Characterization of Candidate Genes for Meat Quality of Indonesian Sheep using RNA Sequencing

Abstract

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 palmitoleic acid (C16:1) and stearic acid (C18:0). The CT genotype was associated with higher levels of meat quality and some fatty acid composition. The association results showed that the ANGPTL2 gene (g.8930776 G>A) was significantly (P<0.05) associated with meat quality traits including tenderness. The GG genotype was associated with higher levels of meat quality. However, mRNA expression of the OLFML3 gene was not different among genotypes. It could be concluded that OLFML3, THOC5, and ANGPTL2 gene are potential to use for candidate marker for meat tenderness in sheep.

Keempukan daging domba merupakan sifat yang sangat penting dalam menentukan kualitas dan tingkat penerimaan masyarakat dalam mengkonsumsi daging. Perbaikan secara genetik terhadap sifat keempukan daging perlu dilakukan dalam upaya menghasilkan daging domba dengan tingkat keempukan tinggi. Perbaikan secara genetik melalui teknologi baru RNA sekuensing dapat digunakan untuk memahami mekanisme hubungan antara sifat keempukan dan kaitannya terhadap kualitas daging domba yang baik. Penelitian ini bertujuan mengkarakterisasi kandidat gen kualitas daging yang meliputi keempukan daging domba, menganalisis polimorfisme kandidat gen, asosiasi dan ekspresi kandidat gen terkait kualitas daging menggunakan RNA sekuensing. Analisis RNA sekuensing profiling dari 5 sampel dengan keempukan tinggi dan 5 sampel dengan keempukan rendah yang telah dipilih dari 140 sampel domba yang telah diukur tingkat keempukannya dilakukan menggunakan Illumina Hiseq 2500. Analisis dilanjutkan dengan pengelompokan gen berdasarkan fungsi dan pathway terkait sifat keempukan daging menggunakan software DAVID. Quantitative Real Time PCR (qRT-PCR) digunakan untuk validasi perbedaan ekspresi gen (DEG). Validasi lebih lanjut dengan menggunakan PCR-RFLP dan ekspresi mRNA untuk mengkonfirmasi efek penanda genetik yang telah diperoleh. Sebanyak 328 gen dapat dideteksi perbedaan ekspresi gen (DEG) yaitu 110 gen terekspresi tinggi dan 218 gen terekspresi rendah. Pengelompokan gen berdasarkan gene ontologi didapatkan secara seluler dan molekuler menghasilkan kluster dominan gen masing-masing extracellular exosome dan calcium banding. Pathway analysis menampilkan steroid hormone biosynthesis sebagai dominan pathway pada perbedaan tingkat keempukan daging domba. Analisis ekspresi gen mengidentifikasi gen kandidat TP53INP1, CYP2E1, HSD17B13, ADH1C, dan LPIN1 menunjukkan ekspresi tinggi dalam memengaruhi kualitas daging, sedangkan gen-gen teratas yang menunjukkan ekspresi rendah diantaranya ANGPTL2, IGFBP7, FABP5, OLFML3, dan THOC5. Pada sampel hati ditemukan 334 SNP dari 328 DEG. Ditemukan multiple SNP (SNP lebih dari satu) pada gen OLFML3, THOC5, dan ANGPTL2. Beberapa kandidat gen yang terpilih untuk validasi lebih lanjut adalah gen OLFML3, THOC5, dan ANGPTL2. Hasil analisis PCR-RFLP menunjukkan bahwa gen OLFML3, THOC5, dan ANGPTL2 bersifat polimorfik. Gen OLFML3, THOC5, dan ANGPTL2 ditemukan tiga genotipe berturut-turut CC, CT, dan TT; CC, CT, dan TT serta GG, GA, dan AA. Hasil asosiasi menunjukkan bahwa gen OLFML3 (g.90317673 C>T) (P<0,05) berasosiasi secara signifikan dengan sifat kualitas daging meliputi keempukan dan susut masak; komposisi asam lemak seperti asam tridekanoat (C13:0), asam palmitoleat (C16:1), asam heptadekanoat (C17:0), asam ginkgolat (C17:1), asam linolenat (C18:3n3), asam arakidik (C20:0), asam eikosanoat (C20:1), asam arakidonat (C20:4n6), asam heneikosilat (C21:0), dan asam nervonat (C24:1); karakteristik karkas meliputi panjang karkas; dan potongan karkas meliputi lemak intermuscular pada loin, otot pada flank dan shank.Genotipe TT menunjukkan tingkat kualitas daging, karakteristik karkas, potongan karkas, dan beberapa komposisi asam lemak yang lebih tinggi. Hasil asosiasi menunjukkan bahwa gen THOC5 (g.68234589 C>T) secara signifikan (P<0,05) berasosiasi secara signifikan dengan sifat kualitas daging meliputi keempukan; komposisi asam lemak meliputi asam palmitoleat (C16:1) dan asam stearat (C18:0). Genotipe CT dikaitkan dengan tingkat kualitas daging dan beberapa komposisi asam lemak yang lebih tinggi. Hasil asosiasi menunjukkan bahwa gen ANGPTL2 (g.8930776 G>A) secara signifikan (P<0,05) berasosiasi dengan sifat kualitas daging meliputi keempukan. Genotipe GG dikaitkan dengan tingkat kualitas daging yang lebih tinggi. Namun, analisis ekspresi mRNA gen OLFML3 tidak berbeda antar genotipe. Hasil penelitian ini dapat disimpulkan gen OLFML3, THOC5, dan ANGPTL2 merupakan kandidat gen yang potensial untuk digunakan sebagai penanda genetik untuk seleksi cepat sifat keempukan dan kaitannya terhadap kualitas daging domba yang baik.