CSC-IPB, College of Agriculture, UP Los Baños Development of Tomato Non-Host to Tomato Virus through Targeted Mutagenomics and Bioinformatics Approaches HF Galvez, R Schafleitner, AO Canama, RB Quilloy, DV Lantican, RV Cortes, LL Tandang and BA Kebasen
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Development of Tomato Non-Host Tomato Virus through ...tgc.ifas.ufl.edu/2013/2013/12 - Galvez.pdfCSC-IPB, College of Agriculture, UP Los Baños Development of Tomato Non-Host to Tomato
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Candidate gene list for recessive resistance genes Criteria: Demonstrated reduced symptome expression after mutating or repressing the expression of a specific gene
Source: Literature Begomovirus: Lozano-Durán et al 2011, Xie et al 1999, McGarry et al 2003, Carvalho et al 2006, Singh et al 2006
CMV: Yoshi et al 1998, Huh et al 2011
CSC-IPB, College of Agriculture, UP Los Baños
CSC-IPB, College of Agriculture, UP Los Baños
DNA Markers through SGN Database and other Bioinformatics Tools
Hayde F. Galvez Genetics Laboratory Crop Science Cluster-Institute of Plant Breeding College of Agriculture University of the Philippines Los Baños
Fulbright Visiting Scholar October 17, 2011 to April 13, 2012
Boyce Thompson Institute for Plant Research
Host Supervisor: Dr. Lukas Mueller (SGN)
DNA Marker Development
(virus-host factors)
CSC-IPB, College of Agriculture, UP Los Baños
CSC-IPB, College of Agriculture, UP Los Baños
CSC-IPB, College of Agriculture, UP Los Baños
AVRDC Training Grant (2 UPLB/Project Staff)
December 2012 (Part-I); February 2013 (Part-II)
AVRDC – The World Vegetable Center, Tainan, Taiwan
Host Supervisor: Dr. Roland Schafleitner
(TILLING in combination with NGS)
Pre-NGS at AVRDC
Institute of Plant Breeding, College of Agriculture, UP Los Baños
Example:
Pool 1 = 97 M2families
Pool 2 = 50 M2 families
Pool 3 = 47 M2 families
I. PART 1 (December 2012)
M2 Family Genomic DNA Pooling
Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR amplification of the target virus-host
factor loci for high throughput DNA
sequencing.
PCR amplification conditions of fifteen (15) TYLCV
and CMV candidate host factors were optimized.
The expected product sizes were amplified in all
candidate host factors except for AGO1-1, a
candidate host factor for CMV infection
Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR amplification 15 TYLCV and CMV
candidate host factors
Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR fragments standardized and precipitated
for NGS (lllumina Hi-Seq2000)
New pool 1
Institute of Plant Breeding, College of Agriculture, UP Los Baños
Sequence data analysis will be done using
appropriate softwares /programs
TILLING experiments
High resolution melting experiments
II. PART 2 (February 2013)
POST NGS, TILLING and HRM
CSC-IPB, College of Agriculture, UP Los Baños
Generation Advance (M3 Seeds)
of Tomato M2 Families
CSC-IPB, College of Agriculture, UP Los Baños
Generation advance of tomato mutants
IPB Experimental Field
CSC-IPB, College of Agriculture, UP Los Baños
Greenhouse - seedlings
CSC-IPB, College of Agriculture, UP Los Baños
CSC-IPB, College of Agriculture, UP Los Baños
EMS- tomato mutants • 600+ M1:2 families (UPLB)
TILLING
Screening against Tomato Viruses
Induced Virus Resistance • Tomato mutants
• DNA markers
DNA Marker Development through BIOINFORMATICS • Host R-genes • Virus avirulence gene sequence • Host factors for virus life cycle
CSC-IPB, College of Agriculture, UP Los Baños
Ultimate Outcome
OPTIMUM FRUIT QUALITY of harvest
Full-maturity of crop in the field
Critical especially for processing tomato
Stable supply and market price
Year-round production including dry months with high
population of insect-vector
Increase net income of farmers by ~P 76,600/Ha
Significant savings on pesticide use & associated costs.
Ex-ante economic analysis (Mamaril, 2009)
Potential of P 25.5M to P 180.5M total economic benefits
(evenly distributed among producers & consumers)
Ex-ante economic analysis (Mamaril, 2009)
CSC-IPB, College of Agriculture, UP Los Baños
Molecular biologists/breeders
Professional and student researchers
Public & private tomato breeding
institutions/seed companies
Eventual COMMERCIALIZATION – release of
ToLCV/TYLCV resistant tomato variety(ies)
Ultimate beneficiaries – tomato farmers
and other industry stakeholders, including
in organic agricultural production
Target Beneficiary
CSC-IPB, College of Agriculture, UP Los Baños
Thank you!
CSC-IPB, College of Agriculture, UP Los Baños
Bulacan Ilocos
Pangasinan Nueva Ecija
Baguio
Los Baños
Tomato ToLCV (Tomato leaf curl virus)
CSC-IPB, College of Agriculture, UP Los Baños
Tomato CMV (Cucumber mosaic virus)
CSC-IPB, College of Agriculture, UP Los Baños
Fulbright Visiting Scholar Program ( No. G-1-00005)