Supplementary material 1
Restriction: Double Digests was carried out with restriction EcoR I/Mse I, The restriction time was 3 h at
37ºC, and 2.5 h at 65ºC. The reagents were prepared according to Table s1.
Adapter-ligation: ligation reaction should be completed as soon as possible after restriction. The ligation
condition was incubating for 2 h at 25ºC or incubating overnight at room temperature. After ligation, the
products were diluted 10 times and pre-amplified. Adapter refolding: the adapter in ligation process should
be refolded before using. The refolding thermal cycling conditions was incubating 10 min at 65ºC, 10 min at
37ºC, 10 min at 25ºC and 25 min at 4ºC. The adapter formulation and ligation reaction system were
detailed demonstrated in Table s2, Table s3.
Pre-amplification: the PCR program was 20 cycles including 94ºC for 30 s, 56ºC for 1 min, 72ºC for 1 min.
After pre-amplification, the reaction products were diluted 50 times for selective amplification.
Pre-amplification reaction system was detailed demonstrated in Table s4.Selective amplification: selective
amplification program applied "touch-down" strategy, and the temperature lowered by 0.7 oC at each cycle
as follows: in 1st cycle, the condition was 94ºC for 30 s, 65ºC 1 min, 72ºC for 1 min; from 2nd to 13th cycle,
the annealing temperature decreased by 0.7ºC each cycle, and the remaining steps were same as the 1st
cycle; from 14th to 36th cycle, the annealing temperature was 56ºC, and the remaining steps were same as
the 1st cycle. The selective amplification reagents were in Table s5.
Table S1. Components of double enzyme digestion system for AFLP (20μL).
Components
Volume (μL)
10* reaction buffer
EcoR ǀ (10 U/μL)
Mse ǀ (10 U/μL)
Genomic DNA(50ng/μL )
Sterilized distilled water
Total volume
2.0
0.3
0.3
5.0
12.4
20.0
Table S2. Formula of adapters.
10μL
10μL
180μL
EcoR ǀ adapter(5uM)
100 uM
100 uM
Sterilized distilled water
EcoR I.1
EcoR I.2
100μL
100μL
Mse ǀ adapter(50uM)
100 uM
100 uM L
Table S3. Components of ligation for AFLP.
Components
Volume (μL)
Adapters buffer
Mse ǀ adapter(50 uMoL/L)
EcoR ǀ adapter(5 uMoL/L)
Sterilized distilled water
T4 ligase (3 U/µL)
Total volume
4.0
1.0
1.0
13.6
0.4
20
MseI.1
MseI.2
Table S4. Reaction system of pre-amplification.
Components
Diluted template DNA
Mse ǀ (25 ng/µL)
EcoR ǀ (25 ng/µL)
MgCl2(50 mmoL/L)
Taq DNA polymerase (5 U/µL)
dNTPs(2 mmol/L)
10* reaction buffer
Sterilized distilled water
Total volume
Volume (μL)
5.0
2.0
2.0
0.6
0.08
1.0
2.0
7.32
20.0
Table S5. Reaction system of selection PCR amplification of AFLP.
Components
Diluted template DNA
EcoR ǀ primer(10 ng/µL)
Mse ǀ primer(10 ng/µL)
MgCl2(50 mmoL/L)
Taq DNA polymerase (5 U/µL)
dNTPs（2 mmoL/L）
10* reaction buffer
Sterilized distilled water
Total volume
Volume (μL)
5.0
2.0
6.0
0.6
0.2
2.0
2.0
2.2
20.0