Association of a non-synonymous SNP of IL17RA gene with litter size traits in Large White and Landrace pigs

Interleukin-17 receptor A (IL17RA) is one of the cytokine receptors of the pro-inflammatory interleukin-17 (IL17) cytokine family. The IL17 and IL17RA genes are involved in inflammatory and immune responses as well as reproductive process of mammals. The purposes of this study were to examine polymorphisms in the porcine IL17RA gene and to assess its effects on litter size traits in Large White and Landrace pigs. Three non-synonymous single nucleotide polymorphisms (SNPs) in the porcine IL17RA gene were verified. The porcine IL17RA c.785C>T (p.Ala262Val) was found to be segregating in the Large White and Landrace pigs. No polymorphisms in the coding region of the porcine IL17RA gene at the two non-synonymous SNPs loci of c.997G>A (p.Val333Ile) and c.1962T>G (p.Asp654Glu) were found. The porcine IL17RA c.785C>T polymorphism was significantly associated with the total number born (TNB) and the number born alive (NBA) in Large White pigs ( P <0.05). Moreover, the porcine IL17RA c.785C>T was significantly associated with the TNB, NBA, total birth weight (TBW), and total weaning weight of piglets at 21 days (TWW) in Landrace pigs ( P <0.05). These results supported the importance of the porcine IL17RA gene in the litter size traits of pigs. Thus, the porcine IL17RA could be used as a potential candidate gene for improving litter size traits in pig breeding.


INTRODUCTION
Litter size trait is one of the major economic traits and plays a significant part in the reproductive performance and the profitableness of the pig production industry (Marantidis et al., 2013;Zhang et al., 2015). Embryonic mortality during the implantation period is a major factor in determining the litter size in pigs (Spötter and Distl, 2006;Lin et al., 2009;Chen et al., 2015). Currently, the cytokine genes are considered to play a key function in the communication process between fetal trophoblasts and maternal decidual immune cells at the feto-maternal interface in the uterus which leads to the acceptance of embryo implantation, embryo expansion, and pregnancy success (Saito, 2001;Bowen et al., 2002;Paria et al., 2002;Schäfer-Somi, 2003;Paulesu et al., 2010). Thus, the cytokines are essential for the embryonic development process and the maintenance of pregnancy in animals and humans (Simón et al., 1994;Chaouat et al., 2007;Ostanin et al., 2007;Paulesu et al., 2010). It has been reported that numerous cytokine genes involved in embryo implantation that affect the relevant litter size traits in pigs such as LIF (Spötter et al., 2005;Norseeda et al., 2021a), EPOR (Vallet et al., 2005), OPN (Kumchoo and Mekchay, 2015), IL4 and IL4R (Norseeda et al., 2021b), and IL6 genes (Yang et al., 2011;Norseeda et al., 2021c). Currently, several studies demonstrated that the IL17 cytokines and its receptors play a crucial role in implantation and maintenance of pregnancy in mice and humans (Saito et al., 2011;Klein, 2016;Wang et al., 2019). The biological function of IL17 and its role in immune response have been described in pigs (Levast et al., 2010;Stepanova et al., 2012;Xiang et al., 2020). However, there has been no report on the role of IL17 cytokines and its receptors in reproductive function in pigs.
All this information indicates that the IL17RA gene is involved in the immune response and related to the reproductive process of embryonic implantation and maintenance of pregnancy. Thus, the IL17RA gene can be regarded as a positional and functional candidate gene for litter size in pigs. Currently, there is no published report on the association of the porcine IL17RA polymorphisms (especially non-synonymous SNPs or missense mutation) with litter size traits in pigs. Therefore, in this present study, we have verified the non-synonymous polymorphisms in the coding sequence of the porcine IL17RA gene, while its effect on litter size traits has also been assessed in Large White and Landrace pig populations.

Animals and DNA extraction
All of the 437 pigs used in this study were raised under commercial conditions of the Betagro Hybrid International Company, Thailand. Blood samples were taken from 178 Large White and 259 Landrace sows. The litter size traits were assessed in 1369 litters of all pigs. The reproductive traits of total number born (TNB), number born alive (NBA), the number of piglets weaned alive (NWA), total birth weight (TBW), and total weaning weight (TWW) of piglets at 21 days were recorded. Moreover, blood samples of Chinese indigenous pigs (Meishan, n=10) were obtained from Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University and were used to evaluate the genotype and allele frequencies of the porcine IL17RA polymorphism. The blood samples were used for DNA isolation by the Chelex procedure (Walsh et al., 2013) and stored at 4°C until being analyzed. The experimental methods were approved by the Animal Ethics Committee of Chiang Mai University, Thailand (2562/AG-0001).

Verification of porcine IL17RA polymorphisms and genotyping
To verify the SNPs of the porcine IL17RA gene, the non-synonymous SNPs of the porcine IL17RA gene in the Ensembl database (ENSSSCT00000033369.3, https://asia.ensembl.org) were selected and examined for polymorphism in various pig breeds by in silico analysis. The nucleotide sequences of the porcine IL17RA gene of 12 pig breeds were retrieved from the Ensembl database, consisting of Large White (ENSSSCT00025073732), Landrace (ENSSSCT00045011988), Berkshire (ENSSSCT00065110244), Hampshire (ENSSSCT00035071005), Pietrain (ENSSSCT00055035992), USMARC (ENSSSCG00070016171), Bamei (ENSSSCT00050011314), Wuzhishan (ENSSSCT00005022403), Jinhua (ENSSSCT00060017210), Rongchang (ENSSSCT 00030063115), Meishan (ENSSSCT00040102728), and Tibetan (ENSSSCT00015103275). The porcine IL17RA nucleotide sequences of these 12 pig breeds were aligned using the multiple sequence alignment program of CLUSTALW (https://www.genome. jp/tools-bin/clustalw) to find out the non-synonymous SNPs. The variant SNPs were selected, based on a high frequency of polymorphisms and the restriction enzyme available, to examine the polymorphisms of the porcine IL17RA gene in pig populations. Thus, the variant SNPs were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The specific primers of the porcine IL17RA gene were designed based on relevant nucleotide sequence data of the GenBank (Accession number: NC_010447.5), as shown in Table 1. A mismatched primer was designed in order to generate a recognition site of the restriction enzyme for genotyping (Table 1). The PCR amplification was performed in a reaction volume of 20 μL consisting of 50 ng of a genomic DNA sample, 1×(NH 4 ) 2 SO 4 buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.4 μM each primer (Table 1), and 0.2 U Taq DNA polymerase (Fermentas). The PCR conditions were started with a denaturation step at 94°C for 3 min, which followed by 35 cycles consisting of 94°C for 30 sec, 58 to 60°C for 30 sec, 72°C for 30 sec, and then 5 min at 72°C to complete the reaction. The PCR products were digested by specific restriction enzymes, as shown in Table 1. The digested PCR products were then separated on 6% polyacrylamide electrophoresis gel and visualized using ethidium bromide staining. Table 1 Primer sequences and restriction enzymes used for genotyping of the porcine IL17RA gene.

Statistical analysis
The genotype and allele frequencies of the porcine IL17RA polymorphism were estimated. The chi-square test was used to examine differences in genotype frequencies between pig breeds and to determine the Hardy-Weinberg equilibrium (HWE) within the pig populations. The association between the porcine IL17RA polymorphism and the litter size traits were analyzed with a statistical model as expressed below: Y ijkl = µ + YS i + P j + G k + e ijkl where Y ijkl is the observations of the phenotype values, μ is the overall mean for each trait, YS i is the fixed effect of year-season (i = 1-8), P j is the fixed effect of parities (j = 1 and ≥ 2), G k is the fixed effect of the IL17RA genotypes (k = 1, 2, 3), and e ijkl is the residual error. Moreover, additive effect of the porcine IL17RA polymorphism was estimated as half difference between the estimated effects of homozygous genotypes and the dominance effect was analyzed as the deviation of the heterozygous genotype effect from the mean effect of the homozygous genotypes (Muñoz et al., 2007). The estimated effects were calculated using t-test on significant deviations from zero.  , rs691493622), and c.2435C>A (p.Ala812Asp, rs699434149). Based on the in silico analysis results, six non-synonymous SNP loci (c.785C>T, c.923A>G, c.997G>A, c.1962T>G, c.2082C>A, and c.2435C>A) of the porcine IL17RA gene were found to be polymorphic among 12 pig breeds ( Figure 1). From these, three non-synonymous SNPs (c.785C>T, c.997G>A, and c.1962T>G) showed a high frequency of polymorphism among 12 pig breeds (42%, 42%, and 58%, respectively). Thus, these three SNPs were selected to verify the polymorphisms of the IL17RA gene in our pig populations. The porcine IL17RA c.785C>T was found to be segregating in the Large White and Landrace pigs. This polymorphic site was detected with the restriction enzyme TaiI. Two specific alleles revealed a 101-bp fragment for allele C and two fragments of 84 and 17-bp for allele T, as shown in Figure 2

Genotype and allele frequencies
The genotype and allele frequencies of the porcine IL17RA c.785C>T locus are shown in Table 2. At the porcine IL17RA c.785C>T locus, three genotypes (CC, CT, and TT) were observed in Landrace pigs, whereas two genotypes (CC and CT) were observed in Large White pigs. The CC genotype had higher frequencies than the CT and TT genotypes, as well as the C allele, which had higher frequencies than the T allele in these pig populations. In addition, the porcine IL17RA c.785C>T locus was fixed as c.785T in Meishan pigs. The chi-square test showed that the difference in genotype frequency distribution between Large White and Landrace pigs was statistically significant (χ 2 = 53.78, P<0.001). Moreover, the genotype distributions of the porcine IL17RA c.785C>T locus within these Large White and Landrace populations deviated from the HWE specifications (P<0.05). Furthermore, the two SNPs at the porcine IL17RA c.997G>A and IL17RA c.1962T>G loci were fixed as c.997A and c.1962T in Large White and Landrace populations and as c.997G and c.1962G in Meishan pigs.

Associations of porcine IL17RA polymorphism with litter size traits
Associations of the porcine IL17RA c.785C>T with litter size traits in Large White and Landrace pigs are shown in Tables 3 and 4, respectively. No significant association of porcine IL17RA c.785C>T with any litter size traits was found in the first parity of Large White and Landrace pig populations. However, a significant association of the porcine IL17RA c.785C>T with litter size traits was found in the later parities of both pig populations. The porcine IL17RA c.785C>T was significantly associated with TNB and NBA traits in Large White pigs and TNB, NBA, TBW, and TWW traits in Landrace pigs. In addition, no significant additive and dominance effects on the litter size traits were observed in the Large White and Landrace breeds. Notably, the sows with the TT and CT genotypes had higher litter size values than those of the sows with the CC genotype. Hence, the porcine IL17RA c.785T allele seems to be a favorable allele for litter size traits in these pig populations.

DISCUSSION
In the current study, we verified the variation of the non-synonymous SNPs in the porcine IL17RA gene and assessed its effects on litter size traits in Large White and Landrace pig populations. Three non-synonymous SNPs (c.785C>T, c.997G>A, and c.1962T>G) of the porcine IL17RA gene revealed a high frequency of polymorphisms (>40%) among various Western and Chinese indigenous pig breeds (Large White, Landrace, Berkshire, Hampshire, Pietrain, USMARC, Bamei, Wuzhishan, Jinhua, Rongchang, Meishan, and Tibetan) based on the in silico analysis. In this study, the porcine IL17RA c.785C>T polymorphism was found to be segregating in our pig populations. However, the porcine IL17RA c.997G>A and IL17RA c.1962T>G loci were fixed as IL17RA c.997A and IL17RA c.1962T in these pig populations. Three genotypes of the porcine IL17RA c.785C>T polymorphism were detected in Landrace pigs, meanwhile, only two genotypes (CC and CT) were found in Large White pigs. The porcine IL17RA c.785C was the major allele in both pig populations. The difference in genotype frequency distribution between these two pig breeds was statistically significant (P<0.001) and these may be caused by the different genetic backgrounds of these two pig breeds. Moreover, the chi-square test revealed that the porcine IL17RA c.785C>T polymorphism in two populations was a significant deviation from the HWE specifications. These results suggest that there are effects of selective mating based on some desirable production traits that are related to the porcine IL17RA c.785C>T polymorphism locus in Large White pigs. In addition, null alleles may be another reason for this pig population deviating from the HWE. Moreover, excess heterozygosity of the  porcine IL17RA c.785C>T loci occurred in the Landrace pig population, that might be due to outcrossing among their parent lines and this could have caused populations to be found deviating from the HWE (Namipashaki et al.,2015;Chen et al., 2017). Although the association of IL17A polymorphism with reproduction traits has been scarcely studied, a previous study has demonstrated that the human IL17RA (rs4819554) polymorphism is associated with the pregnancy disorder of preeclampsia in a Chinese Han population (Chen et al., 2020). Conversely, there is no association of the human IL17RA polymorphism with preeclampsia in Chinese women . However, the downregulated IL17RA expression is related to preeclampsia (Ma et al., 2019). Furthermore, the IL17RA gene is hypermethylated in pregnant women with preeclampsia (Halvatsiotis et al., 2019). There is evidence indicating that the IL17RA gene is expressed in endometrial and conceptus tissues during early pregnancy (Klein, 2016). Thus, the IL17RA gene is assumed to relate to embryonic implantation and maintenance of pregnancy.
In this study, the porcine IL17RA c.785C>T polymorphism was significantly associated with the TNB, NBA, TBW, and TWW traits. The porcine IL17RA c.785T allele is positively related to litter size traits. Interestingly, this IL17RA c.785T allele is also exhibited in the Chinese Meishan pig (Table 2) which is commonly known to be a high prolificacy breed with low embryonic mortality. These findings suggest that the porcine IL17RA c.785T allele seems to be a favorable allele for litter size traits. The porcine IL17RA c.785C>T polymorphism was a non-synonymous SNP leading to a non-conservation amino acid exchange at position p.Ala262Val of exon 9. Although this variant p.Ala262Val of the porcine IL17RA gene has not yet been functionally identified, it is located within the extracellular FnIII D2 of the IL17RA molecule which is involved in binding of the IL17A and IL17F molecules and it is also located close to the N-linked glycosylation sites and disulfide bonds which are involved in signal transduction and post-translation 401 modification process. Moreover, the porcine IL17RA is located (SSC5, 69.4 Mb) within the QTL regions for TNB, NBA, the number of stillborn, and the total litter weight (Cassady et al., 2001;Schneider et al., 2012;Bergfelder-Drüing et al., 2015;He et al., 2017;Zhang et al., 2019). Therefore, it could be expected that the observed amino acid change of the porcine IL17RA (p.Ala262Val) might promote the functional explanation of the reproductive process or might be in the close linkage of disequilibrium with the causal SNPs of a positive effect on litter size traits in pigs.
Moreover, the interaction of IL17RA and its ligand molecules may be involved in the regulation of reproductive function in animals. It has been reported that the mice deficient in IL17RA and IL17RC fail to respond to both IL17A and IL17F (Iwakura et al., 2011), as well as the specific mutations in functional motifs of the IL17RA gene, were exhibited to impair cells' response to IL17A molecule (Gaffen, 2009;Krstic et al., 2015). Moreover, previous studies have demonstrated that the human IL17A and IL17F polymorphisms, coding for ligands of the IL17RA and IL17RC molecules, are associated with recurrent pregnancy loss (Najafi et al., 2014;Zidan et al., 2015). Obviously, the serum circulation of IL17 can induce abortion in mammals. A previous study has exhibited that the exogenous recombinant IL17 induced fetal loss in a regular mouse model, whereas an anti-IL17 neutralizing antibody prevented the fetal loss in an abortion mouse model (Xu et al., 2016). Moreover, higher serum IL17 levels corresponded to the early fetal loss in mice (Li et al., 2013). Similarly, the imbalance expressions of IL17, IL10, and TGF-beta induced chronic endometritis and related to the recurrent implantation failure in women (Wang et al., 2019). However, the IL17 which also expressed in the human placenta may play a key role in angiogenesis, placental development, and/or immunoregulation in the establishment of pregnancy (Pongcharoen et al., 2007). Furthermore, the IL17 and IL17R may have a regulatory role in trophoblast invasion by increasing progesterone secretion (Pongcharoen et al., 2006;Pongcharoen and Supalap, 2009) and these two genes are exhibited in endometrial and conceptus tissues during pre-implantation (Klein, 2016). It is possible that the IL17 acts upon endometrium and contributes to the maintenance of pregnancy by binding to its receptors (Klein, 2016). These pieces of evidence suggest that the IL17RA and its ligand may be necessary for the reproductive process, especially embryonic attachment, implantation, and maintenance of pregnancy, in animals and humans. However, further study is needed in order to clarify the role of IL17RA and its ligand in implantation and embryo survival. Moreover, the association of porcine IL17RA gene including its ligand with litter size traits is required to confirm in various pig populations.

CONCLUSION
The polymorphism of the porcine IL17RA gene was verified and its effects on litter size traits were elucidated in commercial pigs. The porcine IL17RA c.785C>T had obvious effects on litter size traits in Large White and Landrace pig populations. These results promote the emphasis of the porcine IL17RA gene in the litter size traits of pigs. Thus, the porcine IL17RA could be used as a potential candidate gene for improving litter size traits in pig breeding.