1. Aylin ALKAN, Cihan C. CIVAŞ, Erez UZUNER, Hasan O. OTAŞ, Sena ATICI
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

2. 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

3. 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

4. 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

5. 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

6. 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

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

8. 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

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

10. 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)

11. 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

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

13. MATERIALS & METHODS QTL Analysis (Linkage Analysis & Genotyping)
Resistant Sensitive
Markers …
Phenotypes from F6 Generation # # # # # # # # # # # … F1 F2 F3 F4 F5 F6
Dilution of the common characters
Different individuals from F6 line

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

15. 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.

16. 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

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

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

19. 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

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

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

23. 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

24. 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

25. 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

26. RESULTS Fine mapping of highly specific gene region on Chromosom 3

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

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

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

30. 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.

31. 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.

32. 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

33. 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)

34. REFERENCES
1. Riley D.G. & Joseph S.V. (2011). Host Plant Resistance to Tomato spotted wilt virus (Bunyaviridae: Tospovirus) in Tomato. HortScience, 46(12), Angel, C. & Schoelz, J. (2013). A Survey of Resistance to Tomato bushy stunt virus in the Genus Nicotiana Reveals That the Hypersensitive Response Is Triggered by One of Three Different Viral Proteins. MPMI, 26(2), Shi, A. & Vierling, R. (2011). Identification of molecular markers for Sw-5 gene of tomato spotted wilt virus resistance. AJBMS, 1(1), Verlaan M.G., Hutton S.F., Ibrahem R.M., Kormelink R., Visser R.G.F., Scott J.W., Edwards J.D., Bai Y. (2013). The Tomato Yellow Leaf Curl Virus Resistance Genes Ty-1 and Ty-3 Are Allelic and Code for DFDGD-Class RNA– Dependent RNA Polymerases. PLOS Genetics, 9(3):e Chu, M., Desvoyes, B., Turina, M., Noad, R., & Scholthof, H. (2000). Genetic Dissection of Tomato Bushy Stunt Virus p19-Protein-Mediated Host-Dependent Symptom Induction and Systemic Invasion. Virology, 266(1), İşçi B. (2008). Asmada QTL (Kantitatif Karakter Lokus) Analizi. ANADOLU, Journal of the Aegean. Agricultural Research Institute, 18(2), Vouillot, L., Thelie, A., & Pollet, N. (2015). Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases. G3; Genes|Genomes|Genetics, 5(3), Hsu, P., Lander, E., & Zhang, F. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6),