| Frogeye leaf spot,caused by Cercospora sojina(C.sojina)has expanded seriously threatening worldwide soybean production.It has strong infectivity and physiological race differentiation,and primarily occurs on foliage of soybean plants,even though all the aboveground tissue can also become infected.Heilongjiang is a typical soybean production region in the northeast of China with a suitable climate for FLS developing.In recent years,a new highly virulent strain Race15 has emerged,which has led to a loss of resistance in many cultivars in this region.At present,the causes of differences in the virulence of different strains are unknown,and there are few reports on differential virulence genes and intraspecific virulence differentiation of C.sojina.This study is the first to use phenotype-genotype association to prioritize candidate effectors at the genomewide scale,through the careful matching of virulence profiles from nationwide strain surveys of C.sojina the northeast of China.We sequenced the Race15 genome and compared it to the Race1 genome sequence,as its virulence is significantly lower.We then re-sequenced 30 isolates of C.sojina from different regions to identifying differential virulence genes using genome-wide association analysis(GWAS).Finally,virulence-related differentially expressed genes(DEGs)were obtained from the two different virulence strains under nitrogen starvation stress,that could mi mic the physiology of pathogen during infection,then weighted gene co-expression network analysis(WGCNA)was used to find co-expressed gene modules and the relationshi p between gene networks and phenotypes.This study has provided insights into the gene identification,functional characterization,genetic diversity analysis and resistance breeding.The main results of this study are as follows:1.In total,601,794 high quality reads were generated by Pac Bio sequencing,covering 6,038,283,778 bp in total,and having a mean length of 10,033 bp and an N50 length of 13,900 bp.The genome of the Race15 strain of C.sojina(40.12 Mb)consisted of 12 curated contigs,with an N50 length of 4.9 Mb.12,607 coding genes were predicted,at a gene density of approximately 314 genes per Mb.Additionally,non-coding genes were predicted,with 200 t RNA,2 s RNA and 13 sn RNA genes being predicted in the genome of Race15.A total of 680 genes were annotated and classified using the PHI database,340 genes annotated in the CAZy database,777 genes were predicted to be related to Secondary Metabolism and 766 genes were predicted as Secretory Proteins.These virulence-related pathogenic genes are mainly involved in cell wall degrading enzymes,fungal morphology,toxins and pigment biosynthesis.This study found that C.sojina contained a large number of polyketide synthases(PKS)and non-ribosomal peptide synthase(NRPS)genes involved in toxin biosynthesis,and its expression was significantly up-regulated during infection.C.sojina can produce cercospora toxins,at the same time,it was also found that carbohydrate-binding module(CBM1)gene expression was absent during the infection.It is one of the reasons for the slow infection process of C.sojina.2.While the Race15 and Race1 genomes largely corresponded,there were various inversion,translocation,and Tran+Inver(translocation and inversion)ev ents that disrupted the otherwise collinear gene order.A comparison of these two strains showed high coverage and synteny.Synteny analysis revealed that although the two genomes contained most of the honologous genes,in the process of evolution they res pectively experienced a significant volume of distinct genome structure variation.This resulted in some changes in genomic structures,leading to changes in coding genes,and even changes in functional proteins,especially in non-linear areas.There were also many paralogous genes,and these paralogous genes encoded similar functions.Among these clusters,417 clusters contained more than 3 genes.The largest cluster contained 210 genes.Race15 had 245 specific genes,in which 41 genes annotations in virulence-related databases.In particular,the specific gene Vtc4,which has been shown to be associated with high virulence,as this gene plays an important role for phosphate uptake,it influences storage of polyphosphates,morphology of the strain,biosynthesis of melanin and the virulence of the strain.The other specific genes of Race15 may be related to high virulence too.3.Most isolates were collected from sites in the main soybean producing area in Northeast China,the region in China were FLS disease is the most serious.All of these isolates have close ancestral evolutionary relationships,consistent with the geographical location of the samples collected.Race15 had the highest virulence and was most closely related to strains SB,JS and JY.The virulence values of these 3 isolates were also relativel y high(second,third,and fourth,respectively),while the proxi mal isolates of these 4 isolates were not highly virulent.4.The key candidate genes were identified by the intersection of three kinds of genes.Of the five identified key virulence related genes,four were in contig 3 and one was in contig 1,which was consistent with the virulence related areas detected in the Manhattan map.Gene A05389 is annotated as α-L-rhamnoside,which is involved in overcoming saponin-mediated plant defense systems.Gene A00784 annotation is cellobiose dehydrogenase,which has been shown to be involved in the degradation of host cellulose.The other genes were annotated as NRPS,which have been shown to be involved in the biosynthesis of mycotoxins and melanin.These genes play a key role in infecting the host.5.Comparative transcriptomics studies revealed differences between the two strains before nitrogen starvation treatment.Through annotation of DEGs,a total of 54 virulence-related DEGs were identified.Among them,the DEGS scytalone dehydratase and 1,3,8-trihydroxynaphthalene reductase were annotated as increase virulence.They are all related to the biosynthesis of melanin.After nitrogen starvation treatment of strains Race15 and Race1,a total of 31 virulence-related DEGs were identified,among which the up-regulated gene Vtc4 was not only annotated as increase virulence,but also a specia l gene of Race15 and annotated in the green module related to virulence.6.Green and Red module genes were highly expressed in all three samples of Race15 strain under nitrogen starvation stress for 24 h.These two module genes should be related to the function and development of Race15.There were 36 virulence-related DEGs that were identified in 2 highly virulence modules.The important nitrogen starvation-responsive DEGs were involved in the synthesis of melanin,poly P storage in the vacuole,lignocellu lose degradation and cellulose degradation during fungal development and differentiation.A large number of NRPS genes were enriched in the red and green modules associated with virulence and in the Race15-LN vs.Race1-LN group,but no PKS was found.It is speculated that NRPS is also one of the reasons for the difference in virulence.7.Three genes were randomly selected for verification of expression levels.The average relative expression of each group obtained by q PCR is consistent with the average FPKM value of each group obtained by sequencing.The two groups have a strong positive correlation(r> 0.95),which proves that the high-throughput sequencing data is reliable. |
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