1. 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

2. Marker assisted selection
Accelerate breeding
Reduces work load
Enhances accuracy
Lowers costs
SOURCE: Syngenta

3. MAS MAS is said to lower the breeding costs.
But MAS requires considerable investment into:
Human resources
Equipment
Reagents

4. Number of data points produced
Breeding support
Trait discovery
Number of data points
Nowadays, most data points are produced for trait discovery, and much less for breeding support. For entities focusing on section for a few traits, high throughput screening tools only make sense if the sample amount is really huge.
Source: Monsanto

5. Assumption
MAS for a limited number of resistance genes on a moderate number of entries
Investment into high throughput technology not economically viable
Classical MAS costs (100,000 reactions): -Reagents: USD 30 k -HR: 24 person months
5 loci
Some 1,000 genotypes/population
10 populations
2 cycles
100,000 reactions

6. Reducing costs Reduce working costs and reagents Sampling
DNA extraction
PCR
Scoring
Data handling

7. DNA Extraction Test of several simple methods (max. 2 steps)
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
Methods yielding good quality DNA need multiple working steps and require special chemicals or instruments (colums).

8. 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 ????)

9. Saving time and reagents for PCR: Multiplexing
R-gene Marker name Marker type Chromosome Reference
Ty-1 TG178 SCAR 6 Barbieri et al., 2010
Ty-3 P SCAR 6 Ji et al., 2008
Ty-1/3 M2 SCAR 6 AVRDC
Ty-2 T0302 SCAR 11 Garcia et al., 2007
Ty-2 TES0344 SSR 11 Yang et al., 2012
ty-5 AVRDC-TM719 SSR 4 AVRDC
ty-5 SLM4-34 SSR 4 AVRDC
ty-5 SINAC1 (TAQ I) SSR 4 Anbinder et al., 2009
ty-5 AVRDC-TM273 SSR 4 AVRDC
ty-5 AVRDC- TM81 SSR 4 AVRDC
ty-5 AVRDC- TM70 SSR 4 AVRDC
ty-5 AVRDC-TM947 SSR 4 AVRDC
Mi-1 PM3F/R SCAR 6 El Mehrach et al., 2005
Mi-1.2 Mi23 SCAR 6 Seah et al., 2007
Tomato yellow leaf curl and nematode resistance genes are frequently used in breeding. Therefore markers selecting for 3 Ty loci and 1 nematode R locus were chosen for testing multiplex PCR. For some loci, CAPS marker are routinely used for detection. But for multiplex, CAPS are not preferred, therefore either SCAR or SSR markers have been chosen, or developed and tested. All markers here have been tested for being diagnostic in different breeding populations from India, Indonesia and Taiwan.

10. Saving time and reagents for PCR: Multiplexing
Select markers for multiplex testing
Simple and clear banding pattern
Distinct size differences between PCR bands
Locus
Marker
Type
Chr.
Pos.
Frag. length
Reference
Ty-1/3 M2
SCAR 6
30.875
264/252
Present study
Ty-2
T0302 11
~900/791
Garcia et al., 2007
Ty-5
AVRDC- TM273
SSR 4
3.2
~180/173
Mi-1.2
Mi23
2.322
~380/431
Seah et al., 2007

11. 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
Cross-hybridization among primers is less of a problem, especially at high Tm

12. Multiplex PCR R S
Ty-2
Mi-1
Ty-1/3
ty-5

13. Ty-2 Mi-1 Ty-1/3 ty-5 R S Tanya BL982 CLN2498D CLN3024A CLN3205B
CLN3212C
CLN3150A-5
CLN3126A-7
CLN3447G
CLN3070J
CLN3241H-27
CLN3125P
CLN2819B
F8-48
F9-159
CLN3682F
FLA456
CLN3125K
LA1969
LA1932
Marker
Ty2 S R Mi
Ty1,3
ty5 R S
Ty-2
Mi-1
Ty-1/3
ty-5

14. Multiplex PCR Ty-2 Mi-1 Ty-1/3 ty-5S
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

15. 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

16. 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

17. 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)

18. New markers http://www.tomatogenome.net/VariantBrowser/.
Tomato RAD-tags
Line 1 2 3 4 5 6 7 8 9 10 11 12

19. Cost Analysis Virus resistance screening - Activity/expense Cost (US$)
Phenotypic Selection Glasshouse and trial preparation Labor
Glasshouse supplies
G2 stage, 2000 genotypes Seed preparation
Trial planting, inoculation and maintenance Labor 3,796
Pots, potting mix
Inoculum preparation
Incidentals
Trial harvest and cultivar screening Labor
ELISA testing
Trial clean-up Labor
Total cost 6,681
Cost per cultivar
single pentaplex assay
MAS per 96 samples Sample harvesting and DNA extraction Labor
Consumables
G0 stage PCR amplification Labor
Consumables
Product detection and analysis Labor
Consumables
Total cost
Per-sample cost
Cost per data point per cultivar
Slater et al., Mol Breeding (2013) 32:299–310

20. COSTS (100,000 samples) Saves 67% of reagent costs and 70% of labor
Quick DNA & Multiplex
Standard Method
DNA isolation
PCR for MAS for 4 loci
Labor
US$ 15,000
US$ 19,000
26 person months
US$ 5,900 US$ 5,300 6 person months
Costs: 11 cents reagent costs per data point.
Saves 67% of reagent costs and 70% of labor