Poster for CIAT 2009 Knowledge Sharing Week

1. Tagging and genotyping SNPs in crop plants using xMAP technology
Constanza Quintero, Germán Plata , Fausto Rodríguez, Mathias Lorieux and Joe Tohme
CIAT (International Center for Tropical Agriculture), Agrobiodiversity and Biotechnology Project, AA6713, Cali, Colombia;
c.quintero@cgiar.org
INTRODUCTION Figure 1. No effect of reduction in PCR enzyme
7000
Average MFI (called alleles)
Single nucleotide polymorphisms are becoming the marker of choice for a wide variety of 0.025U 0.0125U
6000
organisms, due to their high abundance in genomes, their evolutionary stability and the
5000
availability of high throughput technologies for their detection. Our interest in enhancing
SNP genotyping capacity is the integration of these markers as a cost-effective tool for 4000
selection in breeding programs, through the use of the xMAP technology and the Luminex 3000
analyzer. One of the bottlenecks we faced, was the massive identification of SNPs and 2000
the automated design of multiplex PCR panels to have access to the putative SNPs
1000
located in candidate genes. It was understood that computational tools were needed, since
preliminary assays showed that those activities could be time-consuming. Standardization 0
and adjustment of reagents concentrations in single base extension (SBE) assays were 3 4 11 15 16 24 32
Bead
also considered, in order to reduce the cost per data point but keeping the accuracy and We also concluded that decreasing Thermo Sequenase (USB, Cleveland, Ohio) up to
the reproducibility of the genotyping method. Our research is focused in rice crop model 0.008U and microspheres up to 1000 beads/reaction produced no dramatic differences in
where two main objectives are targeted: a) the identification of SNPs in candidate genes MFI values (Figure 2). Cost per data point has been reduced 6-fold in comparison to the
involved iron metabolism and their integration in the generation of new rice varieties with experimental conditions at the beginning of the project (0.1U of AccuPrime Taq ; 0.128U of
high iron content in the polished grain, and b) the development of a set of 140 SNPs Thermo Sequenase and 3000 beads/reaction).
identified in genes associated with tolerance to abiotic stresses (drought, acid soils,
AVERAGE MFI (called alleles)
mineral deficiencies or toxicities), to validate their utility and to explore their value in 12000
Figure 2. No effect of reduction in SBE enzyme and beads
breeding programs for other cereals. 0.032 Units 0.016 Units 0.008 Units
10000
MATERIALS AND METHODS 8000
SNP identification 6000
414 genes 4000
(iron metabolism + stress related) 2000
0
mRNAs retrieved (GenBank) ds) ds) ds) ds) ds) ds) ds) ds) ds)
bea bea bea bea bea bea bea bea bea
0 00 500 000 000 500 000 000 500 000
3 (2 3 (1 3 (1 6 (2 6 (1 6 (1 5 (2 5 (1 5 (1
SN
P NP NP P1 P1 P1 P1 P1 P1
S S SN SN SN SN SN SN
BLAST against rice genes
SNP genotyping
Retrieval of japonica (TIGR4) and To date, primers for 376 SNP sites have been tested, most of them in SBE reactions
indica (BGI) genomic fragments (3Kb up and downstream) carried out in multiplex of 13 to 19 SBE primers, joined and quantified in a Luminex-100.
Approximately 64% of the putative SNPs were validated as true polymorphisms (Table 1)
BLAST Alignment and SNP detection and, up to 35 extended products are successfully read together (Figure 3).
Table 1. Multiplex kits assayed.
Data storage Experiment design IRON METABOLISM GENES STRESS RELATED GENES
# SNPs # SNPs
Multiplex kit Multiplex kit
Putative Validated Monomorphic Putative Validated Monomorphic
SNP genotyping
0 32 26 0 1 30 21 3
SNP genotyping was carried out with the single base extension (SBE) method using the 1 27 14 4 2 30 20 0
flow cytometer Luminex 100 as platform following the next steps: 2 27 17 1 3 30 17 2
3 27 18 0 4 30 16 2
PCR amplification of SNP-containing DNA fragments 4 27 17 1 5 29 24 0
5 27 17 0 6 29 20 0
6 16 7 1 Total 178 118 7
Enzymatic removal of excess dNTPs and primers 7 11 4 0
8 3 3 0
SBE or minisequencing Total 197 123 7
Figure 3. Simultaneous calling of 35 SNP alleles.
Hybridization of extended SBE primers with the coupled beads G
G
8,000
SNP detection as Mean Fluorescent Intensity with the Luminex 100 7,500
7,000 G
6,500
Allele calling with Masterplex™ GT (Miraibio, Inc) 6,000 G G
G
5,500 G
G G G G
Design of multiplex PCR and SBE panels 5,000
G
G
G
G
MI
F
4,500
Previous work with the bean crop model showed us that large differences in amplicons size 4,000
G G G
G
G
could be a major constraint for multiplex PCR. To avoid this problem we choose a size for 3,500
G
G
G
G
the SNP-containing fragment to be between 80-120bp. PCR primers were designed using 3,000
G
G
G G
G
G G
2,500
Primer3 (Rozen and Skaletsky, 2000) under the following restrictions: length, 19-30 2,000
G
G GG
G
G
G G
G
G G G
nucleotides; GC%, 40-60; Tm, 50-60°C. SBE Primers were designed with SBEPrimer 1,500
G
G
G
G G
G G
G G
G
G
(length, 18-20 nucleotides; Tm, 50-75°C) and false priming was tested with the same 1,000
G G G
500
software (Kaderali et al., 2003). AutoDimer software (Vallone and Butler, 2004) was used
0
for testing primer dimer formation of PCR oligos.
11
12
13
14
19
23
24
25
27
30
31
34
35
39
40
41
42
43
44
45
47
48
49
15
17
18
26
28
29
37
38
2
3
6
8
BEAD
RESULTS CONCLUSIONS
SNP identification and multiplex design The designed platform (Perl modules and MySQL relational database) is flexible
Plata and Rodríguez implemented Perl modules and a MySQL relational database which enough that it can be used for in silico SNP discovery in other crops.
allowed the identification of 7664 SNPs in 414 rice gene sequences. Also, the development 64% of the identified rice putative SNPs were validated as true polymorphisms in
of a multiplexing algorithm, allowed the design of 86 PCR panels (kits) to amplify 1928 multiplex reactions of up to 35 extended products.
SNPs. The different modules of softwares, previously cited, could be run under the same Adjustment of key reagent concentrations allowed a 6-fold reduction in the cost per
platform. The highest number of SNPs that was targeted per PCR panel was 34. data point, which is desirable for marker-assisted selection projects.
Optimizing PCR and SBE assays REFERENCES
Since we are pursuing the integration of SNP markers to breeding programs for marker- Kaderali, L., et. al. (2003). Nucleic Acids Res, 31, 1796-1802
assisted selection, enhancing multiplex capacity together with a decrease in the cost per Rozen, S. and Skaletsky, H. (2000) . Methods Mol Biol, 132, 365-386
data point are required. We found that the reduction of AccuPrime Taq DNA polymerase Vallone, P.M. and Butler, J.M. (2004). Biotechniques, 37, 226-231
(Invitrogen, Carlsbad, Calif.) up to 0.0125U/SNP had no effect in the accuracy of the allele ACKNOWLEDGEMENTS
calling and SNPs continue to be genotyped correctly (Figure 1). Special thanks to Agrosalud Project and the Generation Challenge Program for funding of this
research.