Gene Expression Network Analysis on Angiogenesis Pathway in Low Temperature-Induced Ascites Susceptible Chickens in B-line Pedigree of Iranian Meat-Type Strain, Arian

Ascites syndrome is a metabolic disorder in late ages of meat-type chickens. The only commercial broiler strain of Iran, Arian, has been incurred by this syndrome during a couple of decades ago. Due to susceptibility to ascites and some poor performance issues this strain are wiping out from indigenous market. Prevalence of ascites syndrome in commercial broiler of Arian is nearly 10% in average and in paternal lines it gets more fatality. Role of genetic factors in arising ascites has been proven by former researchers. Fighting against this syndrome through old methods are expensive and time consuming. Since presence of hypertrophy in a tissue needs to make facilities such as blood vessels to providing oxygen and nutrients so studying on angiogenesis pathway and its key important genes was directed. Whereas numerous organs engage the disease and among them heart has key role for initiating and developing the disease, so genomic study dealing with next generation sequencing (NGS) technology has been conducted to identify genes relating to angiogenesis and hypertrophy in right ventricle of ascites susceptible birds. 464 one-day-old chicks from B-line pedigree of Arian strain have been reared up to 21 days at the same rearing and nutritional conditions. At the age of 21 the rearing house was divided to 3 sections. Normal and cold sections at the ends of the house and buffer section at the middle. 464 chickens were transferred from normal to the cold section and kept the ambient temperature lower than 16 degrees for inducing ascites. 120 birds from cold section were selected randomly and slaughtered at the age of 42. Those of birds having ascites symptoms classified to ascites susceptible (As) and the other assigned to healthy (He). Samples of right ventricle was picked up and stored with RNAase-Later in liquid N tank. Then 12 As samples and 12 He samples were chosen for extracting total RNA by Biozol kit. After that every 6 samples in each groups pooled together and finally 2 As and 2 He samples prepared to make cDNA libraries for high throughput sequencer machine, Ilumina Hiseq 2000. RNA integrity score (RIN) were more than 7 for all samples. All small RNAs such as microRNAs, rRNAs and tRNAs were eliminated by oligo dt beads and finally all mRNAs was used for preparing library. cDNAs as long as 200 bp were selected for library. Sequencing has been done by BGI Company. For mapping, aligning, and DE analyzing several softwares were used such as: Tophat, cufflinks, cuffmerge and cuffdiff. Then significant DEGs imported to String for creating gene expression network and use DAVID 6.8 for investigation gene annotation and pathway analysis and finally Cytoscape v. 3.5.1 was used for network and cluster analysis. Nearly 90% of reads mapped on genome reference. In ascites group among 125 DEGs 79 genes was upregulated and 46 loci was down-regulated. Among 125 significant differentially expressed genes (FDR<20%) between As and He birds 59 genes were identified in a pathway in KEGG database. Gene ontology has been shown that the most significant biological process term containing 9 genes in term of gga04512: ECM-receptors interaction. The gene expression network analysis shows that average node neighboring was 3.763, network heterogeneity was 0.9 and network centralization was 0.236. Cluster analysis depicted 3 significant clusters involving key rule in angiogenesis including Cyclin- ependent Kinase1 (CDK1), ECM-receptors interaction and Interleukin 7 (IL7) (p-value<0.05). Up regulating of CDK1 with CCNB1 and CCNB2 may activated maturation promoting factor (MPF) that eventually induce cell division and proliferation in endothelial cells (ECs). Up regulating COL6A2 shows its roles in remodeling of ECM-receptors that in turn involving in proangiogenesis process in right ventricle hypertrophy in As birds. IL7 up regulated significantly (P-value<0.05) in As birds. This factor can induce IL8 and Ang-1 secretion from ECs. These factors have key important role for vascular remodeling and vascular tone in proangiogenesis and angiogenesis process. Studying on gene networks in susceptible bird’s heart cells has been shown that over expression in angiogenesis related genes was seen significantly in susceptible birds especially in ECM-receptor interaction, Cell division and IL-7 pathways as well. All these recent pathways rule actively in angiogenesis process and vascular network remodeling. So after finding these networks and related clusters it can be possible to find more extended studies on down regulated genes in apart or together for using breeding improvement objectives.