Genetic Basis Of Rumen Functional Evolution And Crosstalk With Microorganisms

The study of genetic basis of functional formation in animal complex organ is an important way to understand the formation of life.Ruminants are named by their unique forestomach system and ruminant behavior.As the major organ in the forestomach system,the intensive interactions with microbiomes in the rumen make ruminants obtain a unique evolutionary advantage through superior utilization of energy,which significantly promoted the expansion and diversification of ruminant taxa.Many progress has been made in the expression characteristics of rumen functions.However,the genetic basis underlying the functional evolution of this crucial organ remains largely unknown.Here,we perform comparative transcriptomics as well as ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations.Also,we explore the developmental reprogramming of rumen functions,microbial colonization,and their functional interactions by conducting an integrated analysis of dynamic ruminal transcriptome and metagenome data during development.This study included three parts of identification of genes related to rumen structure and function,identification and functional verification of rumen key candidate genes in evolution,and functional interactions between rumen and microbiomes.1.We collected 897 transcriptomes covered 50 tissues from Cetartiodactyla where the ruminant taxa belong to,covering three suborders of the Cetartiodactyla which evolved multi-chamber stomach structure: sheep and roe deer in Ruminantia,camels in Tylopoda,and Bryde's whale and Indo-Pacific Finless Porpoise in Cetacea.Based on the newly developed comparative transcriptome metric for identifying tissue-specific genes,named E50,we identified 655 relatively highly expressed(RHEs)genes in the rumen.Among these genes,the rumen co-expressed 14.7% genes with the esophagus,and others genes are recruited from a range of other tissues related to integumentary,immune and digestive system.The functional pathways involved in these genes are strongly associated with known rumen functions.By comparing the gene expression profiles of first-chamber(FC)stomach of camels,cetaceans and ruminants,the global gene expression patterns of all the FC stomachs are consistently most similar to the esophagus in all these species.Shared genes among the three FC stomachs are involved in epidermal development and differentiation.However,genes involved in ketone metabolism and microbial regulation pathways were significantly highly expressed in the rumen.Collectively,these results suggest that rumen share a common developmental origin from the esophagus with other two FC stomachs.The rumen not only up-regulated genes from the esophagus,also recruited genes from other tissues to acquire improved functions in the ketone body metabolism,antibacterial activities and epithelial structure.Rumen RHE genes identified in adults and rumen up-regulated genes compared to the esophagus in embryo constitued 846 rumen ‘core' genes.These rumen core genes collectively reflect rumen functions spanning from the embryo to the mature stage,all of which is relevant for characterizing rumen development and evolution.2.We combined evolutionary genomic analyses on the 846 rumen core genes in the context of previous study of ruminant comparative genomics with functional assays.We employed evolutionary genomic analyses on the 846 rumen core genes in the context of previous study of ruminant comparative genomics.Among the 846 rumen core genes identified above,657 genes with RSCNEs nearby and 28 PSGs were identified,which are mainly involved in ketone body metabolism,keratin filament binding,detection of bacterium and establishment of skin barrier.Among the set of 846 rumen core genes,657(77.7%)genes have nearby RSCNEs,of which RSCNEs of 243 genes overlapped with highly accessible chromatin.WDR66 is under positive selection in the common ancestor of Ruminantia and up-regulated in rumen compared to other FC stomach and esophagus.The regulatoy activity of the rumen DAP-associated RSCNE in the intronic region of WDR66 showed significantly higher luciferase transcriptional activation by luciferase reporter vector assay,indicating that it may act as an enhancer.The key ketogenesis rate-limiting gene(HMGCS2)with five ruminant-specific mutations was under positive selection which may induce a change of the protein 3D structure.By synthesizing the proteins of HMGCS2 orthologs with five amino acids replacements mutually in sheep and human,the ruminants exhibit higher HMG-COA synthesis activity than those of other mammals by enzyme activities assay.These results indicated that regulatory changes are the most dynamic venue of evolution,while positive selection on coding regions is also an important mode in the evolution of rumen functional innovations.3.We further collected rumen transcriptome and metagenomic data in seven time points from postnatal goats to tease the functional interactions between the rumen and its microbiomes.By integrating analysis of ruminal transcriptome and metagenome data,we explored the developmental reprogramming of rumen functions,microbial colonization and their functional interactions.Dynamic rumen transcriptome and microbial profiles presented two distinct phases during the early rumen development that the functions shift from a predominantly immune response(d 1-d 14)to nutrient metabolism of rumen wall,and a functional transition from bacteriocin biosynthesis(d 7-d 28)to glycolysis/gluconeogenesis activities of rumen microbiome,respectively.The developmental shift in the rumen transcriptome(at d 21)was earlier than the feed stimulus(at d 25)and the shift of rumen microbiome(at d 42).Based on the rumen transciptomic and metagenomic profiles,15 temporal dynamic rumen gene modules and 20 microbial modules were revealed by co-expression network analysis.Functional correlations between rumen and its microbiome were vividly involving in rumen pH homeostasis,nitrogen metabolism,and immune response.Two newly evolved genes(DEFB1 and LYZ1)reached their highest expression at d 42 during rumen development,while the microbial diversity index decreased to a nadir.We synthesized the proteins of two newly evolved genes in goat and validated the functions by performing inhibition zone assays.These two new genes both showed antibacterial activity to gram-positive bacteria.These results highlight that several important antibacterial functions are evolved in the rumen relative to other similar organs,and that some of these may work by specifically managing the microbiome composition.Our study highlights the multi-facetted genetic mechanisms taking place in the evolution of key functional innovations of the rumen relative to its esophagus ancestor.The identified rumen core genes and their specific mutations provide a starting point for future studies of rumen development gene regulatory network,and for understanding the interactions between rumen and microbiota.This will be key to further improvement of ruminant livestock,e.g.by providing a framework for manipulating the rumen fermentation process.