Appendix S2: Molecular Methods
Mitochondrial amplification
The complete mitochondrial cytochrome-b gene (1140 base pairs) was amplified using universal
primers L14723 and H15915 (Table 1, Irwin et al. 1991). Total reaction volume of 25 µl
included 100-200 ng of DNA, 0.12 µM of each primer, 1.5 mM MgCl2, 0.012 mM each dNTPs,
1X reaction buffer, 0.32 mg/mL Bovine Serum Albumin and 0.625 U GoTaq Taq polymerase
(Promega Corporation, Madison, Wisconsin, USA). The thermal profile included an initial
denaturing step at 95oC for 3 min followed by 34 cycles of 95oC for 1 min, 50oC for 1 min and
72oC for 1 min, followed by a final extension time of 72oC for 10 min.
Nuclear amplification for X- and Y-chromosome markers
Amplicons of approximately 800 bps in length of intron eight of transketolase-like 1 (TKTL1)
gene on the X-chromosome were amplified using unpublished primers (Table 1). Amplicons of
approximately 940 bps in length of intron 11 of Ubiquitously Transcribed Tetratricopeptide
Repeat Containing gene (UTY) were amplified using primers outlined in Hellborg and Ellegren
(2003) (Table 1). The total reaction volume of 25 µl included 100-200 ng of DNA, 0.5 µM of
each primer, 0.125 mM each dNTPs, 1X reaction buffer, 0.15 X DMSO and 1 U Phire Hot Start
II DNA Polymerase (Thermo Fisher Scientific, Pittsburg, PA, USA). The thermal profile
included an initial denaturing step at 98oC for 1 min 45 sec followed by 34 cycles of 98oC for 20
sec, anneal for 20 sec and 72oC for 40 sec. The annealing temperatures selected for each primer
combination were two degrees higher than that estimated for the primer with the lower melting
point (Table 1).
Sanger sequencing
Cycle sequencing was performed with BigDye version 3.1. (Applied Biosystems, Inc., Foster
City, California, USA) and primers listed in Table 1 with reaction conditions following
manufacturers protocols. Sequences were electrophoresed on and ABI 3130 (Applied
Biosystems, Inc., Foster City, California, USA) sequencing platform.
AFLP methods
The AFLP protocol followed Vos et al. (1995) with thermal profile modifications described by
McDonough et al. (2008). Genomic DNA was digested with AseI and EcoRI restriction enzymes
(New England Biolabs Inc., Ipswich, Massachusetts, USA). Preselective PCR was performed
using primers AseI-T and EcoRI-C and selective PCR was performed using seven primer pairs
on all individuals (Table 2). Selective PCR products along with Genescan-400HD ROX size
standard were electrophoresed on an ABI 3100-Avant genetic analyzer (Applied Biosystems,
Inc., Foster City, California, USA). Fragments ranging from 50 to 400 nucleotides were scored
for presence or absence of bands using GeneMapper version 4.0 software (Applied Biosystems).
All ambiguous fragments (i.e., fragments that were weakly amplified in some individuals) were
removed from the final data set. Error rate and reproducibility were evaluated by replicating 10%
of the reactions as suggested by Bonin et al. (2004) and an error was considered as an
amplification that was inconsistent between replicates.
Table 1. Primers used for PCR and sequencing.
Primer Name
CYTB
L14723
H15915R
UTY
UTY11F
UTY11R
TKTL1
PCR
TKTL1_X8Fb
TKTL1_X9Rb
Sequencing
TKTL1_X8Fc
TKTL1_X9Rc
Primer sequence
Tm (DegC)
5'-ACCAATGACATGAAAAATCATCGTT-3'
5'-GGAATTCATCTCTCCGGTTTACAAGA-3'
61.4
63.4
5'-CATCAATTTTGTAYMAATCCAAAA-3'
5'-TGGTAGAGAAAAGTCCAAGA-3'
53.2
50.9
5'-GCCATCTCTCAAATCAACAT-3'
5'-GGCTGCTAAGTAAACAGCAT-3'
53.5
53.4
5'-YGATTGCTGGAAACAGGAAA-3'
5'-CCCAAGGCTTTGTCTTTCA-3'
Table 2. Primers used for selective amplification of amplified fragment length polymorphism
(AFLP) bands. Asterisk indicates fluorescently labeled primer.
Primer Name
EcoRI-CAT*
AseI-TAC
AseI-TAG
AseI-TCT
AseI-TGC
AseI-TGT
AseI-TGA
AseI-TGG
Sequence
5'-GACTGCGTACCAATTCCAT-3'
5'-GATGAGTCCTGAGTAATTAC-3'
5'-GATGAGTCCTGAGTAATTAG-3'
5'-GATGAGTCCTGAGTAATTCT-3'
5'-GATGAGTCCTGAGTAATTGC-3'
5'-GATGAGTCCTGAGTAATTGT-3'
5'-GATGAGTCCTGAGTAATTGA-3'
5'-GATGAGTCCTGAGTAATTGG-3'
References
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Hellborg L, Ellegren H (2003) Y chromosome conserved anchored tagged sequences (YCATS)
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