, RFRP could

act in concert with kisspeptin, a convincing activator

for sexual behaviors [28–30], with opposing effects, to

control seasonal transition of reproductive status by alter-

ing activities of GnRH neurons [12, 31]. RFRP3 and

kisspeptin mRNA expression was influenced by photope-

riod and food availability, respectively, and the two RFa-

mide neuropeptides were important mediators for

integrating seasonal environmental cues and hormonal

signal to govern the precisely-timed LH surge [23, 32].

Furthermore, RFRP was negatively regulated by estradiol

in an indirect manner, which may contribute to estrogen

feedback to the reproductive axis of mice [33], although it

was not in accordance with the case in rats [34] and Syrian

hamsters in short days [25]. So RFRP may play a key role

in reproductive inhibition, especially in seasonal transition

of breeding state.

On the basis of its important role in reproduction, RFRP

gene may be considered as a candidate gene for mamma-

lian reproductive traits. In the present study, thirteen goat

breeds were used for genotypes determination. Among

them, four local breeds in China and two importing breeds

were selected for association analysis, including three

nonseasonal breeds (Jining Grey goat, JG; Guizhou White

goat, GW; Boer goat, B) and three seasonal breeds (Tai-

hang goat, TH; Liaoning Cashmere goat, LC; Saanen Dairy

goat, SD; with sexual activities in short-day). The mean

litter sizes of Jining Grey, Guizhou White, Boer, Saanen

Dairy, Taihang and Liaoning Cashmere goats had been

reported to be 2.94 [35], 2.74 [35], 2.10 [36], 2.0 [35], 1.03

[35] and 1.18 [35]. The Jining Grey goat is an excellent

local breed in China, with significant characteristics of high

prolificacy and year-round estrus [35]. The objectives of

the present study were to acquire and analyze the sequence

of the goat RFRP gene and to determine the relationship

between its polymorphisms and traits related to goat

reproduction.

Materials and methods

Animals and genomic DNA isolation

Blood samples (10 mL, jugular vein, ACD anticoagulant)

were collected from 241 JG does kidded in 2010 (first,

second, or third parity) (Jining Grey Goat Conservation

Base, Jiaxiang County, Shandong Province, P. R. China),

56 GW does (Guizhou White Goat Conservation Farm,

Yanhe Tujia Nationality Autonomous County, Guizhou

Province, P. R. China), 33 B does (Qinshui Demonstration

Farm, Qinshui County, Shanxi Province, P. R. China), 60

SD does (Shaanxi Province, P. R. China), 55 TH does

(Wuzhi County, Henan Province, P. R. China), 82 LC

does (Liaoning Cashmere Goat Breeding Center, Liaoyang

City, Liaoning Province, P. R. China), 37 Wendeng Dairy

goat (WD) does (Wendeng City, Shandong Province,

P. R. China), 59 Shannan White goat (SW) does (Jingbian

County, Shaanxi Province, P. R. China), 53 Matou goat

(MT) does (Shiyan City, Hubei Province, P. R. China), 53

Guanzhong Dairy goat (GD) does (Fuping County, Shaanxi

Province, P. R. China), 58 Chuandong White goat (CW)

does (Yikouxian Goat Breeding Farm, Yunyang County,

Chongqing, P. R. China), 49 Nanjiang Brown goat (NB)

does (Nanjiang Brown Goat Breeding Farm, Nanjiang

County, Sichuan Province, P. R. China), and 60 Inner

Mongolia Cashmere goat (IC) does (Inner Mongolia White

Cashmere Goat Breeding Farm, Etuokeqi, Ordos City, the

Inner Mongolia Autonomous Region, P. R. China). Geno-

mic DNA was extracted from whole blood using phenol–

chloroform method, dissolved in TE buffer (10 mmol/L

Tris–HCl [pH 8.0], 1 mmol/L EDTA [pH 8.0]) and kept at

-20 �C.

The 241 Jining Grey goat does were selected at random

and were the progeny of five goat bucks (n = 44, 46, 49,

50, 52). Because the five goat bucks were sold, their blood

was not collected for genotyping. No selection on litter size

or other fertility traits was conducted in the flock over

previous years. Kidding seasons consisted of 3-month

groups starting with March through to May as season 1

9168 Mol Biol Rep (2012) 39:9167–9177

123

(spring, n = 63), June through to August as season 2

(summer, n = 53), September through to November as

season 3 (autumn, n = 75) and December through to

February as season 4 (winter, n = 50). All experimental

procedures involving animals were approved by the Chi-

nese Ministry of Agriculture and the animal care and use

committee at the respective institutions where the experi-

ments were performed.

Primers and PCR amplification

According to the DNA sequences of RFRP gene of human

(GenBank No. NC_000007), cattle (GenBank No. NC_00

7302), and sheep (GenBank No. EU655707), and the mRNA

sequence of sheep RFRP (GenBank No. EF494241), a total

of nine pairs of PCR primers named as P1 to P9 (Table A1 in

Online Appendix) were designed using Oligo 6.0 to amplify

50 regulatory region, all of the three exons and two introns of

goat RFRP gene. The primers were synthesized by Shanghai

Invitrogen Biotechnology Co. Ltd. (Shanghai, China).

Polymerase chain reactions were carried out in 20 lL

volume containing 0.15 lmol/L each primer, 1 9 PCR

buffer (50 mmol/L KCl, 10 mmol/L Tris–HCl [pH 8.0],

0.1 % Triton X-100), 2.0 mmol/L MgCl2, 0.2 mmol/L

each dNTP, 100 ng caprine genomic DNA, 0.05 U/lL Taq

DNA polymerase (Promega, Madison, WI, USA), and the

rest was ddH2O. Amplification conditions were as follows:

initial denaturation at 95 �C for 5 min; followed by 32

cycles of denaturation at 95 �C for 30 s, annealing for 30 s

at 54–65 �C (Table A1 in Online Appendix), extension at

72 �C for 25–110 s; with a final extension at 72 �C for

5 min on Mastercycler� 5333 (Eppendorf AG, Hamburg,

Germany).

Cloning, sequencing and bioinformatics analysis

The PCR products were separated on 1.5 % agarose gels,

recovered by Geneclean II kit (Promega) and then ligated

into the pMD18-T simple vector (TaKaRa, Dalian, China)

according to the manufacturer’s instructions. The recom-

binant plasmid was then transformed into Escherichia coli

TOP10 competent cells. Positive clones were identified by

restriction enzyme digestion. At least, six positive clones

were sequenced in both directions for each pair of primers

using an ABI3730 automatic sequencer (Perkin Elmer

Applied Biosystems, Foster City, CA, USA) by Shanghai

Invitrogen Biotechnology Co. Ltd. (Shanghai, China).

The sequences were assembled and used to detect

the single nucleotide polymorphisms (SNPs) using SeqMan

II program (DNASTAR6.0). Sequence alignment, molec-

ular homology assessment and phylogenetic analysis were

performed through MegAlign (DNASTAR6.0), BLAST

(NCBI) and Mega 4.0 [37], respectively. Transcription

factor binding site in 50-regulatory region of goat RFRP

gene was predicted using TFSEARCH 1.3 (http://www

.cbrc.jp/research/db/TFSEARCH.html) [38]. Protein struc-

ture and function prediction were performed using Pep-

stats (http://www.ebi.ac.uk/Tools/emboss/pepinfo/), PSO

RT II Prediction (http://psort.hgc.jp/form2.html), Prot-

Comp 9.0 (http://linux1.softberry.com/berry.phtml?topic=

protcompan&group=programs&subgroup=proloc), TMH

MM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/),

SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), PP

Search program (http://www.ebi.ac.uk/Tools/ppsearch/),

SMART (http://smart.embl-heidelberg.de/), respectively.

PCR–RFLP analysis

The PCR products of primer pairs P7-P9 were digested by

AvaI, RsaI and EcoRV (NEB, Beijing, China) at 37 �C

overnight, respectively. After restriction enzyme digestion,

the products were detected by 2.0–2.5 % agarose gels and

genotyped using an AlphaImagerTM 2200 and 1220 Doc-

umentation and Analysis Systems (Alpha Innotech Cor-

poration, San Leandro, CA, USA).

Average daily sunshine duration and latitude data

The sunshine duration data were extracted from ‘The Annual

Surface Climate Normals of International Exchanging Sta-

tions of China (1971–2000)’ in China Meterological Data

Sharing Service System (http://cdc.cma.gov.cn/shuju/inde

x3.jsp?tpcat=SURF&dsid=SURF_CLI_CHN_MUL_MYE

R_19712000CES&ageid=3). The observation stations clos-

est to the farms, from which the blood samples were col-

lected, in the provenance of ten goat breeds were taken as the

data source for correlation analysis between G allele fre-

quency and average daily sunshine duration. There are

sunshine duration data of thirty years for us to utilize. Based

on the locations of the farms, we acquired the latitude data of

ten goat breeds from Google map (http://www.tsov.net/tool/).

The collected sunshine duration and latitude data were shown

in Table A2 (see Online Appendix).

Statistical analysis

Genotype distributions were analyzed using the Chi-

squares test. Correlation analysis between the allele fre-

quency and average daily sunshine duration was carried out

using Pearson correlation analysis (SAS 8.1). The matrix of

genetic distances was constructed by MEGA 4.0 based

on the three polymorphic loci and the matrix of geo-

graphical distances was constructed by JWD software

(version 1.0.0.1). The correlation between two matrixes

was also analyzed by Pearson correlation analysis (SAS

Mol Biol Rep (2012) 39:9167–9177 9169

123

8.1). The correlation analyses related to latitude were all

performed using Pearson correlation analysis (SAS 8.1).

The following fixed effects model was employed for

analysis of litter size in JG and least squares means were

used for multiple comparisons in litter size among the

different genotypes for each primer pair.

yijklm ¼ lþ Si þ KSj þ Pk þ Gl þ eijklm

where yijklm is the phenotypic value of litter size; l is the

population mean; Si is the fixed effect of the ith sire (i = 1,

2, 3, 4, 5); KSj is the fixed effect of the jth kidding season

(j = 1, 2, 3, 4); Pk is the fixed effect of the kth parity

(k = 1, 2, 3); Gl is the fixed effect of the lth genotype

(l = 1, 2, 3), and eijklm is the random residual effect of each

observation.

Analysis was performed using the general linear model

procedure of SAS (Ver 8.1) (SAS Institute Inc., Cary, NC,

USA). Mean separation procedures were conducted using a

least significant difference test.

Results

Sequence analysis of goat RFRP gene

Genomic DNA of goats was successfully amplified using

nine pairs of primers for RFPR gene. The results showed

that the amplification product sizes (Fig A1 in Online

Appendix) were consistent with the target ones (Table A1

in Online Appendix), which could be used for further

analyses, such as SNP detection and PCR–RFLP.

The RFRP genomic DNA sequence of 4,862 bp length

was submitted to GenBank (accession number: JF327669)

after sequence homology analysis. It contains about 1.5 kb

50 regulatory region upstream of the start codon, all of the

three exons and two introns. The splicing of exons and

introns is consistent with the GT-AG rule. Alignments of

goat RFRP sequence with the corresponding region of

bovine and human RFRP gene revealed 95.5 and 67.2 %

identity, respectively.

Goat RFRP has a CDS in size of 591 bp, coding

196 amino acids. The identities in CDS nucleotide and

amino acid sequences of goat RFRP with chicken, rat,

mouse, human, cattle and sheep were 57.1, 72.6, 73.0,

81.7, 96.1, 99.2, 46.2, 55.1, 55.3, 73.5, 94.4, and 98.5 %,

respectively. Furthermore, in order to analyze RFRP

molecular evolutionary relationship, we aligned the CDSs

or deduced proteins of different species, respectively.

Two molecular phylogenetic trees (Fig A2 in Online

Appendix) were constructed using MEGA 4.0, with the

neighbor-joining (NJ) procedure (bootstrap replicates =

1,000). From the NJ trees, we found that the molec-

ular phylogenic relationship was congruent with species

evolution and goat RFRP is closely related to that of

sheep and cattle.

The goat RFRP protein was predicted with Pepstats

(EBI emboss) to have a molecular weight of 22.5 kDa and

a pI of 10.6. The result of PSORT II Prediction indicated

that the residing probability of goat RFRP in the extra-

cellular region (including cell wall) was 55.6 %, but that

was 33.3 % in the cell nucleus. So we also used ProtComp

9.0 and TMHMM 2.0 to predict the sub-cellular localiza-

tion of RFRP and its potential transmembrane helices,

respectively. The results showed that RFRP was an extra-

cellular protein (secreted) without any transmembrane

helices. The signal peptide prediction performed by Sig-

nalP 3.0 showed that RFRP contained a signal peptide of

21 amino acids in its amino terminus. PPSearch program

results (Fig. A3 in Online Appendix) indicated that there

were four PKC phosphorylation sites (5, 37, 101, 121), one

CK2 phosphorylation site (60), one N-myristoylation site

(159) and one segment of cell attachment sequence (48–50)

in goat RFRP protein. SMART results (Fig. A3 in Online

Appendix) showed only a low complexity segment in the

predicted signal peptide. Multiple alignment of RFRP from

different animals, RFRP1-3 amino acid sequences and the

results by bioinformatics application software on web

service were depicted in Fig. A3 (see Online Appendix).

The result of TFSEARCH 1.3 revealed that A712G

mutation was located in a transcription factor binding site

(GTCGGGGA) of MZF1 (Myeloid zinc finger 1) which

was closely adjacent to an S8 (Paired mesoderm homeobox

protein 2) binding site (Fig. 4a). When A was present at

712 locus (GTCAGGGA), the binding site of MZF-1 dis-

appeared (Fig. 4b).

Polymorphism analysis of RFRP gene in thirteen goat

breeds

Sequencing and Seqman II results revealed three nucleo-

tide mutations (shown in Figs. 1b, 2b, 3b; A712G and

T1493C, at the -828th and -47th position upstream of the

start codon ATG, respectively; A3438T, at the 510th

nucleotide of CDS in exon 2 according to Ovis aries RFRP

mRNA sequence [GenBank No. EF494241]) in goat RFRP

gene. A3438T mutation resulted in a conservative amino

acid change (Glu170Asp), the other two (A712G and

T1493C) were in the 50 regulatory region.

T to C transition at the 1,493 locus, A to T transversion

at the 3,438 locus changed the recognition site of restric-

tion endonucleases RsaI and EcoRV, respectively. A to G

transition at the 712 locus changed the created AvaI rec-

ognition site using created restriction site PCR (CRS-PCR).

After PCR–RFLP analysis, A712G mutation gave rise to

three genotypes AA, AG and GG (Fig. 1a); T1493C

mutation gave rise to genotypes TT, TC and CC (Fig. 2a);

9170 Mol Biol Rep (2012) 39:9167–9177

123

A3438T mutation gave rise to genotypes AA, AT and TT

in goat breeds tested (Fig. 3a).

Allele and genotype frequencies of RFRP gene in thir-

teen goat breeds were presented in Table 1. For the 712,

1,493 and 3,438 loci, the G, T and A alleles were dominant

alleles almost in all thirteen goat breeds except for three

breeds (SW, MT and CW) for the 712 locus. Thirteen goat

breeds at three loci were in Hardy–Weinberg equilibrium

(P [ 0.05) except for the JG at the 712 locus (P \ 0.01).

Test of difference for RFRP genotype distribution

among six selective goat breeds

The test result of difference for RFRP genotype distribu-

tion among six selective goat breeds was summarized in

Table 2. For the 712 locus, significant difference existed in

four combinations of seasonal breeds and nonseasonal

breeds (JG/LC, JG/TH, GW/LC and GW/SD). For the

1,493 locus, there were significant differences between

nonseasonal breeds (JG and GW) and seasonal breeds (LC

and SD). For the 3,438 locus, there were significant dif-

ferences between nonseasonal breeds (GW and B) and all

three seasonal breeds (LC, TH and SD), between JG and

SD goats. No significant difference existed in any other

combinations of seasonal breeds and non seasonal breeds.

Therefore, for all pairs of nonseasonal breeds and seasonal

breeds in this study, there were no consistent significant

differences of genotype distribution in each locus.

Correlation between allele frequency and sunshine

duration or latitude

For the three polymorphic loci, Pearson correlations

between allele frequency of G, T, A and the average daily

sunshine duration were examined. The allele frequency of

712G was positively correlated with the average daily

sunshine duration in ten local goat breeds in China

(r = 0.830, P = 0.003). The scatter diagram for A712G

was shown in Fig. 5. There were no significant correlations

between allele frequency and the average daily sunshine

duration for T1493C (r = 0.045, P = 0.903) and A3438T

(r = 0.505, P = 0.165). Our results showed that there

was a moderate correlation (r = 0.69773, P = 0.0249)

between latitude and G allele frequency for A712G locus

(Fig. 6) and no significant correlation existed for other

two loci: T1493C (r = 0.21771, P = 0.5457) and A3438T

(r = 0.34794, P = 0.3245).

712A 712G

1 2 3 4 5 6 7 8 9 Mbp

600 500 400 300

200

100

a

b

Fig. 1 The PCR–RFLP (AvaI) analysis of A712G in goat RFRP gene

and sequences of different genotypes. a PCR–RFLP analysis of the

712 locus in goat RFRP gene using primer P7 (2.5 % agarose gel

stained with ethidium bromide). Only one AvaI site was located at the

29th of the 172 bp amplified fragment by primer P7. Three genotypes:

AA, bands 2 and 7 (172 bp); GA, bands 1 and 9 (172/143/29 bp); GG,

bands 3–6 and 8 (143/29 bp). M DNA Marker I (100–600 bp)

(Tiangen, Beijing). b: Sequence of genotypes AA and GG at the 712

locus in goat RFRP gene

1493T 1493C

M 1 2 3 4 5 6 7 8 9 10 11 12

bp

600500400300200

100

a

b

Fig. 2 The PCR–RFLP (RsaI) analysis of T1493C in goat RFRPgene and sequences of different genotypes. a PCR–RFLP analysis of

the 1,493 locus in goat RFRP gene using primer P8 (2.5 % agarose

gel stained with ethidium bromide). Only one RsaI site was located at

the 134th of the 233 bp amplified fragment by primer P8. Three

genotypes: TT, bands 5, 10–12 (233 bp); TC, bands 1–4, 7 and 9

(233/134/99 bp); CC, bands 6 and 8 (134/99 bp). M DNA Marker I

(100–600 bp) (Tiangen, Beijing). b: Sequence of genotypes TT and

CC at the 1,493 locus in goat RFRP gene

Mol Biol Rep (2012) 39:9167–9177 9171

123

Correlation between genetic and geographical distances

The matrixes of genetic distances and geographical dis-

tances were shown in Tables A3 and A4, respectively.

There was no significant correlation between these two

matrixes (r = 0.09723, P = 0.5727), indicating that no

significant effect of geographical distances on genetic

structure. This suggested that the G allele frequency

(A712G locus) of goat RFRP gene was not influenced by

the genetic drift.

Influence of fixed effects on litter size in Jining Grey

goats

Litter size in JG does was significantly influenced by sire,

kidding season and parity (all P \ 0.05). The least squares

mean and standard error for litter size of different RFRP

genotypes in JG were given in Table 3. There were no

significant differences in litter size among different geno-

types for the three loci.

Discussion

Sequence analysis and polymorphisms of goat RFRP

gene

In the phylogenetic and amino acid identity analysis, the

goat RFRP is shown to be evolutionally conserved among

mammals. Furthermore, the goat RFRP shows the closest

relationship with sheep and cattle, then with human and

rodents (rat and mouse), ultimately with chicken. This

coincides with the fact that goat, sheep and cattle belong to

the same family, Bovidae, while human, rat and mouse

belong to other different orders of mammals. It may indi-

cate that the homologous degree of RFRP among different

species corresponds to their degree of phylogenetic rela-

tionships. Goat RFRP precursor contains RFRP1 and

RFRP3, which both have a LPXRF-amino acid at their

C-termini like chicken GnIH, while RFRP2 with LPLRL at

its C-terminus. Moreover, RFRP2 of all six kinds of

mammals have no LPXRF-amino acid at their C-termini,

even for rodents the RFRP2 sequence doesn’t exist. This

may be the reason why mammalian RFRP contains only

Fig. 4 The predicted

transcription factor binding sites

using TFSEARCH 1.3. R1 and

R2 represent the possible

binding region for the

transcription factors MZF1 and

S8, respectively. a R1 could

bind with MZF-1 when it was G

at 712 locus; b R1 could not

bind with MZF-1 when it was A

at 712 locus

3438A 3438T

M 1 2 3 4 5 6 7 8 9 10 11 12bp

600 500 400 300

200

100

a

b

Fig. 3 The PCR–RFLP (EcoRV) analysis of A3438T in goat RFRPgene and sequences of different genotypes. a, PCR–RFLP analysis of

the 3,438 locus in goat RFRP gene using primer P9 (2.0 % agarose

gel stained with ethidium bromide). Only one EcoRV site was located

at the 105th of the 404 bp amplified fragment by primer P9. Three

genotypes: AA, bands 1–3 and 9–11 (404 bp); AT, bands 4–7 (404/

299/105 bp); TT, bands 8 and 12 (299/105 bp). M DNA Marker I

(100–600 bp) (Tiangen, Beijing). b: Sequence of genotypes AA and

TT at the 3,438 locus in goat RFRP gene

9172 Mol Biol Rep (2012) 39:9167–9177

123

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(25

)0

.26

8(1

5)

0.1

52

(5)

0.0

73

(6)

0.1

09

(6)

0.0

68

(4)

0.4

24

(25

)0

.234

(11

)0

.327

(17

)0

.41

3(2

4)

0.1

32

(7)

0.1

08

(4)

0.0

83

(5)

AG

0.3

04

(73

)0

.44

6(2

5)

0.3

33

(11

)0

.54

9(4

5)

0.4

91

(27

)0

.356

(21

)0

.407

(24

)0

.447

(21

)0

.481

(25

)0

.46

5(2

7)

0.3

02

(16

)0

.243

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67

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)

GG

0.5

92

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86

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76

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(15

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.192

(10

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1(7

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.566

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.649

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.65(3

9)

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0.2

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0.4

91

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18

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0.3

54

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46

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27

0.4

57

0.5

67

0.6

47

0.2

83

0.2

30

.217

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.744

0.5

09

0.6

82

0.6

52

0.6

46

0.7

54

0.3

73

0.5

43

0.4

33

0.3

53

0.7

17

0.7

70

.783

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73

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90

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10

.47

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20

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83

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2.7

6

T1

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CN

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23

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63

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25

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85

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95

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66

0

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52

(19

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(24

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.56

9(3

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.667

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9(3

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81

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(28

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4(2

4)

0.6

35

(33

)0

.333

(12

)0

.467

(28

)

CC

0.1

80

(42

)0

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2(7

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(4)

0.1

02

(6)

0.1

67

(9)

0.0

17

(1)

0.1

02

(6)

0.0

41

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0.0

19

(1)

0.0

17

(1)

0.0

38

(2)

00

.05(3

)

All

ele

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0.5

76

0.6

77

0.7

38

0.5

93

0.7

33

0.7

29

0.8

16

0.7

17

0.7

76

0.6

44

0.8

33

0.7

17

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0.4

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0.4

24

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62

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07

0.2

67

0.2

71

0.1

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0.2

83

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24

0.3

56

0.1

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83

H–

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0.4

0.0

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90

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81

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60

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66

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00

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73

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75

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81

0.4

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0.0

56

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25

H–

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stv2

5.7

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21

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Mol Biol Rep (2012) 39:9167–9177 9173

123

two biologically active forms, RFRP1/3 rather than

RFRP2. Protein prediction showed that RFRP is a secreted

protein with a signal peptide of 21 amino acids in its amino

terminus, which corresponded to its character as a

neurotransmitter.

There are few studies on DNA sequence cloning and

polymorphism scanning of RFRP gene in mammals. Up to

now, only polymorphisms of human RFRP gene have been

detected [39]. Three SNPs were determined in coding

regions of human RFRP gene, which designated as 143G-

[C, 172A-[G and 431C-[T, respectively. The former two

mutations result in non-conservative amino acid substitu-

tions and the 431C-[T alteration does not give rise to

amino acid change. In the present study, we have also

found three SNPs in thirteen goat breeds, which were

A712G, T1493C and A3438T. One mutation (A3438T)

was in the exon 2 of goat RFRP gene and just gave rise to a

conservative amino acid change (Glu170Asp), which may

unlikely affect the function of RFRP gene. The other two

mutations (A712G and T1493C) were in the 50 regulatory

region. Unlike in human, no polymorphisms were found in

exon 1 and 3 of goat RFRP gene in our research.

Relationship of goat RFRP gene with photoperiod

and seasonal reproduction

Generally, goat is a kind of animal with seasonal repro-

duction in temperate regions. They keep regular repro-

ductive activities, such as estrus and ovulation, mainly in

autumn with gradually shortening day length but show

anestrous state in other seasons. Traditionally, seasonal

reproduction is dominantly influenced by photoperiod and

involved in negative feedback action of estradiol on

hypothalamic-pituitary axis. Previous studies on rodents

[23, 25, 26, 40] and sheep [8, 12] revealed that the

expression of RFRP gene was affected by photoperiod [25]

and inversely related with reproductive status [8, 40].

In our study, the significant positive correlation of G allele

frequency with average daily sunshine duration was

determined for the A712G locus in ten Chinese local goat

breeds. Meanwhile, there was no significant correlation

between geographical distances and genetic distances for

local goat breeds. Our results indicated that the difference

of G allele frequency didn’t result from genetic drift but

really a selective pressure in sunshine duration consistent

with studies mentioned above. It was further proved by the

moderate correlation between G allele frequency for

A712G locus and latitude, which highly correlated with

sunshine duration (data not shown). Moreover, our study

revealed that the A712G mutation was located in a MZF-1

transcription factor binding site which was closely adjacent

to an S8 binding site [41, 42]. MZF-1 and S8 are both

influenced by retinoic acid which is crucial for eyes and

expression of photoreceptor [43, 44]. And recent studies

Table 2 Test of difference for 712/1493 loci (above diagonal) and 3,438 locus(below diagonal) genotype distributions of RFRP in six selective

goat breeds

Breed Jining grey goat Guizhou white goat Boer goat Liaoning cashmere goat Taihang goat Saanen dairy goat

Jining grey goat 19.74***/1.53 0.96/1.43 15.78***/9.45** 7.57*/0.24 1.07/9.95**

Guizhou white goat 12.34** 4.85/2.60 9.81**/7.47* 4.87/0.39 13.13**/8.19*

Boer goat 12.60** 0.09 4.81/0.98 2.10/1.43 1.69/5.05

Liaoning cashmere goat 2.90 8.14* 9.15* 0.74/6.09* 5.66/3.59

Taihang goat 3.76 13.14** 14.36*** 2.51 3.59/8.15*

Saanen dairy goat 14. 68*** 32.23*** 32.33*** 17.55*** 9.78**

* P \ 0.05, ** P \ 0.01, *** P \ 0.001

0.3

0.4

0.5

0.6

0.7

0.8

27 29 31 33 35 37 39 41

latitude (°)

712G

alle

le f

requ

ency

Fig. 6 The correlation analysis between 712G allele frequency and

latitudes for ten local goat breeds in China

0.3

0.4

0.5

0.6

0.7

0.8

2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Average daily sunshine duration (hour)

712G

alle

le f

requ

ency

Fig. 5 The correlation analysis between 712G allele frequency and

average daily sunshine duration for ten local goat breeds in China

9174 Mol Biol Rep (2012) 39:9167–9177

123

also revealed that retinoic acid signaling pathway in rodent

was regulated by photoperiod [45, 46]. Mutation of A to G

at 712 locus was predicted to give rise to a MZF-1 binding

site, namely, the A712G mutation influenced the binding

activity of MZF-1 binding site with MZF-1. However,

whether the mutation actually affects the binding activity

of MZF-1 needs to be further confirmed. We preliminarily

presumed that the A712G mutation might be involved in

photoperiodic regulation of biological processes in goats

by altering binding activity of cis-element with MZF-1.

It may be an explanation for why the expression of RFRP

gene could be influenced by photoperiod. Additionally,

human MTNR1B (melatonin receptor 1B) gene was under

the selective pressure of sunshine duration [47], whether it

is a mediator between photoperiod and expression of

melatonin-driven RFRP gene is an interesting question.

Then the genotype frequency distributions for the three

mutations were detected in the six selective goat breeds.

There were significant differences between some of non-

seasonal breeds and seasonal breeds, such as four pairs of

JG/LC, JG/TH, GW/LC and GW/SD for the 712 locus, two

nonseasonal breeds (JG and GW) and two seasonal breeds

(LC and SD) for the 1,493 locus, and two nonseasonal

breeds (GW and B) and all selective seasonal breeds (LC,

TH and SD) for the 3,438 locus. However, no consistent

significant difference in genotype distribution was shown

between all nonseasonal estrous breeds and seasonal

estrous breeds for each locus, indicating that the three loci

may have no definite relationships with seasonal repro-

duction or the effect of three mutations on reproductive

seasonality may just exist in some goat breeds. Test of

difference in genotype distribution may be not a precise

way for measuring reproductive seasonality. More appro-

priate indexes are needed in further studies. Furthermore,

seasonal regulation involving RFRP should be more

efficient when RFRP acted in concert with other important

factors like kisspeptin as mentioned in introduction.

Association between goat RFRP gene and litter size

Numerous studies in sheep and goats have shown that

polymorphisms of BMP15 [48–50], GDF9 [51], especially

BMPR1B gene (FecB mutation) [49, 52] are associated

with litter size, but no similar study of association analysis

between litter size and goat RFRP gene. And RFRP has

been shown to directly influence GnRH secretion, with

further regulating LH synthesis and secretion and ulti-

mately controlling reproductive activities of animals [1, 7,

8, 15–19]. Recently, it has been demonstrated that GnRH I,

GnRH I-receptor and RFRP-3 coexpressed in close vicinity

of mouse ovary and the interaction between GnRH

I-RFRP-3 neuropeptides may be involved in the regulation

of follicular development and atresia [53]. In the present

study, we have also analyzed the effects of goat RFRP gene

mutations on litter size. But there was no significant

association between genotypes and litter size for three

polymorphic loci. The A3438T mutation probably doesn’t

affect the function of protein because it gives rise to a

conservative change of amino acid. Furthermore, it was not

located in the region of cleaved mature protein with

activity. The T1493C mutation was located at 47 bp

upstream of start code ATG but no relationship was found

between this mutation and litter size in goats. Our results

demonstrated that there was possibly no obvious associa-

tion between polymorphisms of RFRP gene and litter size

in goats.

Conclusion

In this work, a fragment of 4,862 bp DNA sequence of

goat RFRP gene was obtained (GenBank No. JF327669).

It contained about 1.5 kb 50 regulatory region upstream of

the start codon, all of the three exons and two introns. Goat

RFRP gene has a CDS of 591 bp, coding 196 amino acids

which was predicted to be a secreted protein with a signal

peptide of 21 amino acids. The sequence of RFRP gene is

highly conservative in mammals. In addition, three SNPs

(A712G and T1493C in 50 regulatory region; A3438T in

exon 2) were found. The frequency of G allele at 712 locus

was positively correlated (r = 0.830, P = 0.003) with

average daily sunshine duration in ten Chinese local goat

breeds. This association was more convincing because no

correlation existed between geographical distances and

genetic distances, which indicated that there was no effect

of genetic drift on the G allele frequency. Additionally, the

effect of A712G mutation on the binding activity of MZF-1

site is worthy of further research. However, there may be

Table 3 Least squares mean and standard error for litter size of

different genotypes of the three loci of RFRP gene in Jining grey

goats

Locus Genotype Number of does Litter size

A712G AA 25 2.46a ± 0.20

AG 73 2.36a ± 0.15

GG 142 2.27a ± 0.11

T1493C TT 88 2.34a ± 0.16

TC 104 2.25a ± 0.12

CC 42 2.44a ± 0.19

A3438T AA 162 2.32a ± 0.10

AT 64 2.29a ± 0.14

TT 15 2.43a ± 0.21

a Least squares means with the same superscript for the same locus

have no significant difference (P [ 0.05)

Mol Biol Rep (2012) 39:9167–9177 9175

123

no definite association between polymorphisms of RFRP

gene and reproductive seasonality in goats. For lack of

functional data in goats, our study drew a preliminary

conclusion and further studies are needed to verify the

photoperiodic regulation effect of goat RFRP gene on

reproduction.

Acknowledgments This work was supported by the earmarked

fund for China Agriculture Research System (CARS-39), National

Key Technology Research and Development (R&D) Program

of China (2008BADB2B01), National Natural Science Foundation of

China (30871773), National High Technology R&D Program of

China (2006AA10Z139), National Key Basic R&D Program of China

(2006CB102105), Special Fund for Basic Scientific Research of

Institute of Animal Science, Chinese Academy of Agricultural Sci-

ences (2010jc-9).

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