Supplementary Materials
1. The CAG Protocol for SNP and InDel detections using agarose gel
1) Collect 1-2 mg of plant material (about two 2 mm-long young fragments of leaves in rice)
and put them directly into each well of a 96-well PCR plate (BIOplastics, cat. no. AB19700,
the Netherlands) that is preloaded with 4 glass beads (2 mm in diameter, Sigma, USA). Add
150 μL of extraction buffer (10 mM Tris, pH 9.5, 0.5 mM EDTA, 100 mM KCl) to each well
and cover the plate with a sealing mat (Axygen, cat. no. AM-96-PCR-RD, USA).
2) Beat the plate vigorously for 1 min with a Mini-Beadbeater-96TM (GlenMills, USA), and
then centrifuge at 2,000 g for 5 min.
3) Use 1 μL of supernatant for a 10-μL PCR containing 0.2 mM dNTP, 0.16 μM forward and
reverse primers, 0.5 unit ExTaq polymerase (TaKaRa, cat. no. DRR100B, Japan) in 1× ExTaq
buffer. PCR program: 95°C for 2 min; 8 cycles of 94°C for 20 sec, (Tm + 3C) for 30 sec
(1°C decrease per cycle), 72°C for 60 sec for every 1 kb of amplicon length; and 35 cycles of
94°C for 20 sec, (Tm - 4C) for 30 sec, 72°C for 60 sec; the then final extension at 72°C for 5
min. Immediately after the PCR program is finished, a complete denaturation-slow annealing
is carried out: 99°C for 10 min; 70 cycles of 70°C for 20 sec (0.3°C decrease per cycle); then
hold at 15°C. Check PCR products by running a 1% agarose gel if necessary.
Critical: Design primers to allow the target SNPs or InDels to be located at least 250 bp away
from the two ends (referred to as “blind ends”), and not in the very middle of the amplicon.
Adherence to these primer design principles will ensure that both of the cleaved products will
be visible on the gel.
4) In parallel, perform another set of reactions using a 1:1 mixture of mutant and wild-type
DNA samples, using the same method described in step 3 above.
5) CELI digestions are performed with re-annealed PCR products from steps 3 and 4 in a 15
μl reaction mixture containing 4 μL PCR product and 1 unit CELI (10 units/μL, home-made)
in CELI buffer (10 mM HEPES, pH 7.5, 10 mM KCl, 10 mM MgSO4, 0.002% Triton X-100
and 0.2 μg/mL BSA) at 45°C for 15 min, followed by the addition of 3 μL 0.5 M EDTA (pH
8.0) to stop the reaction.
Alternatives: MBN digestions are performed in a 15 μL reaction mixture containing 4 μL
re-annealed PCR products from steps 3 and 4. 2 units MBN (10 units/μL, New England
Biolabs, cat. no. M0250S, USA) in a MBN buffer (20 mM Bis-Tris pH 6.5, 10 mM MgSO4,
0.2 mM ZnSO4, 0.002% Triton X-100 and 0.2 μg/ml BSA) at 60°C for 30 min, followed by
the addition of 2 μL 0.2% SDS to stop the reaction.
6) Electrophoresis: digested products are loaded on 2% agarose gel (HydraGene, cat. no.
9012-36-6, USA) containing 0.5 μg/ml ethidium bromide (Amresco, cat. no. 0492, USA) and
1× TAE electrophoresis buffer, and separated at 130 V for 30 to 60 min, depending on the
fragment size. Gel images acquired using standard UV transillumination equipment such as a
BioSpectrum AC Imaging System (UVP, USA).
7) The electrophoresis results are analyzed by comparing the band patterns in the two gels
with (step 4) or without (step 3) the wild-type DNA. A plant is homozygous for the SNP or
InDel if two cleaved bands are only detected in the samples containing wild-type DNA (step 4
reactions). A plant is is a homozygous wild-type if no cleaved PCR product is observed in
either gel. A plant is heterozygous for the SNP or InDel if cleaved PCR products are observed
in both gels.
2. The CCG protocol for SNP and InDel detections using capillary FS96 analyzer
1) Following the same procedure as CAG protocol (above) until step 5.
2) The CELI digested products are diluted to 100 μL with ddH2O. Capillary electrophoresis is
then performed with an FS96 analyzer (Advanced Analytical, USA) at 9 kV for 30 sec for
pre-run, 15 sec for marker injection, 45 sec for sample injection, and 40 min for sample
separation, using the DNF-920 dsDNA reagent kit (Advanced Analytical Technologies, cat.
no. DNF-920-K0500, USA) by following the manufacturer’s manual. Acquire the pseudo gel
pictures using PROSize software (Advanced Analytical, USA).
3) Analyze the results as in step 7 of the CAG protocol above.