Isoform Expression Profile Analysis of Residual Feed Intake for Isfahan Native and Ross Chickens Using RNA-Seq Data

In the past decade, many performance traits in chickens have greatly improved to meet the growing demand for chicken meat. On the other hand, due to the 70% share of feed costs in the total cost of livestock production, feed efficiency has become one of the most important genetic traits in livestock and poultry. Residual feed intake (RFI) is the difference between the actual absorption of animal feed and the expected of the animal absorption based on body weight and its level of performance. Selection to improve feed efficiency through RFI is suggested due to its phenotypic independence from body weight and body weight gain. There is evidence for the genetic basis of variation in productivity in all species. Previous studies have shown that some genes may play an important role in controlling RFI through effects on the digestive system and metabolic processes. It seems that changes in the amount of residual feed intake is the result of changes in the expression of the genes associated with it, and the study of transcript changes in chickens with high RFI helps to identify the mechanisms that affect it. Experiments were conducted on Commercial breeds (high production efficiency) and Isfahan Native Chickens (low production efficiency). From each breed, 60 chicks were harvested and caged from day 24 to 42 days for 19 days. The chicks of both groups were raised under the same management conditions. In order to measure the residual feed intake of each bird in individual cages and daily intake (FI) of the amount of feed consumed in 19 d (from day 24 to 42) divided by number of days, and daily gain (DG) of the weight difference 24. Using these figures, the amount of residual feed consumed will be calculated. Ten samples of liver tissue were then collected from each breed and immediately transferred to liquid nitrogen and transferred to the laboratory for total RNA extraction. Two pooled samples of every breed (each consisting of three extracted RNA sample) were prepared. Prior to Alignment, the quality of the raw readings sequence was evaluated using the Fastqc software. The reference genome for the chicken (Galgal4) and the annotated file were obtained from the Ensembl database. After controlling the quality and Trimming the reads, the Hisat2 software was used to mapping obtain reads from the genome of the reference. Analysis of differential gene expression was performed using Cufflinks.. To identify different isoform of the gene and to measure the expression of isoform, it is necessary to examine the position of microwave splicing between the exons. Cuff compare compares the transcripts that are assembled with a reference genome and categorizes each transcript into known, new, and potential new isoform. The phenotypic results showed that the difference in the mean RFI values between the native (+13.430±5.393 g/day) and commercial (-11.212±4.435g/day) chickens was highly significant (P<0.001). About 83% of the sequenced fragments were matched to the reference genome and Duplex analysis of gene expression was performed..Differential expression analysis for more than 45070 isoform Related to 19003 genes using Cuffdiff showed that 28949 and 16121 isoform were up- and down-regulated (adjusted P≤0.05) in the native breed, respectively. 206 Isoform was identified as a change in expression between the native and commercial chickens. Moreover 21081 novel isoform were identified which have not been included in the gene annotation so far. Moreover, the functional enrichment analysis showed that the down-regulated isoform in the native chickens were mainly involved in the different response to stress،response to oxygen-containing compound،response to cytokine،response to nutrient،response to lipid،animal organ development ،single-organism biosynthetic process،regulation of multi-organism process response to nutrient level and lipid biosynthetic process. The Differentially Expressed Genes and their isoform explored between the chickens of the two breeds led to the identification of some important candidate genes for further breed improvement programs, including RSAD2, IL15, EGR1, and DUSP16. These genes could be related to the higher RFI of the native. Our findings showed that a large number of related genes with metabolism (fat, amino acids, carbohydrates, and vitamins), growth, as well as oxidative stress and immunity, can make a difference in the RFI. Identification of the difference genes and their isoform between two breeds of chickens led to the identification of important candidate genes for breeding programs, including RSAD2, IL15, EGR1 and DUSP16.