Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters

Abstract

Background: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture.

Results: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications.

Conclusion: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

Keywords: Colletotrichum higginsianum; Fungal genome; SMRT sequencing; accessory chromosomes; optical map; secondary metabolism genes; segmental duplication; subtelomeres; transposable elements.

Fig 1

Validation of the C. higginsianum genome assembly by alignment of unitig sequences (orange) against chromosome optical maps (blue). MluI restriction sites are represented in optical maps and unitigs by vertical bars. Chromosomes 7 and 9 show discrepancies between unitigs and optical maps. These optical maps are colour-coded to highlight the break-points

Fig. 2

Schematic representation of selected C. higginsianum secondary metabolism (SM) genes clusters. a Resolution of a former split SM cluster by the PacBio assembly. The new cluster 16 encompasses four contigs from the old assembly [13], two of which contain former clusters 18 and TRC3. Arrowheads: transposable elements b Comparison of cluster 19 and the depudecin cluster of Alternaria brassicicola. Protein identity is high (> 70%) and gene order and orientation are conserved except for the gene DEP6/CH63R_06317. c Comparison of cluster 46 and the fusicoccin cluster from Diaporthe amygdali [74]. In D. amygdali, genes are dispersed at two distinct loci in contrast to C. higginsianum. Protein identity is moderate to high and genes were extensively rearranged. Shading indicates syntenic blocks and genes pairs. Yellow: acetyl-transferase

Fig. 3

Schematic representation of the distribution of secondary metabolism gene clusters and transposable elements across the 12 C. higginsianum chromosomes. The 5' end of unitig_7 containing the ribosomal repeats is fragmented between 13 unitigs that are too small to align with the optical map. Putative locations of the centromeres are indicated where possible

Fig. 4

Waves of expression of secondary metabolism (SM) genes of C. higginsianum during infection of Arabidopsis thaliana. a Heatmap showing the expression profiles of SM key genes. Under-represented transcripts (dark green to bright green) and over-represented transcripts (dark red to bright red) are depicted as log2 relative expression index. The log2 expression levels are presented in the adjoining heatmap colour-coded from white (not expressed) to dark blue (strongly expressed). Red arrowhead: ChPKS38. b Schematic representation of the stage-specific expression of SM gene clusters. The expression of all genes within each cluster was evaluated using the Transcript Per Million (TPM) normalisation method. A cluster was considered expressed if more than 50% of genes had a TPM greater than 1% of the actin gene TPM, and |log2FC| ≥ 2, q-value ≤ 0.01. c Time-course of the expression of the pChPKS38::RFP reporter gene in planta and in vitro (cellophane) using confocal microscopy. All images are overlays of bright field and RFP channels captured with the same settings. RFP channels are projections of 15-25 0.2 μm optical sections. Co: conidium, arrowhead: appressorium, BH: biotrophic hypha, NH: necrotrophic hypha. Bars = 10 μm

Fig. 5

Schematic representation of the predicted domain structure of three families of conserved repeat elements present in the subtelomeric regions of all C. higginsianum chromosomes. DTX-chim_G199 was likely derived from DHX-G198 by the insertion of a DNA transposon, whereas DHX-chim-G203 was derived from DHX-G198 by the insertion of a non-LTR retrotransposon

Fig. 6

Violin plot depicting the frequency distribution of the distance (bp) between genes and the nearest transposable element (TE). The inner box plots represent the median and interquartile range of the distance for each of three gene classes. Genes located within secondary metabolism clusters (SM genes) and genes encoding candidate secreted effector proteins were located significantly closer (p < 0.001) to TEs than a random sample of genes taken from the genome as a whole

Fig. 7

Circos plot showing segmental duplications (SDs). Genes are represented in green and transposable-elements in red. Gene IDs in each duplicated block (grey sectors) are given without the prefix "CH63R_". An entire secondary metabolism gene cluster (shaded blue) is duplicated in SD2. cP450: Cytochrome P450; Eff: Effector protein; Sec: Secreted; TF: Transcription Factor; TS: Terpene Synthase