Intraspecific Comparative Analysis Reveals Genomic Variation of Didymella arachidicola and Pathogenicity Factors Potentially Related to Lesion Phenotype

Abstract

Didymella arachidicola is one of the most important fungal pathogens, causing foliar disease and leading to severe yield losses of peanuts (Arachis hypogaea L.) in China. Two main lesion phenotypes of peanut web blotch have been identified as reticulation type (R type) and blotch type (B type). As no satisfactory reference genome is available, the genomic variations and pathogenicity factors of D. arachidicola remain to be revealed. In the present study, we collected 41 D. arachidicola isolates from 26 geographic locations across China (33 for R type and 8 for B type). The chromosome-scale genome of the most virulent isolate (YY187) was assembled as a reference using PacBio and Hi-C technologies. In addition, we re-sequenced 40 isolates from different sampling sites. Genome-wide alignments showed high similarity among the genomic sequences from the 40 isolates, with an average mapping rate of 97.38%. An average of 3242 SNPs and 315 InDels were identified in the genomic variation analysis, which revealed an intraspecific polymorphism in D. arachidicola. The comparative analysis of the most and least virulent isolates generated an integrated gene set containing 512 differential genes. Moreover, 225 genes individually or simultaneously harbored hits in CAZy-base, PHI-base, DFVF, etc. Compared with the R type reference, the differential gene sets from all B type isolates identified 13 shared genes potentially related to lesion phenotype. Our results reveal the intraspecific genomic variation of D. arachidicola isolates and pathogenicity factors potentially related to different lesion phenotypes. This work sets a genomic foundation for understanding the mechanisms behind genomic diversity driving different pathogenic phenotypes of D. arachidicola.

Keywords: Didymella arachidicola; comparative analysis; genomic variation; intraspecific; lesion phenotype; pathogenicity factors.

Figure 1

Pathogenicity assays and lesion phenotype of 41 D. arachidicola isolates. (A) The disease index of peanut seedlings infected by 41 D. arachidicola isolates at 14 dpi; CON denotes non-infected peanut seedlings. Letters on the bars denote significant differences in disease index at 0.05 level; error bars denote the standard error of the mean. (B) The average disease index of peanut seedlings infected by isolates of different lesion phenotypes at 14 dpi. “**” denotes significant differences at 0.05 level; error bars denote the standard error of the mean. (C) Adaxial and abaxial leaf surfaces of different lesion phenotypes showed at the top and bottom, respectively.

Figure 2

The genomic circos map of D. arachidicola isolate YY187. Circles from inside to outside are (a) chromosome information of the D. arachidicola genome (scale marks in Mb). (b) Gene density on different chromosomes, color-coded from blue to yellow, represents values from low to high. (c) GC content, color-coded from yellow to green, represents values from low to high. Visualization was performed using advanced circos in TBtools v1.098769.

Figure 3

Genome-wide distribution and types of variations (identified by comparison with the reference genome of isolate YY187) within all D. arachidicola isolates. (A) Genome-wide percentage distribution of SNPs and InDels within different genomic regions, respectively. Each bar indicates the average percentage of variations in a certain genomic region among all the isolates, with the error bars showing the standard error of the mean; (B) the proportion of different variation types in the CDS region of D. arachidicola isolates.

Figure 4

Phylogenetic analysis of D. arachidicola population with high-quality SNPs. The SNPs of 40 isolates were called by GATK based on the fact that the genome of isolate YY187 was selected as the reference, which caused the absence of isolate YY187 on the phylogenetic tree. The maximum likelihood (ML) tree was constructed using MEGA X with 1000 bootstrap replicates. The tree was visualized by modification in FigTree v1.4.4. Bootstrap values are shown above the branches. The sub-populations of D. arachidicola were classified and displayed by different colors. The isolates of B type are designated in blue font color.

Figure 5

Chromosomal distribution of 13 differential genes. The green lines represent gene distribution on different chromosomes. G1–G13 refer to the 13 genes and mark the location of genes on different chromosomes using a blue line. TBtools v1.098769 visualized the figure.