Analysis Of Whole Genome Sequencing And Comparative Genomic Analysis,and Establishment Of Protoplast Preparation System Of Alternaria Panax Strain JY15 From Panax Ginseng

Alternaria leaf and stem blight caused by Alternaria panax is one of the important diseases in ginseng production.There is a considerable amount of literatures on the species of A.panax,the occurrence patterns of diseases,comprehensive control measures,and the fungicide resistance of pathogens.However,there has been relatively few research on the pathogenic mechanism of A.panax.Genome sequencing and comparative genomic analysis are the basis for research of the pathogenic mechanism.Therefore,in this study,the A.panax JY15 strain isolated from ginseng was subjected to whole-genome sequencing using the MGIseq2000platform for second-generation sequencing and the Oxford Nanopore Prometh ION sequencer for third-generation sequencing.Gene structure,genome structure,gene function prediction and annotation,as well as comparative genomics analysis were carried out using relevant databases.The aim was to comprehensively understanding A.panax JY15 from aspects such as gene content,gen function,secondary metabolites,unique genes,and unique gene families,in order to lay a foundation for the study of the pathogenic mechanism of the pathogen.The main results obtained are as follows:1.Through whole genome sequencing and bioinformatics analysis,the genome size of A.panax JY15 was determined to be 35,017,728bp,with 12 scaffolds assembled.The length of N50 and N90 was 3,578,011bp and 2,071,204bp,respectively,and the GC content was 51.14%.A total of 11,216 genes were predicted,and among them,NR,GO,COG,Pfam,KEGG,CAZy,PHI-1,PHI-2,P450,DFVF,signal peptides,and secretory proteins were annotated in 11,022,6,038,832,8,528,3,822,1,701,3,832,2,880,1,100,1,900,1,055,2,070,and 1,015 genes,respectively.2.Through comparative genomics analysis of the full genome data of A.panax JY15,A.panax BNCC115425,A.tenuissima FERA1166,A.alternata SRC1lr K2f,A.arborescens FERA 675,A.burnsii CBS107.38,A.rosae MPI-PUGE-AT-0040,Alternaria sp.MG1,A.atra CS162,and A.gaisen FERA 650,a deep analysis was conducted on the genomic characteristics and evolutionary status of A.panax JY15.The analysis showed that A.panax JY15 has the closest genetic relationship with A.panax BNCC115425(isolated from American ginseng),confirming that the sequencing data belongs to A.panax fungus.In order to explore the relationship between the whole genomes of A.panax JY15 and other species of Alternaria,a comparative genomic analysis was conducted with A.atra CS162,which has a closer genetic relationship with A.panax JY15.The analysis revealed that the gene expression level of A.panax JY15 in the t1pks-related gene cluster was the highest,which included 106 genes.A.panax JY15 has 3,171 unique expressed genes and2,601 gene families,among which there are 1,542 unique pathogenic genes and 937pathogenic gene families.3.The expression patterns of 24 putative pathogenic genes,obtained from the whole genome and comparative genomics analysis of A.panax JY15,were investigated at different time points using quantitative real-time PCR,suggesting that22 of these genes are pathogenic genes associated with toxin production.Among these genes,g5089 and g5057 were found to be significantly upregulated and were selected for knockout using a yeast transformation method based on the whole genome information,providing a basis for future gene knockout experiments.4.Establishment and optimization of a preparation system for A.panax JY15were determined in this study.The results showed A.panax was incubated for three days at 150 r/min,25℃in an oscillating incubator to obtain of A.panax mycelium,with the concentration ratios of collapsase,snailase and cytolytic enzyme of 1%,0.1%and 1%,respectively,and 5g/m L of A.panax mycelium was digested at 25℃for 2.5 h at 90 rpm,0.7 mol NaCl as the osmolarity stabilizer,protoplasts of 2.96±0.08×10~7CFU/m L could be obtained.After coating the protoplasts on the regeneration medium,the colony morphology,growth rate and pathogenicity of the regenerated strains were not significantly different from those of the wild type.The quality of the protoplasts prepared under these conditions was able to meet the requirements for genetic transformation.