Supplemental Methods. Microsatellite Marker Development for Platanus racemosa.
Microsatellite Locus Design
Microsatellites (n=28; 27 dinucleotide repeats, 1 trinucleotide repeat) were
developed and tested in Platanus racemosa using the FIASCO (Fast Isolation by AFLP
of Sequences Containing repeats) technique with slight modification to maximize
isolation efficiency (Zane et al. 2002). They were later tested samples from isolated
populations of P. occidentalis and P. orientalis to identify species-specific markers (Lang
2010). This technique involves enrichment of microsatellite containing regions of the
genome, thus increasing the odds of detection. Approximately 100 ng genomic DNA (P.
racemosa) was digested with the restriction enzyme MseI, and ligated using T4 ligase
with a MseI adapter (5’-TACTCAGGACTCAT-3’ / 5’-GACGATGAGTCCTGAG-3’).
The restriction ligation mixture was incubated at 37 C overnight and then diluted in 50
l dH2O. A combined polymerase chain reaction (PCR) was conducted with all four
MseI primers (5’-GATGAGTCCTGAGTAAN-3’) with program parameters of 72 C for
3 min; 20 cycles of 94 C 30 sec, 53 C 1 min, 72 C 2 min; and a final step of 72 C for
7 min. Hot start was not used in order to allow nicks in the ligated DNA to be filled by
the Taq DNA polymerase. Resultant products were visualized on a 1% agarose gel
showing a smear roughly 400 bp – 2,000 bp long. To concentrate the product it was
dried with a vacufuge and resuspended in 43 l dH2O.
The resultant product (43 l) was hybridized to a 5’-biotinylated (AT)17 probe (8
l) with 42 l 10x SSC and 7 l 1% SDS in a 100 l reaction volume at 95 C for 3 min
and 25 C for 15 min. Streptavidin beads (1mg) were washed with TEN100 (10 mM
Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5) three times, re-suspended in 40 l of
TEN100, and unrelated PCR product (10 l) was added. To capture the probe-DNA
complex, the DNA-probe was first diluted with 300 l TEN100 and then combined with
the prepared Streptavidin beads. The mixture was incubated at room temperature for 30
min on a shaker (gentle agitation). Six nonstringency washes were then performed with
400 l TEN100, an incubation of 5 min at room temperature with gentle mixing and
recovering of the DNA by magnetic field separation. Six stringency washes were then
performed with 400 l SSC (0.2 x) and 0.1% SDS, and an incubation of 5 min at room
temperature with gentle mixing. The final wash was stored for further use.
To recover the DNA from the beads-probe complex two denaturation steps were
performed, one with TE and one with NaOH. For the TE elution, 50 l of TE were added
to the Streptavidin beads and incubated at 95 C for 5 min and the supernatant containing
the target DNA was stored. For the NaOH elution, 12 l of 0.15 M NaOH were added
after which the mixture was incubated for 5 min at 95 C. To the supernatant, 7.68 l
0.1667 M acetic acid were added to neutralize the pH, and TE (30.32 l) were added to
reach the final volume of 50 l.
Once recovered from the Streptavidin beads, the DNA was precipitated. One
volume of isopropanol and sodium acetate (0.15 M final concentration) was added to
each of the following: last non-stringency wash (400 l), last stringency wash (400 l),
TE elution (50 l), and NaOH elution (50 l). Each of these should contain DNA
fragments containing the selected repeat (AT)17. The mixtures were kept at –20 C for 30
min then centrifuged at maximum speed for 15 min. The supernatant was discarded and
tubes were placed in a speed-vac to completely dry remaining pellets. Each pellet was
resuspended in 50 l dH2O.
PCR product from the NaOH elution was dried with a speed-vac and resuspended
in 4 l of dH2O to be used for cloning fragments into Escherichia coli. First the DNA
was inserted into the plasmid vector. The 4 l microsatellite enriched DNA was
combined with 1 l salt solution and 1 l Topo vector and gently mixed. The ligation
reaction was incubated for 30 min. To transform the E. coli 25 l of TOP 10 competent
cells was gently mixed with 6 l of vector containing target DNA and incubated on ice
for 10 min. The cells were heat shocked at 42 C for 45 sec and then immediately
transferred to ice. Room temperature SOC medium (250 ul) was added to the cells and
placed on a shaker at 225 rpm and 37 C for 1 hour. Half of the cell culture along with
40 l x-gal (40 mg/ml) was spread onto a LB plate and the remaining culture was spread
on a second plate with x-gal, and incubated overnight at 37 C.
Direct colony PCR was performed from isolated transformed (white) colonies. PCR was
done in 25 l reactions with H2O (13.9 l), 10x buffer (2.5 l), dNTPs (5 l), MgCl2 (1
l), primers T7 and M13R (1.25 l each), and Taq polymerase (0.1 l). The program
used consisted of an initial step of 97 C for 2 min, 30 cycles of 97 C 30 sec, 48 C 1
min, 72 C 2 min (+ 45 sec per cycle), and a final step of 72 C for 7 min. PCR products
were visualized on a 1 % agarose gel and successfully amplified samples were purified
with Exo1 (exonuclease) and Antarctic phosphatase at 37 C for 15 min and 80 C for 15
min. Samples were sequenced and screened for microsatellite loci. A total of 191
colonies were amplified via PCR, 186 of which were screened for microsatellite loci. Of
those, 52 microsatellite loci were discovered, although only 28 of those contained repeats
 4 with flanking regions sufficient to generate both forward and reverse primers. Primers
were developed in the range of 19 to 25 bp in length that produced fragments ranging
from 100-400 bp, for later multiplexing. Primers were tested on a subset of individuals
from each study taxon to check for primer compatibility, 10 of which successfully
amplified (Supplemental Table 1).
Supplemental Table 1. Microsatellite primer sequences for 10 loci developed for P.
racemosa in the full dataset (missing data included). Includes annealing temperature
(Ta), observed size range (bp) for entire data set, number of successfully called genotypes
out of 679 individuals (N), and number of revealed alleles (A).
Size range
Ta (˚C)
(bp)
N
A
(TC)9
61
194 - 234
608
15
(CTT)4
61
313-334
593
4
(TG)10
61
121-135
631
6
(GA)4(GT)5
61
315
596
1
(CA)18
61
115-150
509
8
(CT)11(CA)16
61
203-231
521
12
(AC)6
61
222-230
530
5
(CA)7
61
198-218
614
9
Locus
Primer sequence 5'-3'
Repeat motif
plms29
F: GCCCATTAGATGGGTTGAAA
R: AGCGAATCCATGTGCCTAAT
plms53
F: GCAACTTGGTCTTGGTTGGT
R: CAGCCGATTGGGTATATGGT
plms71
F: ACGGGTGAGCTCCCTACTTT
R: GACATCCTCCACCAAACACC
plms92
F: TCCTTACATCTTTGCCCACA
R: CCCATGAACCTCTCTGATCC
plms109
F: TGATGACAAATACTCAGGGAAA
R: CGATAGCCAAAAGCGAAAGA
plms113
F: GGCAAGCCAGGATTTAGTTG
R: CGGGATAAGAGTTTGTTGAGTTG
plms122
F: CTTCTGTGCTTGTGCCTCAC
R: CTTTGCACCAATGTGCCTTA
plms130
F: TACCACACCAACGTCCTTCC
R: ACCCTCTCAAATATGCCAATTA
plms136
F: GGCACCCTAATTACCCACCT
(GT)9
61
219
533
1
(CA)9
61
259-275
521
7
R: TCTGATCCCGACAAAACCAT
plms176
F: AACAGCAAAACAGCCCACTC
R: AAACCAGCCAATCCAATTCC