Heritability and gene functions associated with sclerotia formation of Rhizoctonia solani AG-7 using whole genome sequencing and genome-wide association study

Sclerotia are specialized fungal structures formed by pigmented and aggregated hyphae, which can survive under unfavourable environmental conditions and serve as the primary inocula for several phytopathogenic fungi including Rhizoctonia solani. Among 154 R. solani anastomosis group 7 (AG-7) isolates collected in fields, the sclerotia-forming capability regarding sclerotia number and sclerotia size varied in the fungal population, but the genetic makeup of these phenotypes remained unclear. As limited studies have focused on the genomics of R. solani AG-7 and the population genetics of sclerotia formation, this study completed the whole genome sequencing and gene prediction of R. solani AG-7 using the Oxford NanoPore and Illumina RNA sequencing. Meanwhile, a high-throughput image-based method was established to quantify the sclerotia-forming capability, and the phenotypic correlation between sclerotia number and sclerotia size was low. A genome-wide association study identified three and five significant SNPs associated with sclerotia number and size in distinct genomic regions, respectively. Of these significant SNPs, two and four showed significant differences in the phenotypic mean separation for sclerotia number and sclerotia size, respectively. Gene ontology enrichment analysis focusing on the linkage disequilibrium blocks of significant SNPs identified more categories related to oxidative stress for sclerotia number, and more categories related to cell development, signalling and metabolism for sclerotia size. These results indicated that different genetic mechanisms may underlie these two phenotypes. Moreover, the heritability of sclerotia number and sclerotia size were estimated for the first time to be 0.92 and 0.31, respectively. This study provides new insights into the heritability and gene functions related to the development of sclerotia number and sclerotia size, which could provide additional knowledge to reduce fungal residues in fields and achieve sustainable disease management.

[1]  A. Pain,et al.  Pangenome Analysis of the Soilborne Fungal Phytopathogen Rhizoctonia solani and Development of a Comprehensive Web Resource: RsolaniDB , 2022, Frontiers in Microbiology.

[2]  Ying-Hong Lin,et al.  Superoxide Initiates the Hyphal Differentiation to Microsclerotia Formation of Macrophomina phaseolina , 2022, Microbiology spectrum.

[3]  E. Stukenbrock,et al.  Genome-wide association and selective sweep studies reveal the complex genetic architecture of DMI fungicide resistance in Cercospora beticola. , 2021, Genome biology and evolution.

[4]  Alicia R. Martin,et al.  Genome-wide association studies , 2021, Nature Reviews Methods Primers.

[5]  Xiaochen Bo,et al.  clusterProfiler 4.0: A universal enrichment tool for interpreting omics data , 2021, Innovation.

[6]  Jianping Xu,et al.  Genome-Wide Association Analysis for Triazole Resistance in Aspergillus fumigatus , 2021, Pathogens.

[7]  P. Bork,et al.  Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation , 2021, Nucleic Acids Res..

[8]  S. Opiyo,et al.  Comparative genome analyses of four rice-infecting Rhizoctonia solani isolates reveal extensive enrichment of homogalacturonan modification genes , 2021, BMC genomics.

[9]  Zejian Guo,et al.  Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi , 2021, Communications Biology.

[10]  Thomas M. Keane,et al.  Twelve years of SAMtools and BCFtools , 2020, GigaScience.

[11]  Zhiwu Zhang,et al.  GAPIT Version 3: Boosting Power and Accuracy for Genomic Association and Prediction , 2020, bioRxiv.

[12]  Wenbo Ge,et al.  Genome-Wide Association for Itraconazole Sensitivity in Non-resistant Clinical Isolates of Aspergillus fumigatus , 2020, bioRxiv.

[13]  Mario Stanke,et al.  BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database , 2020, bioRxiv.

[14]  J. Frisvad,et al.  Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger. , 2020, Fungal genetics and biology : FG & B.

[15]  B. McDonald,et al.  The Genetic Architecture of Emerging Fungicide Resistance in Populations of a Global Wheat Pathogen , 2020, bioRxiv.

[16]  R. Knox,et al.  Genetic and transcriptional dissection of resistance to Claviceps purpurea in the durum wheat cultivar Greenshank , 2020, Theoretical and Applied Genetics.

[17]  Kutubuddin A Molla,et al.  Understanding sheath blight resistance in rice: the road behind and the road ahead , 2019, Plant biotechnology journal.

[18]  Ning Li,et al.  Natural variation in ZmFBL41 confers banded leaf and sheath blight resistance in maize , 2019, Nature Genetics.

[19]  M. Schatz,et al.  GenomeScope 2.0 and Smudgeplots: Reference-free profiling of polyploid genomes , 2019, bioRxiv.

[20]  Steven L Salzberg,et al.  Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype , 2019, Nature Biotechnology.

[21]  Y. Bossé,et al.  Benefits and limitations of genome-wide association studies , 2019, Nature Reviews Genetics.

[22]  Karam B. Singh,et al.  Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen Rhizoctonia solani AG1-IA , 2019, Functional & Integrative Genomics.

[23]  Yu Lin,et al.  Assembly of long, error-prone reads using repeat graphs , 2018, Nature Biotechnology.

[24]  Konstantinos D. Tsirigos,et al.  SignalP 5.0 improves signal peptide predictions using deep neural networks , 2019, Nature Biotechnology.

[25]  Yuheng Yang,et al.  Sclerotinia sclerotiorum Thioredoxin Reductase Is Required for Oxidative Stress Tolerance, Virulence, and Sclerotial Development , 2019, Front. Microbiol..

[26]  Xingyong Yang,et al.  Comparative transcriptome analysis reveals regulatory networks and key genes of microsclerotia formation in the cotton vascular wilt pathogen. , 2019, Fungal genetics and biology : FG & B.

[27]  Xingang Wang,et al.  RaGOO: fast and accurate reference-guided scaffolding of draft genomes , 2019, Genome Biology.

[28]  Zhiwu Zhang,et al.  BLINK: a package for the next level of genome-wide association studies with both individuals and markers in the millions , 2018, GigaScience.

[29]  Kazutaka Katoh,et al.  MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization , 2017, Briefings Bioinform..

[30]  S. Tangphatsornruang,et al.  Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach. , 2019, Genomics.

[31]  M. Karlsson,et al.  Out in the Cold: Identification of Genomic Regions Associated With Cold Tolerance in the Biocontrol Fungus Clonostachys rosea Through Genome-Wide Association Mapping , 2018, Frontiers in Microbiology.

[32]  Y. Xuan,et al.  Transcriptomic evidence for involvement of reactive oxygen species in Rhizoctonia solani AG1 IA sclerotia maturation , 2018, PeerJ.

[33]  R. Branicky,et al.  Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling , 2018, The Journal of cell biology.

[34]  Zhenglu Yang,et al.  dbCAN2: a meta server for automated carbohydrate-active enzyme annotation , 2018, Nucleic Acids Res..

[35]  Adam M. Phillippy,et al.  MUMmer4: A fast and versatile genome alignment system , 2018, PLoS Comput. Biol..

[36]  A. von Haeseler,et al.  UFBoot2: Improving the Ultrafast Bootstrap Approximation , 2017, bioRxiv.

[37]  A. Goesmann,et al.  Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3 , 2017, Archives of Microbiology.

[38]  W. Dong,et al.  Metabolites contributing to Rhizoctonia solani AG-1-IA maturation and sclerotial differentiation revealed by UPLC-QTOF-MS metabolomics , 2017, PloS one.

[39]  Olivier Loudet,et al.  New Strategies and Tools in Quantitative Genetics: How to Go from the Phenotype to the Genotype. , 2017, Annual review of plant biology.

[40]  Thomas K. F. Wong,et al.  ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates , 2017, Nature Methods.

[41]  B. McDonald,et al.  A fungal wheat pathogen evolved host specialization by extensive chromosomal rearrangements , 2017, The ISME Journal.

[42]  Niranjan Nagarajan,et al.  Fast and accurate de novo genome assembly from long uncorrected reads. , 2017, Genome research.

[43]  Tulio de Oliveira,et al.  Microbial genome-wide association studies: lessons from human GWAS , 2016, Nature Reviews Genetics.

[44]  C. Shu,et al.  Survival of Rhizoctonia solani AG-1 IA, the Causal Agent of Rice Sheath Blight, under Different Environmental Conditions , 2017 .

[45]  B. McDonald,et al.  Multilocus resistance evolution to azole fungicides in fungal plant pathogen populations , 2016, Molecular ecology.

[46]  Lilin Zhang,et al.  Characterization of 47 Cys2 -His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae. , 2016, The New phytologist.

[47]  C. Zou,et al.  Bulked sample analysis in genetics, genomics and crop improvement , 2016, Plant biotechnology journal.

[48]  J. Blom,et al.  Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes , 2016, BMC Genomics.

[49]  Evgeny M. Zdobnov,et al.  BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs , 2015, Bioinform..

[50]  R. Bayles,et al.  The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles , 2015, Theoretical and Applied Genetics.

[51]  A. Goesmann,et al.  Improved genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB 7/3/14 as established by deep mate-pair sequencing on the MiSeq (Illumina) system. , 2015, Journal of biotechnology.

[52]  T. Henkel,et al.  How many fungi make sclerotia , 2015 .

[53]  Gaston H. Gonnet,et al.  The OMA orthology database in 2015: function predictions, better plant support, synteny view and other improvements , 2014, Nucleic Acids Res..

[54]  A. von Haeseler,et al.  IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies , 2014, Molecular biology and evolution.

[55]  O. Lepais,et al.  SimRAD: an R package for simulation‐based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches , 2014, Molecular ecology resources.

[56]  Yong Bok Lee,et al.  Proteomic analysis of Rhizoctonia solani AG-1 sclerotia maturation. , 2014, Fungal biology.

[57]  James K. Hane,et al.  Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8 , 2014, PLoS genetics.

[58]  G. Braus,et al.  Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots. , 2014, The New phytologist.

[59]  A. Goesmann,et al.  Establishment and interpretation of the genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB isolate 7/3/14. , 2013, Journal of biotechnology.

[60]  Rachel B. Brem,et al.  Genome Wide Association Identifies Novel Loci Involved in Fungal Communication , 2013, PLoS genetics.

[61]  Alexey A. Gurevich,et al.  QUAST: quality assessment tool for genome assemblies , 2013, Bioinform..

[62]  Heng Li.  Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM , 2013, 1303.3997.

[63]  Jing Zhang,et al.  The evolution and pathogenic mechanisms of the rice sheath blight pathogen , 2013, Nature Communications.

[64]  M. Lind,et al.  A Genome-Wide Association Study Identifies Genomic Regions for Virulence in the Non-Model Organism Heterobasidion annosum s.s , 2013, PloS one.

[65]  C. Kimchi-Sarfaty,et al.  Understanding the contribution of synonymous mutations to human disease , 2011, Nature Reviews Genetics.

[66]  Matko Bosnjak,et al.  REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms , 2011, PloS one.

[67]  Robert J. Elshire,et al.  A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species , 2011, PloS one.

[68]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[69]  G. Karaca,et al.  Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun, Turkey , 2011 .

[70]  C. Georgiou,et al.  Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi , 2010, Journal of applied microbiology.

[71]  K. Aliferis,et al.  Metabolite composition and bioactivity of Rhizoctonia solani sclerotial exudates. , 2010, Journal of agricultural and food chemistry.

[72]  C. Georgiou,et al.  Superoxide radical is involved in the sclerotial differentiation of filamentous phytopathogenic fungi: identification of a fungal xanthine oxidase. , 2010, Fungal biology.

[73]  K. Abd-Elsalam,et al.  First Report of Rhizoctonia solani AG‐7 on Cotton in Egypt , 2010 .

[74]  K. Aliferis,et al.  1H NMR and GC-MS metabolic fingerprinting of developmental stages of Rhizoctonia solani sclerotia , 2010, Metabolomics.

[75]  R. Bain,et al.  Effects of nutrient status, temperature and pH on mycelial growth, sclerotial production and germination of Rhizoctonia solani from potato , 2009 .

[76]  Toni Gabaldón,et al.  trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses , 2009, Bioinform..

[77]  P. VanRaden,et al.  Efficient methods to compute genomic predictions. , 2008, Journal of dairy science.

[78]  Pier Luigi Martelli,et al.  PredGPI: a GPI-anchor predictor , 2008, BMC Bioinformatics.

[79]  Edward S. Buckler,et al.  TASSEL: software for association mapping of complex traits in diverse samples , 2007, Bioinform..

[80]  Manuel A. R. Ferreira,et al.  PLINK: a tool set for whole-genome association and population-based linkage analyses. , 2007, American journal of human genetics.

[81]  P. Cremer,et al.  The α,α-(1→1) Linkage of Trehalose Is Key to Anhydrobiotic Preservation , 2007 .

[82]  Erik L. L. Sonnhammer,et al.  Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server , 2007, Nucleic Acids Res..

[83]  Burkhard Morgenstern,et al.  AUGUSTUS: ab initio prediction of alternative transcripts , 2006, Nucleic Acids Res..

[84]  Amos Bairoch,et al.  ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins , 2006, Nucleic Acids Res..

[85]  R. Bain,et al.  Effects of water potential on mycelial growth, sclerotial production, and germination of Rhizoctonia solani from potato. , 2006, Mycological research.

[86]  C. Georgiou,et al.  Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress. , 2006, Integrative and comparative biology.

[87]  B. Nelson,et al.  Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. , 2006, Molecular plant pathology.

[88]  C. Georgiou,et al.  Effect of the antioxidant ascorbic acid on sclerotial differentiation in Rhizoctonia solani , 2001 .

[89]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[90]  Elias S. J. Arnér,et al.  Physiological functions of thioredoxin and thioredoxin reductase. , 2000, European journal of biochemistry.

[91]  A. Brown,et al.  A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. , 1998, FEMS microbiology letters.

[92]  R. Baird.  First Report ofRhizoctonia solaniAG-7 in Indiana , 1995 .

[93]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[94]  D. Drubin,et al.  Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae , 1993, The Journal of cell biology.

[95]  Naiki Takashi,et al.  Ecological and morphological characteristics of the sclerotia of Rhizoctonia solani Kühn produced in soil , 1978 .

[96]  R. Cooke,et al.  Survival and Germination of Fungal Sclerotia , 1971 .

[97]  E. D. Rudolph.  THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI , 1962 .

[98]  O. T. Page.  THE INFLUENCE OF LIGHT AND OTHER ENVIRONMENTAL FACTORS ON MYCELIAL GROWTH AND SCLEROTIAL PRODUCTION BY BOTRYTIS SQUAMOSA , 1956 .