Convergent Developmental Patterns Between Sheep And Goat Rumen Based On Single-cell Transcriptome And Metagenome

Sheep(Ovis aries)and goats(Capra hircus)have produced several convergent phenotypes over long periods of evolution,with the rumen an important structure for adaptive evolution in both species and a key organ for the digestion of crude fiber.The contents of rumen are rich in a highly dense and diverse microbial community.With the development of animals,the structure and function of the rumen undergo tremendous changes.However,due to the limitations of the sample and technologies,a complete singlecell developmental map has not been successfully constructed so far.Here,based on the phylogenetic tree constructed by mitochondrial D-loop and Cytb sequences,the maternal origin and convergent evolution of sheep and goats were analyzed.Further Chengdu gray goat and Wadi sheep were selected as the research subjects,and single-cell transcriptome profiles were constructed at early embryonic(e45,e60),middle embryonic(e75,e90,e105),and late embryonic(e120,e135),and postnatal non-ruminant(d0,d7,d14),transition(d21,d28,d35,d42,d49)and ruminant(d56,d90)of six key stages during rumen development by using the sc RNA-seq technology,and the correlation between metagenomic data of rumen fluid with host was analyzed.The main results are as follows:(1)Archaeological data results shown that both sheep and goats were domesticated in The Fertile Crescent 10,000 years ago,and phylogenetic tree results constructed based on mitochondrial D-loop and Cytb sequences shown that both sheep and goats had multiple maternal origins(sheep: haplogroup A,B,C,and E,goats: haplogroup A,B,C,and D).(2)Based on HE tissue section,we found that the rumen tissue of sheep and goats was mainly composed of epithelium,pluripotential blastemic tissue before e45,and then differentiated into myometrium after e60,rumen column at e75,and rumen papilla at e90.Rumen papilla increased keratinization after the postnatal transition stage,and the of the lamina propria and submucosal tissue,tunica muscularis layers were gradually thickening.(3)About 300,000 high-quality single-cell transcriptome data from sheep and goat rumen were sequenced.Most cells from the same developmental stage were clustered closer together,and cells in the embryonic and non-rumination stages were clearly separated from cells in the transition and ruminant stages.Rumen epithelial cells mainly included basal cells(BCs),spinous cells(SCs)and granulosa cells(GCs).SCs expressed both BCs and GCs marker genes KRT15 and KRTDAP,and GCs mainly express epithelial differentiationrelated genes(such as SPINK5,KRTDAP and DAPL1).In addition,four subtypes of sheep fibroblast and eight of goat were identified,which were found to be stage-specific,and 12 homologous cell types of sheep and goats were defined.(4)Most of the sampling timepoints were shared differentially expressed genes(no less than 2 sampling points).The total number of up-regulated differentially expressed genes in the early and middle embryonic stages(e45-e75)was much higher than other timepoints.Early and middle embryonic stages(e45-e105),and rumination stages(d56-d90)showed similar patterns of DEGs,but DEGs patterns in late embryo(e120-e135),non-rumination stage(d0-d14)and transition stage(d21-d49)were significantly different.Differentially expressed genes in adjacent stages were hardly shared,indicating the uniqueness of the function of each stage.GO analysis showed that up-regulated DEGs were enriched to the same GO entries in adjacent stages of sheep and goats,such as epithelial differentiation,oxidative phosphorylation,etc.In addition,BCs,SCs,GCs,fibroblasts and neural cells showed significant differences in the number of DEGs at the six stages,and conserved genes of cell types that were not affected by rumen development were found,for example,upregulate genes HSD17B2,KRT5 and KRT15 of BCs,might maintain basic functions of ruminant basal cells.(5)A total of 111 transcription factors(TFs)were predicted in sheep rumen and 99 in goat,of which 23 up-regulated and 23 down-regulated TFs were shared between the two species.In addition,we also identified shared TFs at specific stages,such as JUN in nonruminant stage,CUX1,FOS,JUN,TCF12,TCF21 and YY1 in ruminant stage.Some TFs functioned in different cell types at the same stage,whereas some TFs activated in the same cell type at different stages.Cell communication analysis showed the changes of cell communication between sheep and goats were similar before the transition stage but showed obvious differences after entering the rumination stage.The increased ligand-receptor interactions at adjacent stages were involved in biological processes mainly related to cell proliferation and immunity.(6)Analyzing rumen data from sheep,goats,and cattle downloaded by NCBI,the same rumen epithelial marker gene was found,such as KRT5 for BCs subtype BC type 1 and DAPL1 for BRIFA2C+cg-like SCs.Using downloaded data from humans,mice and monkeys,similar marker gene expression patterns were found in the same cell types,such as PGC and CLDN18 in epithelial cells,COL3A1 and COL1A2 in stromal cells,and JCHAIN and CAP3 in immune cells;a total of 8 TFs specific to ruminants(ATF4,CEBPD,FOS,IRF1,JUN,NFIX,PITX1,and YY1),and 3 TFs shared by 6 species(ATF3,FOSB,and JUNB),these TFs were highly active in different cell types.Besides,4 epidermal growth factor receptors(EGFR)-related ligand-receptor interactions were only expressed in ruminants(especially in rumen epithelial cells),while immune-and inflammation-related ligandreceptor interactions(such as CD44-HBEGF)were expressed much higher in cattle and goats than that of three non-ruminant animals(human,monkey and mouse).(7)Based on rumen fluid metagenomic data,a total of 23 bacterial phyla and 278 bacterial genera were identified.The composition and abundance of rumen microorganisms in sheep and goats were similar at different developmental stages,and the rumen microbial communities from embryonic to non-ruminant stages were mainly composed of the composition of aerobic bacteria,such as Phyllobacterium;after entering the transition and ruminant stages,they were dominated by anaerobic bacteria,such as Prevotella.There were six bacterial genera in rumen embryonic stage(e45-e105)and non-ruminant stage(d0-d14),namely Sphingomonas,Cutibacterium,Phyllobacterium,Agrobacterium,Rhizobium and Pseudomonas,which were strongly positively correlated with 67 development-related DEGs and immune-related pathways.There were mainly 5 bacterial genera in the postnatal transition and ruminant stages(d21-d90),namely Prevotella,Butyrivibrio,Clostridium,Succiniclasticum and Alloprevotella,which were negatively correlated with 26 DEGs,and mainly enriched in biological processes such as energy metabolism,cell migration,immunity,and fatty acid metabolism.(8)Based on the results of correlation analysis,it was found that rumen cell types with microbial composition and metabolic functions were similar between sheep and goats.The results showed that cell types were stably present at the six stages(T cells,B cells,endothelial cells)or appearing relatively later(such as basal cells subtype basal＿CA1＿high,spinous cells subtype spinous＿KRT15＿high,etc.)during rumen development were strongly positively correlated with these seven bacterial genera(Alloprevotella,Bacteroides,Butyrivibrio,Clostridium,Mannheimia,Prevotella,and Succiniclasticum).Overall,based on convergent evolutionary using mitochondrial genetic analysis,this study annotated the major cell types at all six developmental stages of rumen in sheep and goats,constructed the most comprehensive single-cell transcriptome profiling of rumen to date,found DEGs,transcriptional regulatory networks,and convergent expression between cell communication between sheep and goat,described the similarities of microbial types between species in the microbial community at six developmental stages of rumen in sheep and goats,and found the correlation between rumen microbial composition,microbial biological function with the expression patterns of rumen cell types.