Comparative genomics of Botrytis cinerea strains with differential multi-drug resistance

DATA REPORT published: 28 April 2016 doi: 10.3389/fpls.2016.00554 Comparative Genomics of Botrytis cinerea Strains with Differential Multi-Drug Resistance Michael Chatzidimopoulos 1*, Fotis Psomopoulos 2 , Emmanouil E. Malandrakis 3 , Ioannis Ganopoulos 4 , Panagiotis Madesis 4 , Evangelos K. Vellios 1 and Pavlina Drogoudi 5 1 Laboratory of Plant Pathology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Volos, Greece, 2 Department of Electrical and Computer Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3 Department of Ichthyology and Aquatic Environment, University of Thessaly, Volos, Greece, 4 Centre for Research and Technology Hellas, Institute of Applied Biosciences, Thessaloniki, Greece, 5 Department of Deciduous Fruit Trees in Naoussa, Institute of Plant Breeding and Plant Genetic Resources, Hellenic Agricultural Organization ‘Demeter’, Naoussa, Greece Keywords: Botrytis cinerea, anilinopyrimidines, next-generation sequencing (NGS) whole genome sequencing, multi-drug resistance, INTRODUCTION Edited by: Alessio Mengoni, Università degli Studi di Firenze, Italy Reviewed by: Jacob A. Tennessen, Oregon State University, USA Luana Presta, University of Florence, Italy *Correspondence: Michael Chatzidimopoulos Specialty section: This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Plant Science Received: 17 March 2016 Accepted: 11 April 2016 Published: 28 April 2016 Citation: Chatzidimopoulos M, Psomopoulos F, Malandrakis EE, Ganopoulos I, Madesis P, Vellios EK and Drogoudi P (2016) Comparative Genomics of Botrytis cinerea Strains with Differential Multi-Drug Resistance. Front. Plant Sci. 7:554. doi: 10.3389/fpls.2016.00554 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. MATERIALS AND METHODS 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 Frontiers in Plant Science | www.frontiersin.org 1 April 2016 | Volume 7 | Article 554 Chatzidimopoulos et al. Comparative Genomics of Botrytis cinerea 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 InhibitorsSDHIs; Bos), cyprodinil (class: Anilinopyrimidines-Ani), fludioxonil (class: Phenylpyrroles-Phen), carbendazim (class: Benzimidazoles-Ben), and iprodione (class: DicarboximidesDic) 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 QoIR BosR AniR BenR DicR , resistant (R) to five fungicides; and (3) Phenotype HydR QoIR BosR AniR PhenR BenR DicR , resistant to all seven fungicides tested. ends using End Repair Mix 2 (Illumina). In order to verify the size of PCR enriched fragments, the template size distribution was checked on a Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip. The DNA sequence of each cluster on a flow cell determined with the TruSeq SBS Kit v3 (Illumina). The generation of raw data performed with the HiSeq Control Software v2.2 (Illumina). 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). 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 (...truncated)