Association of rs81471943 and reproductive traits reveals EXOC4 as 1 novel molecular breeding marker in pigs 2

Background: In mammals, the exocyst complex component 4 ( EXOC4 ) gene has often been reported 30 to be involved in vesicle transport. The rs81471943 (C/T) locates at the intron of porcine EXOC4 , and 31 six quantitative trait loci (QTLs) within 5-10 Mb around EXOC4 are associated with ovary weight, 32 teat number, total born alive, and corpus luteum number. However, the molecular mechanisms between 33 EXOC4 and reproductive performance of pigs remains incompletely elucidated. Results: In this study, rs81471943 was genotyped from a total of 994 Duroc sows, and the genotype 35 and allele frequency of rs81471943 (C/T) were statistically analyzed. Then the associations between 36 rs81471943 and four reproductive traits including number of piglets born alive (NBA), litter weight at 37 birth (LWB) ， number of piglets weaned (NW), and litter weight at weaning (LWW) were determined. 38 Besides, the sanger sequencing and PCR-restriction fragment length polymorphism were that frequency CC was significantly higher than that CT TT, and TT was the favorable genotype NW P =0.01) and 41 LWW P <0.01). Moreover, luciferase a positive transcription regulatory element in rs81471943 activity 1781 G/A P53 ELK1 45 zinc finger ( MZF1 Conclusions: and/or MZF1 . These findings provide useful information for identifying the molecular marker of EXOC4 -assisted selection in pig breeding. (two-tailed) for experiments.

LWW (P<0.01), and the haplotypes of -1781G/A and rs81471943 significantly affect the transcription 48 activity of EXOC4, which might influence the binding of potential cis-acting elements P53, ELK1 49

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Background 53 Pigs have experienced a particularly long history of haplotype changes through artificial selection from 54 domestication to modern breeding practices [1,2]. In porcine production, improvements in 55 reproductive performance of sows are slow due to the low heritability of reproductive traits [3]. The 56 recent development of sequencing and genotyping technology for pigs has enabled the exploration of 57 genomic evidence of selection and the detection of candidate genes associated with some target traits. 58 Studies have found that the positive selections of Duroc are associated with specific genes related to 59 lactation [4], reproduction [5] , meat quality [6], and growth traits [7]. Other studies have reported that 60 the reproductive traits such as number of piglets born alive (NBA) [8], lactation capacity, litter weight 61 at weaning (LWW) [9], and number of piglets weaned (NW) [10] all have been genetically improved 62 through phenotypic or selective signatures. 63 Most of porcine reproduction performance is evaluated using quantitative traits, which are mainly 64 affected by minor genes. The minor genes can be identified by quantitative trait locus (QTLs) mapping 65 [11,12]. According to PigQTLdb (https://www.animalgenome.org/cgi-bin/QTLdb/SS/index), there are 66 29,865 QTLs associated with 688 different traits. On chromosome 18, there are three, three, two and 67 five QTLs significantly associated with ovary weight [13], teat number, total born alive, and corpus 68 luteum number traits [14][15][16], respectively. In the commercial single-nucleotide polymorphism (SNP) 69 array, one SNP (rs81471943) locates on the exons of exocyst complex component 4 (EXOC4) gene, 70 and six QTLs associated with reproduction traits are identified within 5-10 Mb around EXOC4 on 71 chromosome 18, suggesting that EXOC4 may be highly associated with reproductive traits. EXOC4 72 belongs to the exocyst complex gene family, which connects vesicles to the cell membrane and 73 tissues, with slightly higher expression in the ovary, skeletal muscle, spleen, and hypothalamus [17]. 75 However, the underlying relationships between rs81471943 and reproductive traits as well as 76 transcription mechanism of EXOC4 are still unclear in pigs. 77 To explore the relationship between rs81471943 and reproduction traits, in this study, we acquired the 78 genotype frequencies of rs81471943 in Durocs, and the associations between rs81471943 and 79 reproductive traits such as NW, LWW, NBA, and litter weight at birth (LWB) were determined. Then 80 5'-deletion and luciferase assay were utilized to identify whether rs81471943 associates with the 81 transcription of EXOC4. The study contributes to the understanding of the regulatory mechanisms of 82 the EXOC4 and the molecular marker-assisted selection in pig breeding. 83

Materials and Methods 84
Animals 85 The ear samples of 994 Duroc sows prepared for SNPs genotyping were obtained from a breeding herd 86 in Fujian China and were collected from 2009 to 2017 and were taken from a previous study [18]. The 87 ear samples were collected into 75% alcohol immediately and stored at -20 °C. TaKaRa MiniBEST  88 Universal Genomic DNA Extraction Kit (Ver 4.0) was applied to extracted the genomic DNA was from 89 ear tissues. The A260/280 ratios of DNA samples were determined with NanoDrop 2000 (Thermo 90 Scientific), when the DNA samples with A260/280 ratio from 1.7 to 2.0 were genotyped using Illumina 91 PorcineSNP60 BeadChip (Illumina, San Diego, CA, USA). Four reproductive traits, including NBA, 92 LWB, NW, and LWW were recorded. In which, NBA and LWB were measured in 24 hours after 93 delivery, and LWW and NW were recorded after weaned. 94 Table 1. PCR products were purified by gelatinization, and the addition of "AAA" tail to ligated with 124 PMD-18T, then sequenced by Sanger Sequencing (Beijing Aoboxingke, China). The DNA sequences 125 of PCR products were determined by using DNASTAR software. Each deletion fragment digested with 126 Hind III and MIu I was cloned into the eukaryotic expression vector pGL3-vector, which was also 127 digested with Hind III and MIu I restriction endonuclease. Then, according to the manufacturer's 128 instructions for the dual-luciferase reporter assay kit (Promega, Madison, WI, USA), we used the 129 BioTek Synergy 2 multifunctional microplate reader (BioTek, Winooski, VT, USA) for fluorescence 130 detection. The ratio expression of firefly luciferase to renilla luciferase of each deletion fragment was 131 calculated. 132

Identification of SNP and transcription binding sites 142
The DNA of porcine ears was extracted and used as a template for PCR amplification. PCR was 143 performed by PrimerSTAR® high fidelity enzyme (TaKaRa, Dalian, Liaoning, China) to obtain the 144 fragment containing rs81471943 of EXOC4 to a length of 640 bp; the primers are listed in Table 1  145 under EXOC4-SNP. Then, the PCR product was linked to the T-vector and sequenced by Sanger 146 Sequencing (Aoboxingke, Beijing, China). The DNA sequences of PCR products were determined 147 using DNASTAR software. To identify the SNP, the alignment sequence and reference sequence were 148 aligned using BLAST. Then, the potential transcription factor binding region was predicted by 149 TFBIND [21],), and Jaspar [22]. 150

Statistical analysis 151
The phenotypic data of Duroc pigs were used for the association analysis of rs81471943 and 152 reproductive traits, including NBA, LWB, LWW, and NW. Estimated breeding values (EBVs) of all 153 pigs and the reliabilities of EBVs were imputed using animal model best linear unbiased prediction 154 [20] and obtained from the in-farm genetic evaluation software Herdsman swine management platform 155 research were tested by a single marker regression mixed linear model via SAS software. 157 The molecular experiment data were expressed as mean ± standard deviation (SD) of repeated 158 experiments. Each experiment was repeated at least three times independently. The significance of 159 differences in means between two groups was analyzed using Student's t-test (two-tailed) for molecular 160 experiments. * indicates P < 0.05; ** indicates P < 0.01. 161

Polymorphisms of rs81471943 163
The genotype frequencies of rs81471943 on EXOC4 gene of the 994 Duroc pigs were calculated and 164 counted ( Table 2). EXOC4 contained three genotypes, whereas CC was the dominant genotype with 165 genotype frequency 0.715, which was higher than that of CT (0.258) and TT (0.027). The dominant 166 allele was C, whose allele frequency was 0.844, which significantly higher than that of T allele (0.156). 167 The χ2 valued 0.001 (χ20.05 (2) =5.99 ， P>0.05) confirmed that the frequency distribution of 168 rs81471943 was in accordance with the Hardy Weinberg equilibrium law in the selected Duroc pig 169 population. 170

Association Between rs81471943 and Reproduction Traits 171
The description statistics for phenotypes of 994 Duroc sows were provided in Table 3 as well as EBVs  172 shown in Table 4. The relationship between the genotype of rs81471943 and reproductive traits was 173 further explored and analyzed. The NBA, LWW, and NW of individuals with TT was higher than that 174 with CC and CT, and the LWB of individuals with CC was higher than that with CT and TT (Table 3). 175 no significant difference among the three genotypes. The EBVs of LWB (P=0.02) of individuals with 177 CC was significantly higher than that with CT and TT. The EBVs of NW (P=0.01) and LWW (P<0.01) 178 of individuals with TT was significantly higher than that with CC and CT (Table 4). 179 These observations suggested that TT was the favorable genotype on NW (P=0.01) and LWW (P<0.01), 180 while CC was the favorable genotype on LWB (P=0.02). 181

Isolation of rs81471943 on EXOC4 182
Target fragments of EXOC4 contained rs81471943 were amplified by extracted DNA from eight Duroc 183 pigs ( Figure 1A) and identified by Sanger sequencing ( Figure 1B, C, D). Compared with the sequence 184 of EXOC4 in the Ensembl genome browser (release 89), a polymorphic mutation base C/T located on 185 EXOC4 (Chromosome 18: 16,079,412), which is in line with rs81471943 in the commercial SNP array. 186 To determine the polymorphic loci rs81471943 of EXOC4, 10 pigs were used in PCR-RFLP detection. 187 As shown in Figure 2, three genotypes CC, CT, and TT were identified by restriction endonuclease 188 BsrB I. 189

Transcription Activity Analysis of the EXOC4 Promoter 190
To investigate effects of rs81471943 on the expression of EXOC4, we tried to explore whether there is 191 any SNP maker, which links with rs81471943, at the promoter of EXOC4. 5'-deletion and a luciferase 192 assay were first used to identify regulatory elements on EXOC4 promoter. Six fragments with 5'-193 deletion of EXOC4 promoter were amplified ( Figure 3A) and cloned into pGL3-vector ( Figure 3B

Transcription activity analysis of different haplotypes on EXOC4 gene 207
To further determine the relationship between rs81471943 and transcription activity of EXOC4 in pigs, 208 the fragment -1826/-1551 of EXOC4 was amplified and sequenced on Duroc pigs. Interestingly, after 209 comparing the sequences published on Ensembl genome browser (release 89), one SNP was identified 210 and located on -1781G/A of EXOC4, where also was the potential transcription factor-binding fragment. 211 As the linkage between -1781G/A and rs81471943, four haplotypes were defined as HA-1(GC), HA-212 2(AC), HA-3(GT), and HA-4(AT ). The 5' deletion fragments EXOC4-P2 (-1914/+134) of four 213 haplotypes were amplified and cloned into the eukaryotic expression vector pGL3-vector. Luciferase 214 assays were used to detect the effect of different haplotypes on transcription of EXOC4. As shown in 215 Figure 5, we found that the luciferase activity of HA-1 was significantly higher than that of HA-2 (P< 216 rs81471943 might link with -1781G/A, and -1781G showed a favorable to affect the expression of 218 EXOC4. Moreover, many potential binding sites of transcription factors were predicted on -1781G/A 219 of EXOC4 (Table 6), and P53 and ETS transcription factor (ELK1) might bind at -1781A, and myeloid 220 zinc finger 1 (MZF1) might bind at -1781G. In this study, we found rs81471943 (C/T) located in the seventh intron of the EXOC4 gene. After 237 correlation between reproductive traits and EXOC4 genotype in Duroc pigs (Table 3). There were three 239 genotypes (CC, CT, and TT) exist in the Duroc populations, while C was the beneficial allele. Moreover, 240 TT was the favorable genotype on NW (P=0.01) and LWW (P<0.01), while CC was the favorable 241 genotype on LWB (P=0.02). 242 Then association between SNP and phenotypes confirmed that TT was the favorable genotype on NW 243 (P=0.01) and LWW (P<0.01) in Duroc pigs (Table 3 and Table 4). Previous studies have shown that 244 there are three QTLs were significantly associated with teat number [14-16] on chromosome 18, which 245 indicated that EXOC4 could affect the lactation performance of commercial pig. In this study, we also 246 found TT was significantly associated with NW and LWW, but not NBA and LWB. In addition, 247 previous studies demonstrated that the reproductive traits were highly correlated with each other, such 248 as NBA is highly genetically correlated with LWB [29]. In this study, we found that while CC was the 249 favorable genotype on LWB (P=0.02), but not NBA. This observation might be caused by the limited 250 population size used in this study, and it was likely that CC will be significantly associated with NBA 251 with large population size [30]. Collectively, although the results were got from the small population, 252 to the certain extent, the association of rs81471943 and lactation capacity NW and LWW could provide 253 useful information for EXOC4-mediated reproduction in pigs. [31][32][33]. 254 To explore effects of rs81471943 on the expression of EXOC4, the SNP makers which link with 255 rs81471943 at the promoter of EXOC4 were further investigated. In this study, regulatory elements of 256 EXOC4 promoter were first identified (Figure 4

Conclusions 283
In Duroc pigs, we found that the CC genotype frequency was significantly higher than that of CT and 284 TT in rs81471943, and TT was the favorable genotype on NW (P=0.

Availability of data and materials 304
The SNPs genotyping date were taken from a previous study [18].