The Differential Expression and Snp Analysis of the Ovoinhibitor Gene in the Ovaries of Laying Duck Breeds ( Anas Platyrhynchos )

Ovoinhibitor (OIH) is the main proteinase inhibitor in the egg white. In the present study, real-time quantitative PCR and Western-Blot were used to analyze different expression pattern of OIH in ovaries as a candidate gene of reproductive traits in Jingjiang ducks (JJ ducks) and Shaoxing ducks (SX ducks) during three laying stages. To study the polymorphism of the OIH gene in those two duck populations, we designed five pairs of primers to detect SNPs of exon 3-5, 5-6, 14-16 and intron 7, 9 of the OIH gene by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) and DNA pool sequencing methods. The results showed that OIH expression increased during the laying stage in the ovaries of both duck breeds. The relative expression levels of OIH in the egg at hatch and 180days of age were lower in JJ ducks than in SX ducks, but higher in JJ ducks than SX ducks at 500 days of age. Only exon 5-6 locus had a novel SNP. One variation (389G>A) was detected in the two tested duck populations and it was associated with some laying traits, such as body weight of hatch, age at first egg, weight at first egg, egg number at 72weeks of age. The AG genotype was associated with inferior body weight of hatch and superior weight at first egg, age at first egg and egg number at 72weeks of age. Therefore, these results suggest that OIH may be a strong candidate gene related to some laying traits in ducks.


IntRoduCtIon
Ovoinhibitor (OIH) is the main proteinase inhibitor in the albumen (Kinoshita et al., 2004).It is produced in the tubular gland cell of the oviduct, which is regulated by estrogen and progesterone (Liu et al., 1971).It has a broad inhibitory activity of other proteinases present in the blood plasma and in the egg white of chickens (Laskowski & Kato, 1980).Słowińska et al. (2014) reported that the ovoinhibitor in the turkey reproductive tract maintains a microenvironment for the sperm in the epididymis and ductus deferens.Gao et al.(2016) reported that protease inhibitors, such as ovoinhibitor, may play a key role in the degradation of egg yolk proteins.Other authors suggested that ovoinhibitors may be related to the reproductive performance of poultry (Zhu et al., 2011).
There are literature reports on the impact of OIH in poultry; however, studies on the function of OIH in the ovaries of different duck breeds are rare.Therefore, as a candidate gene for reproductive traits, the objective of this study was to determine the expression patterns of OIH in the ovaries of Jingjiang ducks(JJ duck) and Shaoxing ducks (SX ducks) during the three different laying stages, and to identify the SNP of the OIH gene associated with laying traits.

Animal source and samplespreparation
The Jingjiang (JJ) and Shaoxing (SX) are two local varieties of ducks in China.The SX duck has a small body size, early maturity (about D102) and high laying performance (over 300 eggs per year) (Zhao et al., 2005).The JJ duck is an indigenous species of the Hubei Province of China, and has small body size, early maturity (about D100) and low laying performance (about 200 eggs per year) (Ding et al., 2004).
In this study, 100 JJ, 100 SX, 250 JJ line I, and 226 JJ line II female ducklings (JJ lines I and II were two lines derived from the JJ breed) were reared from 0 to 500daysof age.All ducks were housed in individual cages in the same room with the controlled temperature (25±3°C), and fed a commercial diet during the experiments.At age at first egg, and at 180 days (egg production peak) and 500 days of age (egg production rapidly decreases at this age), six JJ ducks and six SX ducks were anesthetized with a mixture of 79% CO 2 and 21% O 2 for 1 min and then decapitated.The ovaries were collected after dissection, immediately frozen in liquid nitrogen, and stored at -80ºC until further analyses.
Blood samples and performance measurements (body weight at hatch, and at 4 weeks, 8 weeks, 12 weeks, and 72 weeks of age, and at first egg, age at first egg, egg weight and number of eggs laid at 72weeks of age) were collected from the 476 ducks (JJ lines I and II).Genomic DNA was obtained according to standard procedures and stored at -20 °C.

RNA isolation and cDNA synthesis
Total RNA was extracted using TRIZOL reagent, according to manufacturer's protocol (TaKaRa, Dalian, China).Total RNA samples were separately pooled per duck and ovary.cDNA was synthesized from 1 μg total RNA, derived from the RNA pools, using PrimeScript RT-reagent kit (TaKaRa, Dalian, China) with random hexamers.

Real-time quantitative PCR
All PCR primers for real-time quantitative RT-PCR are shown in Table 1.Primers were chosen to generate specific products, of around 200 bp of β-actin and OIH.The β-actin (EF667345) and OIH (NM_001030612) sequences for the designed primers were retrieved from GenBank.Real-time quantitative PCRs were performed in a 20-μL mixture containing 10 μL 2×SYBR Green/Fluorescein qPCR Master Mix (TaKaRa, Dalian, China), 1 μL template cDNA (1μg/μL), 0.4 μM forward and reverse primers, and 8.2 μL dd H 2 O. Realtime quantitative PCR was performed on an ABI 7500 real-time quantitative PCR thermal cycling instrument (Applied Biosystems, USA).The real-time quantitative PCR amplification was performed in triplicate.The cycling conditions wereas follows: 1 cycle of 50°C for 2 min, 95°C for 10min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s.

Western-Blotting Analysis
The OIH protein from each tissue was extracted and separated in 12% gradient SDS polyacrylamide gels.The separated OIH protein was then transferred onto a polyvinylidene difluoride (PVDF) membrane.The specific antibody for OIH was used at 1:1000 dilution.All incubations were implemented in TBSE solution (containing 5% dry milk).Immunodetection was executed using Pierce chemiluminescence (Thermo, USA).The signal intensity was determined by the density measurement method.

PCR amplification, SNP identification, and genotyping
According to duck OIH gene sequences (NC_006100), five pairs of primers (shown in Table 1) were designed to amplify the OIH gene exon3 to16 regions and to detect their SNP.
The PCR assays were performed in an Eppendorf Mastercycler gradient (Eppendorf, Germany).The final volume was15 μL, and contained 7.5 μL 2×Taq PCR mix (Dongsheng, Guangdong, China), 10 pM of each primer, and 50 ng genomic DNA.The cycling condition consisted of an initial denaturation step at 95 °C for 4 min followed by 35 cycles of 95 °C for 30 s, annealing at X °C (Table 1) for 35 s, 72 °C for 30 s, and a final extension at 72 °C 10 min.
Genomic DNA of JJ lines I and II was used as template and the sequenced sequences from different samples were aligned to search for base variations.
The PCR-RFLP was used to analyze the polymorphisms in duck OIH.It was performed by mixing 8 μL of the PCR products (with primer exon 5-6), 10 U Taq I restriction enzyme (New England Biolabs, USA) and 1 μL 10 × reaction buffer.The mixtures were incubated at 37 °C for 12 h and separated at 150 v in 3% agarose gel.

Statistical analyses
The real-time quantitative PCR data were calculated using the 2 −∆∆Ct comparative CT method.The results are expressed as mean ± standard deviation.Ct values were tested for normality (Kolmogorov-Smirnov test) prior to analysis.Statistical analysis of data was performed by one-way analysis of variance followed by LSD post-hoc test.
The gene frequencies were determined for each population by direct counting.Hardy-Weinberg equilibrium (HWE) was tested using χ 2 -test of PopGene32 (version 3.2) in each population.The association analyses between OIH genotypes and laying traits (including body weight at hatch, and 4, 8, 12,72 weeks of age, weight at first egg, age at first egg, egg weight and egg number at72 weeks of age) were analyzed according to the general linear model (GLM) procedure of the software SPSS (version 18.0).All analyses were performed first using a full animal model, and next using a reduced animal model.The full animal model included fixed effects of SNP genotypes, populations, and random effects (including permanent environment, animal and residual).The effect associated with populations was not matched in the linear model, and the preliminary analyses indicated that this effect had no significant influence on variability of the traits in the analyzed populations.
Therefore, the following reduced linear model was used for final analysis: Where Y ij is the trait measured on each of ijth animal; μ was the overall population means; G i is the fixed effect associated with the ith genotype; and e ij is the random error.The least square analysis was used to determine the difference between genotypes.

Expression of duck OIH
The mRNA expression of OIH in ovaries of JJ and SX ducks collected at the three different ages was determined by real-time quantitative PCR.As Figure1 shows, the mRNA of OIH was detected in all examined ovary samples.The relative expression of the OIH mRNA transcript in the ovaries continuously increased during the laying period (from the first egg to 500 days of age).The relative expression level of OIH mRNA in the ovaries (p<0.05) was significantly different between the two breeds from at age at first egg and 180 days of age, but not at 500 days of age (p>0.05).Furthermore, in the ovaries, the relative mRNA expression of OIH between age at1 st egg and 180 days of age was lower in JJ ducks compared with SX ducks, but was higher in in JJ ducks than in SX ducks at 500 days of age.The method of Western-Blot was used to examine whether the OIH protein could be detected in ovary samples.The predicted duck OIH protein weight was 49kDa.The results are shown in Figure 2. The OIH protein was detected in all ovaries of both SX and JJ

The Differential Expression and Snp Analysis of the Ovoinhibitor Gene in the Ovaries of Laying Duck Breeds (Anas Platyrhynchos)
ducks, and the results did not show any significant differences (p>0.05) in them RNA expression of OIH between the two breeds at any of the evaluated periods.This may be probably due to the fact that part of the mRNA of OIH was not translated into the protein in the ovaries at the different stages.

Identified SNP
The sequences amplified with all primers (P1, P2, P3, P4 and P5ofexon 3-5, exon 5-6, intron 7, intron 9 and exon 14-16) were aligned between JJ duck lines 1 and 2. No mutations were detected in exon 3-5, intron 7, intron 9, or exon 14-16.There was one variation locus in exon 5-6, G→A at the 389-nucleotide position (389G>A), which was detected by restriction endonucleases TaqI.This variation was a synonymous mutation and did not cause any amino acid changes.This SNP was detected by PCR-RFLP using the amplification product of exon 5-6.We submitted the sequences of two homozygotes of exon 5-6 to GenBank, and gain accession numbers KC977993 (for the GG genotype) and KC977994 (for the AA genotype).
As Figure 3 shows, the G to Amutation at locus 389 expressed three genotypes: GG, GA and AA, and the partial sequences of three genotypes are shown in Figure 4.

Allele and genotype of OIH gene exon5-6
The frequencies of allele and genotypes of OIH gene exon 5-6 in the two duck populations are shown Table 2.In the 389 locus of exon 5-6, the G allele was

The Differential Expression and Snp Analysis of the Ovoinhibitor Gene in the Ovaries of Laying Duck Breeds (Anas Platyrhynchos)
the preponderant allele in the evaluated populations, and the JJ duck line II population deviated from Hardy-Weinberg equilibrium (HWE) (p<0.01).

Association analysis of OIH gene exon 5-6
We analyzed the association of the SNP with laying traits of two duck populations (JJ lines I and II).The results are presented in Table 3.In JJ duck line I, heterozygotes (AG) were significantly younger at first egg than those with genotypes AA and GG (p<0.05), and egg number at 72 weeks of age was significantly different among the AG, GG and AA genotypes (p<0.05).In JJ duck line II, the genotype AA was significantly heavier at hatch, and produced a higher number of eggs at 72weeks of age (p<0.05)than genotypes GG and AG.Moreover, body weight at first egg of the genotype GG was significantly lower compared with genotype AG (p<0.05).

dISCuSSIon
Ovoinhibitor (OIH) is a Kazal-type serine inhibitor.It is the main protease inhibitor found in the chicken plasma, and known to inhibit the activity of trypsin, chymotrypsin, and elastase (Zahnley, 1975;Shechter et al. 1977;Vered et al., 1981).OIH is a three-domain protein and shares its structure with the ovomucoid protein (Moore et al., 2004).It is reported that OIH is highly expressed in the magnum and liver of chickens, followed by the uterus, egg yolk, and eggshell precursors, respectively, and that its expression increases in the liver during sexual maturation and subsequently decreases in mature hens (Bourin et al., 2011).However, to date, the expression pattern of OIH in ovaries of laying ducks has not been not detected.Real-timequantitative PCR has become a standard method to measure gene expression by evaluating the amount of mRNA produced (Ong et al., 2002).So, in this study, the expression of OIH mRNA in the ovaries at age at first egg, and 180 days and 500 days of age were researched using real-time quantitative PCR.
In this study, OIH mRNA expression level increased from age at1 st egg until 500 days of age in the ovaries of both JJ and SX ducks, and was significantly different between age at first egg and 180 days.These results are inconsistent with the OIH expression levels reported in the liver of mature chickens (Bourin, et al., 2011), and suggest that the profile of OIH expression is different among species and tissues.
SX and JJ ducks are both local Chinese breeds, with SX ducks presenting high egg production and JJ ducks low egg production.In this study, we found that the OIH mRNA expression in JJ ducks was lower than in SX ducks at age at first egg and 180 days of age, whereas at 500 days of age, the OIH mRNA expression in JJ ducks was higher than in SX ducks.In addition, the Western-Blot results have showed similar OIH mRNA expression pattern.These results indicate that OIH is closely related to the laying performance of ducks and may play an important role in the ovary during the egg-laying period.
The candidate gene method is a very powerful method to detect associations of gene polymorphisms Pan AL, Shen J, Pu YJ, Du JP

The Differential Expression and Snp Analysis of the Ovoinhibitor Gene in the Ovaries of Laying Duck Breeds (Anas Platyrhynchos)
with economic traits in farm animals (Schwerin, 2003).In view of the function of OIH, in this research, the OIH gene was chosen as a candidate gene to study the association between gene polymorphisms and laying traits in ducks.Goliasova & Wolf (2004) reported that in populations subjected to selection, in general, deviations of genotype frequencies from the Hardy-Weinberg equilibrium are expected in some loci that impact traits under selection.The results of this study indicate that the JJ duck II population deviated from the Hardy-Weinberg equilibrium (HWE) (p<0.05) for the OIH gene.One of the reasons may be that the evaluated population was subjected to intensive selection during commercial breeding.
In this study, one novel mutation was found in exon 5-6 of the OIH gene using PCR-RFLP methods.We analyzed the associations between genotypes and laying traits, including hatching weight, weight at 4, 8,12, and 72weeksof age, weight at first egg, age at first egg, egg weight, egg number at 72 weeks of age.The results indicated the AG genotype presented inferior hatching weight and superior weight at first egg, age at first egg, and egg number at72 weeks of age.This is in accordance with Wang (2015), who suggested that the SNP of OIH gene introns 10 and 11 may be related with some laying traits in ducks.Therefore, we postulate that the OIH gene may have a quantitative trait locus (QTL), which controls some laying traits in the exon 5-6,and that the genotype AG may be used as a SNP marker for inferior hatch weight and superior weight at first egg, age at first egg, and egg number at 72 weeks of age.Nevertheless, we concluded that, in order to prove the associations of the mutation of the OIH gene exon 5-6, more samples and further analyses should be carried out.

ACknowledgeMentS
This study was supported by the open project of Animal Embryo and Molecular Breeding of Hubei Key Laboratory (Grant No. 2017ZD141) and Technical innovation special project of Hubei Province (Grant No. 2016ABA117).

Figure 1 -
Figure 1 -Development changes of the relative expression levels of OIH mRNA in the ovaries of Jingjiang and Shaoxing ducks.Note: Different lowercase letters (a and b) indicate significant difference (p<0.05) between Jingjiang and Shaoxing ducks; different uppercase letters (A, B and C) indicate significant age of first egg, 180-day and 500-day differences (p<0.05) in the same duck population.

Figure 2 -
Figure 2 -Western-blot results of OIH in the ovaries of Jingjiang and Shaoxing ducks.Note: 1 is stage of age at first egg; 2 is stage of 180 days of age; 3 is stage of 500 days of age; (a) Western blot results of OIH; (b) relative expression of OIH in the ovaries of JJ and SX ducks

Table 1 -
Primers used for real time quantitative PCR and SNP detecting of duck OIH gene

Table 3 -
Least square means and standard errors of the laying traits in two duck populations