Supplementary Information File 2
Offspring-parent assigments and sexing
Offspring-parent assigments and sexing were carried using DNA-based methods. For this
purposes, total genomic DNA was extracted from ethanol preserved fin clips using 96well plate method proposed by Ivanova et al. [1]. All individuals used in the experiments
were genotyped at ten microsatellite loci previously shown to be polymorphic in threespined sticlebacks (Table S2). Polymerase chain reactions were carried out in 8µl final
volume that contained 4 µl 2xPhusion® Flash High-Fidelity PCR Master Mix (Thermo
Scientific), 1µl primer mix (2 pmol), 2 µl H2O and 1 µl of diluted DNA (10 ng/µl).
Thermal cycling protocol consisted of 1 min of initial denaturation at 98°C, 34 cycles of
98°C for 1s, annealing at 60°C for 15 s and extension at 72°C for 20s, followed by the
final extension for 1 min at 72°C. PCR products were diluted 1:280 and screened in ABI
3730 DNA analyzer (Applied Biosystems) using 500 ROX (Applied Biosystems) as an
internal size standard. Alleles were scored with Genemapper V. 4.1 software (Applied
Biosystems).
Genotypes were checked for typographical and genotyping errors as well as for null
alleles with Micro-checker software [2]. Using 10 000 simulations and confidence
interval of 99% no loci showed evidence for null alleles. All samples (n = 282; 45 parents
and 237 offspring) were genotyped at seven or more loci with 95 % of all samples being
genotyped at 9 to 10 loci. Parental genotypes (15 fathers and 30 mothers) were used to
calculate microsatellite statistics (Table SI2) and allele frequencies in Cervus 3.0 version
[3]. Offspring assignment to parents (30 families) was conducted using maximumlikelihood (ML) method implemented in Cervus. Suggestions about parentage with a
certain level of confidence in Cervus are made when likelihood ratio (LOD score) of
parental pair-offspring exceeds a certain threshold. This critical value was estimated
based on 10 000 simulations with genotyping error rate of 1%. Simulations were run
assuming that all fathers and all mothers were sampled, as it was the case in our
experiment. All offspring, but two were assigned to parent pair with strict (95%)
confidence. Because of a half-sib experimental design, we were able to check if the most
likely parent pair suggested by Cervus was indeed crossed. Therefore, in case of one
offspring that was not assigned to parental pair right away, but was assigned only a father
with 95% confidence, we ran a separate analysis using the same parameters as described
above, and were able to determine the most likely mother out of two candidates.
Offspring sex was identified by amplifying a part of 3’UTR of the NADP-dependent
isocitrate dehydrogenase (Idh) gene [4]. This molecular sexing protocol yields two bands
for three-spined stickleback males and one band for females. Final volume of PCR
reactions was 10 µl and consisted of 1 µl 10 x NH4 reaction buffer, 0.3 µl MgCl2
(50mM), 0.08 µl dNTPs (25 mM of each), 0.32 µl forward and reverse primers (10
pmol), 0.05 µl Biotaq polymerase (5U/µl) (Bioline), 6.93 µl H2O and 1 µl of diluted
DNA (10 ng/µl). The following thermal cycling protocol was used: 94°C for 3 min, 38
cycles with 94°C 30 s, 94°C for 30 s, 56°C 30 s, 72 °C 1 min and 5 min at 72°C for final
extension. Amplicons were separated by agarose gel electrophoresis, visualized under
UV light and scored.
Table SI2. Microsatellite loci used in the study with their GenBank accession numbers,
number of alleles per locus (NA), expected heterozygosity (HE) and the source of primer
sequences.
Locus
Stn3
Stn23
Stn30
Stn42
Stn96
Stn110
Stn168
Stn174
Stn195
Stn223
GenBank accession
number
G72128
G72137
G72241
G72148
G72176
G72182
G72209
G72310
G72221
BV102499
NA
HE
Source
10
15
13
17
9
15
22
10
11
28
0.727
0.758
0.852
0.907
0.783
0.881
0.903
0.802
0.87
0.951
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[5]
[6]
References
1. Ivanova NV, Dewaard JR, Hebert PDN. 2006 An inexpensive, automation-friendly
protocol for recovering high-quality DNA. Mol. Ecol. Notes 6, 998–1002.
2. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P. 2004 MICROCHECKER:
software for identifying and correcting genotyping errors in microsatellite data. Mol.
Ecol. Notes 4, 535–538.
3. Kalinowski ST, Taper ML, Marshall TC. 2007 Revising how the computer program
CERVUS accommodates genotyping error increases success in paternity assignment.
Mol. Ecol. 16, 1099–1006.
4. Peichel K, Ross JA, Matson CK, Dickson M, Grimwood J, Schmutz, J, Myers RM,
Mori S, Schluter D, Kingsley DM. 2004 The master sex determination locus in three
spine sticklebacks in a nascent Y chromosome. Curr. Biol. 14, 1416–1424.
5. Peichel CL, Nereng KS, Ohgi KA, Cole BL, Colosimo PF, Buerkle CA, Schluter D,
Kingsley DM. 2001 The genetic architecture of divergence between threespine
stickleback species. Nature 414, 901–905.
6. Colosimo PF, Peichel CL, Nereng K, Blackman BK, Shapiro MD, Schluter D,Kingsley
DM. 2004 The genetic architecture of parallel armor plate reduction in threespine
sticklebacks. PLoS Biol. 2, E109