| Plant bacterial soft rot is a kind of disease with a high degree of harm and no effective biological control.Pectobacterium carotovorum,one of its main pathogens,has attracted much attention because of its wide distribution and diversity.In this study,Pectobacterium carotovorum subsp.carotovorum PC1 was used as the research object.Firstly,third-generation sequencing technology of SMRT was used to sequence its whole genome,and the sequencing data obtained by Pac Bio RS II sequencer was used to assemble a high-quality and unspaced PC1 whole genome.The total length of the sequence is 5 303 871 bp,the GC content is 51.7%,including a complete closed-loop chromosome.The results of the second-generation sequencing of PC1 on NCBI were used as the reference sequence(NC＿012917.1)to adjust and determine the starting site of the PC1 strain sequence and annotate the reference genome.The results of methylation analysis of offline data showed that there were three kinds of m6 A methylation modification in PC1.Secondly,the conventional genome annotation process was used to annotate the PC1 genome and draw the genome circle map.In the genomic composition analysis,5 060 CDS,78 t RNA,22 r RNA and 48 tandem repeats were predicted.Basic genome alignment and functional annotation were conducted with six major databases(NR,Swiss-Prot,Pfam,Egg NOG,GO and KEGG).Mobile element analysis predicted 17 genomic islands,3pre-bacteriophages and 9 CRISPR-Cas regions.In the metabolic system analysis,144 genes related to carbohydrate active enzymes and 5 groups of secondary metabolite synthesis gene clusters were obtained.Pathogenic system analysis predicted 575 virulence factor genes,304 drug resistance genes,774 pathogen genes,7 secretory systems,422 secretory proteins,1 103 transporters,1 109 transmembrane proteins and 22 pairs of two-component regulatory system information.In addition,there were 6 genomic islands,1 prophage,7 carbohydrate enzyme related genes,18 virulence genes,5 drug resistance genes,15 pathogen-host interaction related genes,2 different types of secretory system genes and 1 pair of two-component regulatory system among the 3 gap coding genes during the comparison with the reference genome.Finally,six strains of related bacteria were selected from subspecies and species level,and the comparative genomes of PC1 were analyzed from various aspects.Results of average nucleotide consistency(ANI)analysis showed that the ANI value between PC1 and BP201601.1 of the same subspecies was the highest and the genetic relationship was the closest.The evolutionary classification tree was drawn based on the results of ANI value,which showed that four strains of Pectobacterium carotovorum were classified into the same cluster.Results of collinear alignment showed that the collinearity of PC1 genome was better than that of other subspecies at the subspecies level.The collinearity difference between BP201601.1 and other subspecies was the smallest,and the overall genome arrangement was the most similar.Cluster analysis of gene families were conducted with two levels,and visual views were drawn.Among them,28 gene families were unique to PC1 at two levels.According to the analysis of key pathogenic genes,three kinds of genes related to cell wall degrading enzymes were obtained,including 19 genes encoding Pectinase function,25 genes encoding cellulase synthesis and 22 genes encoding protease synthesis.Cell wall degrading enzyme related genes are highly conserved in Pectobacterium.In this paper,a variety of bioinformatics analysis methods were used to annotate the whole genome of PC1 genome.Among them,the important functional genes not involved in the second-generation annotation,the specific genes and key pathogenic genes obtained by comparative genome analysis can be used as the key research objects of follow-up experiment analysis.This study not only provides a reference for sequence splicing and genome annotation for Pectobacterium carotovorum researchers,but also provides a theoretical basis for the development and research of specific biological fungicides for soft rot. |
