Judul: Characterization of Candidate Genes for Meat Quality of Indonesian Sheep using RNA Sequencing
Abstrak: Lamb tenderness is a very important characteristic in determining the quality and level of public acceptance in consuming meat. Genetic improvement of the tenderness of the meat needs to be done in an effort to produce lamb with a high level of tenderness. Genetic improvement through the RNA sequencing technology can be used to understand the mechanism of the relationship between tenderness traits and its relation to good quality lamb meat. This study aims to identify the candidate genes for meat quality including tenderness of lamb meat, study polymorphism of candidate genes, association and expression of candidate genes related to meat quality. Analysis of RNA sequencing profiling of 5 samples with high tenderness and 5 samples with low tenderness from 140 sheep that have value of tenderness were analyzed using the Illumina Hiseq 2500. The analysis was continued with gene grouping based was carried out using the Illumina Hiseq 2500. The analysis was continued with gene grouping based on function and pathway related to meat tenderness using DAVID software. Quantitative Real Time PCR (qRT-PCR) was used to validate differences in gene expression with RNA sequencing. Further validation using PCR-RFLP and mRNA expression to confirm the effect of the genetic markers that have been obtained. A total of 328 genes could be detected differences in gene expression (DEG), namely 110 genes with high expression and 218 genes with low expression. Gene grouping based on gene ontology obtained cellular and molecularly resulted in the dominant gene clusters, each of which was extracellular exosome and calcium banding. Pathway analysis showed steroid hormone biosynthesis as the dominant pathway for differences in the tenderness level of lamb meat. Gene expression analysis identified candidate genes TP53INP1, CYP2E1, HSD17B13, ADH1C, and LPIN1 showed high expression in influencing meat quality, while the top genes that showed low expression included ANGPTL2, IGFBP7, FABP5, OLFML3, and THOC5. In the liver sample, 334 SNPs from 328 DEG were found. Multiple SNPs (more than one SNP) were found in the OLFML3, THOC5, and ANGPTL2 genes. Several candidate genes that were selected for further validation are OLFML3, THOC5, and ANGPTL2 genes. The results of PCR-RFLP analysis showed that the OLFML3, THOC5, and ANGPTL2 genes were polymorphic. The OLFML3, THOC5, and ANGPTL2 genes were found to have three genotypes, CC, CT, and TT; CC, CT, and TT; GG, GA, and AA. The association results showed that the OLFML3 gene (g.90317673 C>T) (P<0.05) was significantly associated with meat quality traits including tenderness and cooking loss; fatty acid composition such as tridecanoic acid (C13:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), ginkgolic acid (C17:1), linolenic acid (C18:3n3), arachidic acid (C20:0), eicosenoic acid (C20:1), arachidonic acid (C20:4n6), heneicosylic acid (C21:0), and nervonic acid (C24:1); carcass characteristics including carcass length; and the retail meat cut including intermuscular fat on the loin, muscle on the flank and shank. The TT genotype showed a higher level of meat quality, carcass characteristics, retail meatcut, and some fatty acid composition. The association results showed that the THOC5 gene (g.68234589 C>T) was significantly (P<0.05) associated with meat quality traits including tenderness; fatty acid composition such as
Keyword: gene, meat quality, RNA sequencing, sheep, tenderness