An overview of research project done for PHRO Krzeszowice that turned into my MSc. thesis.

1. Selection of tomato plants
resistant to a local isolate
of Tomato Spotted Wilt Virus
(TSWV)

2. • 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

3. Taxonomy
• Family Bunyaviridae
• Class Tospovirus

4. • Lipid envelope
glycoproteins G1 &
G2
• N protein
• L protein
(polymerase)
• 3 ss RNA forming
loop (hairpin or
panhandle)
(Jansen, Lenoir, 1998)
Virus particle

5. Viral proteins expression
strategy

6. Transmission
• Thrips – transmited
(Frankiniella
occidetalis)

7. Signs of infection
• Growth inhibition
• Necrotic spots
• Spots on fruits
• Chlorotic patterns

8. 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

9. 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

10. 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)

11. SCAR 421 identification
scheme

12. SCAR 421 identification
Standards
• 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

13. SCAR 421 identification
Standards
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

14. SCAR 421 identification
breeding 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.

15. SCAR 421 identification
results
• A - resistant and susceptible homozygotic plants
• B - resistant homozygotic plants
• C - resistant heterozygotic plants

16. 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

17. Greenhouse test
principles
• 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.

18. Greenhouse test
results
• First signs of thrips feeding were observed
on leaves after a week of exposure in the
test chamber. They developed in time.

19. Greenhouse test
results
Thrips feeding evidence.

20. Greenhouse test
results
• 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

21. Greenhouse test
results
• Left – plant with
TSWV disease
symptoms
• Right – plant with no
TSWV disease
symptoms

22. Greenhouse test
results
no symptoms deformed leaf severe chlorosis

23. Greenhouse test
results
no symptoms brown spots zoomed

24. Greenhouse test
results
• 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.

25. ELISA tests
principles
• 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.

26. ELISA tests
results
• 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

27. ELISA tests
results

28. Biological tests
review
Accession
SCAR 421
pattern
Physiological
symptoms
Presence 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

29. 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

30. Team
Andrzej Stefan Czech
prof. dr hab. Kazimierz Strzałka
Zakład Fizjologii i Biochemii Roślin,
Instytut Biologii Molekularnej i Biotechnologii,
Uniwersytet Jagielloński
dr Marek Szklarczyk
prof. dr hab. Barbara Michalik
Katedra Genetyki Hodowli i Nasiennictwa,
Wydział Ogrodniczy, Akademia Rolnicza w Krakowie
mgr Zbigniewowi Gajewskiemu
prof. dr hab. Tadeuszowi Kobyłko
Zakład Botaniki,
Wydział Ogrodniczy, Akademia Rolnicza w Krakowie
dr Ewie Żukowskiej
PHRO w Krzeszowicach