development of tomato non-host tomato virus through ...tgc.ifas.ufl.edu/2013/2013/12 -...

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


(2011 – 2014)

Philippines (DOST)-Taiwan (NSC)Cooperative R&D Project

Philippines: HF Galvez, AO Canama, RB Quilloy, DV Lantican, RV Cortes Crop Science Cluster-Institute of Plant Breeding

College of Agriculture, UP Los Baños

LL Tandang, BA Kebasen IPB-UPLB-BSU Highland Crop Research Station, Benguet State University

TILLING of tomato for multiple virus resistance

Taiwan Counterpart:

R Schafleitner, L Kenyon, P Hanson AVRDC – The World Vegetable Center


Targeting Induced Local Lesions IN Genome

(TILLING)

Till BJ, SH Reynolds, EA Greene, CA Codomo, LC Enns, JE Johnsons, C Burtner, AR Odden,

K Young, NE Taylor, JG Henikoff, L Comai and S Henikoff. 2003. Large-scale discovery of

induced point mutations with high-throughput TILLING. Gen Res 13:524-530.


Project Framework 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


• Amplify mutant + reference DNA • Hybridize DNA hetero- and homoduplex formation

• Digest hetero- with single-strand DNA nuclease

• Fragment analysis

M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis

Mutagen (EMS and gamma ray)

Phenotyping ofsegregating M2 plants

“Knock-outs” Interesting mutants

M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis

Mutagen (EMS and gamma ray)Mutagen (EMS and gamma ray)



M1:2 plants

Mutational (molecular) analysis SNPs detection

M2:3 generation of confirmed mutants

M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M2:3 disease screening against ToLCV and CMV

Plant Virus

Resistance

and TILLING

Targeting Induced Local Lesions IN

Genome (TILLING)

Generate mutants as series of

variants (allele) for any target gene

Identify plants with point-mutation

at virus resistance gene sequences

Screen confirmed mutants for

resistance to target virus diseases


• Amplify mutant + reference DNA • Hybridize DNA hetero- and homoduplex formation

• Digest hetero- with single-strand DNA nuclease

• Fragment analysis

M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M1:2 plants

Mutational (molecular) analysis SNPs detection

M2:3 generation of confirmed mutants

M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M0 generation seed

(Hawaii 7996)

Heterozygous

M1 plants

M2 plants

BWR screening

Collect seeds

Molecular analysis




M2:3 disease screening against ToLCV and CMV

Year 1 to 2

Grow M1:2 plants; harvest leaf samples for DNA (UPLB)

Develop DNA markers & mutation screen of M1:2 for target R-genome sequences (UPLB & AVRDC)

Year 3

Select confirmed M1:2 mutants; grow M3 families (UPLB & AVRDC)

Screen M3 tomato mutants against ToLCV/CMV strains (UPLB & AVRDC)

UPLB/IAEA Research Project

(Galvez et al., 2009)

600+ M1:2 - EMS chemical mutagen

400 M1:2 – Gamma ray irradiation

1,000+ M1:2 families generated


Expected Output

Allelic mutants of tomato with induced resistance against ToLCV/CMV (Year 3)

Point-mutations/SNPs as DNA markers tagging the induced virus resistance (Year 3)

Functional genomics characterization of virus resistance mechanism in tomato (Year 2)

Induced vast germplasm of tomato for virus resistance

DNA MARKERS tagging the induced resistance for MAS


Workplan

Y1 Y2 Y3

Objectives Expected Output Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

X X X X X

X XX X X X X X

X X X X XX X X X XTo screen for loss-of-

susceptibility mutants of

tomato against

TYLCV/TolCV, CMV

To develop DNA markers

tagging the viral resistance

gene sequences

Allelic variants of tomato

with induced virus

resistance

Molecular characterization

of induced resistance gene

sequences

Functional genomics

characterization of virus

resistance in tomato

Point-mutations and SNPs

as DNA markers tagging the

induced virus resistance


Just completed generation advance (M3 seeds) of

large tomato M2 families.

Genomic DNAs processed from pooled M2 families;

individual plant DNA extraction on-going.

Initial set of DNA markers available to mine candidate

virus-host factors in tomato. These were designed

using relevant bioinformatics tools.

Accomplishment Highlights


Next generation sequencing (NGS) of amplified candidate

genes/loci is on-going with 15 DNA markers and using pooled

M2 families

Analysis of NGS results and search for interesting SNPs using

appropriate bioinformatics facility will proceed after NGS.

High-throughput phenotyping identifies interesting visible

mutations.

o High frequency of mutation observed in leaf morphology

and plant habit.

Accomplishment Highlights


Theoretical Background/

Rationale


TOMATO (Philippines)

High-value commodity - everyday ingredient of many dishes

Fresh or processed: rich source of vitamins, nutrients & Lycopene

2010 (BAS, Philippines) Tomato

Prod Area (Ha) 17,663 (2nd)

Prod Volume (MT) 204,272 (3rd)

Yield (MT/Ha) 11.56

Value of Production (USD) ($1 = P 42)

50 M

Background and Rationale


Major Production Constraint : VIRUS DISEASES

Mainly Tomato Leaf Curl Virus (ToLCV) (Sta. Cruz et al. 2008)

ToLCV infection can reach 90-100%, with 50-100% yield loss

(DA-BAR Chronicle, 2010)

Philippine varieties : susceptible to tolerant (Sta. Cruz et al. 2008)

Production USD

($1 = P 42)

Value (BAS, 2010) 50 M

Ave. Yield Reduction (%) 50

Value of Potential Yield 100 M

Losses (virus disease) 50 M


Monopartite Begomovirus (family Geminiviridae)

DNA-A like genome (circular ssDNA)

C4 and C2 proteins - suppressor of RNA silencing

(PVX-mediated transient assay)

Whitefly vector (Bemisia tabaci Genn.)

– circulative manner

At least 4 strains of ToLCPV

2 in Luzon and all 4 in Mindanao

Tomato leaf curl Philippine virus (ToLCPV)*

* Dolores & Bajet 1995-Philippine J Phytopathol 31; Kon et al. 2002 -J Phytopathol 150; Matsuda et al. 2008 -

Arch. Virol 153; Tsai et al. 2010 –Ann Appl Biol; Sharma et al. 2011–Arch Virol 156


V1 = CP; V2 = Pre-CP

C1 = Rep

C2 = TrAP

C3 = Ren

C4 = small ORF in Rep, different reading frame

http://www.ncbi.nlm.nih.gov


Tomato leaf curl Philippines betasatellite (ToLCPHB)*

DNAβ (betasatellite)

Requires helper begomovirus DNA (eg. ToLCPV)

- Replication, Encapsidation

- Insect transmission, Movement in plants

Pathogenecity determinant

- Induce severe symptom – co-inoculated with

ToLCPV (PVX-mediated transient assay)

~ half size & little sequence similarity with ToLCPV DNA

Conserved hairpin structure and a TAATATTAC loop

βC1 protein – suppressor of RNA silencing

* Sharma et al. 2011–Arch Virol 156


http://www.ncbi.nlm.nih.gov


Phytosanitation - rouging

Insect vector control- insecticide sprays

Cultural methods

Host plant resistance

Conventional breeding

Induced resistance

Genetic engineering

Virus Management


Status of Host Plant Resistance and Breeding

Resistance to CMV is not available in tomato germplasm

No resistant lines have been developed yet by conventional

approaches (AVRDC Publication 04-610, 2004)

CMV resistant transgenic lines available but not effective to

Philippine strains of CMV (Galvez et al., unpublished data).

TYLCV resistant or tolerant tomatoes commercially available

BUT NOT EFFECTIVE with Philippine ToLCV

TYLCV and ToLCV – different virus species

(Zitter, 1991; Sta. Cruz et al., 2008; Tsai et al. 2010)

Tolerant tomato varieties for Philippine ToLCV (ToLCPV)

Fresh market-tomato hybrids available in private seed co.

Processing tomato: tolerant variety being bred by NFC-DA

(DA-BAR Chronicle, 2010)


Characterized resistance genes (Ty loci) – Ty1 to Ty5

Reyes et al. unpublished data:

o Ty1 or Ty2 tomato lines – very susceptible to Phil ToLCV

o Ty3 + unknown R-gene(s) – moderate resistance

AVRDC - different combinations of Ty-1 to Ty-3 (Hanson 2009)

o Reduced symptom severity (TOLERANCE)

o DNA markers and MAS breeding established

o Best Ty2 + Ty3 tomato lines recommended (Hanson 2011)

Efforts continuing to identify and map other resistance

sources/loci (Hanson 2009)

Breeding for broad TYLCV/ToLCV resistance

Also resistance to insect vector

Status of Host Plant Resistance and Breeding


Development of Tomato Non-Host to

Tomato leaf curl (Philippine) virus


Virus Resistance Mechanisms in Plants

Kang BC et al., Annu. Rev. Phytopathol. (2005)


• Viruses require host factors to complete their life cycle

• Absent or mutated host factors may cause recessive virus resistance

Kang et al., 2005


Whitham S.A. and Wang Y. Curr Opin Plant Biol. (2004)

Summary of host proteins implicated in plant virus infection cycles


Recessive Virus Resistance Genes

Maule A.J. et al., Molecular plant pathology (2007)

eIF4E eIF(iso)4E

eIF4E eIF4E

eIF4E


TILLING for Crop Improvement

Crop/Plant sp. Trait Gene Reference Remarks

For Virus Resistance

Tomato potyviruses i.e. PVY, TEV and PepMoV eIF4E1, eIF4E2, eIF(iso)4E, eIF4G, eIF(iso)4G Piron et al. 2010 Immunity to two potyvirues; publish

the different primers designed to

amplify target genes in tomato

Melon MNSV and CVYV eIF4E, eIF4G Nieto et al. 2007

Watermelon potyviruses i.e. ZYMV, PRSV, WMV, SqVYV eIF4E Levi et al. (USDA) 2010 -

http://www.nationalwatermelonassociation.

com

Barley Bymovirus (BaMMV-BaYMV complex)

resistance

Hv-eIF4E (Rym 4/5 Locus) http://194.47.52.113/janlars/partnerskapAlna

rp/ekonf/20091022/SvenGottwalds.pdf

Other Traits

Tomato fruit quality traits, disease & stress response

i.e. necrosis, wilting

RIN, Gr, Rab11a, Exp1, PG, Lcy-b, Lcy-e Minoia et al. 2010 TILLING platform publicly available via

web for mutation screening services

Tomato various potentially all "The Genes That Make Tomatoes" Watanabe et al. 2007 TILLING platform in micro-Tom in SOL

wheat, soybean, maize,

lettuce, tomato, rice,

various various Slade and Knauf 2005

Soybean oil content, protein content, allergen-free various http://www.ars.usda.gov/is/AR/archive/jul0

5/genes0705.htm

Melon fruit shelf life ACC oxidase, L124F Dahmani-Mardas et al. 2010

Wheat waxiness waxy genes i.e. Wx-A1, Wx-D1 Dong et al. 2009

Wheat decreased or absent amylose fraction GBSSI or starch synthase I Slade et al. 2005 in commercial application

Wheat various various Parry et al. 2009

Barley semi-dwarfism/lodging resistance, powdery

mildew resistance, two-rowed spike

sdw1, mlo, HvHox1 (Vrs1 Locus) http://194.47.52.113/janlars/partnerskapAlna

rp/ekonf/20091022/SvenGottwalds.pdf

Barley powdery mildew resistance mlo, mla resistance genes Mejlhede et al. 2006

Africa orphan crop e.g. Tef semi-dwarfism/lodging resistance plant height genes, green revolution genes www.atdforum.org/journal/html/2009-34/5/

Arabidopsis, maize, lotus,

barley, wheat

various various Till et al. 2007 proof-of-concept to production in crop

improvement

various various various Till et al. 2007


Piron F. et al., PLoS ONE 5(6) 2010

Piron F. et al., PLoS ONE 5(6) 2010

Immune to two potyviruses

Primers designed to

amplify eIF4E1, eIF4E2, eIF(iso)4E, eIF4G, eIF(iso)4G in tomato

Watanabe et al. Plant

Biotech 24(33:38) 2007

TILLING platform in micro-Tom in SOL database

Tomato TILLING


Tomato leaf curl virus

CSC-IPB, College of Agriculture, UP Los Baños Lozano-Duran et al. 2011.


Lozano-Duran et al. 2011.

Heat shock protein cognate 70 (HSC70)

70% reduced TYLCSV accumulation Coatomer delta subunit (deltaCOP)

100% abolished or 0% virus accumulation


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


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)


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


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


PCR amplification 15 TYLCV and CMV

candidate host factors


PCR fragments standardized and precipitated

for NGS (lllumina Hi-Seq2000)

New pool 1


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


Generation Advance (M3 Seeds)

of Tomato M2 Families


Generation advance of tomato mutants

IPB Experimental Field


Greenhouse - seedlings


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


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)


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


Thank you!


Bulacan Ilocos

Pangasinan Nueva Ecija

Baguio

Los Baños

Tomato ToLCV (Tomato leaf curl virus)


Tomato CMV (Cucumber mosaic virus)


Fulbright Visiting Scholar Program ( No. G-1-00005)

development of tomato non-host tomato virus through ...tgc.ifas.ufl.edu/2013/2013/12 -...

Documents