A novel synonymous SNP (A47A) of the TMEM95 gene is significantly associated with the reproductive traits related to testis in male piglets

Transmembrane protein 95 (TMEM95) is located on the acrosomal membrane of the sperm head involved in the acrosome reaction; thus, it is regarded as affecting spermatogenesis and reproduction traits. The aim of this study was to explore the novel single nucleotide polymorphisms (SNPs) within the pig TMEM95 gene as well as to evaluate their associations with the testicular sizes in male Landrace (LD) and Large White (LW) breeds. After pool sequencing and bioinformatics analysis, only one novel coding SNP was found in exon 1, namely NC_010454.3: g.341T >C, resulting in a synonymous mutation (A47A). This SNP could be genotyped using the StuI polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. The minor allelic frequencies (MAFs) were 0.259 and 0.480 in the LD and LW breeds. Their polymorphism information content (PIC) values were 0.310 and 0.375. The LW population was at the Hardy–Weinberg equilibrium (HWE) (p> 0.05), whereas the LD population was not (p< 0.05). Association analyses demonstrated that a significant relationship was found between this A47A polymorphism and testis weight at 40 days of age in the LW population (p= 0.047), and the heterozygote individuals showed lower testis weight than those with other genotypes. Moreover, this SNP was significantly associated with three testis measurement traits at 15 days of age in the LW population (p< 0.05); the individuals with genotypes TT and TC showed consistently superior testis measurement traits than those with genotype CC. These findings demonstrate that the A47A polymorphism had a significant effect on testis measurement traits, suggesting that the TMEM95 gene could be a candidate gene associated with reproductive traits. These results could contribute to breeding and genetics programs in the pig industry via DNA marker-assisted selection (MAS).


Introduction
During the past 20 years, there have been great, increasing improvements in the global pig industry, but reproduction still remains a severe issue.Although Large White (LW) and Landrace (LD) pigs are the most popular breeds in many countries, especially in western China, they have some reproductive trait defects (Bergfelder-Drüing et al., 2015), e.g., reproductive barriers.It is well known that reproduction traits are determined by both females and males (Beerda et al., 2008;Pausch et al., 2014); however, to date, numerous studies on reproduction traits of female pigs have been published, but the study of male reproductive traits is limited.Owing to the more important role of males in reproduction in the pig industry, male reproduction should be emphasized in the current pig industry (Mack et al., 2014).
It is generally known that the male reproduction traits are complex quantitative traits controlled by numerous minor Published by Copernicus Publications on behalf of the Leibniz Institute for Farm Animal Biology.
genes based on polygenic hypothesis, and DNA markers can further improve genetic selection.Therefore, many breeders prefer the use of DNA markers to traditional methods for selection and breeding of pig, as well as characterization and conservation of genetic resources (Fontanesi et al., 2012;Fischer et al., 2015).Currently, identifying single nucleotide polymorphisms (SNPs) of potential candidate genes and their associations with male reproductive traits is of importance in the DNA marker-assisted selection (MAS) approach (Uimari et al., 2011;Zhang et al., 2009;Ren et al., 2017).However, to date, limited information about the candidate genes affecting male pig reproductive traits is available.Thereby, it is crucial to determine effective DNA markers from candidate genes associated with male reproductive traits in breeding and genetics of pig.At present, candidate family genes (such as the TMEM family) are usually used to identify candidate genes influencing reproductive traits in order to enhance male reproductive traits in pigs using MAS.
As a member of the TMEM family, the transmembrane protein 95 (TMEM95) gene is located on chromosome 17 in Homo sapiens (human), on chromosome 17 in Pan troglodytes (chimpanzee), on chromosome 11 in Mus musculus (house mouse), on chromosome 10 in Rattus norvegicus (Norway rat), on chromosome 19 in Bos taurus (cattle), on chromosome 11 in Ovis aries (sheep) and on chromosome 12 in Sus scrofa (pig).TMEM95 is located on the acrosomal membrane of the sperm head involved in the acrosome reaction (Pausch et al., 2014).Spermatozoa of mt/mt animals, deficiency of TMEM95, demonstrated no fluorescence at the acrosomal membrane, implying that successful fertilization by spermatozoa of mt/mt animals might be compromised, which was consistent with the equatorial segment of the acrosome and contact of the spermatozoon with the cell membrane of the oocyte (Bedford et al., 1979;Palermo et al., 1997;Pausch et al., 2014;Ramasamy et al., 2014).Therefore, TMEM95 is hypothesized to affect spermatogenesis, and it will be possibly associated with male reproductive traits.
However, until now, there had been no information about the precise function of TMEM95 except bovine TMEM95.Bovine TMEM95 has a highly conserved single-pass type I transmembrane protein consisting of 183 amino acids (aa) with a predicted extracellular N-terminal signal peptide (Zhang et al., 2016), a 23-aa transmembrane domain and an 8-aa intracellular C-terminal domain (Pausch et al., 2014).Importantly, the premature stop codon (C161X) of bovine TMEM95 is located within the predicted transmembrane domain and truncates the protein, thus this mutation could cause idiopathic male subfertility in cattle (Pausch et al., 2014).However, its real function and novel genetic variations are unknown in many other species.
To date, no novel mutations have been reported in the pig TMEM95 gene.Moreover, whether the novel SNPs of the pig TMEM95 gene (if present) is significantly associated with male pig reproduction remains elusive.Herein, we firstly re-port the identification of a synonymous mutation (A47A) at the pig TMEM95 gene and describe a method based on a StuI PCR-RFLP for the detection.In addition, the relationship between this SNP and the testicular measurement traits are firstly evaluated, which would be of benefit in identifying candidate genes related to reproductive traits in order to increase reproduction traits related to testis in pig as well as to aid the pig industry using MAS.

Animal sources and data collection (testicular traits)
A total of 289 testis samples were obtained from male piglets belonging to two breeds: Landrace (LD) and Large White (LW) piglet herds, which are located at a national pig breeding farm, Ankang, Shaanxi, China (Chen et al., 2016;Ren et al., 2017).Among these, all LD male piglets (n = 99) were 40 days old, 32.63 % of LW piglets (n = 62) were 40 days old, and 67.37 % of LW piglets (n = 128) were 15 days old.Data about testis weight (TW), testis long circumference (TLC) and testis short girth (TSG) were obtained from the testicular tissues, which were used for association evaluation analysis (Chen et al., 2016;Ren et al., 2017).

Genomic DNA isolation and DNA pool construction
Genomic DNA of 289 samples was isolated from testis tissue following the procedure as described by Lan et al. (2007).The quantification of genomic DNA concentration was assayed, and the working solution of each DNA samples was 50 ng µL −1 (Wu et al., 2014;Jia et al., 2015).A total of 50 DNA samples from each breed were randomly selected to construct genomic DNA pools.The genomic DNA pools were used as template for PCR amplification and exploring genetic variation of the TMEM95 gene.

Primer design, PCR amplification and sequencing
Based on Sus scrofa TMEM95 gene sequences (NC_010454.3,GI:347618782), a total of four pairs of primers (P1-P4, Table 1) were designed to amplify the entire exon 1-7 and their flanking regions within the TMEM95 gene, which covering all coding region of this gene.
The PCR was carried out in 50 µL of reaction volume containing 2.0 µL pool genomic DNA, 1.0 µL of each primer (forward and reverse primer), 32 µL 2× Eco Taq PCR super mix (+ dye) and 14 µL ddH 2 O.The touch-down PCR protocol was as follows: 5 min at 95 • C; two cycles of 94 • C for 30 s, annealing from 68 to 52 • C by 2 • C decreases for 30 s, and 72 • C for 30 s; 30 cycles of 94 • C for 30 s, 50 • C annealing for 30 s and 72 • C for 30 s; a final extension at 72 • C for 10 min; and subsequent cooling to 4 • C.
The total 50 µL of PCR products was gel-purified using the EasyPure Quick Gel Extraction Kit (TransGene Biotech, Beijing, China).The purified fragments were inserted into the pGEM-T easy vector (Promega, USA).The colony PCR was used to verify the positive colonies and those were sequenced via a sequencing service (Genscript, Nanjing, China) (Yang et al., 2016).

Genotyping the A47A SNP of the pig TMEM95 gene by the StuI PCR-RFLP
After pool DNA sequencing and BLAST analysis, only one novel coding SNP was determined (NC_010454.3:g.341T > C), which resulted in a synonymous mutation.Interestingly, this SNP could be genotyped by the StuI PCR-RFLP assay.Hence, for this SNP locus, using the above P1 primer, volume and protocol of the pig TMEM95 gene, PCR amplification was carried out to detect all male piglet individuals in this study.Aliquots of 10 µL PCR products with the pig TMEM95 gene were digested with 5 U StuI (MBI, Vilnius, Lithuania) following the supplier's directions for buffer condition.The digested products were detected by electrophoresis in 2.0 % agarose gel stained with ethidium bromide (Wu et al., 2014).

Statistical analysis
Genotypic and allelic frequencies were directly calculated.
Association tests of the polymorphism (A47A) with three reproduction traits (TW, TLC and TSG) were considered at two different growth periods (15 days old/40 days old) in LW piglets, and one period (40 days old) in LD piglets.These association analyses were performed by the procedure of analysis of variance (ANOVA) of the software SPSS (Version 18.0) if data agreed with the characteristic of normality and homogeneity of variances.If data did not agree, a nonparametric test (Kruskal-Wallis) was conducted using software SPSS (18.0).The ANOVA applied the general linear model (GLM) and the reduced linear model was as follows: , where Y ij k = the observation of the reproduction trait (e.g., testis weight) evaluated on the ith level of the fixed factor age (α i ), the j th level of the fixed factor genotype (β j ), where µ = the overall mean for each trait and ε ij k is the random error for the ij kth individual (Henderson et al., 1986;Zhao et al., 2004).Moreover, additive effects were calculated as the mean of the difference between homozygotes, using the least squares means (Falconer et al., 1996).Dominance effects were calculated as the deviation of heterozygotes from the mean of the homozygotes (Short et al., 1997).Allele substitution effects were estimated by using linear regression techniques, regressing phenotypes on the number of copies of the mutant alleles (0, 1, and/or 2) of this locus (Rothschild et al., 1996).Notably, multiple tests were not corrected at p = 0.05 or p = 0.01.

Results
After the pool sequencing and bioinformatics analysis, the only one novel coding SNP within the pig TMEM95 gene was detected (Fig. 1), namely NC_010454.3:g.341T > C, which was different from other species, e.g., human and bovine.This SNP was found in exon 1, and resulted in synonymous mutation (GCT (47Ala) > GCC (47Ala)).
As could be seen in Table 2, the genotypic and allelic frequencies of the A47A SNP in the LD and LW breeds were evaluated.The minor allelic frequencies (MAFs) were www.arch-anim-breed.net/60/235/2017/Arch.Anim.Breed., 60, 235-241, 2017  In the two breeds, the associations between this SNP and the pig testis measurement traits were investigated (Table 3).No significant relationship was observed between this novel synonymous SNP and testis measurement traits at 40 days of age in LD pigs.However, a significant relationship was observed between this SNP locus and testis weight at 40 days of age in LW pigs (p = 0.047).The heterozygote individuals showed lower testis weight than those with other genotypes.Moreover, three significant relationships were observed between this SNP locus and testis measurement traits at 15 days of age in LW pigs (p < 0.05).Consistently, at the 15-day-old stage, individuals with genotypes TT and TC showed superior testis measurement traits than those of genotype CC in LW breeds.Moreover, the results based on the additive effect, dominant effect, and allele substitution (α) effects are also shown in Table 4.There were no significant additive effects, dominant effects, and allele substitution (α) effects between the A47A polymorphism and testis measurement traits in LD breed.However, several significant additive, dominant, and allele substitution (α) effects of this SNP and traits were also revealed (p < 0.05).

Discussion
Previous studies have reported that TMEM95 has a significant association with acrosomal reaction, and it influences sperm fertilizing an egg cell.Therefore, TMEM95 plays an important role in fertility and it is activated and induced during the process of fertilization.To date, no report has described the significant relationships between polymorphisms in the TMEM95 gene and testis measurement traits in pigs.Therefore, it was crucial and necessary to evaluate the associations between the novel SNP of the pig TMEM95 gene and testis measurement traits.
In this study, only one novel coding SNP in the pig TMEM95 gene was firstly reported.For this locus, LD male population was not at Hardy-Weinberg disequilibrium (p < 0.05), implying that the rapid, powerful and effective selection strategies might change the allelic balance of this locus.Statistically, the observed homozygote CC was significantly higher than its expectation.For the LW population, it was at Hardy-Weinberg disequilibrium (HWE) (p > 0.05), imply-  The values with different letters ( a and b ) within the same column differ significantly at p < 0.05.
ing that this population was suitable for evaluating the relationship between this SNP and reproduction.Differences in the distributions of genotypic frequencies for the A47A SNP, based on the χ 2 test (χ 2 = 42.037,df = 2, p < 0.001), suggesting that there were significantly difference between LW and LD populations.Furthermore, allelic distributions showed significant differences in two breeds, based on the χ 2 test (χ 2 = 26.512,df = 1, p < 0.001).Since the tested breeds represented different reproduction types (such as the LD breed with reproductive barriers, while the LW breed had strong reproduction performance), the genotypic and allelic distributions were demonstrated to have a significant association with reproduction performance.For instance, when compared with the LD breed, the LW breed possessed higher frequencies of the "C" allele, suggesting that this allele was possibly associated with the stronger reproduction traits.Therefore, the pig TMEM95-A47A mutation was assumed to have probable effects on reproduction traits.
Although the novel A47A mutation in the pig TMEM95 gene was a synonymous mutation, it may impart an effect on fitness, splice regulation, and miRNA binding (Parmley et al., 2007).Several computational analyses have also indicated that synonymous mutations impact mRNA stability (Chamary et al., 2005;Quax et al., 2015;Lorenz et al., 2011).Synonymous mutations affected protein folding and function (Parmley et al., 2007;Gartner et al., 2013).Therefore, the relationships between the SNP locus (A47A) and testis measurement traits in piglet were carried out.
Based on the association analysis, the SNP-StuI locus was found to be significantly associated with the testis size in LW pigs (p < 0.05).Moreover, genotype CC individuals was consistently inferior compared to the other genotypes' individuals in LW testis weight and size, suggesting that the allele T of the pig TMEM95 gene had positive effects on testis measurement traits in this breed.Ghorbankhani (2014) showed that testicular circumference influenced the reproduction of Sanjabi growing ram lambs, independent of nutritional status (Ghorbankhani et al., 2015).In some livestock, testicular volume could reflect spermatogenesis (Gouletsou et al., 2008).Therefore, the identification of individual variation is essential to select and sort the animals for high sperm production potential.There have been some studies on bulls and rams that have shown that testicular morphometry can  serve as an indicator of fertility, thus providing high correlations with sperm production (Rege et al., 2000;Devkota et al., 2008).
In the modern pig industry, ultrasonography is generally recognized as the most accurate method for measuring testicular dimensions in England, but it is expensive.However, if we use candidate genes or DNA markers via MAS to improve the testicular size, all costs will be significantly lowered and the accuracy will significantly increase (Schiff et al., 2004).Hence, according to the close relationship between testis measurement traits and reproduction, we suggest that the allele T of the pig TMEM95 gene had positive effects on reproduction traits.

Conclusions
We have identified and genotyped a novel A47A mutation within the pig TMEM95 gene, and this SNP was found to significantly affect testis measurement traits, suggesting that TMEM95 could be a candidate gene associated with reproductive traits.These findings could contribute to breeding and genetics programs in the pig industry via MAS.

Figure 2 .
Figure 2. Electrophoresis pattern of the StuI locus within the pig TMEM95 gene.

Table 1 .
Amplification PCR primer sequences of the pig TMEM95 gene.

Table 2 .
Genotypic and allelic frequencies and population indexes for the A47A SNP of the pig TMEM95 gene.

Table 3 .
Relationship between the SNP-StuI of the TMEM95 gene and reproduction traits in pig (least squares means, LSM a ± standard error, SE) (p < 0.05).