Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains

Bradshaw, Rosie E.; Sim, Andre D.; Chettri, Pranav; Dupont, Pierre‐Yves; Guo, Yanan; Hunziker, Lukas; McDougal, Rebecca L.; Van der Nest, Ariska; Fourie, Arista; Wheeler, David; Cox, Murray P.; Barnes, Irene

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Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains

Bradshaw, Rosie E.; Sim, Andre D.; Chettri, Pranav; Dupont, Pierre‐Yves; Guo, Yanan; Hunziker, Lukas; McDougal, Rebecca L.; Van der Nest, Ariska; Fourie, Arista; Wheeler, David; Cox, Murray P.; Barnes, Irene

URI: http://hdl.handle.net/2263/75669

Date: 2019-06

Abstract:

Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.

Description:

Supplementary Material: Fig. S1 Predicted duplications and deletions in chromosomes 1‐14 for 18 strains of D. septosporum.

Fig. S2 Initial evidence for a reciprocal chromosome translocation in the NZE10 genome. (A) Assembled contigs from the SLV genome were aligned with NZE10 reference chromosomes (scaffolds). Two contigs (circled) mapped to both chromosomes 5 and 13 of the NZE10 reference genome. This was found in many of the other genome sequences. (B) Visualisation of reads from the ALP3 genome mapped onto a region of chromosome 13 show a gap, in which mate pairs are mapped to chromosome 5.

Fig. S3 A reciprocal translocation involving chromosomes 5 and 13 in the NZE10 genome. (A) The reciprocal translation was centred on an identical sequence (GCGCGGT) found at positions 1459800‐1459806 in NZE10 chromosome 5 and 717926‐717932 in chromosome 13. Chromosomes 5 and 13 are shaded grey and pale blue respectively with ends coloured to distinguish the two arms in each case. Coloured sequences surrounding the breakpoint indicate which arm they are from. (B) In strains from regions other than Australasia, the two long sections of NZE10 chromosomes 5 and 13 are joined to make a 2.2 Mb chromosome and two short sections to make a 1.4 Mb chromosome. Sequences around the common 7 bp sequence are shown for strain ALP3 as an example. (C, D) Pairs of divergently transcribed genes straddle the breakpoints on NZE10 chromosomes 5 (C) and 13 (D). A GC content of about 70% was seen at the breakpoint regions (50 bp sliding window) as shown by the %GC (blue) profiles.

Fig. S4 Alignment of pathway regulator AflR from 19 D. septosporum strains. Amino acid changes compared to strain NZE10 are highlighted in blue (these sites are also variant between AflR sequences of D. septosporum , Cladosporium fulvum , Aspergillus parasiticus and Aspergillus nidulans ; (Chettri et al ., 2013)) or in green (at sites conserved between those four species). The Zn2Cys6 zinc binuclear domain is highlighted in pink; the linker sequence thought to determine DNA binding specificity in grey; the acidic glutamine rich motif in yellow and C terminal arginine residues implicated in AflJ binding in red.

Fig. S5 Secondary structure predictions for AflR from D. septosporum NZE10 and ALP3. Pairwise alignment predicted by HHpred. The arrow indicates the location of the N349K polymorphism in ALP3.

Table S1 Transposable elements in the Dothistroma septosporum genomes.

Table S2 Genes deleted in the 18 genomes compared to Dothistroma septosporum NZE10.

Table S3 Genes deleted from chromosome 14 and their expression levels in NZE10.

Table S4 Single Nucleotide Polymorphisms (SNPs) in dothistromin genes, grouped by dothistromin gene loci.

Table S5 Deleted genes on Dothistroma septosporum chromosome 12.

Table S6 Gene duplications predicted by CNV (copy number variant) analysis.

Table S7 (a) Polymerase Chain Reaction (PCR) primers used for verification of 5:13 translocation (b) Primers used for copy number variant (CNV) verification (quantitative PCR [qPCR]).

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