Aylin ALKAN, Cihan C. CIVAŞ, Erez UZUNER, Hasan O. OTAŞ, Sena ATICI 26.05.2016 Thursday Molecular Genetics of Eukaryotes IdentIfIcatIon of resIstance genes agaInst major vIruses In Solanum caose usIng molecular markers Aylin ALKAN, Cihan C. CIVAŞ, Erez UZUNER, Hasan O. OTAŞ, Sena ATICI Department of Molecular Biology and Genetics, Faculty of Science, Izmir Institute of Technology, TR 35430, İzmir, TURKEY

INTRODUCTION Viruses Small genomes Code for a very limited amount of proteins Large yield losses Hypersensitive cell death Response (HR)  DEFENSE MECHANISM Pathogen Plant recognize pathogen Triggering HR Activation of related genes

INTRODUCTION Tomato bushy stunt virus (TBSV) Small icosahedral virus Single copy of a positive sense single-stranded RNA genome with ~4800 nucleotides p19 and p22 resistance genes Leaf crinkling Necrosis Deformation of tomato fruit and leaves Stunting and bushy growth Chlorotic spots

INTRODUCTION Tomato yellow leaf curl viruses (TYLCV) Begomovirus genus (Geminiviridae) The MOST widespread 3rd important plant viruses worldwide Single-stranded, circular and bidirectionally organized DNA genome Ty-1 and Ty-3  allelic resistant genes

INTRODUCTION Tomato spotted wilt virus (TSWV) - (genus Tospovirus; family Bunyaviridae) sw2, sw5, sw6 resistant genes Small brownish ringspots Bronzed appearance Purpling and upward rolling of leaves Stunting of leaves and plants Yellow ringspots on fruits

INTRODUCTION Whitefly High incidence of diseases Control measures in infected areas  insecticides or physical barriers Large populations Marker assisted selection (MAS)  selecting disease resistant by identifying genetic markers that are linked to specific genes/alleles or combination of multiple resistant genes/alleles Single nucleotide polymorphisms (SNPs) Abundance and high-throughput scoring potential Genome mapping - Association studies - Diversity analysis

INTRODUCTION Genome Editing Tool PAM, Protospacer Adjacent Motif seqeunces Targeting with single guide RNA Cas9 double strand breaks Non-Homologous End Joining Homology Directed Repair

MATERIALS & METHODS Work Flow of Identification of Resistant Genes Plant Material Whitefly Inoculation Segregation Analysis DNA Extraction Genotyping- SNPs and QTL Statistical Analysis Design of sgRNA and Plasmid Construction for CRISPR Application Plasmid Transformation into Agrobacterium and Agrobacterium Transfection CRISPR Screening with T7 Endonucleases

MATERIALS & METHODS X6 B A Sensitive Resistant Solanum caose Plant Materials 500 Progenies 500 Progenies Sensitive Resistant A B Solanum caose Genotypically highly similar resistant (A) and sensitive (B) plants

MATERIALS & METHODS Whitefly Inoculation Viruses Infected Whiteflies in terms i ii iii 48h inoculation to Plants Removal of Whiteflies TYLCV TSWV TBSV Translocation of viruses from White flies to the same tomatoes Imidacloprid (neurotoxic insecticide)

MATERIALS & METHODS QTL (Segregation Analysis) Non-inoculated plants of the same line  Negative control Virus-free whitefly inoculation  Positive control Multiple Virus Whitefly Inoculated Plants Sensitive Plants F6 resistant fruit F1 resistant fruit F6 sensitive fruits

MATERIALS & METHODS QTL Analysis (DNA Extraction) For pure DNA, A260/280 = 1.8-2.

MATERIALS & METHODS QTL Analysis (Linkage Analysis & Genotyping) Resistant Sensitive Markers 1 2 3 4 5 6 7 8 9 10 11 12 … Phenotypes from F6 Generation #1 + + - + + + + + + + + + + #2 + + + + - + - - - + + - - #3 - - - - - - - + - - + + - #4 - - + - - - - + - - + + + #5 + + - + - + - + - - + + - #6 - - - - + - + + - - + - + #7 - + - - - + - + + + + - + #8 + + + + - + - + + - + + - #9 + + - + + + + - + - + - + #10 + + + + - - - + - + + + - #11 - - + - + + + - - - + - + … F1 F2 F3 F4 F5 F6 Dilution of the common characters Different individuals from F6 line

MATERIALS & METHODS QTL Analysis (Particular SNP Marker Sites) SNP marker sites that are on the 12 tomato chromosomes

3.6 significance threshold Positions of the markers as triangles MATERIALS & METHODS QTL Analysis (Mapping & Statistical Analysis) 3.6 significance threshold Positions of the markers as triangles Composite interval mapping evaluates the probability that an interval between two markers is associated while simultaneously controlling for the effects of other markers on the trait.

MATERIALS & METHODS QTL Analysis (Highly Specific PCR Primers) Primer Name Sequence ( 5’  3’ ) Gene Location Prm f 1002 AAAAGTGAACTTTAAC Chromosome 3 Prm r 1002 CCTAAAGTGGTGAGAA Prm f 2165 AACATGGATAGGACCA Chromosome 11 Prm r 2165 TTGGCCAGAGTGGAAA Designed primers that are used for specifying large-scaled candidate regions

MATERIALS & METHODS Guide RNA Design High GC contents 20 nt sequences Add restriction enzyme cutting site according to vector

MATERIALS & METHODS Vector Construction Guide RNA Ligation Cut both vector and gRNA oligos with EcoRI and BamHI Ligation with T4 ligase

MATERIALS & METHODS Electroporation and Agrobacterium tumefaciens Transfection Leaf piece growth on the transfected Agrobacterium tumefaciens After the callus formation, new plant was growth with adding growth factors Constructed Plasmid transports to Agrobacterium tumefaciens using the electroporation technique

MATERIALS & METHODS CRISPR Screening Genomic PCR Heteroduplex Formation Cut with T7 Endonucleases Agarose Gel Analysis

Percent incidences of sensitive and resistant plants for all offspring RESULTS Percent incidences of sensitive and resistant plants for all offspring

RESULTS Tomato bushy stunt virus infection Tomato yellow leaf curl virus infection Tomato spotted wilt virus infection Features that are used for separation of resistant and sensitive plants

Most possible candidate sites for resistance genes after QTL analysis RESULTS Candidate gene locus on chromosome3 Candidate gene locus on chromosome11 Most possible candidate sites for resistance genes after QTL analysis

QTL analysis results in respect of LOD values 3,6 Red line indicates threshold for LOD value QTL analysis results in respect of LOD values

RESULTS Fine mapping of highly specific gene region on Chromosom 3

RESULTS %39 similarity %34.6 similarity Sequence similarities between sw-5 and gene-1 Sequence similarities between sw-5 and gene-2

RESULTS The views of the Gel Electrophoresis which belong to PCR and T7 endonucleases

RESULTS The disease symptoms of the variations that knocked-out of the genes respectively

DISCUSSION Disease symptoms mostly seen in non-resistant plants, however some resistant plants show symptoms. Resistance are provided by multiple genes because missing genes can cause becoming sensitive to these viruses Designed SNP markers and QTL analysis were most useful for determining gene location on chromosomes.

DISCUSSION Chromosome 3 and Chromosome 11 were found as having high value of LOD scores Linked resistance genes which are found as gene-1 on Chromosome 3 and gene-2 on Chromosome 11 One of them is not enough for providing resistance, they cooperate with each other for resistance mechanism.

DISCUSSION CRISPR/Cas9 system provides high efficiency knock out After the knock out of any genes, virus resistance disappears. However, when both genes are knocked out, amount of virus damages increases According to BLAST results, gene-1 and gene-2 do not belong to NB-LRR class

DISCUSSION Future Studies: Determining mechanism of resistance genes (localization, molecular function, interaction and defense mechanism) Nuclear binding NB-LRR(leucine rich motif) family (gene for gene interaction AVR-R ) (virus-plant) Developing more favorable tomatoes using these gene regions, Increasing yield by using gene transformation to other crops which found in same family( tomato, eggplant, pepper, potato)

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