| The development and maturity of genome sequencing technology had a profound impact on the field of life science research,which turned distributed genetic markers into in-depth understanding and mining the information of genome sequence.For many years,our country have been using too many chemical fertilizers and pesticides on the agriculture production,which significantly affect the sustainable agricultural development.One of the most important measures to resolve the problem is applying rhizosphere bacteria to the field efficiently.Currently,Bacillus spp.and Trichoderma spp.have become the research highlight as they play very important role in biocontrol and plant growth promoting.Our laboratory isolated and sequenced three Bacillus strains from three different rhizosphere separately,which were Bacillus amyloliquefaciens SQR9,NJN-6 and Bacillus subtilis HJ5,which were all beneficial to plant growth.We annotated the genomes of the three Bacillus strains and tried to understand their genetic background which related to biocontrol function.Besides the three genomes,we collected 28 bacillus amyloliquefaciens&bacillus subtilis genomes from NCBI.We divided the 31 strains into two groups according to their isolation,one group was Plant-Associated(PA,isolated from rhizosphere or plant),the other group was non-Plant-Associated(nPA,industrial fermentation strain or type strain).We compared the differences between the two groups to investigate the evolving trends of genome during the evolution using comparative genomic methods.In another aspect,we set up an open-source genome annotation pipeline for Trichoderma genomes.We isolated another fungi Trichoderma guizhouense NJAU 4742,which was widely applied in agricultural production.We annotated the genome of NJAU 4742 and compared it with other Trichoderma spp.The main results obtained are listed as follows.1.To understand deeply the genetic background related to plant beneficial function of Bacillus amyloliquefaciens SQR9,NJN-6 and Bacillus subtilis HJ5,we finished the sequencing of the genomes of three species and annotation.They all had single circular chromosome without plasmid.The genome size of SQR9,NJN-6 and H5 was 4.12 Mb,4.05 Mb and 4.01 Mb,respectively,while the number of protein-coding genes of three strain was 4,078,3,894 and 3,917.As a model PGPR in our laboratory for unravelling rhizosphere ecological behavior and plant-microbe interactions,we investigated the genome of SQR9 deeply,the main results were as follows.Compared with genome of Bacillus amyloliquefaciens FZB42,DSM7,YAU B9601-Y2 and Bacillus subtilis 168,there were 3,014 orthologous genes,while the pan-genome contained 5,643 orthologous genes,besides SQR9 contained 309 specific genes.We found 11 genomic islands inside the genome of SQR9 using Island Finder and SeqWord,while the third island related to synthesis of polyketide was specific to other Bacillus strains.There were 7.9%of genome related to synthesis of antibiotics.Compared with FZB42,we systemized the gene and gene cluster related to the degradation of plant polysaccharides,cell motility and chemotaxis,NRPS/PKS and plant growth promotion.At last,all of the annotation was integrated into the circular map and pathway of SQR9 genome and a server of genome browser was set up for remote access.2.In order to understand the adaptive evolutionary process of the genome of Bacillus amyloliquefaciens and subtilis spp.,which showed the ability of plant growth-promotion,we collected 16 genomes of Bacillus amyloliquefaciens spp.and 12 genomes of Bacillus subtilis.Together with SQR9,NJN-6 and HJ5,we used the comparative genomics,phylogenomics and dynamic evolution to analyze the 31 genomes.The main results are as follows.(1)The phylogeny and hierarchal clustering based on the 31 core genomes together indicated that strains isolated from the rhizosphere are clearly separated from nPA strains in both B.amyloliquefaciens and B.subtilis.This finding suggested that important changes in the core genome of these Bacillus strains had occurred during adaptation to different habitats;more precisely a co-evolution with plants showed the same evolving trends.(2)According to the results of species tree,we divided the 31 strains into Plant-Associated and non-Plant-Associated.The comparison of the core genome between the two groups,based on the COGs analysis,revealed that the former were more enriched in genes involved in intermediary metabolism and biosynthesis of secondary metabolites.Enrichment of the above mentioned genes could have the advantageous results of increasing the capacity to utilize various plant-derived substrates and synthesizing antibiotics,which would contributed to competitive edge and rhizosphere adaptation.In addition,a further PA-specific gene analysis also indicated that these two groups of genes are probably the most important characteristics that contributed to rhizosphere adaption of PA strains and their differences from nPA stains.(3)We performed a dynamic evolutionary AnGST analysis to explore the gene gain/lost events that occurred at the divergence of these two subspecies of Bacillus spp.It was found that the major horizontal genes acquired by both BA-PA and BS-PA were involved in metabolism,transcription/signal transduction,and synthesis of secondary metabolites,which were all potentially important for rhizosphere survival and competition.(4)In summary,the results illustrated that the ability of adaptation in plant rhizosphere for Bacillus strains were obtained by multiple horizontal gene transfer events.It should be emphasized that rhizosphere competence cannot be solely attributed to the action of one or several genes,but to the overall system and interactions of genes involved in rhizosphere adaptation.Our study also showed that rhizosphere competence was not obtained by a single event but is the result of several subsequent evolutionary process.3.Due to the highly plastic and diverse genomes of fungal species,the accuracy of genome structure annotation was not very high.To get better accuracy and reliability of Trichoderma genome annotation,we optimized the annotation methods and designed a pipeline for annotating Trichoderma genome "trichoCODE".The whole f The work flow of trichoCODE is described as bellow.Stage 1,training preparation.To integrate all the possible evidences(like homology protein sequences,ab initio predicted genes and transcripts)into high quality gene training set.Stage 2,training and prediction.Using the gene training set to train Augustus and SNAP and use them to make gene prediction.Stage3,combination.Using EVM tool to combine all the predicted gene sets and evidences into consensus gene models.Stage 4,make-up.Update the predicted gene models with UTRs and alternative spliced isoforms.Finally,comparing with other annotation software to the published Trichoderma genomes,the results showed that trichoCODE gave the reliable predicted gene models.4.Trichoderma guizhouense NJAU 4742 is a very important strain used for suppressing multiple plant pathogenic fungi.We sequenced the genome of NJAU 4742,and used trichoCODE to finish the annotation.The results of genomes analysis showed,the genome size was 38.9 Mb and contained 11,297 protein-coding genes.The phylogeny analysis showed NJAU 4742 was very close to Trichoderma virens,which all had mycoparasitic activity.Compare with other Trichoderma genomes,we found the in NJAU4742 there were abundant aspartic acid proteases,cysteine protease,metallo proteinases and serine protease,etc.Additionally,as the same as T.virens,NJAU 4742 contained abundant secondary metabolites.Interestingly,the number and types of PKS genes in NJAU 4742 was more than the other strains.These data should be important molecular support for transforming NJAU 4742 into new type of biocontrol agent. |
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