Comparative Genomic,Transcriptomic And Evolutionary Analyses Of HSP And Fn-? Protein Gene Families In Buffalo

The buffalo has been domesticated around 3000–6000 years ago and remained underutilized for their genetic development prior to having substantial economic significance as meat,dairy,and draught animal.Exploring the buffalo genetic architecture would help to manage available genetic variability,which is ultimately used for selective breeding and genomic selection.Recent advancements in high-throughput technologies and bioinformatics analysis are being employed to detect genetic variants,gene regulation,gene functioning,and single nucleotide polymorphism,which offer a wide range of whole-genome data with high coverage to genomic,epigenomic,transcriptomic,and proteomic information related to cellular interactions,functioning,and behavior.In buffaloes,candidate gene studies used available genetic resources to uncover the functional candidate genes and their association to evolutionary history,genetic diversity,functional elements,and performance traits.Thus,transcriptome and candidate gene approach to next-generation data could help to elucidate the inheritance of the complex traits,that should pave the way for genetic improvement in the climatic resilience,disease resistance,and production performance of buffalo populations globally.The first experiment was designed with the aim of transcriptomic profiling of 24 different tissues of swamp buffalo(brain vs non-brain tissues),through analyzing the differentially expressed genes(DEGs)to identify novel transcripts and their functional annotation with particular emphasis on Heat Shock Protein(HSPs),fibronectin type III domain(FN-III)proteins gene families and housekeeping genes.We obtained 178.57 Gb clean transcriptomic data with GC contents 52.77%,and an alignment of 95.36% to reference genome with 88.49% exon coverage.The SNPs ratio was 456941 with 25.04% heterozygosity.We incur 26363 m RNAs transcripts including 5574(34.57%)novel genes,among which 55.58%(3,999)were up-regulated and 44.41%(3,195)down-regulated.Additionally,138(42 upregulated and 14 down-regulated)and 133(57 up-regulated and 42 down-regulated)transcripts belonging to HSP and FN-III protein genes,respectively were also identified.Moreover,2076 housekeeping genes including 24 novel transcripts,199 up-regulated,344 down-regulated,and1533 normal transcripts were also observed.The functional annotation revealed that DEGs,HSPs,FN-III,and housekeeping genes were associated with cellular metabolic activities,signal transduction,cytoprotection,structural and binding activities.The related functional pathways included cancer pathway,PI3k-Akt signaling,axon guidance,adhesion,and regulation of actin cytoskeleton,JAK-STAT signaling,basic cellular metabolism,disease regulation,thermogenesis,and oxidative phosphorylation.Heat-shock proteins(HSP)are conserved ATP-dependent chaperones classified into HSP10,HSP40,HSP70,HSP90,HSPB1,HSPD,and HSPH1 family based on their molecular mass,involved in protein degradation,maturation,and refolding.The second study was conducted to analyze the HSP proteins gene family to better understand these proteins at the molecular level.Physiochemical properties,multiple sequence alignment,and phylogenetic analysis were performed for 64 HSP genes in the buffalo genome.Four genes were identified as belonging to the HSP90 family,10 to HSP70,39 to HSP40,8 to HSPB,and one for each HSPD,HSPH1,and HSP10,respectively.The aliphatic index was higher for HSP90 and HSP70 as compared to the HSP40 family,indicating their greater thermostability.Grand Average of hydropathicity Index values indicated the hydrophilic nature of HSP90,HSP70,and HSP40.Multiple sequence alignment indicated the presence of highly conserved consensus sequences that are plausibly significant for the preservation of structural integrity of proteins.Additionally,the phylogenetic tree revealed that buffalo HSPs are more closely related to Capra hircus and distantly associated with Danio rerio.In mammal's fibronectin type III(FN-III)domain proteins are usually involved in cellular growth,migration,differentiation,and adhesion.The FNDC5/irisin regulates energy metabolism and is present in different tissues such as the liver,brain,testis,etc.The third experiment aimed to investigate the physicochemical characteristics,FN-III proteins evolution and FNDC5/irisin as a ligand targeting the gonadal receptors including androgen(AR),DDB1 and CUL4 associated factor 6(DCAF6),estrogen-related receptor ?(ERR-?),estrogen-related receptor ?(ERR-?),kruppel like factor 15(KLF15),and nuclear receptor subfamily 3 group C member 1(NR3C1)to elucidate its possible role in folliculogenesis and spermatogenesis.Our study presented the molecular structure and function of 29 FN-III genes widely distributed in the buffalo genome.Phylogenetic analysis,motif,and conserved domain pattern demonstrated that all the FN-III proteins are evolutionary well conserved with a variety of stable to unstable,hydrophobic to hydrophilic,and thermostable to thermo-unstable proteins.Comparative structural configuration for FNDC5 predicted the variation in amino acid residues but the FNDC5 structure of humans,buffalo,and cattle were similar to each other.For the first time,we predicted the binding scores and interface residues of FNDC5/irisin as a ligand for six representative receptors,with a functional role in energy homeostasis and significant involvement in the folliculogenesis,and spermatogenesis in buffalo.The genomic and transcriptomic data analyzed in the present study would not only help to understand the genetic architecture of different phenotypic traits but also the regulation of their gene expression in buffalo.The results of the study laid a good working foundation for clarifying the law of differentially expressed genes in different tissues of buffalo and breeding of buffalo resistant to heat stress.