Genomic Sequencing And Analysis Reveals Adaptive Evolution To Hypoxia Of Plateau And Mandarin Voles

High altitude hypoxia and subterrenean hypoxia are two types of typical environmental hypoxia in terrestrial mammals.During the long-term evolution,the species living under hypoxic conditions have evolved a series of stable adaptive mechanisms at the morphological and molecular levels.A systematic study on the adaptive evolution to hypoxia in mammals may provide a basis for the treatment of human hypoxia-related diseases.Plateau vole(Neodon fuscus)is a typical plateau rodent which live on the ground,distributed in the alpine meadow at an altitude of 3,700m-4,800m on the Tibetan Plateau;Mandarin vole(Lasiopodomys mandarinus)is a typical subterranean rodent,distributed in the farmland of North China;Brandt's vole(L.brandtii)is a closely related species of Mandarin vole which yet live on the ground,mainly distributed on the Mongolian plateau at an altitude of about 1,000m.In fact,all these 3 voles belong to the subfamily of Arvicolinae.Therefore,they are pretty good animal models for studying plateau hypoxia and subterrenean hypoxia.In this study,Plateau vole,Mandarin vole and Brandt's vole were conducted to sequence the whole genome by high throughput second-generation sequencing in different library strategies.And then the reads were assembled to genomes of 3 voles,respectively.Annotation of the 3 genomes were performed comprehensively by identifying the genome-wide repetitive sequences,non-coding RNA genes,and the structure and function of coding genes.Meanwhile,the systematic evolutionary history analysis including the estimation of divergence time,evolution rate and the population history were conducted in 3 voles by analyzing their whole genome sequence.In addition,we compared the genomes of Plateau vole and Brandt's vole to study the adaptive evolution mechanism of high altitude hypoxia in Plateau vole.In order to study the adaptive evolution to high altitude hypoxia in mammals in further,we also did analysis of the genome sequence through 4 plateau mammals.In other hand,we compared the genomes of Mandarin vole and Brandt's vole to uncover the mechanism of adaptive evolution to subterranean hypoxia in Mandarin vole.And we similarly analyzed the genome sequence through four kinds of subterranean mammals to study the adaptive evolution to subterranean hypoxia in mammals.The main results are summarized as follows:1.Whole genome sequencing and assembly385.90 Gb genomic data of Plateau vole was obtained.The genome size was 2.32 Gbp predicted by 17K-mer method.The sequencing depth was 166.34 ×.The length of assembled genome was 2.24 Gbp.Contig N50 and scaffold N50 were 45.59 kb and 9.52 Mbp.BUSCO and CEGMA evaluation were 95.60%and 97.18%,respectively.324.9 Gb genomic data of Mandarin vole was obtained.The genome size was 2.25 Gbp predicted by 17K-mer method.The sequencing depth was 172.85 ×.The total length of the assembled genome was 2.15 Gbp.Contig N50 and scaffold N50 were 51.15 kb and 6.15 Mbp.BUSCO and CEGMA evaluation were 94.70%and 95.97%respectively.470.39 Gb genomic data of Brandt's vole was obtained.The genome size was 2.32 Gbp predicted by 17K-mer method.The sequencing depth was 202.75 ×.The total length of the assembled genome was 2.23 Gbp.Contig N50 and scaffold N50 were 50.79 kb and 0.68 Mbp.BUSCO and CEGMA evaluation were 94.50%and 95.16%,respectively.2.Genome-wide annotationThe repetitive sequences annotation for genomes of Plateau vole,Mandarin vole and Brandt's vole were 34.99%,33.93%and 36.82%,respectively.The copy numbers of miRNA,tRNA,and snRNA in 3 voles by the non-coding RNA annotation were equivalent while the rRNA copy number of Brandt's vole was higher.The structural annotation of coding genes for 3 voles were consistent.The functional annotation ratio was 97.98%(20,319/20,738)for Plateau vole,93.27%for Mandarin vole(19,801/21,229),and 97.66%(20,049/20,530)for Brandt's vole.3.Evolutionary history of 3 volesThree voles could be gathered together in phylogenetic tree constructed from single-copy orthologous gene families of 12 species.Plateau vole belonging to Neodon,evolved independently at about 7.4MYA.Both Mandarin vole and Brandt's vole belong to Lasiopodomys,which separated into 2 species above at about 5.8 MYA.The evolution rate of 12 species were significant different,in which the 3 voles were faster than other species.Mandarin vole was the fastest one,Brandt's vole was the second,and Plateau vole was the third.The Qinghai-Tibet Plateau uplift movement and glaciation may have different effects on evolutionary history of 3 voles.Plateau vole is affected by both the plateau uplift and glaciation.Brandt's vole is affected more by glaciation and less by the plateau uplift.And Mandarin vole was affected minimally by glaciation and plateau uplift.4.Adpative evolution of Plateau vole and plateau mammals to plateau hypoxic environment1)The expanded gene families of Plateau vole and Brandt's vole were 175 and 138,and the unique genes were 74 and 23,respectively,mainly related to protein synthesis,and angiogenesis promotion through the EGF gene of HIF-1 signaling pathway.The contracted gene families were 981 and 705,missing genes were 558 and 457,respectively,mainly related to the senses and metabolism such as olfaction and vision.The positive selected genes(PSGs)of Plateau vole and Brandt's vole included MAPK signaling pathway-related genes and protein kinase B,which can regulate HIF-1 signaling pathway.The PSGs enrichment evolution are mainly related to oxygen-regulated energy metabolism function.The PSGs from Plateau vole are mainly related to interleukin,coenzyme and vitamins while which from Brandt's vole mainly related to immune function.2)Plateau vole mainly through FGF7 gene to regulate MAPK signaling pathway and protein kinase B signaling for promoting the expression of HIF-1?,which can indirectly regulate HIF-1 signaling pathway.It also regulates the cardiovascular system through EGF and CYCS genes,responding to external cell stimuli through the SERPINE1 and multiple EIF2 families,and increases the aerobic respiration capacity through CYCS,also enhances sugar metabolism pathways by CDK1 and HSPA9.In addition,through the key genes RIPK1,CYCS and IL12B,Plateau vole could regulate several other pathways:immune and neurological diseases,anti-cancer pathways,cytoplasmic DNA sensing pathways,nucleotide excision repair,NF-kappa B signaling,RIG-I-like receptor pathways,and tryptophan metabolism.3)The four different plateau species,Plateau vole,north american rabbit,black snub-nosed monkey and yak had no common gene family for expansion,only 4 common positive selection genes,whose function includes promoting angiogenesis and remodeling anaerobic pathway.Plateau species also adapt to plateau hypoxia in the species-special evolutionary way,in which,Plateau vole improves energy metabolism,yaks evolved through sugar metabolism,north American rabbits evolved through DNA repair,and black snub-nosed monkeys evolved through the genes related to synapses.5.Adpative evolution of Mandarin vole and subterrean mammals to subterrnean hypoxic environment1)There are 178 and 138 gene families expanded in Mandarin vole and Brandt's vole,respectively,of which 69 and 23 are unique.These genes are mainly related to protein synthesis,by the GAPDH gene in HIF-1 signaling pathway,elevating anaerobic metabolism and reducing oxygen consumption.The contracted gene families were 724 and 705,and missed gene families were 596 and 457,respectively.The genes were mainly related to olfaction,vomeronasal organs,and metabolism.The PSGs of Mandarin vole and Brandt's vole including CDKN1B,EPO,and VHL regulate cell proliferation and oxygen transport capacity in the HIF-1 signaling pathway.The enriched PSGs could be classified into different functional categories:mitochondria,DNA repair and other items related to hypoxic adaption.The PSGs of Mandarin vole include IL6R and IFNGR2 genes which can regulate the HIF-1 signaling pathway.And the enrichment was mainly related to energy metabolism and DNA repair while Brandt's vole' mainly related to immune function.2)Mandarin vole regulates PI3K-Akt and AMPK signaling pathways through GAPDH,PRKAG3 and PDGFB;regulates the cardiovascular system through FOXP3,IL7R and CD7,responding to external stimuli through MDM2 and NUP85;improves glucose metabolism,redox,and mitochondrial function through GAPDH and PEX10 regulates several other pathways:hematopoietic cell lineage,melanoma,cell cycle,peroxisomes and pathways related to energy metabolism through several key genes MDM2,CDK2 and IL7R.3)Golden mole,star-nosed mole,blind mole rat,and Bathyergidae(damara mole rat and naked mole rat)did not share expanded genes and positive selection genes.However,the expanded genes of four subterrnean mammalian all have more immune functions than their close relatives,and also undergo positive selection evolution in terms of vision.For hypoxic adaptation,there are more expanded genes in golden mole,blind mole rat,and Bathyergidae,and golden mole,star-nosed mole,and blind mole rat all evolved adaptation to the subtermean hypoxic environment by improving energy metabolism and DNA repair.There are other species-specific ways that may be related to differences in soil environment,climate,and living habits:star-nosed moles regulate their respiratory system,blind mole rats regulate their oxidative stress response,Bathyergidae regulate the HIF-1? signaling pathway.The main conclusion:1)By de novo sequencing and assembly,2.24 Gbp,2.15 Gbp and 2.23 Gbp genomes for Plateau vole,Mandarin vole and Brandt's vole were obtained,of which 34.99%,33.93%and 36.82%for the repeated sequences,4 kinds of non-coding RNA,the number of coding genes is 20,738,21,229 and 20,530 respectively.2)The Tibet-Plateau uplift movement and the galaciation have different effects on the population history of the 3 voles.The Plateau vole is affected by both the Tibet-Plateau uplift movement and the galaciation,the Brandt's vole is affected more by galaciation and less by Tibet-Plateau uplift movement,the Mandarin vole was affected minimumally by galaciation and Tibet-Plateau uplift movement.3)Plateau vole and Mandarin vole have evolved specific adaptive mechanisms to their respective hypoxic environments.?In terms of regulating HIF-1 signaling pathway,Plateau vole mainly regulates MAPK signaling pathway and protein kinase B signaling through FGF7,which can indirectly promote the expression of HIF-1?;the expanded gene EGF in HIF-1 signaling pathway promotes angiogenesis.Mandarin vole directly regulates the expression of HIF-1? mainly through VLH,IL6R and IFNGR2,and CDKN1B in HIF-1 signaling pathway which can regulate cell proliferation,EPO can regulate oxygen transport capacity,and expanded gene GAPDH can increases anaerobic metabolism to reduces oxygen consumption.? In terms of other pathway regulation,Plateau vole mainly through RIPK1,CYCS and IL12B genes to regulate immune and neurological diseases,anti-cancer pathway,cytoplasmic DNA sensing pathway,nucleotide excision repair and tryptophan metabolism pathway.Mandarin vole mainly through MDM2,CDK2 and IL 7R genes to regulate hematopoietic cell lineage,melanoma,cell cycle,peroxisomes and pathways related to energy metabolism.?In other regulatory systems,Plateau vole mainly through CYCS and EGF to regulate the cardiovascular system,and Mandarin vole mainly through FOXP3,IL7R and CD 7;Plateau vole responds to the stimulation of outside cell mainly through SERPINE1 and EIF2 gene families,and Mandarin vole mainly through MDM2 and NUP85.Plateau vole mainly through CYCS,CDK1 and HSPA9 to improve aerobic respiration and glucose metabolism,Mandarin vole mainly through GAPDH and PEX10 to improve glucose metabolism,redox reaction and mitochondrial function.4)Plateau species evolved adaptation to the plateau hypoxia through the species-specific way.Subterrenean mammals evolved adaptation to the subterrenean hypoxia through the convergent categories of PSGs rather than the PSGs' structure themselves.In conclusion,Plateau vole and Mandarin vole could regulate HIF-1 signaling pathway indirectly and directly,thereby regulate cardiovascular system,stress response or energy metabolism to adapt to their specific hypoxic environment.This reflects the consistency of evolutionary adaptation to hypoxia among close relatives.