Integrating a genome-wide association study with transcriptome analyses to 1 identify candidate genes and pathways for feed conversion ratio in Yorkshire 2 pigs 3

Background: Feed conversion ratio (FCR) is an important productive trait that largely affects 15 profits in pig industry. Elucidating the genetic mechanisms underpinning the FCR potentially 16 promote the efficiencies of improving FCR through artificial selection. In this study, we integrated 17 a genome-wide association study (GWAS) with transcriptome analyses in different tissues in 18 Yorkshire pigs (YY), aimed at identifying key genes and signaling pathways significantly 19 associated with FCR. 20 Results: A total of 61 significant single nucleotide polymorphism (SNPs) were detected by 21 GWAS in YY. All of these SNPs are located on porcine chromosome (SSC) 5 and the covered 22 region was considered as a quantitative trait locus (QTL) region for FCR. Some genes that 23 distributed around these significant SNPs were considered as the candidates for regulating FCR, 24 including TPH2, FAR2, IRAK3, YARS2, GRIP1, FRS2, CNOT2 and TRHDE. According to the 25 transcriptome analyses in hypothalamus, TPH2 exhibits abilities of regulating the intestinal 26 motility by a serotonergic synapse and an oxytocin signaling pathway. In addition, GRIP1 is involved in a glutamatergic and GABAergic signaling pathway, which regulates FCR through 28 affecting the appetite in pigs. Moreover, GRIP1, FRS2, CNOT2, TRHDE regulates the 29 metabolism in various tissues by a thyroid hormone signaling pathway. 30 Conclusions: Synthesizes results from GWAS and transcriptome analyses, TPH2, GRIP1, FRS2, 31 TRHDE, CNOT2 genes were considered as candidate genes for regulating FCR in Yorkshire pigs. 32 These findings help to improve the understandings of the genetic mechanism of FCR and 33 potentially optimize the design of breeding schemes. 34

FCR were implemented in previous studies in our lab [29]. Subsequently, the genes identified by 138 ISwine and GWAS were integrated analyzed with transcriptome results. Database for Annotation,139 Visualization and Integrated Discovery software (DAVID bioinformatics resources: 140 https://david.ncifcrf.gov/) was used for functional classification and pathway analysis for all the 141 identified genes. 142 143 Results 144

Genome-wide association analyses for FCR 145
In total, 61 SNPs reached the significant thresholds of 5.796, which was calculated as the 146 Bonferroni correction (=-log10(0.05/31326)) [30]. All the significantly associated SNPs (61 SNPs) 147 are located on SSC 5. Among these SNPs, most of them (54 SNPs) are located within the region of 148 36.1-44.3 Mb on SSC 5, while 5 SNPs are located within the region of 47.1-47.8Mb and 2 SNPs 149 are located within the region of 33.4~34.5Mb. 150

LD block, associated regions analysis and candidate genes identified for FCR 151
Several linkage disequilibrium (LD) blocks were detected in the regions where the 61 significantly 152 associated SNPs located: 3 LD blocks were detected in the region of 33.4-34.5Mb on SSC 5; 3 LD 153 blocks were detected in the region of 36.1-44.3Mb on SSC 5 and 1 LD block was detected in the 154 region of 47.1-47.8Mb on SSC 5 ( Figure 2). The region 33.  Mb on SSC 5 aligned to the Sscrofa 11.1 genome assembly by NCBI 157 Remap. Then, pigQTLdb [31] was used to identified QTLs in these regions, and the results 158 showed these regions contained QTLs regulating the traits of days to 110 kg, feed intake, average 159 daily gain, body weight, loin percentage, intramuscular fat content, average backfat thickness, etc. 160 (Table S1). Feed intake and growth traits are tightly related to the performance FCR. Thus, these 161 regions were also considered as crucial QTL regions associated with FCR. 162 All the detailed information of the significantly associated SNPs identified by GWAS and the 163 putative candidate genes in this QTL region is shown in Table S2. Among the identified 61 164 significantly associated SNPs, 26 SNPs are located within some different genes. These significant 165 SNPs together with their corresponding genes are shown in Table 1. Some other genes located in 166 the 0.5 Mb genome region flanking the significantly associated SNPs were also considered as 167 candidate genes, including revealed fibroblast growth factor receptor substrate 2 (FRS2), 168 tryptophan hydroxylase 2 (TPH2), thyrotropin releasing hormone degrading enzyme (TRHDE), 169 GLI pathogenesis related 1 (GLIPR1) and fatty acyl-CoA reductase 2 (FAR2) etc. ISwine platform 170 [28] was also used to identify candidate genes for FCR in pigs. All the candidate genes identified 171 by ISwine platform are shown in Table S3. Based on the results from ISwine, TRHDE, TPH2,  172 FAR2, FRS2, GLIPR1 genes were confirmed as candidate genes for regulating FCR in Yorkshire 173 pigs. 174

175
To clarify the genetic mechanisms that involved in the regulation of FCR in pigs, we integrated 176 the GWAS results with a previously published transcriptome data of FCR, by using DAVID [32]. 177 The discovered signaling pathways and possible major genes are showed in Figure 3. It showed 178 that a mutation in TPH2 gene may influence the expression of neurotransmitter serotonin (5-HT), 179 which mediates colonic motility by the secretion of hypothalamic oxytocin (Figure 3a). In addition, 180 a mutation in GRIP1 gene may influence the aggregation of GABA and glutamate, which 181 mediates appetite of pigs ( Figure 3c). Notably, a thyroid hormone signaling pathway, which is 182 regulated by GRIP1, FRS2, CNOT2, TRHDE genes, was significantly differently expressed in 183 pigs with high or low FCR. The thyroid hormone signaling pathway participates in the regulation 184 of metabolism in various tissues ( Figure 3b). 185 were detected as the candidate genes for feed efficiency [33,34]. These genes mainly related to 197 lipid metabolic process, inositol phosphate metabolism and insulin signaling pathways. In the current study, we implemented a genome-wide association analysis for FCR in a large Yorkshire 199 population. Our analyses identified a series of novel significant SNPs located in the 33.  Mb on SSC 5. LD analysis showed these regions are highly linked, and 201 many QTLs related to feed intake and growth traits were located in these regions. Logically, these 202 regions were considered as candidate QTL regions for FCR. Genes located within 1Mb of the 203 significantly associated SNPs, including Fatty acyl CoA reductase 2 (FAR2), Interleukin-1 204 receptor-associated kinase-3 (IRAK3), and tyrosyl-tRNA synthetase 2 (YARS2), were inferred as 205 candidate genes regulating FCR in our study. 206 FAR2 gene spanned from 44.38Mb to 44.55Mb on SSC5. It is a key gene for fatty acid β 207 -oxidation, acetyl-CoA translocation, peroxisome biogenesis, and the glyoxylate cycle [35]. 208

Discussion
Moreover, FAR2 were associated with insulin resistance [36]. Previous studies reported that lipid 209 metabolism can explain the variation of FCR [14,37,38]. Therefore, the gene FAR2 might be a 210 candidate gene for FCR. 211 IRAK3 belongs to serine-threonine kinases and it is negatively correlated with mitochondrial 212 oxidative stress marker SOD2. It has been reported that high IRAK3 and low SOD2 cause weight 213 loss [39,40]. Previous studies reported that decreased IRAK3 was associated with increased 214 mitochondrial reactive oxygen species (ROS) [41] and some other studies reported that ROS can 215 decrease muscle mass by regulating mitochondrial biogenesis and the expression of antioxidant 216 gene [42,43]. Mitochondrial energy metabolism is a potential factor affecting the Feed conversion 217 ratio in pigs [15]. Therefore, IRAK3 is worthy to be further functionally investigated. 218 YARS2 is a key gene binds tyrosine to the homologous mt-tRNA for the synthesis of 219 mitochondrial proteins. The mutations of YARS2 can lead to mitochondrial respiratory chain 220 complex deficiencies and are related to mitochondrial myopathy [44,45]. YARS2 has not been 221 functionally characterized in pigs. However, since its function involves mitochondrial protein 222 synthesis and mitochondrial respiratory, it might be an important candidate gene for FCR in pigs. 223

GRIP1 control appetite through glutamatergic and GABAergic signaling 224
In this study, we integrated GWAS results with transcriptome analyses, aiming at identifying 225 candidate genes and biological pathways of FCR in pigs. The performances of feed intake have 226 been found significantly different in FCR divergent selection pigs, meanwhile feed intake is a 227 major physiological process associated with variations of FCR [46][47][48]. GABA ( γ-amino-butyric 228 acid ) and glutamate, which express in hypothalamic neurons, can promote feeding and weight 229 gain, while GRIP1 can interact with the C termini of AMPA receptors and clustered at both