Selection of tomato plants resistant to a local isolate of Tomato Spotted Wilt Virus (TSWV)
Jul 25, 2015
Selection of tomato plants resistant to a local isolate
of Tomato Spotted Wilt Virus (TSWV)
• 1 bln $/year lost worldwide.• 800 species of plants in 80
families serves as host • susceptible plants: tomato,
tobacco, peanuts, dahlia, chrysanthemum and weeds
• no chemical control available
Why investigate TSWV resistance?
Peanuts
Resistant Susceptible
Taxonomy
• Family Bunyaviridae
• Class Tospovirus
• Lipid envelope glycoproteins G1 & G2
• N protein• L protein
(polymerase)• 3 ss RNA forming
loop (hairpin or panhandle)
(Jansen, Lenoir, 1998)
Virus particle
Viral proteins expression strategy
Transmission
• Thrips – transmited (Frankiniella occidetalis)
Signs of infection
• Growth inhibition
• Necrotic spots
• Spots on fruits
• Chlorotic patterns
Resistance
• First resistant to TSWV tomato plants were found in 1920-1930
• First stable resistant line were obtained in 1964 by Stevens. (L. peruwianum x L. esculentum)
• Backcrossing and molecular tests showed that the resistance is single loci encoded
• Resistance gene named Sw-5 is a dominant gene
Resistance marker
With a use of RAPD a specific sequence linked to the resistance were found. Sequencing resulted in obtaining SCAR 421 molecular marker.
SCAR 421 (sequence characterized amplification region) is in the distance < 1 cM from Sw-5 gene
Aims of research
• Identification of resistant plants as a base for new breeding lines. (resistant homozygote)
• Identification of resistant plants among crossed plants
• Verification of plant resistance by biological test
All tests were done for PHRO Krzeszowice(marker assisted breeding program)
SCAR 421 identification scheme
SCAR 421 identificationStandards
• As resistant standards Stevens and Sw-5 lines
• As susceptible standards Alisa Craig and Mercury F1
• F1 generation obtained by crossing Stevens and Alisa Craig was used as heterozygotic standard
SCAR 421 identificationStandards
M - GeneRuler 1 kbp.
S/S – homozygote susceptible – single 0.9 kbp band
R/R – homozygote resistant – single 0.94 kbp band
R/S – heterozygote – two bands 0.9 kbp and 0.94 kbp
SCAR 421 identificationbreeding lines
• cultivar Maresme
• commercial line Sw-5, Mm1567, Mm1578, Mm1583, Dr-3
• hybrids - Ms x Sw-5, Br x Stevens, Dr-3 x Sw-5 and Dr-1 x Sw-5.
SCAR 421 identificationresults
• A - resistant and susceptible homozygotic plants• B - resistant homozygotic plants • C - resistant heterozygotic plants
SCAR 421 identification
• Maresme cultivar - heterozygote R/S
• lines Sw-5, Mm1567, Mm1578, Mm1583, homozygotes resistant R/R
• line Dr-3 - homozygote susceptible S/S
• hybrids - Ms x Sw-5, Br x Stevens, Dr-3 x Sw-5 i Dr-1 x Sw-5 - heterozygotes R/S
Greenhouse testprinciples
• Susceptibility to the virus was tested on 12 plants from 10 analysed accession.
• Small plants (two leaves stage) were placed in isolated greenhouse chamber with thrips and plants infected by TSWV
• The TSWV isolate originated from one of local tomato breeders.
Greenhouse testresults
• First signs of thrips feeding were observed on leaves after a week of exposure in the test chamber. They developed in time.
Greenhouse testresults
Thrips feeding evidence.
Greenhouse testresults
• First symptoms of the TSWV disease appeared after 6 weeks, although growth had been inhibited earlier.
• Necrotic spots, streaking, ring spots, stunting, wilting and shoot deformation
Greenhouse testresults
• Left – plant with TSWV disease symptoms
• Right – plant with no TSWV disease symptoms
Greenhouse testresults
no symptoms deformed leaf severe chlorosis
Greenhouse testresults
no symptoms brown spots zoomed
Greenhouse testresults
• TSWV disease observed for most plants from line Dr-3.
• Some noticed for two hybrid plants Br × Stevens.
• The remaining plants did not give any signs of the disease although numerous thrips feeding scars were visible.
ELISA testsprinciples
• All plants used for the greenhouse test were tested.
• Microplates coating: rabbit polyclonal antibodies against TSWV proteins (Anti-Virus-IgG).
• Leaf tissue: crushed in PBS-TPO buffer, two replicates.
• Detection: rabbit polyclonal antibodies against• TSWV, conjugated with alkaline phosphatase
(Anti-Virus-IgG-AP-conjugate).
• A405 measured in automatic plate reader, 1 h after addition of 4-nitrophenyl-phosphate.
ELISA testsresults
• No virus was detected for the plants without TSWV disease symptoms
• No virus was detected for the 2 plants with TSWV disease symptoms
• Virus was detected for 11 plants with TSWV disease symptoms
ELISA testsresults
Biological tests review
AccessionSCAR 421
patternPhysiological
symptomsPresence of
TSWV particles
Mm cultivar R/S 0/12 0/12
Sw-5 R/R 0/12 0/12
Dr-3 S/S 11/12 10/12
Mm1567 R/R 0/12 0/12
Mm1578 R/R 0/12 0/12
Mm1583 R/R 0/12 0/12
Ms x Sw-5 R/S 0/12 0/12
Br x Stevens R/S 2/12 1/12
Dr-3 x Sw-5 R/S 0/12 0/12
Dr-1 x Sw-5 R/S 0/12 0/12
Summary
• General consistency of data generated by PCR, greenhouse and ELISA testing confirms that the Sw-5 gene confers resistance to the TSWV isolate from Poland.
• New breeding lines (homozygotic) resistant to TSWV were selected for further breeding program
• This was the first case of genetic marker assisted breeding program in polish agricultural company
TeamAndrzej Stefan Czechprof. dr hab. Kazimierz StrzałkaZakład Fizjologii i Biochemii Roślin,Instytut Biologii Molekularnej i Biotechnologii, Uniwersytet Jagielloński
dr Marek Szklarczykprof. dr hab. Barbara MichalikKatedra Genetyki Hodowli i Nasiennictwa,Wydział Ogrodniczy, Akademia Rolnicza w Krakowie
mgr Zbigniewowi Gajewskiemu prof. dr hab. Tadeuszowi KobyłkoZakład Botaniki, Wydział Ogrodniczy, Akademia Rolnicza w Krakowie
dr Ewie Żukowskiej PHRO w Krzeszowicach