TILLING in tomato: development of a EMS mutant collection ... · TILLING in tomato: development of...
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TILLING in tomato: development of a EMS mutant collection and molecular screening for point mutations INTRODUCTION We are applying TILLING strategy (1) to tomato for the development of tomato lines carrying agronomic and nutritional traits of interest. In the present work we show results concerning the development and phenotypic characterization of a tomato mutant collection originated by treating tomato seeds (cv Red Setter) with two different concentrations (0.7% and 1%) of the chemical mutagen EMS. Furthermore preliminary data of the molecular screening for point mutations of interest will be presented. MATERIAL and METHODS EMS mutagenesis Tomato seeds (cv Red Setter) were treated with two different concentrations (0.7% and 1%) of the chemical mutagen EMS (ethyl methane sulfonate) for 18h at RT with gentle shaking. Then, seeds were extensively washed with H 2 O, dried and sown in compost in 96 well seed trays, which allowed an accurate determination of germination frequency. Plant growth M1 and M2 tomato plants were either field and greenhouse soil transplanted and grown to maturity for seeds harvest. DNA extraction and analysis Genomic DNA was prepared from individual plants by using the “ChargeSwitch gDNA Plant Kit” (Invitrogen). The mismatch was cleaved with the endonuclease ENDO I and the detection was performed on denaturing acrylamide gels by using the Licor 4300 DNA analizer. RESULTS and DISCUSSION Roughly 13000 M2 progeny of 6667 EMS-mutagenized M1 tomato plants were grown in open field and scored for their mutant traits. For each mutant phenotype, data and images were recorded and organized in classes and subclasses corresponding to different plant development stages (i.e. seed, seedling) or to different parts of the plant such as leaf morphology or fruits size. Out of 13000 analysed M2 plants, 5000 resulted to show at least a mutant trait and of these 1834 M2 plants resulted to fall in more than one phenotype class. A high number of the mutations affected cotyledons, leaf morphology, size and habit of the plant. A low number of plants showed mutations affecting morphology and colour of the flower as well as fruit colour. In Figure 1 the percentage of defined phenotype classes and some examples of mutant traits are shown. M3 seeds were harvested from individual M2 plants. Most of the tomato M2 plants (9000) produced seed number per fruit like control plants. 1650 (12.8%) M2 plants showed total sterility while 2125 M2 plants resulted to be partial sterile since they produced less then 50 seeds per fruit (Figure 2). All phenotype data are being stored and analyzed with the Microsoft Access 2000 software. Soon after data recording completion, the database, comprising all the mutant information including photographs, will be publicly available. Genomic DNA was extracted from 2000 M2 plants and 1000 M3 plants. For high-throughput screening, we worked on gDNA pooled eightfold. We first begin to screen the rab11a gene (ACC. AJ245570), a gene of the GTPase family thought to be involved in the control of protein trafficking within cells (2). We analysed a 407 bp rab11a exonic fragment on 1000 plants. The mismatch was cleaved with the endonuclease ENDO I and the detection was performed on denaturing acrylamide gels (Figure 3). In these 1000 plants we found 6 mutations. Therefore, with a target fragment size of 1 kb, we estimate an average of 12.5 mutations per 1000 plants. Ongoing activities are being focused on rab11 mutant phenotype characterization and on tomato mutant population screening for genes involved in fruit quality, fruit ripening, and pathogen resistance. References 1) C.M. McCallum, L. Comai, E.A. Greene and S. Henikoff (2000), Plant Physiology 123:439-442 2) C. Lu, Z. Zainal, G. A. Tucker and G. W. Lycett (2001), The Plant Cell 13: 1819-1833 Filomena Carriero (1), Angelo Petrozza (1), Olimpia D’Onofrio (1), Giovanni Sozio (1), Silvia Minoia (2), Florence Piron (2), Abdelhafid Bendahmane (2), Michel Caboche (2), Francesco Cellini (1) (1) Metapontum Agrobios, S.S. Jonica 106, km 448.2, 75010 Metaponto (MT) Italy (2) URGV/INRA, 2 rue Gaston Crémieux, 91057 Evry cedex France E-mail :[email protected] Figure 1. Percentage of defined phenotype classes and examples of tomato mutant traits. Flower morphology 1,68% Cotyledons 15% Fruit morphology 1,8% Disease and stress response 4,8% Flowering 5% Plant size 11,7% Leaf colour 7,0% Fruit colour 0,11% Seed germination into fruit 7,8% Leaf morfology 13% Plant habit 14,4% Fruit size 15% Inflorescence 1,0% Flower colour 0,7% Plant GEM5 – Venice, 11-14 October 2006 0 1000 2000 3000 4000 5000 6000 without seeds 1-10 11-30 31-50 51-125 126-200 >200 seed class intervals number of plants ACKNWLEDGMENTS The authors would like to thank Rosalba Grillo, Giuseppe Festa and Giuseppe Lauria for their technical assistance in genomic DNA preparation. This work is part of the Italyco project under evaluation for financial support by Italian Minestry of University and Research and part of the European project EU-SOL. Figure 2. Histogram showing the number of M2 plants that produced M3 seeds falling in our defined seed class intervals. Figure 3. Example of point mutations detection with the rab11 gene specific primer analysis. The boxed numbers indicate the individual plants having the mutation. 4786 5086 5136 5142 - 5158
Transcript of TILLING in tomato: development of a EMS mutant collection ... · TILLING in tomato: development of...
TILLING in tomato: development of a EMS mutant collection and molecular screening for point mutations
INTRODUCTIONWe are applying TILLING strategy (1) to tomato for the development of tomato lines carrying agronomic and nutritional traits of interest. In the present work we show results concerning the development and phenotypic characterization of a tomato mutant collection originated by treating tomato seeds (cvRed Setter) with two different concentrations (0.7% and 1%) of the chemical mutagen EMS. Furthermore preliminary data of the molecular screening for point mutations of interest will be presented.
MATERIAL and METHODSEMS mutagenesisTomato seeds (cv Red Setter) were treated with two different concentrations (0.7% and 1%) of the chemical mutagen EMS (ethyl methane sulfonate) for 18h at RT with gentle shaking. Then, seeds were extensively washed with H2O, dried and sown in compost in 96 well seed trays, which allowed an accurate determination of germination frequency. Plant growthM1 and M2 tomato plants were either field and greenhouse soil transplanted and grown to maturity for seeds harvest. DNA extraction and analysisGenomic DNA was prepared from individual plants by using the “ChargeSwitch gDNA Plant Kit” (Invitrogen). The mismatch was cleaved with the endonuclease ENDO I and the detection was performed on denaturing acrylamide gels by using the Licor 4300 DNA analizer.
RESULTS and DISCUSSIONRoughly 13000 M2 progeny of 6667 EMS-mutagenized M1 tomato plants were grown in open field and scored for their mutant traits. For each mutant phenotype, data and images were recorded and organized in classes and subclasses corresponding to different plant development stages (i.e. seed, seedling) or to different parts of the plant such as leaf morphology or fruits size. Out of 13000 analysed M2 plants, 5000 resulted to show at least a mutant trait and of these 1834 M2 plants resulted to fall in more than one phenotype class. A high number of the mutations affected cotyledons, leaf morphology, size and habit of the plant. A low number of plants showed mutations affecting morphology and colour of the flower as well as fruit colour. In Figure 1 the percentage of defined phenotype classes and some examples of mutant traits are shown. M3 seeds were harvested from individual M2 plants. Most of the tomato M2 plants (9000) produced seed number per fruit like control plants. 1650 (12.8%) M2 plants showed total sterility while 2125 M2 plants resulted to be partial sterile since they produced less then 50 seeds per fruit (Figure 2). All phenotype data are being stored and analyzed with the Microsoft Access 2000 software. Soon after data recording completion, the database, comprising all the mutant information including photographs, will be publicly available.Genomic DNA was extracted from 2000 M2 plants and 1000 M3 plants. For high-throughput screening, we worked on gDNA pooled eightfold. We first begin to screen the rab11agene (ACC. AJ245570), a gene of the GTPase family thought to be involved in the control of protein trafficking within cells (2). We analysed a 407 bprab11a exonic fragment on 1000 plants. The mismatch was cleaved with the endonuclease ENDO I and the detection was performed on denaturing acrylamide gels (Figure 3). In these 1000 plants we found 6 mutations. Therefore, with a target fragment size of 1 kb, we estimate an average of 12.5 mutations per 1000 plants. Ongoing activities are being focused on rab11 mutant phenotype characterization and on tomato mutant population screening for genes involved in fruit quality, fruit ripening, and pathogen resi stance.
References1) C.M. McCallum, L. Comai, E.A. Greene and S. Henikoff (2000), Plant Physiology 123:439-4422) C. Lu, Z. Zainal, G. A. Tucker and G. W. Lycett (2001), The Plant Cell 13: 1819-1833
Filomena Carriero (1), Angelo Petrozza (1), Olimpia D’Onofrio (1), Giovanni Sozio (1), Silvia Minoia (2), FlorencePiron (2), Abdelhafid Bendahmane (2), Michel Caboche (2), Francesco Cellini (1)
(1) Metapontum Agrobios, S.S. Jonica 106, km 448.2, 75010 Metaponto (MT) Italy (2) URGV/INRA, 2 rue Gaston Crémieux, 91057 Evry cedex France
E-mail :[email protected]
Figure 1. Percentage of defined phenotype classes and examples of tomato mutant traits.
Flower morphology
1,68%
Cotyledons15%
Fruit morphology1,8%
Disease and stress response
4,8%Flowering
5%
Plant size11,7%
Leaf colour7,0%
Fruit colour0,11%
Seed germination into fruit
7,8%
Leaf morfology13%
Plant habit14,4%
Fruit size15%
Inflorescence1,0%
Flower colour0,7%
Plant GEM5 – Venice, 11-14 October 2006
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1000
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3000
4000
5000
6000
withoutseeds
1-10 11-30 31-50 51-125 126-200 >200
seed class intervals
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mb
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f p
lan
ts
ACKNWLEDGMENTS
The authors would like to thank Rosalba Grillo, Giuseppe Festa and Giuseppe Lauria for their technical assistance in genomic DNA preparation. This work is part of the Italyco project under evaluation for financial support by Italian Minestry of Un iversity and Research and part of the European project EU-SOL.
Figure 2. Histogram showing the number of M2 plants that produced M3 seeds falling in our defined seed class intervals.
Figure 3. Example of point mutations detection with the rab11 gene specific primer analysis. The boxed numbers indicate the individual plants having the mutation.
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