Deok Gyeong Kang

Identification of Differentially Expressed Genes Associated with Litter Size in Berkshire Pig Placenta

Improvement in litter size has become of great interest in the pig industry because fecundity is directly related to sow reproductive life. Improved reproduction has thus been achieved by elucidating the molecular functions of genes associated with fecundity. In the present study, we identified differentially expressed genes (DEGs) via transcriptomic analysis using RNA-sequencing (RNA-Seq) in Berkshire pig placentas from larger (LLG, mean litter size >12) and smaller (SLG, mean litter size < 6.5) litter size groups. In total 588 DEGs were identified (p < 0.05, > 1.5-fold change), of which 98 were upregulated, while 490 were downregulated in the LLG compared with the SLG. Gene Ontology (GO) enrichment was also performed. We concluded that 129 of the 588 DEGs were closely related to litter size according to reproduction related genes selected based on previous reports, as 110 genes were downregulated and 19 upregulated in the LLG compared with the SLG. RT-qPCR utilizing specific primers targeting the early growth response 2 (EGR2), pheromaxein c subunit (PHEROC) and endothelial lipase (LIPG) genes showed high accordance with RNA-Seq results. Furthermore, we investigated the upstream regulators of these three genes in the placenta. We found that WNT9B, a Wnt signaling pathway molecule, and IL-6, known inducers of EGR2 and LIPG, respectively, were significantly increased in LLG compared with SLG. We believe that the induction of IL-6 and LIPG may play an important role in increasing nutrition supply through the placenta from the sow to the piglet during gestation. These results provide novel molecular insights into pig reproduction.

DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta

Increasing litter size is of great interest to the pig industry. DNA methylation is an important epigenetic modification that regulates gene expression, resulting in livestock phenotypes such as disease resistance, milk production, and reproduction. We classified Berkshire pigs into two groups according to litter size and estimated breeding value: smaller (SLG) and larger (LLG) litter size groups. Genome-wide DNA methylation and gene expression were analyzed using placenta genomic DNA and RNA to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs) associated with litter size. The methylation levels of CpG dinucleotides in different genomic regions were noticeably different between the groups, while global methylation pattern was similar, and excluding intergenic regions they were found the most frequently in gene body regions. Next, we analyzed RNA-Seq data to identify DEGs between the SLG and LLG groups. A total of 1591 DEGs were identified: 567 were downregulated and 1024 were upregulated in LLG compared to SLG. To identify genes that simultaneously exhibited changes in DNA methylation and mRNA expression, we integrated and analyzed the data from bisulfite-Seq and RNA-Seq. Nine DEGs positioned in DMRs were found. The expression of only three of these genes (PRKG2, CLCA4, and PCK1) was verified by RT-qPCR. Furthermore, we observed the same methylation patterns in blood samples as in the placental tissues by PCR-based methylation analysis. Together, these results provide useful data regarding potential epigenetic markers for selecting hyperprolific sows.

Effects of LEP, GYS1, MYOD1, and MYF5 polymorphisms on pig economic traits

Abstract In the present study, we examined the effect of single nucleotide polymorphisms (SNPs) of leptin (LEP), skeletal muscle glycogen synthase (GYS1), myogenic differentiation 1 (MYOD1), and myogenic factor 5 (MYF5) genes on economic trait association in pigs. LEP/HindIII, MYOD1/DdeI, MYF5/FokI, and GYS1/FokI genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from 466 pigs comprised of Duroc, Landrace and Yorkshire breeds. The LEP/HindIII polymorphism differed significantly with respect to average daily gain (ADG) in Duroc pigs (P<0.05). However, the GYS1/FokI polymorphism was not significantly associated with any trait. The MYOD1/DdeI polymorphism was significantly associated with both ADG and meat percentage (MP) in Duroc pigs, and ADG, backfat thickness (BFT) and feed efficiency (FE) in Landrace pigs, whereas the MYOD1/DdeI polymorphism was not significantly associated with any trait in Yorkshire pigs. In addition, the MYF5/FokI polymorphism revealed a close relationship with ADG in Duroc pigs. In conclusion, we believe that the SNPs within LEP, MYOD1 and MYF5 in certain pig breeds play important roles as potential genetic markers for economic traits of pigs.

Effect of Single Nucleotide Polymorphisms in IGFBP2 and IGFBP3 Genes on Litter Size Traits in Berkshire Pigs

ABSTRACT Litter size is among the most important traits in swine breeding. However, information on the genetics of litter size in pigs is lacking. In this study, we identified single nucleotide polymorphisms (SNPs) in the insulin-like growth factor binding protein 2 and 3 (IGFBP2 and IGFBP3) genes in Berkshire pigs and analyzed their association with litter size traits. The IGFBP2 SNP was located on chromosome 15 intron 2 (455, A > T) and the IGFBP3 SNP was on chromosome 18 intron 2 (53, A > G). The AT type of IGFBP2 and the GG type of IGFBP3 had the highest values for all litter size traits including total number born (TNB), number of pigs born alive, and breeding value according to TNB. Homozygous GG pigs expressed higher levels of IGFBP3 mRNA in the endometrium than pigs of other genotypes, and a positive correlation was observed between litter size traits and IGFBP3 but not IGFBP2 expression level. These results suggest that SNPs in the IGFBP2 and the IGFBP3 gene are useful biomarkers for increasing the reproductive productivity of Berkshire pigs.

Squalene epoxidase plays a critical role in determining pig meat quality by regulating adipogenesis, myogenesis, and ROS scavengers

In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.

Effects of a non-synonymous CBG gene single nucleotide polymorphism (SNP) on meat-quality traits in Berkshire pigs

Abstract: Corticosteroid-binding globulin (CBG) is a plasma transport protein that has glucocorticoid-binding activity. In the present study, we identified CBG gene expression in several tissues of four pig breeds: Berkshire, Duroc, Landrace, and Yorkshire. Expression of CBG mRNA was detected in the liver of all four breeds, and was the highest in Berkshire pigs. We also found single nucleotide polymorphisms (SNPs) in the CBG gene from Berkshire pigs, including SNP c.919G>A, which corresponds to 307G>R. We analyzed the relationships between this CBG variant and various meat-quality traits. The SNP was significantly associated with backfat thickness, post-mortem pH24h, meat color [CIE a* (redness), CIE b* (yellowness)], water-holding capacity, fat content, moisture content, protein content, cooking loss, and shear force. However, the effects differed by gender: the values were significantly associated with almost all traits in gilts, whereas only cooking loss and shear force were shown significantly in barrows. The variant G allele was associated with decreases in backfat thickness, post-mortem pH24h, CIE a*, fat content, and cooking loss, but with increases in CIE b*, water-holding capacity, moisture content, protein content, and shear force. Because the general correlation between meat-quality traits were not validated in the present study, we suggest that certain SNP might be used in the restrictive application to distinguish meat-quality traits.

Identification of a Bromodomain-containing Protein 2 ( BRD2 ) Gene Polymorphic Variant and Its Effects on Pork Quality Traits in Berkshire Pigs

Abstract Bromodomain-containing protein 2 (BRD2) is a nuclear serine/threonine kinase involved in transcriptional regulation. We investigated the expression and association of the BRD2 gene as a candidate gene for meat quality traits in Berkshire pigs. BRD2 mRNA was expressed at relatively high levels in muscle tissue. Statistical analysis revealed that the c.1709G>C polymorphism of the BRD2 gene was significantly associated with carcass weight, meat color (a*, redness), protein content, cooking loss, water-holding capacity, carcass temperatures 4, 12 and 24 h postmortem, and the 24 h postmortem pH in 384 Berkshire pigs. Therefore, this polymorphism in the porcine BRD2 gene may be used as a candidate genetic marker to improve meat quality traits in pigs.

Association between an electron transfer flavoprotein alpha subunit polymorphism (rs321948383) and the meat quality of Berkshire pigs

ABSTRACT The electron transfer flavoprotein α subunit gene (ETFA) encodes a protein that forms part of the ETF enzyme. This enzyme is normally active in mitochondria, the energy-producing centres of cells, to donate electrons derived from fatty acid oxidation to ETF- ubiquinone oxidoreductase. In the present study, we identified a non-synonymous single-nucleotide polymorphism (nsSNP) (rs321948383, c.569G > A) in the porcine ETFA gene and analyzed the association between this nsSNP and meat quality traits in 405 Berkshire pigs. The pigs with rs321948383 G/G had a higher meat quality from the aspects of meat colour (L*, p = 0.013), water-holding capacity (p = 0.001), drip loss (p = 0.000), carcass temperatures (T1, p = 0.000; T4, p = 0.000; T12, p = 0.000; T24, p = 0.001), and pH24 (p = 0.014). Therefore, this nsSNP (c.569G > A) could play an important role in improving the meat quality of pigs.

Association of single-nucleotide polymorphisms in NAT9 and MAP3K3 genes with litter size traits in Berkshire pigs

Abstract Litter size is an economically important trait in the pig industry. We aimed to identify genetic markers associated with litter size, which can be used in breeding programs for improving reproductive traits. Single-nucleotide polymorphisms (SNPs) of Berkshire pigs in the N-acetyltransferase 9 (NAT9) and Mitogen-activated protein kinase kinase kinase 3 (MAP3K3) genes were from RNA sequencing results, and already exist in the databank (NCBI), and were confirmed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). A total of 272 Berkshire sows were used to examine the genotype, and their association with litter size traits was analyzed. The NAT9 SNP was located in chromosome 12 exon 640 mRNA (A > G) and the MAP3K3 SNP was located in chromosome 12 intron 11 (80, C > T). Association analysis indicated that the GG genotype of NAT9 and the CT genotype of MAP3K3 had the highest values for litter size traits. The GG genotype expressed higher levels of NAT9 mRNA in the endometrium than the other genotypes did, and a positive correlation was found between litter size traits and NAT9, but not MAP3K3 expression level. These results indicate that the NAT9 and MAP3K3 can be used as candidate genes applicable in breeding program for the improvement of litter size traits in Berkshire pigs.

Identification of relationship between pork colour and physicochemical traits in American Berkshire by canonical correlation analyses

This study was carried out to predict the relationship between the colour and physicochemical traits in pork by using canonical correlation analysis. The variables of pork colour traits were lightness (L*), redness (a*) and yellowness (b*), whereas the variables of physicochemical traits were post-mortem pH24 h, water-holding capacity (WHC), collagen content, fat content, moisture content, protein content, drip loss, cooking loss and shear force. The canonical correlation coefficient (0.819) between the first pair of canonical variates, V1 and W1, was significant (P < 0.01). According to cross loadings, drip loss, cooking loss and fat content provided the relatively high positive correlations with the variates of colour traits (V1), while pH24 h, WHC and moisture content displayed negative relationships with the variates. Otherwise, L* and a* strongly contribute to the variates of physicochemical traits (W1). In addition, a redundancy index of 0.256 suggests that 25.6% of the variance in V1 is explained by W1. Therefore, in order to obtain the reddish pink colour in pork that is preferred by consumers depending on the relationships between the pork quality traits, we suggest that producer leads to the proper maintenance of post-mortem pH24 h, higher WHC, lower drip loss and cooking loss in pork.