Laboratory Protocol Used to Generate Amplified Fragment Length Polymorphisms (AFLPs) for
Oeneis melissa semidea
Genomic DNA (50 – 100 ng) was digested with MseI and EcoRI restriction enzymes and
ligated to MseI and EcoRI adaptors (Table S1) simultaneously in a total reaction volume of 11 μl
(see Tables S2 and S3 for detailed restriction-ligation protocol). Restriction-ligation (R-L)
fragments were then diluted with 89 μl of TE0.1 buffer and stored at -20°C. Diluted R-L
fragments were amplified in a pre-selective PCR using the primers Eco-A and Mse-C (Table S1)
in a total reaction volume of 10 μl (8 μl master mix + 2 μl diluted R-L template; see Table S4 for
detailed pre-selective PCR protocol). The pre-selective primers are complementary to the
adaptors with one additional nucleotide (A for EcoRI and C for MseI) at the 3’ end. Only
restriction fragments with bases complementary to these additional nucleotides (i.e., T and G)
next to the restriction sites will be amplified, reducing the number of fragments to approximately
1/16 of the original amount (Meudt and Clarke 2007). Pre-selective products were diluted with
12.5 μl of TE0.1 buffer and stored at -20°C. Diluted pre-selective products were then amplified
by PCR using selective primers (Eco-AXX and Mse-CXX; Table S1) in a total reaction volume
of 10 μl (8.5 μl master mix + 1.5 μl diluted pre-selective PCR product; see Table S5 for detailed
selective amplification protocol). The selective primers contain two additional nucleotides at the
3’ end, which further reduces the number of fragments to approximately 1/256 of the initial
amount. Six selective primer pairs (EcoRI-AAC + MseI-CAC, EcoRI-AAC + MseI-CTC,
EcoRI-AAG + MseI-CAG, EcoRI-ACA + MseI-CAA, EcoRI-ACC + MseI-CTA, EcoRI-ACG +
MseI-CAA) were used for the selective amplification of Oeneis melissa semidea (Table S1).
Prior to fragment analysis, selective PCR products were diluted 25x with water. Negative
controls were included in each step of the protocol to ensure that no contamination had occurred.
Table S1. Oligonucleotides used for the AFLP analysis of Oeneis melissa semidea.
Protocol Step
Restriction-Ligation
Oligonucelotide
*EcoRI Adaptor 1
EcoRI Adaptor 2
Sequence (5'-3')
CTCGTAGACTGCGTACC
AATTGGTACGCAGTCTAC
Size (bp)
17
18
MseI Adaptor 1
MseI Adaptor 2
GACGATGAGTCCTGAG
TACTCAGGACTCAT
16
14
Eco+A
Mse+C
GACTGCGTACCAATTCA
GATGAGTCCTGAGTAAC
17
17
Eco+AXX
GACTGCGTACCAATTCAAC
GACTGCGTACCAATTCAAG
GACTGCGTACCAATTCACA
GACTGCGTACCAATTCACC
GACTGCGTACCAATTCACG
19
19
19
19
19
Pre-Selective PCR
Selective PCR
†
Mse+CXX
GATGAGTCCTGAGTAACAA
GATGAGTCCTGAGTAACAC
GATGAGTCCTGAGTAACAG
GATGAGTCCTGAGTAACTC
GATGAGTCCTGAGTAACTA
*EcoRI and MseI adaptors must be annealed separately before use in restriction-ligation
reactions (Table S2).
†
All Eco-AXX primers were fluorescently-labelled with FAM dye.
19
19
19
19
19
Table S2. Protocol for annealing EcoRI and MseI adaptors for use in restriction-ligation
reactions.
Annealing Reaction
EcoRI
Reaction Component
EcoRI Adaptor 1
EcoRI Adaptor 2
water
*T10E1
Total
MseI Adaptor 1
MseI Adaptor 2
water
*T10E1
Total
*T10E1 = 10 mM Tris, 1 mM EDTA
MseI
Concentration
1000 μM
1000 μM
Volume (μl)
1
1
108
90
200
1000 μM
1000 μM
10
10
90
90
200
Each master mix was prepared separately, placed in a thermalcycler for 8 min at 94°C followed
by 10 min at 22°C, and centrifuged at 1400 g for 10 s.
Table S3. Protocol for restriction-ligation (R-L) reactions.
Reaction Component
water
T4 DNA ligase buffer with ATP
NaCl
BSA
MseI Adaptor*
EcoRI Adaptor*
MseI (1 U)
EcoRI (5 U)
T4 DNA ligase
genomic DNA (μl)
Total
Concentration
10x
0.5 M
1 mg/ml
100 U
500 U
1U
5U
100 Weiss U
10 - 50 ng
Volume (μl)
0.55
1.1
1.1
0.55
1
1
0.1
0.05
0.05
5.5
11
*MseI and EcoRI adaptors must be annealed as described in Table S2 before
use in R-L reaction.
R-L reactions were held at 24°C for 16 h in a thermalcycler, diluted with 89 μl TE buffer, and
stored at -20°C.
Table S4.Protocol for pre-selective PCR amplification.
Reaction Component
water
betaine
PCR buffer
MgCl2
dNTPs
Mse-C primer
Eco-A primer
*Platinum® Taq DNA polymerase
diluted R-L product
Total
Concentration
3M
10x
25 mM
10 mM
10 μM
10 μM
5 U/μl
-
Volume (μl)
1.5
3.5
1
0.65
0.25
0.5
0.5
0.1
2
10
*produced by Life TechnologiesTM
Preselective PCR thermalcycling conditions: one cycle of 72°C for 2 min; 25 cycles of 94°C for
20s, 56°C for 30 s, and 72°C for 2 min; and a final hold of 60°C for 30 min.
Table S5. Protocol for selective PCR amplification.
Reaction Component
water
PCR buffer
MgCl2
dNTPs
*Platinum® Taq DNA polymerase
Eco+AXX primer
Mse+CXX primer
diluted pre-selective PCR product
Total
*produced by Life TechnologiesTM
Concentration
10x
25 mM
10 mM
5 U/μl
10 μM
10 μM
-
Volume (μl)
4.4
1
1.75
0.25
0.1
0.5
0.5
1.5
10
Selective PCR thermalcycling conditions: one cycle of 94°C for 2 min; 94°C for 20 s, 66°C for
30s decreasing by 1°C/cycle, and 72°C for 2 min for 10 cycles; 94°C for 20 s, 56°C for 20 s, and
72°C for 2 min for 22 cycles; and a final hold of 60°C for 30 min.