Repeat-Induced Point Mutation and Gene Conversion Coinciding with Heterochromatin Shape the Genome of a Plant-Pathogenic Fungus

Abstract

Meiosis is associated with genetic changes in the genome-via recombination, gene conversion, and mutations. The occurrence of gene conversion and mutations during meiosis may further be influenced by the chromatin conformation, similar to the effect of the chromatin conformation on the mitotic mutation rate. To date, however, the exact distribution and type of meiosis-associated changes and the role of the chromatin conformation in this context are largely unexplored. Here, we determine recombination, gene conversion, and de novo mutations using whole-genome sequencing of all meiotic products of 23 individual meioses in Zymoseptoria tritici, an important pathogen of wheat. We confirm a high genome-wide recombination rate of 65 centimorgan (cM)/Mb and see higher recombination rates on the accessory compared to core chromosomes. A substantial fraction of 0.16% of all polymorphic markers was affected by gene conversions, showing a weak GC-bias and occurring at higher frequency in regions of constitutive heterochromatin, indicated by the histone modification H3K9me3. The de novo mutation rate associated with meiosis was approximately three orders of magnitude higher than the corresponding mitotic mutation rate. Importantly, repeat-induced point mutation (RIP), a fungal defense mechanism against duplicated sequences, is active in Z. tritici and responsible for the majority of these de novo meiotic mutations. Our results indicate that the genetic changes associated with meiosis are a major source of variability in the genome of an important plant pathogen and shape its evolutionary trajectory. IMPORTANCE The impact of meiosis on the genome composition via gene conversion and mutations is mostly poorly understood, in particular, for non-model species. Here, we sequenced all four meiotic products for 23 individual meioses and determined the genetic changes caused by meiosis for the important fungal wheat pathogen Zymoseptoria tritici. We found a high rate of gene conversions and an effect of the chromatin conformation on gene conversion rates. Higher conversion rates were found in regions enriched with the H3K9me3-a mark for constitutive heterochromatin. Most importantly, meiosis was associated with a much higher frequency of de novo mutations than mitosis; 78% of the meiotic mutations were caused by repeat-induced point mutations-a fungal defense mechanism against duplicated sequences. In conclusion, the genetic changes associated with meiosis are therefore a major factor shaping the genome of this fungal pathogen.

Keywords: epigenetics; gene conversion; meiosis; meiotic mutation; repeat-induced point mutation (RIP); tetrad analysis.

FIG 1

Comparison of recombination rates, gene conversion rates, and crossover interference for various genomic compartments. (A) Recombination rates for the genome and on core and accessory chromosomes. (B) Correlation between recombination rates and chromosome length. (C) Crossover (CO) interference. Distribution of distances between adjacent CO events (CO-CO distance) in the entire genome (pink), on core chromosomes (blue), and on accessory chromosomes (yellow). The red line represents the fitted gamma distribution for the observed CO-CO distances. γ values higher than 1 indicate positive CO interference, i.e., a lower than expected number of small CO-CO distances. (D) Gene conversion rates for the genome and core and accessory chromosomes. (E) Violin plot of non-crossover-associated gene conversion (NCO-GCs) and crossover-associated gene conversion (CO-GCs) tract lengths for core and accessory chromosomes. The tract lengths of the gene conversion events spanning TEs are excluded (different letters depict significantly different groups with a P value of <0.05 in the Wilcoxon test with Bonferroni correction). In panels A and D, P values of paired Wilcoxon tests are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

FIG 2

Correlation between crossover frequency and gene conversion rates with histone modifications, GC-biased gene conversion, and gene conversion rate per chromosome. (A and B) Correlation between (A) crossover frequencies or (B) gene conversion rates and chromatin modifications. The gene conversion rate is calculated as the number of converted markers per genomic compartment divided by the total number of markers in the genomic compartment. A genomic compartment is defined by all regions of the genome that share the presence/absence of the indicated histone modifications. The presence/absence of the specific chromatin modification (H3K4me2, H3K9me3, or H3K27me3, respectively) in the genomic compartment is depicted with + or – in the table below the x axis. χ²-test P values for comparing the indicated genomic compartments with the genomic compartment lacking all the indicated histone modifications are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant). (C) GC-biased gene conversion in Z. tritici. The stacked barplot shows the proportion of AT to GC converted markers and the proportion of GC to AT converted markers. Binomial test P values are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). (D) Gene conversion rate per chromosome calculated as the proportion of converted markers from the total number of markers on the respective chromosome. The numbers above box plots show the number of tetrads with gene conversion detected on the respective chromosome. Box plots display center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, rate per tetrad.

FIG 3

Genome-wide distribution of de novo mutations associated with meiosis in Z. tritici. (A) CircosPlot of the meiotic SNP distribution in the genomic features of Z. tritici (orange lines, TEs; blue dots, meiotic SNPs outside the TEs; lilac dots, meiotic SNPs inside the duplication on chromosome 3; green dots, meiotic SNPs inside the TEs). (B) RIP-like mutations in the duplicated 14 kb region on chromosome 3. The top line graph shows the difference in normalized coverage between IPO323 and the IPO94269 parent in the region on chromosome 3. The distributions of mutations in the duplicated chromosome 3 region and 10 kb upstream and downstream regions are shown in the lolliplot below the line graph. The start and the end on the x axis of the lolliplot designate the start and the end of the duplicated region. Rectangles in different colors depict the genes located in this region. Each lollipop represents a single mutation. (C) Number of meiotic mutations in and out of the RIP active regions represented as their relative frequencies. CG:TA transitions are colored in light blue, and TA:CG transitions and transversions are colored in red. Fisher exact test P values are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). (D) Number of de novo mutations in different classes and families of repeats (light yellow, long interspersed nuclear element [LINE]; dark yellow, long terminal repeats [LTR]; marine blue, HELITRON; dark blue, tandem inverted repeats [TIR]; white, noCat). (E) Distribution of meiotic SNPs along TEs. Each TE was divided into 40 equal-sized windows. Each black rectangle on the x axis represents a window inside a TE representing 2.5% of the TE length. Beige rectangles represent windows in the regions directly adjacent to TEs. Dots above rectangles represent one mutation in each window (yellow dots, AT:GC transitions; blue dots, CG:TA transitions, red dots, transversions).