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Neutral and selective processes shape MHC gene diversity and expression in
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stocked brook charr populations (Salvelinus fontinalis)
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Fabien C. Lamaze, Eric Normandeau, Scott A. Pavey, Gabriel Roy, Dany Garant, and Louis
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Bernatchez
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6
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APPENDIX / SUPPLEMENTARY MATERIAL
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Table S1. Descriptive genetic statistics for the MHC IIβ locus. Included are the year of sampling, the category of population
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(Cat.) either heavily (HS), moderately (MS), or non-stocked (NS) lakes (Marie et al. 2010) or reference population (Lamaze et
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al. 2013), population names and their location either Portneuf Wildlife Reserve (PN) or Mastigouche wildlife Reserve (MA)
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(see Fig.1, (Marie et al. 2010)), number of individuals sampled (N), number of fish successfully genotyped (Ng), number of
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alleles (A), allelic richness corrected for the smallest population (Ar) unbiased expected heterozygosity (Nei 1978) (He),
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observed heterozygosity (Ho) and FIS (Weir & Cockerham 1984). (*) Indicate significant departure from Hardy-Weinberg
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equilibrium.
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Year
Cat.
2007-2008 HS
MS
NS
Population names
Amanites Lake (AMA)
Belles-de-Jour Lake (BEL)
Méthot Lake (MET)
Average
Arcand Lake (ARC)
Rivard Lake (RIV)
Veillette Lake (VEI)
Average
Caribou Lake (CAR)
Main de fer Lake (MAI)
Sorbier Lake (SOR)
Average
2009
HS
Méthot Lake (MET9)
MS Petit st. Bernard Lake (BER9)
2007
Ref
Jacques Cartier Hatchery (JC)
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Location
PN
PN
PN
N
24
48
72
Ng
18
44
69
A
Ar
He
Ho
FIS
7
6.833
0.751
0.389
0.489*
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10.849
0.78
0.455
0.420*
21
13.439
0.848
0.623
0.267*
14.7 10.374 ± 1.922 0.793 ± 0.029 0.489 ± 0.070 0.392 ± 0.066
6
5.658
0.671
0.7391
-0.105*
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7.943
0.86
0.722
0.164
6
5.781
0.711
0.736
-0.037
6.7 6.461 ± 0.742 0.747 ± 0.058 0.732 ± 0.005 0.007 ± 0.081
6
5.586
0.56
0.476
0.153
6
5.895
0.635
0.5
0.216*
8
8.000
0.841
0.588
0.307*
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6.827 ± 1.090 0.679 ± 0.084 0.521 ± 0.034 0.225 ± 0.045
PN
PN
PN
24 23
24 18
24 19
PN
PN
PN
24 21
24 24
24 17
PN
MA
43 43
47 47
17
13
12.025
10.099
0.861
0.801
0.651
0.511
0.246*
0.365*
-
48 41
16
10.777
0.756
0.561
0.259*
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Table S2. Sequencing Primers, Real-Time PCR Primers, and Taqman MGB Probe for Each Candidate Gene. Product
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sizes are given for Salvelinus fontinalis partial cDNA sequences generated with the sequencing primers.
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Genes
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Sequencing primers (5’ – 3’)
Forward
Reverse
Product
size (bp)
Real-Time PCR Primers (5’–3’)
Forward
Reverse
 Actin
AGATGAAATCGCCGCACTGGTT
CTCGTTGTAGAAGGTGTGATGCCA
278
GCTGTCTTCCCCTCCATCGT
TCTCCCACGTAGCTGTCTTTCTG
MHC Iα (UBA)
CAGGTTTCTACCCCAGTGG
ACAACAACAGCAACGACGAG
443
CATTGAGTGGCTGAAGAAGTA
TCTTCTGGAGCAGAGACACTG
MHC II (DAB)
(SP4501) CCTGTATTTATGTTCTCCTTTC
(SP4502) TAAGTGTTGCTACGGAGCC
350
GCGCCGTACTGGATAAGACAGTT
TCAGCATGGCAGGGTGTCT
Taqman MGB Probe (5’–3’)
TCGTCCCAGGCATC
ACTATGGGAAGAGCACTC
TGAGCTCAGTGACTCC
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Table S3. Parasites abundance for 2008 and 2009. Included are year, the sampling region: either Portneuf Wildlife Reserve
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or Mastigouche wildlife Reserve (see Fig.1, (Marie et al. 2010), the lake, the number of individuals sampled (N), the stocking
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category of lakes (Cat.): either heavily (HS), moderately (MS), or non-stocked (NS) lakes (Marie et al. 2010), and the mean
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number of parasites per fish ± 1 standard error (SE). (g) Indicate that individuals were genotyped at the MHC IIβ. (NA) not
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available as only the stomach was screened in 2008 (see materials and methods).
Year
2008
2009
29
30
31
Echinorhynchus sp.
(Acanthocephala)
Mean ± SE
Eubothrium sp.
(Cestoda)
Mean ± SE
Crepidostomum sp.
(Trematoda)
Mean ± SE
NA
NA
NA
NA
NA
Sterliadochona sp.
(Nematoda)
Mean ± SE
9.7 ± 2.0
0.4 ± 0.4
2.6 ± 0.9
7.1 ± 2.2
324.6 ± 70.3
NA
NA
NA
NA
NA
Region
Mastigouche
Lake
Brochard (BRO)
Hollis (HOL)
Petit St-Bernard (BER)
Chamberlain (CHA)
Moyen (MOY)
N Cat.
10 HS
10 HS
10 MS
10 MS
4 NS
Portneuf
Amanites (AMA) g
Belles de Jour (BEL) g
Méthot (MET) g
Caribou (CAR) g
Main de Fer (MAI) g
11
10
11
11
10
HS
HS
HS
NS
NS
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
277.6 ± 61.5
0.0 ± 0.0
0.0 ± 0.0
1.9 ± 1.7
93.5 ± 16.3
NA
NA
NA
NA
NA
Mastigouche
Portneuf
Petit St-Bernard (BER9) g
Méthot (MET9) g
47
43
MS
HS
3.1 ± 2.6
0.0. ± 0.0
0.02 ± 0.02
2.6 ± 0.6
2.4 ± 0.6
0.07 ± 0. 07
50.7 ± 13.9
249.9 ± 40.1
NA
NA
NA
NA
NA
Figure S1: Neighbournet of the 29 MHC II beta 1 domains observed in Salvelinus
fontinalis. Blue indicates sequences with the deletion of an amino acid at position 59.
Red indicate sequences with an amino acid insertion at position 60. “r” and “s” stand for
resistant (allele 21) or susceptible allele (allele 6) to Aeromonas salmonicida infection
(Croisetière et al. 2008).
Figure S2: Amino acid alignment of the 29 MHC IIβ alleles of Salvelinus fontinalis.
The nomenclature of the allele six and 21 refer to (Croisetière et al. 2008). (*) Indicate
sites under positive selection after controlling for recombination.
Figure S3: Allelic frequency distribution at the MHC IIβ gene. MHC IIβ allele
frequency per lake for three heavily stocked (HS) populations (AMA, BEL, MET),
moderately stocked (MS) populations (ARC, RIV, VEI), three non-stocked (NS)
populations (CAR, MAI, SOR) and one reference domestic strain (JC).
46/53
24/31
48/55
1/9
49/56
4/12
5/13
83/90
82/89
78/85
21/28
53/60
17/24
74/81
71/78
59/66
77/84
Figure S4: The simulated three-dimensional structure model of the beta 1 domain of
Salvelinus fontinalis MHC class II. The tertiary structure prediction was based on the
most frequent allele (allele 29) of 31% homology with the human sequence sp|P04440
(PDB hit: 3lqzB) in the Protein Data Bank (http://www.rcsb.org/pdb/explore/explore.do).
Amino acid residues under significant positive selection in S. fontinalis and
corresponding to antigen binding sites or homodimerization patch in humans are
highlighted in red or dark red, respectively. Residues shown in yellow and orange were
under significant positive selection in S. fontinalis but do not correspond with antigen
binding sites in humans. Residues in orange are conserved among salmonids species
investigated thus far. Residues in blue are human antigen binding sites that were not
found to evolve under positive selection in S. fontinalis. The 85 amplified exon 2 codons
are numbered, only for sites under episodic selection. Those correspond to codons 9-92
of the mature protein. The first and second numbers separated by a dash represent codons
under positive selection in the S. fontinalis and the corresponding codon number of the
mature protein, respectively.
Figure S5: Box plot presenting MHC IIβ expression in brook charr head kidney as
function of minisatellite repeat number in MHC IIβ intron 2 and temperature, (8°C
or 20°C). Grey and white correspond to long or short minisatellite repeat motifs (32bp).
Comparisons of the distribution for the four groups were done with the least significant
difference test with a Bonferroni correction (α = 0.05). The black dot represents the mean
value.
Appendix S6: Supplementary material and methods
MHC sequencing
The forward primer for the 454 amplicon preparation consisted of a nucleotide sequence
containing (from 5’ to 3’) the primer A, the key, the MID and the SaCo_F primer as
described by the manufacturer (Roche). We also designed a degenerate reverse primer as
a SNP located at the 3’ end of our reverse SaCo_R primer was found in a previous study
(Croisetière et al. 2008). The reverse primer for the 454 amplicon was composed from 5’
to 3’ of the B primer and the SaCO_Rde primer (5’-AGCCCTGCTCACCTGTCTTR-3’)
as described by the manufacturer (Roche). Following amplification, samples were
visualized on a 1% agarose gel. Reactions that yielded tight, strong bands at the expected
size were purified using AMPure beads (Beckman Coulter Genomics) with a 96-well
plate following the manufacturer’s instructions. Samples were quantified with Picogreen
reagent (Invitrogen) on a Fluoroskan Ascent FL flourometer (Thermo Labsystems), prior
to combination in an equimolar fashion for a final DNA quantity of 30 ng/µl in three
libraries containing 151 samples each. Then, the libraries were sent to the Plateforme
d’analyses biomoléculaires (Institut de Biologie Intégrative et des Systèmes, Université
Laval, Québec, Canada) to perform the pyrosequencing using the 454 GS-FLX DNA
Sequencer with the Titanium Chemistry (Roche) using the procedure described by the
manufacturer. Each library was pyrosequenced on 1/8 of a plate.
In a first step of quality control of the 454 sequencing, 27 individuals were rerun,
representing at least two individuals per population. To control for quality between 454
sequencing and Sanger sequencing, we also Sanger sequenced MHC IIβ in 90 individuals
from the 2009 populations, using the forward SP4501 and reverse SP4502 primers
(Croisetière et al. 2008). PCR was carried out in a final volume of 12.5 μL containing 512ng of DNA using the GoTaq® Flexi DNA polymerase kit (Promega). The PCR
protocol had a 95°C initial denaturation step for 2min, followed by 35 cycles of
denaturation at 94°C for 30s, an annealing step for 30s at 47°C, and an elongation step at
72°C for 1min. A final extension step at 72°C for 10min and a cool-down step at 10°C
were added. PCR products were directly sequenced on an ABI 3100 (Applied
Biosystems) after running samples on a 1% agarose gel to check for the presence of tight
and strong bands at the expected molecular weight. Cloning was not used, as it is prone to
introduce artifacts (e.g. Longeri et al. 2002). All chromatograms were visually inspected
for quality control.
MHC genotyping
During phase one, the internal branch length threshold value, defining the minimum
branch length to define a cluster and an appropriate threshold value for the minimal
proportion of reads used to define a cluster was set to 0.20 and 0.05, respectively.
Putative individual consensus alleles were aligned with Sanger sequences of homozygote
individuals (n = 10) from 2009 samples and six Salvelinus fontinalis MHC class IIβ
alleles (Croisetière et al. 2008), with MUSCLE (Edgar 2004). The Sanger sequences were
assumed to be free of sequencing error, and gaps in the resulting alignment were removed
from all sequences. Phase two of genotyping pipeline was then performed on the putative
alleles for each individual with a minimum internal branch length of 0.08, which
represents the difference between two alleles of 255bp in length with a single SNP, and a
minimum number of two sequences to define a cluster. The result of this step represents
the putative consensus alleles for all populations. In the third step, we assigned one or
two global alleles to each individual, and excluded alleles that were likely artifactual.
Cleaned aligned sequences from each individual were BLASTed to the individual global
consensus alleles that were the output of phase two of genotyping pipeline. This resulted
in a count of the number of sequences that blasted to a consensus allele for each
individual. We then plotted the number of individuals an allele received with at least one
BLAST hit against the number of sequences representing the allele only in the individuals
where it is found. By examining each allele in this fashion, we found that a natural break
in this relationship could be determined visually. Then, a minimal threshold number of
sequences per individual were established to genotype the allele to that individual. For
most of the alleles, this threshold was 50 reads per individual but for alleles 8, 9, 11, 26
the number was 15.
Finally, for genotype call confirmation and data quality control, the genotype of
two individuals per population were run twice and compared. Also individuals that were
Sanger sequenced were used to confirm the 454 genotype status of each fish found to be
either a homozygote or heterozygote and check the allelic concordance for homozygote
individuals.
Appendix Literature Cited
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resistance/susceptibility alleles to Aeromonas salmonicida in brook charr (Salvelinus
fontinalis). Molecular Immunology, 45, 3107–3116.
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Research, 32, 1792–1797.
Lamaze FC, Garant D, Bernatchez L (2013) Stocking impacts the expression of candidate
genes and physiological condition in introgressed brook charr (Salvelinus fontinalis)
populations. Evolutionary Applications, 6, 393–407.
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stocked brook charr (Salvelinus fontinalis). Molecular Ecology, 21, 2877–2895.
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