Identification of chicken FSHR gene promoter and the correlations between polymorphisms and egg production in Chinese native hens

Contents Egg production is an important economic trait in poultry, and it is of great significance to study the key genes and functional SNPs that affect egg laying performance. Follicle‐stimulating hormone (FSH) plays an important physiological role in the reproductive performance of humans and animals by binding to its receptor (FSHR). Studies have shown that there are many transcriptional regulatory elements in the 5′ flanking region of the FSHR gene that interact with transcription factors to regulate FSHR transcription. In this study, DNA sequencing was used to identify SNPs in the FSHR promoter sequence in both Dongxiang and Suken chickens. To detect the activity of the chicken FSHR gene promoter, we analysed the characteristics of the sequence and constructed three deletion vectors. We confirmed that the region (−18/−544) was the core promoter. Furthermore, five polymorphisms, including a 200‐bp indel at −869, C−1684T, C−1608T, G−368A and T−238A, were detected in both the Dongxiang and Suken chickens. The age at first egg (AFE) for different genotype of −869 indel in Suken chicken was significantly different (p < 0.01). For SNP C−1684T in Dongxiang chickens, the CC genotype had higher egg number at 43 weeks of age (E43) than that of the TC genotype (p < 0.05). For SNP C−1684T in Suken chickens, the TC genotype had higher AFE than that of the CC genotype (p < 0.05). For SNP C−1608T in Suken chickens, the CC genotype had higher AFE than that of the TC genotype (p < 0.05). For SNP G−368A in Suken chickens, the AG genotype had higher AFE than that of the GG genotype (p < 0.05).

throughout the egg laying cycle, and its egg production peak lasts for approximately 25 days.
Follicle-stimulating hormone (FSH) is a glycoprotein "synthesized and secreted by the gonadotropic cells of the anterior pituitary gland (Pierce & Parsons, 1981)" that plays a vital role in gonadal function and fertility (George, Dille, & Heckert, 2011). After FSH is released into circulation, it plays a physiological function by binding to the specific transmembrane receptor (FSHR) located on the target cell (Heckert & Griswold, 1991). In 1996, the cDNA sequence of FSHR was first successfully cloned from chicken ovarian tissue (You, Bridgham, Foster, & Johnson, 1996). The sequence analysis and integrated results of the chicken FSHR gene were demonstrated in 2005 (Wicker et al., 2005).
Although there are many studies on the regulation of mammalian FSHR promoters, the mechanism regulating transcription of chicken FSHR is not yet clear. In this study, the promoter region and sequence of the chicken FSHR gene were obtained by PCR and DNA sequencing. The transcription factor binding site (TFBS) of the chicken FSHR gene was predicted by online software, and the core promoter region was identified by a luciferase activity assay. Mice developed follicular dysplasia after FSHR gene knockout in granulosa cells (Kumar, Wang, Lu, & Matzuk, 1997). The FSHR gene promoter can regulate the transcription initiation site, time and expression level. Thus, in females, the SNPs occurring in the FSHR promoter region may affect the expression of the FSHR gene and influence reproductive performance. Researchers had found that the polymorphisms at the −278 site in the promoter region of the FSHR gene in Chinese Holstein cows significantly affected both the number of follicles and the number of transferred embryos (Yang et al., 2010).
Egg production is an important economic trait in poultry.
Endocrine (Kim, Seo, & Ko, 2004) and many environmental factors (Lewis & Gous, 2006;Liu, Lilburn, Koyyeri, Anderson, & Bacon, 2004), such as photoperiod and different supplements, can affect egg laying performance. However, genetic factors play a decisive role in egg laying performance. Egg laying performance is controlled by multiple genes, and heritability is low. Furthermore, the laying performance of poultry in different periods is also very different (Emsley, 1997;Luo, Yang, & Yang, 2007). In poultry breeding, egg number at 43 weeks of age (E43) is usually an effective indicator of total egg production (Xu et al., 2011). There are obvious differences in egg laying performances of different breeds, including age at first egg (AFE), total egg number and egg weight. Because of the importance of the FSHR gene for reproductive performance, variations in FSHR gene expression may result in distinct reproductive performances in different chicken breeds.
In addition, polymorphisms in the chicken FSHR gene promoter may also influence the transcription of FSHR and affect egg production in chickens. Accordingly, in this study, we detected nucleotide polymorphisms in the promoter of the FSHR gene of Dongxiang and Suken chickens by PCR-RFLP. We then found that several polymorphisms among the five total polymorphisms were associated with E43 or AFE.

| MATERIAL S AND ME THODS
Ethics Committee approval was obtained from the Institutional Ethics Committee of Nanjing Agricultural University to the commencement of the study.

| Animals and DNA extraction
The chicken populations used for the experiment were Dongxiang (n = 116) and Suken chickens (n = 434) from Jiangsu Xincao Farm.
The chicken was bred in cages (one chicken per cage) with the same feeding and management conditions. We recorded the age at first egg (AFE) and egg number at 43 weeks of age (E43) of every chicken.
We collected total 550 blood samples (116 for Dongxiang chicken and 434 for Suken chicken) from chicken wings and stored it at −20°C.

| Primers
Ten pairs of primers shown in Table 1 were designed for the experiment.
All primers were synthesized by JinWeiZhi Biotechnology Co., Ltd., China.

| PCR amplification and sequencing
PCR was performed in a 20 µl mixture containing 10 µl of 2X Taq Mix (Takara Biotechnology Co. Ltd., Dalian, China), 10 pmol of upstream and downstream primers and 100 ng of chicken genomic DNA. The following reaction conditions were used: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, X°C (X was the annealing temperature shown in Table 1) annealing for 30 s and a 72°C extension for 30 s (depending on product length, 1 kb = 1 min) for 35 cycles. The PCR products of P1, P2 and P3 were separated by 1.5% agarose gel electrophoresis and sequenced. SNPs were identified by sequence traces.

| Analysis software
The promoter and transcription factor binding sites were predicted and analysed by Promoter Scan, Genomatix and Methprimer (Table 2).

| Construction of the FSHR promoter luciferase plasmids
The purified promoter fragments of the chicken FSHR gene were amplified by three specific primers containing KpnI and HindIII restriction enzyme cleavage sites and then cloned into the pGL3-basic vector digested with KpnI and HindIII restriction enzymes. The primers used to amplify the desired promoter fragments of the chicken FSHR gene are shown in Table 1.

| Genotyping of polymorphisms
Using the PCR-RFLP method to detect genotypes, we selected the appropriate endonuclease for enzyme digestion of the target genes of the tested chickens. Enzyme reaction conditions followed the enzyme product specifications. Genotypes were detected by the gel bands graphic of 1.5% agarose gel electrophoresis. Electrophoresis conditions included the following specifications: 1X TBE, 120 V/30 min.

| Statistical analysis
Allele and genotype frequencies were calculated by direct counting. The chi-squared test was used to examine the Hardy-Weinberg equilibrium of the SNPs. Association analyses of SNPs with E43 and AFE were performed using spss version 20.0.

| 5′ regulatory sequence analysis of the chicken FSHR gene
A few common cis-elements were predicted in the chicken org/methprimer/; Figure 3).

| Promoter activity analysis of the chicken FSHR gene
PCR product electrophoresis is shown in Figure 4a. Three special plasmids, pFSHR-1479, pFSHR-812 and pFSHR-544, were constructed to identify the promoter activity of the chicken FSHR gene, and the translation start site (ATG) was defined as +1. The constructed plasmids were identified by double digests (Figure 4b).
The plasmids were transiently transfected into chicken ovarian granulosa cells, and luciferase activity assays were performed to identify the promoter activity of the chicken FSHR gene. As shown in Figure 5, the luciferase activity of the promoter pFSHR-544 was significantly higher than that of pFSHR-812, pFSHR-1479 and the negative control pGL3-basic (p < 0.01). In contrast, no significant difference was observed between pFSHR-1479, pFSHR-812 and pGL3-basic (p > 0.05).

| Genotype frequency and allele frequency
We found that four restriction sites exist at C  (Table 3).

| Association analysis of SNPs of the chicken FSHR gene with E43 and AFE
The association analyses of SNPs with E43 and AFE was performed using SPSS version 20.0 (Tables 4 and 5).

Dongxiang chickens with the CC genotype of SNP C−1684T
had higher E43 compared with that of chicken with the TC genotype (p < 0.05) ( Table 4). The AFEs of the SNP −869 indel and SNP G−368A genotypes were significantly different (p < 0.01). The AFEs of the SNP C−1684T and C−1608T genotypes were also significantly different (p < 0.05; Table 5).

| D ISCUSS I ON
Gene promoters play a key role in transcriptional regulation by con-   , 1997), which indicating that the regulatory mechanism of the FSHR gene promoter may differ interspecifically.
A recently research indicated that miR-4281, an miRNA specifically expressed in hominids, directly interacting with the TATA-box motif in the human FOXP3 promoter could efficiently and specifically upregulates FOXP3 expression (Zhang et al., 2018). Overexpression

of CAAT/enhancer-binding protein (C/EBP) in Spodoptera litura-221
(Spli-221) cells increased the promoter activity 5.57-fold, while mutation of the C/EBP CRE abolished the binding of the C/EBP with the CRE (Liang, Zhang, Zeng, Zheng, & Feng, 2015). These findings validate the important role of TATA-box and CAAT-box in promoter regulation.
The E-box was mutated in the promoter of the rat FSHR gene, which resulted in a significant reduction of FSHR promoter activity in MSC-1 cell lines (Heckert et al., 1998). Two E-box sites were predicted to be present in the promoter of the chicken FSHR gene, whether the E-box regulating the FSHR promoter requires further identification. Furthermore, multiple transcription factor binding sites (TFBS) were predicted in the chicken FSHR gene promoter with Genomatix software, including AP1, GATA, SF1, YY1, as well as others. It is noteworthy that the transcription factors E2F, Smad3 and ETS were involved in the transcriptional regulation of the FSHR gene in previous reports (Brune, Adams, & Gromoll, 2010;Heckert, 2001;Kim & Griswold, 2001). However, we did not predict these transcription factor binding sites in the chicken FSHR gene promoter, further illustrating that different promoter regulatory mechanisms are likely to exist in the FSHR genes in different species.
Researchers have identified that the region (−1,195/−598) was the core promoter of the porcine FSHR gene (Wu et al., 2015). In order to identify the core promoter region of the chicken FSHR gene, The sequencing results showed that eleven SNPs exist in the promoter of the chicken FSHR gene. It has been widely reported that mutations in the FSHR gene have a genetic effect on reproductive traits in humans and other animals (Lussiana et al., 2008).
Researchers had found that the FSHR promoter polymorphism FSHR −29G>A influences the androgen levels of human small antral follicle (hSAF; Borgbo et al., 2017). The activity of FSHR promoter is significantly affected by the 29th site G → A mutation that will weaken promoter activity and result in poor response to FSH (Dan, Jing, Liangbin, Ting, & Ying, 2015). We analysed the FSHR gene in muscovy duck detected that the SNP C320T is significantly associated with egg production at 59 weeks of age (p < 0.05), whereas the SNP A227G is significantly associated with age at first egg stage (p < 0.05) (Xu et al., 2017). However, the mechanisms by which these polymorphisms make their effects require to be further researched.  In conclusion, in this study, we identified the core promoter region of the chicken FSHR gene and predicted several transcription factor binding sites. Moreover, a total of five polymorphisms of the FSHR promoter region were detected, and we found that all of them were associated with egg number at 43 weeks of age (E43) or age at first egg (AFE).

CO N FLI C T O F I NTE R E S T
None of the authors have any conflict of interest to declare. Note. In the same group, different superscripts mean significant difference (p < 0.05).