A gapless genome sequence of the fungus Botrytis cinerea

Abstract

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

Keywords: SMRT sequencing; genetic map; grey mould; optical map.

Figure 1

Linkage map for a subset of the 18 identified linkage groups (LGs). LG13 (chromosome 2, Chr2) shows normal linkage patterns. The top arm of LG1 (Chr6) is identical to LG10, whereas the rest of LG1 is identical to LG8, indicative of a translocation.

Figure 2

Examples of alignments between the optical map (orange, top) and the in silico digest of the sequence assembly (grey, bottom) for three selected chromosomes. Each vertical line reflects the presence of a BstEII restriction site, either experimentally determined (optical map) or predicted from the assembly. The red boxes highlight regions in the optical map which are not represented in the sequence assembly.

Figure 3

Reads per kilobase per million (RPKM) values for a subset of chromosomes in sexual progeny from the cross 09Bc11 × B05.10. Each panel depicts RPKM values (y‐axis) over one entire chromosome (indicated by their number) in 70 individual progeny as well as parental isolate 09Bc11. Each isolate is represented by one vertical bar in the graph, with isolate 09Bc11 on the extreme right. Plots for chromosomes 5–16 (Chr5–16) look similar to those for Chr2 and Chr3.

Figure 4

Mapping of two fungicide resistance loci to chromosome 1 (Chr1). The x‐axis represents sequence assembly coordinates. The y‐axis represents the subtraction of frequencies at which a single nucleotide polymorphism (SNP) is found in respective fungicide resistant (R) minus sensitive (S) bulk. Frequencies were calculated for each position in the R or S bulk using the formula: total number of reads representing the SNP at position/total number of reads covering the position in bulk. The y value for any SNP position can vary between zero for unlinked positions to +1 for positively linked positions (SNP linked to resistance) or to −1 in the case of a negative linkage (SNP linked to sensitivity). Blue circles represent synonymous SNPs or SNPs outside coding sequences and green circles represent non‐synonymous SNPs, exclusively. Red circles represent the SNPs that confer resistance. (A) Mapping of the boscalid resistance locus. (B) Mapping of the iprodione resistance locus.

Figure 5

Relationship between physical and genetic distances for chromosomes 2 and 15 (Chr2 and Chr15). The x‐axis displays the genetic distance in centimorgan (cM) and the y‐axis displays the chromosome coordinates in base pairs (bp). The correlation between physical and genetic distances and the correlation coefficient are given in the top right‐hand corner of the graph. Each blue diamond represents a marker group [cluster of single nucleotide polymorphisms (SNPs) fully co‐segregating in all progeny]. The green hatched arrow indicates the approximate position of the centromere. Black arrows indicate regions with high recombination rates.

Figure 6

Validation of gene predictions by proteome analysis. The numbers in the Venn diagram represent the numbers of peptides (derived from tryptic digests of soluble mycelium proteins) mapping to the three versions of the predicted proteome of Botrytis cinerea B05.10.