Transcript Title of presentation

Reducing the costs of marker-assisted selection
through simplified DNA extraction and multiplex PCR
assays for tomato
ROLAND SCHAFLEITNER, CHEN-YU LIN, PETER HANSON
Sept. 09, 2014
ASRT 2014, Sep 9-10, 2014, Bangalore, India
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Marker assisted selection
• Accelerate breeding
• Reduces work load
• Enhances accuracy
• Lowers costs
SOURCE: Syngenta
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MAS
MAS is said to lower the breeding costs.
But MAS requires considerable investment into:
 Human resources
 Equipment
 Reagents
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Number of data points
Number of data points produced
2007
Source: Monsanto
2014
Breeding support
Trait discovery
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Assumption
MAS for a limited number of resistance genes
on a moderate number of entries
5 loci
Some 1,000 genotypes/population
10 populations
2 cycles
100,000 reactions
 Investment into high throughput technology not economically viable
 Classical MAS costs (100,000 reactions):
-Reagents: USD 30 k
-HR: 24 person months
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Reducing costs
Reduce working costs and reagents
 Sampling
 DNA extraction
 PCR
 Scoring
 Data handling
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DNA Extraction
 Trade-off between DNA quality and extraction effort
 DNA from simple methods may not amplify in PCR or may
not be storable
Test of several simple methods (max. 2 steps)
for storability and multiplex PCR
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DNA Extraction
Alkaline extraction (Wang et al., 1993) works best:
 Extract 10 mg fresh tissue with 0.5 N NaOH
 Dilute extract 100-fold with 100 mM Tris-HCl pH 8.0
 Use 1 ml directly for PCR
 Store at 4.0 °C (1 month) or at -20 °C ( up to ????)
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Saving time and reagents for PCR: Multiplexing
R-gene
Marker name
Marker type
Chromosome
Reference
Ty-1
Ty-3
Ty-1/3
Ty-2
Ty-2
ty-5
ty-5
ty-5
ty-5
ty-5
ty-5
ty-5
Mi-1
Mi-1.2
TG178
P6-25
M2
T0302
TES0344
AVRDC-TM719
SLM4-34
SINAC1 (TAQ I)
AVRDC-TM273
AVRDC- TM81
AVRDC- TM70
AVRDC-TM947
PM3F/R
Mi23
SCAR
SCAR
SCAR
SCAR
SSR
SSR
SSR
SSR
SSR
SSR
SSR
SSR
SCAR
SCAR
6
6
6
11
11
4
4
4
4
4
4
4
6
6
Barbieri et al., 2010
Ji et al., 2008
AVRDC
Garcia et al., 2007
Yang et al., 2012
AVRDC
AVRDC
Anbinder et al., 2009
AVRDC
AVRDC
AVRDC
AVRDC
El Mehrach et al., 2005
Seah et al., 2007
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Saving time and reagents for PCR: Multiplexing
Select markers for multiplex testing
• Simple and clear banding pattern
• Distinct size differences between PCR bands
Locus
Ty-1/3
Ty-2
Ty-5
Mi-1.2
Marker
M2
T0302
AVRDC- TM273
Mi23
Type
SCAR
SCAR
SSR
SCAR
Chr.
6
11
4
6
Pos.
30.875
51.0878
3.2
2.322
Frag. length
264/252
~900/791
~180/173
~380/431
Reference
Present study
Garcia et al., 2007
Present study
Seah et al., 2007
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Adapt assay conditions
• Apply 150 nM instead of 200 nM of each primer
• Increase dNTP from 200 to 250 nM
• Separate fragments on 4% instead of 6% non-denaturing
polyacrylamide gels to increase the resolved size range
11 of x
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Multiplex PCR
12 of x
R
S
Ty-2
S
R
Mi-1
R
S
Ty-1/3
R
S
ty-5
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BL982
CLN2498D
CLN3024A
CLN3205B
CLN3212C
CLN3150A-5
CLN3126A-7
CLN3447G
CLN3070J
CLN3241H-27
CLN3125P
CLN2819B
F8-48
F9-159
CLN3682F1-10-156-1-3
FLA456
CLN3125K
LA1969
LA1932
S
S
R
R
S
S
R
R
R
R
R
R
R
S
S
R
S
R
S
S
Mi
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
Ty1,3
S
R
S
S
R
S
S
R
S
R
R
R
R
S
S
R
S
S
R
R
ty5
S
S
S
S
S
R
R
S
R
S
S
S
S
R
R
S
R
S
S
S
R
S
Ty-2
S
R
Mi-1
R
S
Ty-1/3
R
S
ty-5
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13 of x
Marker
Tanya
Ty2
Multiplex PCR
Ty-2
R
S
Mi-1
S
R
Ty-1/3
R
S
ty-5
R
S
• Reliable detection of “R” and “S” alleles at 4 loci in homozygous and heterozygous plants
• Mirror bands in heterozygous Mi-1 and ty-5 loci appeared, but did not affect the
diagnostic capacity of the test
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Flexibility of the multiplex assay
BW12 marker
(SLM12-2)
Ty-2 (P1-16)
BW-12 (SLM12-2)
ty-5 (TM273)
Ty-1/3 (–k)
• Introducing the bacterial wilt resistance marker BW-12 (instead of Mi)
• Modified (shortened) Ty-1/3 marker to better accommodate the 4 markers on the gel
15 of x
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Flexibility of the multiplex assay
Ph-3 [S] (R2M1S)
Ty-2 [R] (P1-16)
Ph-3 [R] (R2M1S)
Ty-2 [S] (P1-16)
Ty-1,3 (Ty1,3 –M2)
ty-5 (TM273)
• Introducing the late blight resistance marker Ph-3 (instead of Mi)
• Modified (shortened) Ty-2 marker to better accommodate the 4 markers on the gel
16 of x
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Multiplex assay
Multiplex assays for tomato MAS can be easily modified to
accommodate different sets of markers
• 6 assays for 7 loci tested
• Interactions between primers do not affect the assay
• Fragment size of the markers needs to be adapted for multiplexing:
▪ Easy redesign of SCAR or SSR markers based on available
Tomato WGS
▪ RAD-seq tags for most important tomato lines available at AVRDC to
identify new markers
▪ 100 tomato re-sequencing project (Aflitos et al., 2014)
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New markers
Tomato RAD-tags
http://www.tomatogenome.net
/VariantBrowser/.
Line
1
2
3
4
5
6
7
8
9
10
11
12
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Cost Analysis
Virus resistance screening - Activity/expense Cost (US$)
Phenotypic Selection
G2 stage, 2000 genotypes
MAS per 96 samples
G0 stage
Glasshouse and trial preparation
Labor
Glasshouse supplies
Seed preparation
Trial planting, inoculation and maintenance Labor
Pots, potting mix
Inoculum preparation
Incidentals
Trial harvest and cultivar screening
Labor
ELISA testing
Trial clean-up
Labor
Total cost
Cost per cultivar
Sample harvesting and DNA extraction
PCR amplification
Product detection and analysis
Total cost
Per-sample cost
Cost per data point per cultivar
Slater et al., Mol Breeding (2013) 32:299–310
Labor
Consumables
Labor
Consumables
Labor
Consumables
705
100
5
3,796
540
160
100
345
900
130
6,681
222.7
single
132.66
228.44
57.89
82.53
50.65
179.62
731.79
7.70
7.70
pentaplex assay
132.66
228.44
57.89
83.45
50.65
179.62
732.71
7.71
1.54
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COSTS
(100,000 samples)
Standard Method
 DNA isolation
 PCR for MAS
Quick DNA & Multiplex
US$ 15,000
US$ 19,000
US$ 5,900
US$ 5,300
26 person months
6 person months
for 4 loci
 Labor
Saves 67% of reagent costs and 70% of labor
20 of x
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21 of x
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