Study On Sequencing And Analysis Of The Genome And Developmental Transcriptome Of The Giant Panda Roundworm Baylisascaris Schroederi

Wildlife is our valuable natural resource,and wildlife conservation is our common responsibility.The giant panda(Ailuropoda melanoleuca),the national treasure of China and a flagship species for wildlife conservation,is one of the world's most recognized and endangered species on earth.Baylisascaris schroederi is the most common intestinal parasite found in the giant pandas and represents a significant threat to both wild and captive populations.Unfortunately,due to the scarcity of foundational researches on this parasite,until now knowledge related to its pathogenic mechanisms,growth and developmental regulations,parasitism adaption,genetics and evolution,host interplays,and immune invasion is still lacking.Herein,the aim of the present study was to uncover these scientific issues at the molecular level by sequencing and analyzing the genome and developmental transcriptome of B.schroederi.These results should not only provide new insights into strategies and theories of prevention and control of baylisascariasis in pandas but also enrich and improve knowledge of conservation biology of the giant panda.In this study a second generation sequencing platform was used to(i)achieve the assembly and annotation of the draft genome of B.schroederi;(ii)decode gene expression profiles of four key developmental stages of B.schroederi,including egg,egg-second stage larvae(L2),intestinal fifth stage larvae(L5),and female adults;(iii)analyze and compare composition and organization features between B.schroederi and Ascaris suum.Toxocara canis and other related nematodes on a genome-wide scale,and further probe the evolutionary status of B.schroederi by reconstructing a phylogenetic tree in Nematoda;(iv)further develope a parasitic stage-specific secretome of B.schroederi based on genomic and transcriptomic data through a well-trained bioinformatic prediction pipeline which was designed for secreted proteins that led to identification of numerous promising candidates for future vaccines and diagnostic antigens;and finally,(v)confirm the vaccine potential of a recombinant inorganic pyrophosphatase antigen of B.schroederi which was obtained from our omics-based secretome dataset using a mice immunization and challenge model for the first time.The main results are summarized as follows:(1)The B.schroederi draft genome was sequenced at 124-fold coverage,producing a final draft assembly of 249 Megabase(Mb),with the contigs N50 of 135 kilobase(kb)and scaffold N50 of 666kb.The longest scaffold is up to 3900kb.The entire genome contains 37.7%GC content and covers 95%core eukaryotic genes(CEG).(2)The B.schroederi genome contains-7.6 Mb repeat sequence,accounting for 3.1%of the entire genome in length,and the repeat sequences comprise tandem repeats,interspersed repeats,and un-annotated repeats according to sequence characteristics.Moreover,this genome also encodes 18,360 protein-coding genes(PCGs),552 tRNAs,155 microRNAs,and other small RNAs,such as small nuclear RNA(snRNA),RNA component of signal-recognition particle(SRP),and small nucleolar RNAs(snoRNA).Analysis of gene structures shows that the length distributions of exons and introns of B.schroederi PCGs have high similarities to counterparts of A.suum and T.canis,respectively.Further annotations by gene structure domain,Gene ontology(GO),and KEGG pathway reveals that B.schroederi PCGs mainly encode kinases,phosphatases,receptors,transporters,and ion channels,similar to those found in both A.suum and T.canes.(3)The gene expression profiles of egg,egg-L2,intestine-L5,and female adult stages of B.schroederi indicate that among a 18,360 gene set inferred from the genome,13,755 genes are expressed at one of these four stages,accounting for 74.92%of entire gene set;11,186(81.32%)of these genes are expressed across these four stages and 2,569(18.68%)genes belong to stage-specific expression.Further functional annotation among highly expressed genes shows that the genes involved in biological processes including cell division,extra-and intra-cellular acid-base regulation,detoxification,and antimicrobial immune predominate in the egg stage;genes involved in biological processes including expression regulation,synthesis and transportation of ribosomal proteins,dynein synthesis,the cellular signal sensing and transduction,and energy storage predominate in the egg-L2 stage;genes involved in biological processes including synthesis of collagen and cuticle proteins which relates to nematode molting predominate in the intestine-L5 stage;and genes involved in biological processes including reproduction and antibacterial immune responses predominate in the adult stage.(4)Analysis of comparative genomics finds that B.schroederi shares high genomic gene presented here confirm that bioinformatics-based secretomic screening data and secretome-originated vaccine and immune diagnostic antigen molecules can be identified and applied to our field practices.