Kajian Genomik dan Transkriptomik Gen-Gen Potensial Sifat Ketahanan Penyakit Ayam IPB-D2

Abstract

Peningkatan kualitas ayam IPB-D1 dapat dilakukan dengan beberapa cara, salah satunya yaitu pembentukan calon galur ayam IPB-D2. Ayam IPB-D2 diseleksi berdasarkan sifat ketahanan penyakit. Ayam IPB-D2 diseleksi dari ayam IPB-D1 dengan kriteria konsentrasi IgY ≥ 10 mg mL-1 dan titer antibodi ND ≥ 3 log2 HI unit. Selain seleksi secara konvensional dengan seleksi konsentrasi IgY dan titer antibodi ND, juga dilakukan secara molekuler. Seleksi molekuler dilakukan dengan memanfaatkan gen-gen potensial hasil RNA sekuensing. Tujuan umum penelitian ini adalah mengkaji gen-gen potensial pada sifat ketahanan penyakit ayam IPB-D2. Tujuan khusus penelitian ini yaitu: 1) mengevaluasi sifat ketahanan penyakit ayam IPB-D2; 2) menganalisis gen-gen potensial sifat ketahanan penyakit ayam IPB-D2 dengan RNA sekuensing dan 3) menganalisis keragaman, ekspresi dan asosiasi gen-gen potensial gen BLB2 serta CD4 terhadap sifat ketahanan penyakit ayam IPB-D2. Penelitian ini menggunakan ayam IPB-D2 G2 berumur 21 minggu. Sampel darah diambil dan digunakan untuk uji ketahanan penyakit dan kajian genomik gen BLB2 dan CD4. Analisis transkriptomik menggunakan jaringan seka tonsil ayam IPB-D2 G2 dengan perbedaan konsentrasi titer antibodi ND tinggi dan titer antibodi ND rendah. Uji ketahanan penyakit ayam IPB-D2 dilakukan dengan melakukan beberapa metode. Konsentrasi IgY dianalisis dengan metode indirect ELISA, titer antibodi ND dianalisis dengan metode HI, profil leukosit dan diferensiasinya dianalisis menggunakan metode Giemsa, populasi CD4+ dan CD8+ dianalisis menggunakan uji flowcytometry. Hasil analisis menunjukkan ayam IPB-D2 memiliki sifat ketahanan tubuh seperti konsentrasi IgY 12,60±1,96 mg mL-1, titer antibodi ND 1,55±1,4 log2 HI unit, leukosit 15.29±4.28 x 106 mm-2, limfosit 50,96±11,91 %, heterofil 42,76±12,23 %, monosit 4,85±0,95 %, eosinofil 1,61±0,9%, dan populasi CD4+ yang lebih tinggi dibandingkan populasi CD8+. Ayam IPB-D2 memiliki sistem kekebalan spesifik dan non-spesifik yang seimbang. Kajian gen-gen potensial pada sifat ketahanan penyakit dilakukan dengan 2 metode yaitu studi literatur hasil RNA sekuensing pada ayam SenSi dan analisis RNA sekuensing pada ayam IPB-D2. Analisis RNA sekuensing pada ayam IPB-D2 menunjukkan terdapat beberapa gen-gen potensial pengontrol titer antibodi ND seperti ZBTB38, CCR9, TLR2A, CD44 dan CDC42BPA. Hasil studi literatur pada hasil RNA sekuensing ayam SenSi menyatakan terdapat beberapa gen yang berhubungan dengan kemampuan imunitas pada ayam SenSi. Salah satu gen yang berpotensi yaitu gen BLB2 yang dianalisis lebih lanjut pada penelitian ini. Kajian genomik gen BLB2 dan CD4 dilakukan dengan analisis keragaman gen. Keragaman gen BLB2 dan CD4 pada ayam IPB-D2 dianalisis menggunakan metode sanger sekuensing. Keragaman dan asosiasi gen BLB2 dan CD4 dianalisis menggunakan SNPStats. Hasil penelitian menunjukkan terdapat 13 SNP gen BLB2 dan 4 SNP gen CD4 pada ayam IPB-D2. Sebanyak 4 SNP gen BLB2 yang berasosiasi dengan konsentrasi IgY yaitu yaitu g.458 T>A, g.485 G>C, g.486 T>G dan g.548 G>C. Kajian transkriptomik gen BLB2 dan CD4 dilakukan dengan analisis ekspresi gen. Analisis ekspresi gen dilakukan dengan mesin qRT-PCR dan dilanjutkan dengan uji T. Hasil penelitian menunjukkan bahwa level ekspresi gen BLB2 dan CD4 pada ayam IPB-D2 dengan titer antibodi ND tinggi lebih tinggi dibandingkan dengan ayam IPB-D2 dengan titer antibodi ND rendah namun tidak berbeda secara signifikan (P>0,05).

The improvement of IPB-D1 chicken can be done in several ways, one of which is the formation of IPB-D2 chicken strains. IPB-D2 chicken is selected from IPB-D1 chicken with the criteria of IgY concentration ≥ 10 mg mL-1 and ND antibody titer ≥ 3 log2 HI units. In addition to conventional selection with IgY concentration and ND antibody titer selection, molecular selection was also conducted. Molecular selection is done by utilizing potential genes from RNA sequencing analysis. The general objective of this research was to study potential genes in disease resistance traits of IPB-D2 chicken. The specific objectives of this research are: 1) evaluate the disease resistance traits of IPB-D2 chicken; 2) analyze the potential genes of IPB-D2 chicken disease resistance traits by RNA sequencing and 3) analyze the polymorphism, expression and asociation of potential genes of BLB2 and CD4 genes on disease resistance traits of IPB-D2 chicken. This study used 21-week-old IPB-D2 G2 chickens. Blood samples were collected and used for disease resistance tests and genomic studies of BLB2 and CD4 genes. Transcriptomic analysis was performed using caecal tonsil tissue of IPB-D2 G2 chickens with different concentrations of high ND antibody titer and low ND antibody titer. The disease resistance test of IPB-D2 chickens was carried out by conducting several method. IgY concentration was analyzed by indirect ELISA method, ND antibody titer was analyzed by HI method, leukocyte profile and differentiation were analyzed using Giemsa method, CD4+ and CD8+ population were analyzed using flow cytometry test. The results of the analysis showed that IPB-D2 chickens had immune properties such as IgY concentration of 12.60 ± 1.96 mg mL-1, ND antibody titer of 1.55 ± 1.4 log2 HI units, leukocytes 15.29 ± 4.28 x 106 mm-2, lymphocytes 50.96 ± 11.91%, heterophils 42.76 ± 12.23%, monocytes 4.85 ± 0.95%, eosinophils 1.61 ± 0.9%, and a higher CD4+ population than the CD8+ population. IPB-D2 chickens have a balanced spesific and non-spesific immune system. The study of potential genes in disease resistance traits was carried out by 2 methods, namely literature study of RNA sequencing in SenSi chickens and RNA sequencing analysis in IPB-D2 chickens. RNA sequencing analysis on IPB-D2 chickens showed that there are several potential genes controlling ND antibody titers such as ZBTB38, CCR9, TLR2A, CD44 and CDC42BPA. While the results of the literature study on the results of RNA sequencing of SenSi chickens stated that there are several genes associated with the ability of immunity in SenSi chickens. One of the potential genes is the BLB2 gene which will be further analyzed in this study. Genomic studies of BLB2 and CD4 genes were conducted by analyzing gene diversity. The polymorphism of BLB2 and CD4 genes in IPB-D2 chickens was analyzed using the sanger sequencing method. The polymorphism and association of BLB2 and CD4 genes were analyzed using SNPStats. The results showed that there were 13 BLB2 gene SNPs and 4 CD4 gene SNPs in IPB-D2 chickens. A total of 4 BLB2 gene SNPs associated with IgY concentration are g.458 T>A, g.485 G>C, g.486 T>G and g.548 G>C. Transcriptomic studies of BLB2 and CD4 genes were carried out by gene expression analysis. Gene expression analysis was performed by qRT-PCR machine and followed by T test. The results showed that the expression level of BLB2 and CD4 genes in IPB-D2 chickens with high ND antibody titer was higher than IPB-D2 chickens with low ND antibody titer but not significantly different (P>0.05).