Figure 1.
Global survey of diversity in accessory chromosomes of Zymoseptoria tritici.
A) Presence or absence of chromosomal segments assayed by PCR in a global collection of four field populations located in Australia, Israel, United States and Switzerland (total n = 98). Green bars represent the number of chromosomal segments found within the populations. Core chromosomes 10 and 13 were included for comparison with the accessory chromosomes 14–21. B) Population differentiation based on the presence of chromosomal segments calculated by Wright's FST. C) The physical location of each gene used for the PCR assays is shown on schematics drawn for each accessory chromosome. The variation of GC-content along the chromosomes is shown in red. D) Content of short direct repeats assessed in 20 kb segments. E) Location of coding regions according to the reference genome [46]. The location of probes used for Southern hybridizations to CHEF gels are indicated in red. F) Gene ontology terms for genes comprised in the PCR assay. Only genes on accessory chromosomes 14 and 16 were described by gene ontology terms [46].
Figure 2.
Electrophoretic karyotype diversity in accessory chromosomes among eight resequenced Swiss Zymoseptoria tritici isolates compared to the reference isolate IPO323.
Chromosomal bands were separated by CHEF gel electrophoresis. The size marker is Saccharomyces cerevisiae chromosomes (Sc).
Table 1.
Illumina resequencing and assembly statistics of Zymoseptoria tritici isolates from the Swiss population.
Figure 3.
Genome resequencing of nine Zymoseptoria tritici isolates from a Swiss population.
Illumina sequencing reads were mapped to annotated coding regions of the reference genome IPO323. The height of the bars represents the number of coding regions found in 20 kb segments in the reference genome. Horizontal rows show the nine different resequenced isolates. For each resequenced isolate black segments of the bars indicate the sum of present coding sequences per 20 kb segments. White segments of the bars indicate absent coding sequences.
Figure 4.
Comparisons of synteny among different variants of accessory chromosome 14 in related species.
Scaffolds from de novo assemblies of the Z. tritici isolates 1E4 (n = 17), 9G4C (n = 16), 3D1 (n = 11) and A26b (n = 7) aligning to chromosomes 14 of the reference isolate IPO323 (horizontal axis) are shown. Scaffolds from isolates of related species Z. pseudotritici (5.9.1; n = 9) and Z. passerinii (P63; n = 4) aligning to chromosomes 14 of the reference isolate IPO323 are shown below. Orange bars show conserved deletions shared among different isolates and species in comparison to the reference chromosome 14. The grey bar indicates a putative deletion in Z. passerinii not spanned by a scaffold. Scaffolds are differentiated by color.
Figure 5.
A) Pulsed-field gel electrophoresis of accessory chromosomes found in the Zymoseptoria tritici isolates IPO323 (reference isolate), 3D1, 9G4C, 26b and the Z. passerinii isolate P63.
The size marker (Sc) represents chromosomes of Saccharomyces cerevisiae. Asterisks represent chromosome 14 variants identified by Southern hybridization with a chromosome specific probe. B) Southern hybridization with a chromosome 14 specific probe. C) Chromosomal dotplot of chromosome 14 in the reference genome of IPO323. Orange blocks indicate the identified deletions compared to other isolates of Z. tritici revealing a palindrome between 500 and 550 kb. D) Multiple sequence alignments of genomic scaffolds spanning breakpoints A–D are shown for Z. tritici isolates IPO323, A26b, 3D1 and 9G4C. In addition, we aligned the genomic scaffold found in the isolate P63 of Z. passerinii. Exact locations of the breakpoints A–D are shown in parentheses and refer to chromosome 14 of the reference genome IPO323.
Table 2.
Primer sequences used for the Southern hybridization probes specific for chromosome 14 and 17, respectively.
Figure 6.
Genomic characterization of chromosome 14 in the Zymoseptoria tritici reference isolate IPO323.
Orange shades indicate the alignment breakpoints identified among isolates of Z. tritici. A) Repeat density (for repeats up to a period size of 50 bp). B) GC content is shown in sliding windows with a window length of 5 kb. C) Numbers of genes are reported for each 5 kb window.
Figure 7.
Remnants of transposable elements and predicted gene functions on chromosome 14 insertions.
A) Identification of remnant long terminal repeat (LTR) and non-LTR transposable elements. Orange regions on chromosome 14 show the three inserted sequences flanked by alignment breakpoints A–F (as on Figure 4 and 5). The palindrome (shaded in brown; see Figure 5C) identified on chromosome 14 is flanked by two outwards facing Copia-1 type LTRs. Similarly, the inserted sequence at 700 kb (E–F) is flanked by two outwards facing non-LTR transposable elements. Related non-LTR transposable elements were also found near the flanking regions (A and D) of the large central insertion and the shorter insertion at 230 kb. B) Enlarged view of the large inserted sequence (between C and D) in chromosome 14. Black segments show the location of annotated genes. Genes with a functional prediction by Gene Ontology terms are indicated according to Goodwin et al. [46].
Figure 8.
Variability in accessory chromosomes among progeny of three crosses between Zymoseptoria tritici isolates.
A) Progeny of a cross between isolates 3D7 and 1A5 (Cross 3). Chromosomal segments of core chromosomes 10 and 13 and accessory chromosomes 14–21 were assayed by PCR. The two top rows indicate the two parental genotypes. The green bars show the number of individual chromosomal segments among the 48 progeny. B), D) and F) Test for random segregation of chromosomal segments that are present in only one of the two parental isolates. The −log10 transformed p values were corrected for non-independence and the horizontal bar represents the Bonferroni-corrected significance threshold (p<0.0007). C) Chromosomal segment numbers among 48 progeny from a cross between isolates 1E4 and 1A5 (Cross 2). E) Chromosomal segment numbers among 48 progeny from a cross between isolates 9B8B and 9G4C (Cross 1).
Figure 9.
Pulsed-field gel electrophoresis of accessory chromosomes in the parental isolates 1E4 and 1A5, as well as their progeny A2.2 and A66.2.
The isolate IPO323 was added as a reference. Both progeny showed a new chromosomal band at 0.9 Mb that is absent in either parental isolate and other screened progeny. Sc represents chromosomes of Saccharomyces cerevisiae added as a size marker. A) Pulsed-field gel electrophoresis of medium-sized chromosomes (up to approx. 3 Mb) of parental isolates 1E4 and 1A5 and 7 progeny. Progeny A2.2 and A66.2 showed a new chromosomal band at 0.9 Mb indicated by an asterisk. B) Pulsed-field gel electrophoresis of accessory chromosomes of parental isolates 1E4 and 1A5 and progeny A2.2 and A66.2. Asterisks identify chromosome 17 variants identified by hybridization. C) Southern hybridization of a chromosome 17 specific probe on chromosomes separated by pulsed-field gel electrophoresis of parental isolates, progeny and the reference isolate IPO323 as in B).
Figure 10.
Initiation of a breakage-fusion-bridge cycle of Zymoseptoria tritici chromosome 17 during meiosis.
A) Genome resequencing of two progeny (A2.2 and A66.2) from a cross between the parental isolates 1E4 and 1A5. Parent 1A5 carries accessory chromosome 17 (parent 1E4 is missing chromosome 17). Illumina sequencing reads were mapped to known coding regions of chromosome 17 on the reference genome IPO323. Black and white segments of the bars represent presence and absence, respectively, of particular coding sequences in 20 kb sections along chromosomes 17. B) Variation in read density along chromosome 17 of parental isolate 1A5 and progeny A2.2 and A66.2. C) Variation in read density among accessory chromosomes in the offspring A2.2 and A66.2. Illumina sequencing reads were mapped to coding sequences of the reference genome IPO323. Read density is reported as fold-difference between the offspring and the parental isolate 1A5. As a reference, the mean fold-difference on core chromosomes is reported as a horizontal line. Both offspring showed a near two-fold higher read density on chromosome 17 compared to other accessory chromosomes. D) Illumina sequencing of an excised chromosomal band at 0.9 Mb identified by PFGE in the offspring A2.2. Illumina sequencing reads were mapped to coding sequences of all chromosomes of the reference genome IPO323. E) Schematic illustration of the hypothesized non-allelic homologous recombination between inverted repeats that generated chromosome 17 in offspring A2.2 and A66.2. The resulting isodicentric chromosome can initiate a breakage-fusion-bridge cycle while the acentric chromosome will be lost during successive rounds of cell division.