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Whole genome resequencing of Botrytis cinerea isolates identifies high levels of standing diversity.

Atwell S, Corwin JA, Soltis NE, Subedy A, Denby KJ, Kliebenstein DJ - Front Microbiol (2015)

Bottom Line: A high level of genetic diversity was found within the 13 isolates.This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity.This suggests that B. cinerea does not display an elevated spontaneous mutation rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California, Davis Davis, CA, USA.

ABSTRACT
How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.

No MeSH data available.


Genomic distribution of polymorphism amongst the 14 isolates. (A) A sliding window analysis of genomic polymorphisms per 10 kb across the genome is shown. (B) Shown is a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4. (C) Again a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4, but with polymorphisms per gene given a binary value. This enabled clusters of seven or more genes in 10 gene windows affected by major effect polymorphisms to be visually determined more easily. Thousands of permutations of the major effect polymorphisms established a significance threshold of six genes per window, which is drawn for reference. No one isolate is key cause of these clusters.
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Figure 3: Genomic distribution of polymorphism amongst the 14 isolates. (A) A sliding window analysis of genomic polymorphisms per 10 kb across the genome is shown. (B) Shown is a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4. (C) Again a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4, but with polymorphisms per gene given a binary value. This enabled clusters of seven or more genes in 10 gene windows affected by major effect polymorphisms to be visually determined more easily. Thousands of permutations of the major effect polymorphisms established a significance threshold of six genes per window, which is drawn for reference. No one isolate is key cause of these clusters.

Mentions: Given the high level of genomic diversity in B. cinerea, we sought to determine if this diversity was randomly distributed across the genome or showed evidence of clustering. The chromosomes of B. cinerea have not been delineated, as such the program PAGIT (Swain et al., 2012) was used to superficially overlay all contigs on chromosomes by comparison to Sclerotinia sclerotiorum (http://www.broadinstitute.org/sclerotinia_sclerotiorum/maps). This fungus is highly co-linear to B. cinerea with an identical chromosome number (Shirane et al., 1989; Amselem et al., 2011), and has an almost complete chromosome map. This created a visualization framework by placing the contigs on 15 chromosomes, with all unplaceable contigs being assigned into artificial chromosome 16 (as they were in the S. sclerotiorum used). The distribution of polymorphisms amongst contigs, placed on these superficial chromosomes, displayed regions or peaks of high density polymorphism, across all chromosomes (Figure 3A and Table S2). Change rates varied greatly per isolate and per contig, with the highest being one polymorphism every 40 bp in contig 15.3 of Grape and the lowest one change every 27,839 bp in contig 4.1 of Katie Tomato (Table S2).


Whole genome resequencing of Botrytis cinerea isolates identifies high levels of standing diversity.

Atwell S, Corwin JA, Soltis NE, Subedy A, Denby KJ, Kliebenstein DJ - Front Microbiol (2015)

Genomic distribution of polymorphism amongst the 14 isolates. (A) A sliding window analysis of genomic polymorphisms per 10 kb across the genome is shown. (B) Shown is a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4. (C) Again a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4, but with polymorphisms per gene given a binary value. This enabled clusters of seven or more genes in 10 gene windows affected by major effect polymorphisms to be visually determined more easily. Thousands of permutations of the major effect polymorphisms established a significance threshold of six genes per window, which is drawn for reference. No one isolate is key cause of these clusters.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Genomic distribution of polymorphism amongst the 14 isolates. (A) A sliding window analysis of genomic polymorphisms per 10 kb across the genome is shown. (B) Shown is a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4. (C) Again a 10 gene sliding window analysis of the number of genes with major effect polymorphisms based on comparison to the isolate T4, but with polymorphisms per gene given a binary value. This enabled clusters of seven or more genes in 10 gene windows affected by major effect polymorphisms to be visually determined more easily. Thousands of permutations of the major effect polymorphisms established a significance threshold of six genes per window, which is drawn for reference. No one isolate is key cause of these clusters.
Mentions: Given the high level of genomic diversity in B. cinerea, we sought to determine if this diversity was randomly distributed across the genome or showed evidence of clustering. The chromosomes of B. cinerea have not been delineated, as such the program PAGIT (Swain et al., 2012) was used to superficially overlay all contigs on chromosomes by comparison to Sclerotinia sclerotiorum (http://www.broadinstitute.org/sclerotinia_sclerotiorum/maps). This fungus is highly co-linear to B. cinerea with an identical chromosome number (Shirane et al., 1989; Amselem et al., 2011), and has an almost complete chromosome map. This created a visualization framework by placing the contigs on 15 chromosomes, with all unplaceable contigs being assigned into artificial chromosome 16 (as they were in the S. sclerotiorum used). The distribution of polymorphisms amongst contigs, placed on these superficial chromosomes, displayed regions or peaks of high density polymorphism, across all chromosomes (Figure 3A and Table S2). Change rates varied greatly per isolate and per contig, with the highest being one polymorphism every 40 bp in contig 15.3 of Grape and the lowest one change every 27,839 bp in contig 4.1 of Katie Tomato (Table S2).

Bottom Line: A high level of genetic diversity was found within the 13 isolates.This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity.This suggests that B. cinerea does not display an elevated spontaneous mutation rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California, Davis Davis, CA, USA.

ABSTRACT
How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.

No MeSH data available.