Study on the Patterns of Genetic Variations Using Molecular and Transcriptomic Analysis of the Differentially Expressed Genes of Jeju Native Pig

Abstract

Pork is a major source of animal protein for humans. The subcutaneous, intermuscular and the intramuscular fat (IMF) are the factors responsible for meat quality. RNA-seq is being rapidly adopted for the profiling of the transcriptomes in different areas of biology, especially in the studies related to gene regulation. The discovery of differentially expressed genes (DEGs) between adult Jeju Native Pig (JNP) and Berkshire breeds of Sus scrofa, are of particular interest for the current study. For the better understanding of the gene expression profiles of fat, liver and longissimus dorsi muscle, DEGs were identified via RNA-seq. Sequence read numbers were obtained from Ilumina HiSeq2000 and mapped to the pig genome using Tophat2. Total 153 DEGs were identified in fat and among the 71 annotated genes have BLAST matches in the non- redundant database. Similarly 169 and 39 DEGs were identified in fat, liver and muscle of JNP respectively, by comparison with Berkshire breed. Out of all identified genes, 41 genes in liver and 9 genes in muscle have given significant expression. Gene ontology (GO) terms of developmental process and KEGG pathway analysis showed that metabolic, immune response and protein binding were commonly enriched pathways in the three tissues. Further the heat map analysis by ArrayStar has shown the different levels of expression in JNP with respect to the Berkshire breed. The validation through real time PCR and western blotting also confirmed the differential expression of genes in both the breeds. Genes pertaining to metabolic process and inflammatory and immune system are more enriched in Berkshire breed. This comparative transcriptome analysis of two tissues suggests a subset of novel markers genes which expressed differently between the JNP and Berkshire. In our study, biological adhesion, cellular, developmental and multicellular organismal processes in fat were up-regulated in JNP as compare to Berkshire. Multicellular organismal process, developmental process, embryonic morphogenesis and skeletal system development were the most significantly enriched terms in fat of JNP and Berkshire breeds (p = 1.17E-04, 0.044, 3.47E-04 and 4.48E-04 respectively). The differential expression of genes was also investigated between piglets of Jeju Native Pig (JNP) and Berkshire breeds. Paired end reads of the sequences those passed the quality filters were aligned to Sus scrofa genome using Tophat2 (v2.0.2). In the current study, 65% (muscle), 20% (fat) and 54% (liver) genes have shown higher expression in the piglets of JNP than Berkshire. Gene ontology and signaling pathway showed that immune response and lipid metabolisms were commonly enriched pathways in all the three tissues. It was found that the genes pertaining to body growth and immune system are significantly (P<0.01) more expressive in Berkshire piglets. DEGs explored between the piglets of two breeds might influence the identification of the genetic markers for further breed improvement programmes. Further, the differential expression of a novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) has been identified in the liver tissue. ACAT2 is onvolved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. The study was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P < 0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs. The demand of pig meat is increasing day by day and during the last decade the quality of pork has become one of the main selection criteria. MyBPH has significantly higher transcript level in the longissimus dorsi muscle of JNP as compare to Berkshire. The current study targeted the role of the MyBPH in the muscle growth of JNP. The systematic in-silico annotations of SNPs and in-vitro functional analysis of MyBPH and other muscle regulatory factors have been performed. Different levels of polymorphism among the SNP sites were confirmed in MyBPH by sequence- and structure-based algorithms. More than 80% of total nsSNPs have been predicted as to be highly deleterious in JNP. In silico analysis by I-Mutant3 and HOPE tool has indicated that misfiling and intermolecular interaction of the MyBPH can significantly (P<0.05) affect the structures and functions of the protein. Therefore, such kind of changes due to nsSNPs target the functioning of MyBPH and inspite of higher transcript levels, MyBPH is still not able to potentiate the myogenesis in JNP. The sharing of specific myogenic features associated with MyBPH and MRFs through ToppCluster indicates that MyBPH can be a potent candidate gene to potentiate biological processes. The mutations in MyBPH can alter the muscle-specific biological and pathophysiological processes involved in muscle growth and metabolism. Our findings reveal MyBPH as a candidate gene for the muscle specific biological and pathophysiological processes. The current study is one of the pioneer transcriptomic studies being conducted in JNP. This study is the first transcriptional analysis for the detection of DEGs from RNA-seq data generated from fat, liver and longissimus dorsi muscle tissue samples. This analysis can be used as stepping stone to understand the difference in the genetic mechanisms that might influence the identification of novel transcripts, sequence polymorphisms, isoforms and noncoding RNAs. Our findings give a new horizon for understanding and identification of the candidate genes that are involved in the altered biological functions. Moreover, transcriptome analysis makes it easier to understand the differences between genetic mechanisms of breeds.