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