The effect of SNP rs400827589 in exon 2 of the MTNR1B gene on reproductive seasonality and litter size in sheep

Abstract In mammals, the melatonin receptor gene has been widely studied since it has a great influence on reproductive traits. However, little is known about the association between polymorphism of the coding region of the MTNR1B gene and year‐round oestrus or the litter size in Small Tail Han sheep. To better understand the effects of single nucleotide polymorphism (SNP) rs400827589 in MTNR1B, a population polymorphism analysis was conducted using genotyping data in 45 sheep breeds around the world. The results indicated that TT was the dominant genotype in all sheep breeds. The associations of this SNP with reproductive seasonality and litter size in Small Tail Han sheep showed rs400827589 was correlated with fecundity as assessed by reproductive seasonality and litter size (p < .05). Bioinformatics analysis indicated the change in amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. The present results demonstrate that rs400827589 could be used in the marker‐assisted selection of the litter size in Small Tail Han sheep.

To date, numerous studies have explored the relationship between polymorphism of MTNRs and litter size or reproductive seasonality traits in different mammals (Calvo et al., 2018;Chu, Cheng, Liu, Fang, & Ye, 2006Chu et al., 2007;Gunwant et al., 2018;Hua, 2006;Liu, Wang, Zhou, Pang, & Wang, 2018;Notter, Cockett, & Hadfield, 2003;Pelletier et al., 2000;Wang, Liu, Wu, et al., 2012). However, there were few studies about the polymorphism of MTNRIB. Interestingly, some studies in birds found that melatonin receptor subtypes were identified in ovaries (He et al., 2014;Sundaresan et al., 2009;Wang et al., 2008), which indicated that melatonin may directly affect ovarian function through activating of its receptors. For example, the expression levels of MTNR1A, MTNR1B and MTNR1C initially increased and later decreased during the follicular development cycle in geese, suggested that melatonin receptors participated in activating small follicles to develop into subsequent higher hierarchical follicles (He et al., 2014). Besides, in a recent study, it was demonstrated that melatonin could directly modulate bovine ovarian function through MTNR1B, melatonin and MTNR1B were involved in the BCL2 family and CASP3-dependent apoptotic pathways in bovine granulosa cells Wang, Liu, Wu, et al., 2012). In addition, authors also found rs10830963 and rs10830962 SNPs in MTNR1B gene were associated with female polycystic ovary syndrome (PCOS) (Li, Shi, You, Wang, & Chen, 2011;Yang, Yang, & Cheng, 2016), this result suggests that SNPs in MTNR1B gene may affect integrity of ovarian function to regulate reproductive activity.
The prolificacy and year-round oestrus of sheep are crucial economic traits. Small Tail Han sheep, a local sheep breed in China, is characterized by high productivity and year-round oestrus (Liu, Jiang, & Du, 2003), and the breed has been considered as a good source of ovine reproductive genes in China. In the early studies, studies reported that the relationship between polymorphism of MTNR1A and litter size and reproductive seasonality traits in different breeds of ruminants (Gunwant et al., 2018), such as sheep (Calvo et al., 2018;Chu et al., 2006Chu et al., , 2008Mateescu, Lunsford, & Thonney, 2009;Notter et al., 2003;Pelletier et al., 2000;Posbergh, Murphy, & Thonney, 2017), goat (Abdolahi, Shokrollahi, Saadati, & Morammazi, 2019;Chu et al., 2007;Hua, 2006) and water buffalo (Gunwant et al., 2018). However, there are few reports about MTNR1B. In our early study on variation of the genome in 89 sheep, we found a significant single nucleotide polymorphism rs400827589 in the re-sequencing data , and several other significant SNPs from which have been published in our early reports Zhou et al., 2018). Therefore, in order to better understand the function of this mutation in MTNR1B, we explored the polymorphic distribution using a large population and then investigated the association with litter size and reproductive seasonality in sheep.

| Ethics statement and sample preparation
All animals used in this study were approved by the Science Research Jugular vein blood samples were collected from 737 ewes in six sheep breeds for DNA isolation using the phenol-chloroform method. The information of sheep breeds included in the study was shown in Table 1.

| Genotyping the candidate SNP in MTNR1B gene
After DNA extraction, the primers used for genotyping the rs400827589 were designed using the MassARRAY Assay Design v.

| Statistical analysis
The calculations of allele frequencies and genotype frequencies and the Hardy-Weinberg equilibrium tests were performed by using Popgene (version 1.31) (Chong, Huang, Liu, Jiang, & Rong, 2018). The association analysis between polymorphisms of the MTNR1B gene and the litter size or year-round oestrus was conducted by using General Linear Model in SAS (v 9.2) (SAS Institute Inc.). p values less than .05 were considered to be significant. The model was described in the previous study , which was as follows: y ij where y ijn is the phenotypic value of litter size; μ is the population mean; P i is the fixed effect of the ith parity (i = 1, 2, 3); G j is the fixed effect of the jth genotype (j = 1, 2, 3); I PG is the interaction effect of parity and genotype and e ijn is the random residual.

| Bioinformatics analysis
The coding sequences of the MTNR1B gene were obtained from

| Population polymorphism analysis of polymorphism in the MTNR1B gene
In this study, rs400827589 in MTNR1B exon 2 was selected for genotyping, the different alleles resulted in amino acid changes.

| Association of polymorphism of rs400827589 with litter size in Small Tail Han sheep
The association analysis between rs400827589 with litter size in Small Tail Han sheep was performed using data from our previous study , the results are shown in Table 5. Whether in the first, second or third parity, individuals with TT and TG genotypes had a larger litter size than those with GG genotype (p < .05).

| Bioinformatics analysis of MTNR1B
To analyze the changes before and after the mutation of rs400827589, the transmembrane domain was predicted using the amino acid (AA) sequence. The results indicated that there were seven transmembrane domains, a mutation from T to G led to no change in the transmembrane domains (Figure 1). In addition, the protein secondary structure before and after the mutation was predicted using the AA sequence (Figure 2).

| D ISCUSS I ON
Melatonin is a highly lipophilic circulating hormone. In addition to regulating insulin secretion and glucose levels, it controls circadian rhythms and reproductive processes (Carla Cristina et al., 2013;Jaworek et al., 2007;Sack, Blood, & Lewy, 1992). The function of melatonin is mediated by its receptors MTNR1A or MTNR1B in mammals (Li et al., 2013;Wang, Liu, Wu, et al., 2012). For example, melatonin can regulate the secretion of progesterone by binding to melatonin receptors in granulosa cells, and the gene expression of LH receptor and gonadotropin-releasing hormone receptor (Li et al., 2011). MTNR1B belongs to the 7-transmembrane G-protein coupled receptor superfamily member in human, widely expressed in the hypothalamus, pituitary gland, ovary, uterus, fallopian tubes and testis tissues, most importantly, with the highest expression in the ovaries (Yang et al., 2016).
Recent studies demonstrated that melatonin could directly modulate bovine ovarian function through MTNR1B, melatonin and MTNR1B are involved in the BCL2 family and apoptotic pathways in bovine granulosa cells     (Table 2), we found TT was the dominant genotype, but GG was also detected in many domestic and foreign sheep breeds. These results indicate the SNP mutation might be at an early stage in evolution which provides information for us to further study its functions.
Transmembrane domains are integral components enabling numerous proteins to function (Nadir, Hassan, Daniel, & Walid, 2012;Tarasova, Rice, & Michejda, 1999). Transmembrane domains can also affect reproduction. For example, two mutations of the FSH receptor in the transmembrane domain can cause primary ovarian failure (Bramble et al., 2016). Our early studies in BMP2 found a transmembrane domain change after a mutation in BMP2 at g.48462350C > T, and this can significantly reduce the ovine litter size in third parity . There was no difference in the seven trans- Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures.
As a key link in the melatonin signalling pathway, polymorphisms of MTNR1B gene may affect melatonin signalling by altering MTNR1B structure and expression, with changes in ovarian function (Yang et al., 2016). Ovarian development has significant effects on subsequent oestrus, ovulation and litter size. Therefore, we divided the six sheep breeds into two groups according to the oestrous characters, and found genotype frequency and allele frequency were significantly different between the two groups (p < .01) in SNP rs400827589 (

| CON CLUS IONS
The TT genotype in MTNR1B rs400827589 was the dominant genotype in sheep around the world. Polymorphism of MTNR1B in rs400827589 can affect reproductive seasonality and was associated with litter size in some Chinese native sheep. In addition, bioinformatics analysis indicated the change of amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. Findings in the present study also indicate that MTNR1B might use to select for litter size in sheep.

CO N FLI C T O F I NTE R E S T
We certify that there is no conflict of interest with any organization regarding the material discussed in the manuscript.