Comparative Genomics of Botrytis cinerea Strains with Differential Multi-Drug Resistance

Botrytis cinerea is a ubiquitous fungus difficult to control because it possess a variety of attack modes, diverse hosts as inoculum sources, and it can survive as mycelia and/or conidia or for extended periods as sclerotia in crop debris. For these reasons the use of any single control measure is unlikely to succeed and a combination of cultural practices with the application of site-specific synthetic compounds provide the best protection for the crops (Williamson et al., 2007). However, the chemical control has been adversely affected by the development of fungicide resistance. The selection of resistant individuals in a fungal population subjected to selective pressure due to fungicides is an evolutionary mechanism that promotes advantageous genotypes (Walker et al., 2013). High levels of resistance to site-specific fungicides are commonly associated with point mutations. For example the mutations G143A, H272R, and F412S leading to changes in the target proteins CytB, SdhB, and Erg27 are conferring resistance of the pathogen to the chemical classes of QoIs, SDHIs, and hydroxyanilides, respectively (Leroux, 2007). Multidrug resistance is another mechanism associated with resistance in B. cinerea which involves mutations leading to overexpression of individual transporters such as ABC and MFS (Kretschmer et al., 2009). This mechanism is associated with low levels of resistance to multiple fungicides including the anilinopyrimidines and phenylpyrroles. However, a subdivision of gray mold populations was found to be more tolerant to these two classes of fungicides (Leroch et al., 2013). 
 
Previous reports have clearly demonstrated that the resistance to anilinopyrimidines has a qualitative, disruptive pattern, and is monogenically controlled (Chapeland et al., 1999). In order to elucidate the mechanism of the resistance, the whole genome of three different samples (gene pools) was sequenced, each containing DNA of 10 selected strains of the same genotype regarding resistance to seven different classes of fungicides including anilinopyrimidines. This report presents the publicly available genomic data.


INTRODUCTION
Botrytis cinerea is a ubiquitous fungus difficult to control because it possess a variety of attack modes, diverse hosts as inoculum sources, and it can survive as mycelia and/or conidia or for extended periods as sclerotia in crop debris. For these reasons the use of any single control measure is unlikely to succeed and a combination of cultural practices with the application of site-specific synthetic compounds provide the best protection for the crops (Williamson et al., 2007). However, the chemical control has been adversely affected by the development of fungicide resistance. The selection of resistant individuals in a fungal population subjected to selective pressure due to fungicides is an evolutionary mechanism that promotes advantageous genotypes (Walker et al., 2013). High levels of resistance to site-specific fungicides are commonly associated with point mutations. For example the mutations G143A, H272R, and F412S leading to changes in the target proteins CytB, SdhB, and Erg27 are conferring resistance of the pathogen to the chemical classes of QoIs, SDHIs, and hydroxyanilides, respectively (Leroux, 2007). Multidrug resistance is another mechanism associated with resistance in B. cinerea which involves mutations leading to overexpression of individual transporters such as ABC and MFS (Kretschmer et al., 2009). This mechanism is associated with low levels of resistance to multiple fungicides including the anilinopyrimidines and phenylpyrroles. However, a subdivision of gray mold populations was found to be more tolerant to these two classes of fungicides (Leroch et al., 2013).
Previous reports have clearly demonstrated that the resistance to anilinopyrimidines has a qualitative, disruptive pattern, and is monogenically controlled (Chapeland et al., 1999). In order to elucidate the mechanism of the resistance, the whole genome of three different samples (gene pools) was sequenced, each containing DNA of 10 selected strains of the same genotype regarding resistance to seven different classes of fungicides including anilinopyrimidines. This report presents the publicly available genomic data.

Isolation-Determination of Fungicide Resistance Profile
Pure cultures of B. cinerea were obtained on sterilized PDA media from infected lettuce plants by slight touching a flamed wire loop onto a freshly sporulating lesion. All isolates obtained from lettuce plants in a commercial lettuce glasshouse located at Krokion, Magnesia, Greece on February 26th, 2012. From each sample a single isolate was made. For all purposes single-spore isolates properly prepaired. The sensitivity of the isolates to the fungicides fenhexamid (class: Hydroxyanilides-Hyd), pyraclostrobin (class: Quinone outside Inhibitors-QoIs), boscalid (class: Succinate De-Hydrogenase Inhibitors-SDHIs; Bos), cyprodinil (class: Anilinopyrimidines-Ani), fludioxonil (class: Phenylpyrroles-Phen), carbendazim (class: Benzimidazoles-Ben), and iprodione (class: Dicarboximides-Dic) was determined by the point inoculation method using the discriminatory concentrations of each fungicide as defined by Chatzidimopoulos et al. (2013). The isolates were then classified in three major groups according to their respective fungicide resistance profile, i.e., (1) Wild type, sensitive to all seven fungicides reported previously; (2) Phenotype QoI R Bos R Ani R Ben R Dic R , resistant (R) to five fungicides; and (3) Phenotype Hyd R QoI R Bos R Ani R Phen R Ben R Dic R , resistant to all seven fungicides tested.

Nucleic Acid and Library Preparation-Sequencing
High quality genomic DNA was extracted from 10 selected single-spore strains of each group (30 in total) by applying a CTAB based protocol (Chatzidimopoulos et al., 2014). Then, three different gene pools were generated containing the DNA of the selected strains according to the fungicide resistance profile. The quantity of DNA was estimated by picogreen (Invitrogen) method using Victor 3 fluorometry. The condition of DNA was checked by a gel electrophoresis method and the purity was assessed on a NanoDrop instrument.
Library preparation performed with the TruSeq Nano DNA Kit (

Sequence Clean-Up, Alignment, and Variant Calling
In order to consistently apply quality and adapter trimming to the sequences, the Cutadapt and FastQC tools were applied through the Trim Galore! wrapper application (Andrews, 2010;Martin, 2011;Krueger, 2012). The sequences were consequently aligned on the B. cinerea B05.10 reference genome, as retrieved from Fungi Ensembl (http://fungi.ensembl.org/Botrytis_cinerea/Info/ Index). After building the reference index files the reads were aligned to the reference genome by using Bowtie 2 (Langmead and Salzberg, 2012) and the produced alignments were parsed using SAMTools (Li et al., 2009). In order to identify potential SNPs and INDELs further analysis was performed using GATK's UnifiedGenotyper (DePristo et al., 2011) and SnpEff v 4.2 (Cingolani et al., 2012).

RESULTS
A total of 12.8, 13, and 11.2 GB sequence data was obtained from the wild type (UTH.PPL.WT5), the 5-resistance variant (UTH.PPL.CR55), and the 7-resistance variant (UTH.PPL.MDR7) respectively. Low quality reads and adapter sequences were removed using FastQC and Cutadapt tools. After quality assessment, there was a loss of ∼0.53, 0.45, and 0.46%, respectively (i.e., high quality sequences). Detailed information of the aligned reads is summarized in

AUTHOR CONTRIBUTIONS
This project was planned and executed by MC under the supervision of PD. The data processing performed jointly by both FP and EM. IG, PM, and EV assisted substantially on the technical part of this work.

FUNDING
The research project was funded under the Action "Research & Technology Development Innovation projects (AgroETAK), " MIS 453350, in the framework of the Operational Program "Human Resources Development." It was co-funded by the European Social Fund and by National Resources through the National Strategic Reference Framework 2007(NSRF 2007 coordinated by the Hellenic Agricultural Organization "DEMETER"/Institute of Plant Breeding and Genetic Resources.