Polymorphisms of melatonin receptor genes and their associations with egg production traits in Shaoxing duck

Objective In birds, three types of melatonin receptors (MTNR1A, MTNR1B, and MTNR1C) have been cloned. Previous researches have showed that three melatonin receptors played an essential role in reproduction and ovarian physiology. However, the association of polymorphisms of the three receptors with duck reproduction traits and egg quality traits is still unknown. In this test, we chose MTNR1A, MTNR1B, and MTNR1C as candidate genes to detect novel sequence polymorphism and analyze their association with egg production traits in Shaoxing duck, and detected their mRNA expression level in ovaries. Methods In this study, a total of 785 duck blood samples were collected to investigate the association of melatonin receptor genes with egg production traits and egg quality traits using a direct sequencing method. And 6 ducks representing two groups (3 of each) according to the age at first eggs (at 128 days of age or after 150 days of age) were carefully selected for quantitative real-time polymerase chain reaction. Results Seven novel polymorphisms (MTNR1A: g. 268C>T, MTNR1B: g. 41C>T, and g. 161T>C, MTNR1C: g. 10C>T, g. 24A>G, g. 108C>T, g. 363 T>C) were detected. The single nucleotide polymorphism (SNP) of MTNR1A (g. 268C>T) was significantly linked with the age at first egg (p<0.05). And a statistically significant association (p<0.05) was found between MTNR1C g.108 C>T and egg production traits: total egg numbers at 34 weeks old of age and age at first egg. In addition, the mRNA expression level of MTNR1A in ovary was significantly higher in late-mature group than in early-mature group, while MTNR1C showed a contrary tendency (p<0.05). Conclusion These results suggest that identified SNPs in MTNR1A and MTNR1C may influence the age at first egg and could be considered as the candidate molecular marker for identify early maturely traits in duck selection and improvement.


INTRODUCTION
Melatonin (Nacetyl5methoxytryptamine) is an important hormone that is synthesized mainly in the pineal gland, and has a profound effect on serval physiology process includ ing circadian rhythm and reproduction through its special receptors in birds [13]. Three melatonin receptor subtypes, MTNR1A (alias MT1, Mel1a), MTNR1B (alias MT2, Mel1b), and MTNR1C (alias Mel1c), which belong to the superfamily of G proteincoupled receptors [4], have been cloned in birds [5,6].
In chicken, melatonin receptor subtypes were identified in ovaries [7,8], suggesting that melatonin directly affects ovarian function through activating of multiple receptors. In geese, the expression levels of MTNR1A, MTNR1B, and MTNR1C initially increased and later decreased during follicular development cycle, indicating that melatonin receptors participated in activating small white follicles and small yellow follicles to develop into subsequent greater hierarchical follicles [9]. In addition, in situ hybridization of MTNR1C mRNA combined with immunocytochemistry for gonadotropin inhibitory hormone (GnIH), a key neurohormone controlling avian reproduction by inhibiting gonadal development [10], revealed a clear cellular colocalization of MTNR1C mRNA and GnIH in paraventricular nuclei [11]. It seems that melatonin most likely acts directly on GnIH neurons through MTNR1C to induce GnIH expression and regulate avian reproduction.
Shaoxing duck is a Chinese dominant layer breed, charac terized by small body size, early maturity and high productivity. Modern Shaoxing duck achieve 50% egg production by 140 days of age, and the mean number of eggs at 500 days old was more than 300 [12]. Shaoxing ducks have been considered as a good source of duck eggs in China, and improving its egg production performance is of primary interest to breeders and farmers. The identification of single nucleotide polymor phisms (SNP) in candidate genes associating with economically important traits has become a powerful tool for genetic im provement of animal selection and production.
Numerous studies have investigated the relationship be tween melatonin receptor subtypes and egg production traits in different species, and made melatonin receptor genes poten tial candidate genes for QTLs [13,14]. However, whether or not nucleotide polymorphisms of MTNR1A, MTNR1B, and MTNR1C are associated with the egg production traits and egg quality traits in ducks is still unknown. Therefore, this experiment aimed to detect the SNPs in MTNR1A, MTNR1B, and MTNR1C and explore their associations with egg produc tion traits and egg quality traits. The expression levels of these genes in ovary of ducks at different age at first egg were also determined.

Performance traits and tissue collection
A total of 785 female Shaoxing ducks were randomly selected and raised in separate cages under similar environmental con ditions and diet. The egg production traits of all ducks were recorded throughout the egg production process in terms of age at first egg, egg weight at 34 weeks old and total number of eggs at 34 weeks old and 72 weeks old. Egg collection for egg quality measurements took place at 34 weeks old. The fol lowing traits were recorded: egg shape index, shell thickness, shell strength, albumen height, Haugh unit score and eggshell color.
The blood samples of all the 785 individual ducks were collected from the wing vein using vacuum tubes containing dipotassium ethylene diamine tetraacetate (EDTAK 2 ) as an anticoagulant for further SNP analysis. To investigate the ex pression of the multiple forms of melatonin receptor mRNA, 6 ducks representing two groups (3 of each) according to the age at first egg (at 128 days of age or after 150 days of age) were carefully selected at age of 150 days. Ovaries were sampled after slaughter. These experiments were conducted in accordance with the Law of the People's Republic of China on Animal Protection.

SNP discovery and genotyping
Based on the complete DNA sequence of Anas platyrhynchos genomic DNA sequence (NCBI accession no. NW_004676748.1), four pairs of primers were designed to amplify the target regions for SNP genotyping using the Primer Premier 6.0 software. Primer sequences are listed in Table 1.
Polymerase chain reaction (PCR) was carried out in a to tal volume of 15 μL consisting of 1 μL genomic DNA, 0.15 μL (10 μM) of each primer, 1.5 μL 10× buffer, 1.5 μL (25 mmol/L) MgCl 2 , 0.3 μL (10 mmol/L) dNTPs, and 1.5 U Taq DNA poly merase. PCR conditions were as follow: 95°C for 3 min, followed by 35 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, and a final extension of 72°C for 10 min. The PCR products were sequenced commercially using ABI 3730XL automated se quencer (Applied Biosystems, Carlsbad, CA, USA) after being purified and extended. SNPs were identified by looking for multiple peaks at the same base pair.

Statistical analysis
The genotypic frequencies were calculated for each individual and the HardyWeinberg equilibrium was analyzed using the Chisquare test of PopGene Version 1.32. Pairwise tests for linkage disequilibrium (LD) were performed for each SNP using the SHEsisPlus online software platform (http://she sisplus.biox.cn/SHEsis.html). The egg production traits were compared among the genotypes. The association between the SNPs and different traits in 785 ducks were analyzed using SPSS 22 with the model Y = μ+G+L+G×L+e, where Y is the dependent variable (analyzed traits), μ is the overall mean, G is the genotype with a variation for the candidate gene, L is the fixed effect of breed, G×L is the interaction between the geno type and duck population (a fixed effect), and e is the random error. The significance of the least squares means was tested with lest significance difference test.

Quantitative real-time polymerase chain reaction
Total RNA was extracted from ovary samples using Trizol reagent according to the manufacturer's protocol. cDNA was synthesized from the total RNA according to the manufac turer's protocol of TransScript FirstStrand cDNA Synthesis SuperMix (TransGen, Beijing, China). Quantitative realtime PCR was performed using ABI 7500 (Applied Biosystems, Foster City, CA, USA). The mRNA expression levels were determined by Applied Biosystems realtime PCR using the MTNR1A, MTNR1B, and MTNR1C mRNAspecific primers and the β-acting gene as the internal control ( Table 1). The 20 μL amplification reaction contained 10 μL of SYBR Green Universal PCR Master Mix, 2 μL cDNA, 0.4 μM of each primer and nuclease free water up to 20 μL. Thermal parameter used to amplify the template started with an initial denaturation at 94°C for 3 min followed by 40 cycles of 94°C for 10 s and annealing at 60°C for 30 s. The relative expression levels of the genes test were calculated using the 2 -ΔCt method. Groups with different letters were significantly different at p<0.05.

Genetic polymorphism of melatonin receptor genes
Seven SNPs were identified in all three melatonin receptor gene exons respectively by direct sequencing (Figure 1), their corresponding allele and genotype frequencies are presented in Table 2. HardyWeinberg equilibrium tests showed that the alleles of MTNR1A g. 268C>T, MTNR1C g. 10C>T, and MTNR1C g. 108C>T were in HardyWeinberg equilibrium (p>0.05), and others deviated from the HardyWeinberg equi librium (p<0.05) ( Table 1). The LD tests of Shaoxing duck population showed that two SNPs (g. 41C>T, g. 161T>C) iden tified in MTNR1B gene were completely linked (D' = 1.0 and r 2 = 1.0) (Supplementary Table S1), providing only three possi ble haplotypes.

Association of SNPs with egg production traits and egg quality traits
The association between the SNPs and egg production traits are shown in Table 3. For the MTNR1A g. 268C>T locus, age at first egg of the genotype CT was significantly less than that of the genotype TT (p<0.05). The g. 108C>T locus of MTNR1C was significantly associated with age at first egg and total num ber of eggs during 34week egglaying period (p<0.05). For MTNR1C g. 108C> T, the egg number of the genotype CT at 34 weeks old was significantly higher than that of the geno type CC (p<0.05), and ducks with the genotype TT exhibited significantly earlier age at first egg than that of the genotype CC (p<0.05). No significant association was found among other SNPs and egg weight, total number of eggs at 34 weeks   old, total number of eggs at 72 weeks old or age at first egg (p>0.05). As shown in Table 4, there were no significant association of 7 SNPs with egg quality trait. Furthermore, relation of SNPs with the color of eggshell was analyzed using chisquare test. When white eggshell was compared with blue eggshell, no significant differences in genotypes of MTNR1B or MTNR1C were observed except MTNR1A (p>0.05). The genotype TT and CT of g. 268C>T in MTNR1A were more common in Shaoxing ducks laying white eggshell compared with blue egg shell (Supplementary Table S2), suggesting MTNR1A g. 268C>T SNP may affect the eggshell color.

DISCUSSION
Age at first egg, an important trait indicating sexual matura tion and egg production performance, was negatively correlated with number of eggs [1517]. However, age at first egg was controlled by polygenes [17] with low to moderate estimated heritability ranging from 0.13 to 0.20 [1820], making the con ventional breeding method ineffective. Therefore, molecular assisted selection becomes a powerful tool for improving egg production traits and raising economic benefits.
As previously reported in other avian species, melatonin regulates gonadal maturation by suppressing luteinizing hor mone (LH) secretion, and stimulating GnIH and gonadotropin releasing hormone secretion [2124]. MTNR1C mRNA colo calized with GnIH neurous [11] indicating that melatonin participates in gonadal maturation through binding its recep tors. Therefore, melatonin receptors would be possible markers for selecting an early maturing breed. Although there were many studies seeking correlations between markers of can didate genes and age at first egg, such as growth hormone, prolactin, neuropeptide Y, folliclestimulating hormone recep tor, and SH3domain GRB2like 2 [2529], little was known Our study demonstrated that g. 268C>T of MTNR1A gene shows a strong association with age of first egg. Beside, g. 268C>T of MTNR1C gene was found to be associated with total number of eggs at age of 34 weeks and age at first egg. A study in chickens also showed that SNPs (JQ249890:g. 384T>C, JQ249891:g. 387 T>C) locating at MTNR1A and SNP (JQ249896:g. 294 G>A) locating at MTNR1C were sig nificantly linked with age at first egg [14]. The novel SNPs which was founded in our study, indicated that melatonin receptor genes may affect age at first egg and involve in sexual mature of ducks.
The exact role of melatonin receptors in different species during the reproductive process is not well understood. It was reported that melatonin receptor density showed a striking downregulation in brain when songbirds were less than fully reproductively mature, and subsequent resumed during pho torefractory state [30]. While Abd et al [31] reported that MTNR1B and MTNR1C expression increased accompany ing a delay in sexual maturity of Japanese quails. In our study, the expression of MTNR1C mRNA in latemature group was significantly decreased compared to the earlymature group. Summarizing the results of the above researches, it is reason able to suggest that MTNR1C activates avian sexual mature by downregulating its expression level. The MTNR1A mRNA expression in our experiment showed a contrary tendency with MTNR1C mRNA, suggesting melatonin receptors reg ulate ovarian function through different mechanisms. Further studies are continuing to elucidate the exact role of melatonin receptors in avian sexual maturity.
Collectively, the results of present study suggest that the MTNR1A and MTNR1C genes play an important role in egg production of ducks, but also SNPs in these genes could be used as markers in molecular markerassisted selection for duck reproduction traits.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.