Supplementary Material
Behavioural tests
Mice trapped as adults in the field were maintained in quarantine under controlled
conditions (12/12 photoperiod, food ad libitum) for two to four months in malefemale pairs before being tested. Preference of males and females was assessed during
two-way choice tests carried out at the end of the light phase. The test apparatus was a
Y-shaped transparent tubular Plexiglas device connected to three boxes, the start box
and two peripheral boxes (Nunes et al. 2009; Latour et al. 2014). A few days before
each test the mouse was left (~10min) to explore the entire empty apparatus, to reduce
stress, neophobia and spatial investigation not directed towards the stimuli during the
experiment.
Urine stimuli (10µl each) were spread over 2cm² delimited areas at the extremity of
each peripheral box. The tubes containing urine were labelled so that behaviour
recording was blind. The left and right positions of the two stimuli were shifted
between tests to avoid any effect of laterality. A given test mouse was introduced in
the start box, separated from the rest of the apparatus with a perforated transparent
sliding door; the slide door was then opened and the test started as soon as the mouse
entered the Y maze. A choice test lasted 5 minutes during which the time spent by the
mouse in contact sniffing or touching the stimuli were recorded with ‘The Observer’
5.0.31 software. The apparatus was thoroughly cleaned between each test.
Urine samples were obtained directly upon handling of a mouse or after leaving it to
urinate in a cleaned box from where urine drops were pipetted and kept in Eppendorf
tubes at -20°C before being used. Sampling took place over several days and times of
the day to capture diurnal variation in urine composition. Urine donors were adult
mice of the two subspecies trapped at the border of the hybrid zone, maintained in
similar standardised conditions to avoid any odour heterogeneity due to differences in
housing conditions or food. The stimuli were pools of urine from 8-9 mice sampled in
distinct sites. Pooling was per sex and subspecies. All females were tested while in
oestrus; pregnant females were neither tested nor used to obtain urine for the stimuli.
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The difference in time spent in close contact with the musculus stimulus versus the
domesticus one was used to compare patterns of preference across sexes and
geography (two-ways ANOVA after checking residuals distribution and
homoscedasticity), and to test whether assortative preference occurred (Student t test
with H0: X = sniff(mus-dom) = 0).
Genomic regions and markers analysed
Search of microsatellite loci and design of PCR primers
We used an automated pipeline (adapted from the msfinder Perl pipeline originally
developed by Dr. Till Bayer from the GEOMAR Helmholtz Center for Ocean
Research) to select in silico the microsatellite loci to study and to design PCR primer
pairs flanking them. Microsatellite loci were searched in the regions of interest (the 49
clusters defined above, sequence obtained from build 37 of the mouse reference
genome) using program Tandem Repeats Finder (TRF) (Benson 1999). We selected
only loci with repeat motif sizes between 2 and 8 nucleotides, with 5 to 50 repeat
motifs, and an alignment match (as compared to a perfect repeat) of at least 80%. We
then defined PCR primers flanking each of these loci, using program PRIMER 3.0
(Untergasser et al. 2012), with default settings, except for PCR product size. The
program was run four times with four different PCR product size ranges (50-150,
151-250, 251-350 and 351-450 base pairs). Each primer pair for a given PCR product
size range was tested by electronic PCR on the whole mouse genome using the e-PCR
program (Rotmistrovsky et al. 2004). When more than one PCR product was thus
predicted, a different primer pair was searched in the same size range, and again
tested by electronic PCR. If this second attempt failed, the locus was discarded for
this size range. For each retained primer pair, the predicted PCR fragment was
searched for tandem repeats other than the focal microsatellite, again using TRF
program. This was intended to eliminate PCR primer pairs encompassing additional
tandem repeats potentially interfering with the focal microsatellite. Tandem repeats
with more than a certain fraction of overlap with the focal microsatellite (measured as
the ratio of the overlap to the size of the focal microsatellite) were considered not
interfering (i.e., part of the same tandem array). We used a threshold of 90% for this
step. This is intended not to eliminate slightly imperfect microsatellites, which are
rather common. Repeats with non-null overlap smaller than this threshold were
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considered interfering. Those with no overlap were considered interfering if at least
one of the following conditions was met: (i) distance to the focal microsatellite less a
certain value (10 base pairs was used); (ii) match to a perfect repeat greater than a
certain value (100% was used, so the condition was never met); (iii) number of
repeats greater than a certain number (5 was used). The PCR primer pairs generating
fragments containing interfering repeats were discarded.
The PCR primer search section of the above algorithm was run once using the
genomic sequence as template, and a second time on the sequence masked for
repeated sequences. All steps other than primer search were identical. For each
combination of locus and PCR size range, we selected only one primer pair, if
possible from the masked template. When the pair had to be chosen from the
unmasked template, we checked for potential overlap of the primers with repeated
sequences, and discarded those pairs for which both primers overlapped.
Choice of loci to PCR-amplify
The in silico process described above recovered all microsatellite loci in the genomic
regions of interest predicted to be amplifiable by PCR, based on the reference genome
sequence, and obeying the various criteria described. In each of the chromosome
segments of interest (“clusters” in the main text), we applied the following strategy to
choose a subset of loci to study experimentally. For each gene in the chromosome
segment (excluding pseudogenes), we chose the closest microsatellite. After removing
these loci, as well as those overlapping with them, the process was reiterated until we
reached the planned size of the experiment, 1,248 loci. In fact this was enough to
saturate the regions of interest, i.e. additional rounds of the choice process would add
loci all lying outside of the interval between the first and last genes of the genomic
fragment considered. Of these 1,248 microsatellite loci, 531 had one of the two
primers overlapping with a repeated element.
Genotyping
General methods
For each microsatellite locus, we chose one PCR size range among the possibilities
offered by the in silico design, while distributing equally (and randomly) the loci
among the four ranges. The corresponding primer pairs were synthesized, with the
addition of an 18-bp 5’tail (5’TGTAAAACGACGGCCAGT3’) to one primer of each
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pair. Since the in silico parameters used to design the primers were identical for all
loci, we used a single PCR condition. An initial denaturation for 5 min at 95°C was
followed by 35 cycles of amplification. Denaturation during each cycle lasted 30 s at
95°C and elongation 30 s at 72°C. Annealing lasted 1.5 min at a temperature that
decreased from 62°C to 56°C by steps of 0.3°C during the first 20 cycles, and was
maintained at 56°C for the remaining 15 cycles. A final elongation at 60°C was
applied for 30 min. The reactions were performed in 384-well plates in a total volume
of 10 µL per well, containing a 1:2 dilution of the commercial Qiagen « Type-it
Microsatellite PCR Kit », 30 ng of genomic DNA, and the primers. The latter
included the locus-specific primer with the 18-bp tail (0.5 pmol), the other locusspecific primer without tail (2 pmol) and a fluorescently labelled primer with the
sequence of the 18-bp tail (1.5 pmol). Each of the four fragment size ranges received
a different fluorescent label for this latter primer (FAM, NED, PET and VIC from the
smallest to the largest size range, respectively). This provided a way to fluorescently
label the PCR products of the many loci to be typed in the main experiment (1,248)
using only four fluorescently labelled oligonucleotides (method described for instance
Schuelke 2000), rather than one specific labelled primer for each locus, as is usually
done in this type of experiment but would have represented a prohibitive cost for our
experiment (1,248 different labelled primers).
For each sample, the PCR products were multiplexed four by four in equal volumes,
combining loci with different colours (also corresponding to the four different PCR
range sizes), and diluted 1:100 in water. A 3 µL aliquot of this dilution was added into
7 µL of formamide denaturating solution, together with 0.2 µL of a fluorescent size
marker (600 LIZ, ABI), and the result was loaded onto an ABI 3130 16-capillary
automated sequencer. In this way the loci were separated both by size and colour in
the electrophoregrams, to avoid as much as possible interference between loci, given
the complexity of the profiles expected from the pooled samples. Although the PCR
product size ranges targeted during the design were adjacent (50-150, 151-250, 251350 and 351-450 bp), the actual product sizes tended to naturally cluster at the upper
bound of their ranges (since longer flanks offer more possibilities of primer design),
and were thus overall well separated across ranges. We were however careful not to
pool together loci with close PCR product sizes, by ranking loci inside each size range
according to predicted PCR size, and pooling loci with identical ranks.
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The numerous pipetting steps needed, from handling of genomic DNA, primers and
reagents to loading of the reactions on the sequencer were performed using pipetting
robots (Tecan Genesis RSP, TM) equipped with washable pipetting needles and for
low volume manipulation. We used two robots in different rooms for unamplified and
amplified DNA, respectively. We thus minimized the risk of cross-contamination, of
errors in sample and locus identification along the process, and of errors in the
preparation of reaction mixes.
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