Supplementary information
Figure S1 Alignment of the amino acid sequences deduced from the Aime-MHC-I
genes. The boxed symbols represent types of intron 2 sequences, as shown in Figure
S2. The underlined sequence shows the putatively classical but monomorphic Aime-F
gene [1], and the light- and dark-grey regions indicate the two wild-specific
haplotypes and two nonclassical loci uninvolved in this study, respectively. These five
sequences were from GenBank (JX987008, JX987023, EU162656, EU162657 and
EU162661). Dots indicate identities and gaps represent missing amino acids to the
first sequence. Crosses represent putative antigen-binding sites, corresponding to the
method of Bjorkman et al. [2].
1
2
Figure S2 Inter-haplotype sharing of intron 2 sequences (A) and nucleotide sequence alignment
(B) of the Aime-MHC-I genes. The black and gray lines represent classical and nonclassical loci,
respectively (A). Dots indicate identities and gaps represent missing amino acids to the first
sequence (B). The shaded intons were from GenBank (EU162656, EU162657 and EU162661).
The Aime-C*09 (JX987008) and Aime-L*07 (JX987023) were not detected in this study but
shared the intronic sequences with other haplotypes.
3
Table S1 Haplotypic frequencies and heterozygosities of the three MHC class I loci.
Haplotype
Aime-C
Aime-I
Aime-L
Mean
01
0.011
0.156
0.089
/
02
0.344
0.522
0.433
/
03
0.100
0.089
0.122
/
04
0.033
0.144
0.089
/
05
0.222
0.011
0.022
/
06
0.267
0.044
0.244
/
07
0.011
0.033
/
/
08
0.011
/
/
/
Na
8
7
6
7
HO
0.667*
0.667*
0.644*
0.659
HE
0.758
0.679
0.729
0.722
Note: Na is the number of haplotypes. Asterisks indicate significant deviation from
Hardy-Weinberg equilibrium (P < 0.05). The figures in bold represent the
predominant haplotype for each locus.
4
Table S2 Nonsynonymous and synonymous substitutions at selected sites (positive or
purifying) within the three Aime-MHC-I genes using REL methods.
Locus
Aime-C
Aime-I
Aime-L
Codon
E[dS]
E[dN]
Normalized
Posterior
Bayes
E[dN-dS ]
probability
factor
70+
0.53
4.78
4.25
0.97
73.67
73+
0.18
1.42
1.24
0.99
345.34
76+
0.43
1.42
0.99
0.97
55.38
80+
0.42
1.41
0.99
0.97
55.78
83
0.34
1.43
1.09
0.97
82.32
94
0.43
1.42
0.99
0.96
55.23
118*
8.83
0.22
-8.61
0.99
205.39
119*
8.88
0.19
-8.69
0.99
191.40
152+
0.91
15.52
14.61
0.99
281.12
63+
0.98
1.77
0.79
0.95
84.41
66+
0.77
1.77
1.00
0.98
165.08
70+
0.81
1.75
0.94
0.96
103.89
73+
0.55
1.77
1.22
1.00
7164.58
74+
0.87
1.76
0.88
0.96
91.33
81+
1.04
1.77
0.73
0.94
71.34
97+
0.78
1.76
0.98
0.97
130.06
103
0.62
1.76
1.14
0.98
272.08
114+
0.73
1.77
1.04
0.98
203.41
152+
0.90
1.77
0.87
0.96
109.15
163+
0.92
1.77
0.85
0.96
99.91
23
1
3.50
2.50
0.90
59.16
24+
1
3.54
2.54
0.91
66.12
116+
1
3.81
2.81
0.99
483.55
156+
1
3.78
2.78
0.98
284.68
160
1
3.78
2.78
0.98
281.97
5
Notes: E[dS] and E[dN] indicate posterior means of the synonymous and
nonsynonymous substitution rates at each site. E[dN-dS] indicates the posterior mean
of the dN-dS difference. The asterisks and crosses represent the purifying selection and
antigen-binding sites, respectively.
6
Table S3 Nonsynonymous and synonymous substitutions within the three
Aime-MHC-I genes using MEGA 4.0.
Locus
Aime-C
Aime-I
Aime-L
Position
N
dN
dS
dN / dS
P
ABS
57
0.104 (0.028)
0.034 (0.018)
3.059
0.004
Non-ABS
125
0.007 (0.004)
0.024 (0.013)
0.292
1.000
All
182
0.036 (0.008)
0.027 (0.010)
1.333
0.240
ABS
57
0.101 (0.025)
0.066 (0.023)
1.530
0.142
Non-ABS
125
0.008 (0.003)
0.037 (0.014)
0.216
1.000
All
182
0.036 (0.008)
0.046 (0.011)
0.783
1.000
ABS
57
0.045 (0.016)
0.017 (0.011)
2.647
0.025
Non-ABS
125
0.010 (0.004)
0.030 (0.011)
0.333
1.000
All
182
0.021 (0.006)
0.026 (0.008)
0.808
1.000
Notes: Standard errors (in parentheses) were obtained through 1000 bootstrap
replicates. N is the number of codons, and P-value shows statistical significance of
differences between dN and dS.
7
Data S1 Materials and Methods
Sampling
We collected 46 blood samples during routine medical examination of giant
pandas in the China Research and Conservation Center for the Giant Panda (Wolong).
Genomic DNA was extracted using the conventional phenol-chloroform method [3].
Amplification and genotyping
The exons 2 and 3 of MHC class I genes encode domains responsible for
antigen presentation. Thus, we amplified the exon 2–intron 2–exon 3 using the
locus-specific primer pairs published by Zhu et al. [1]. We cloned the PCR products
and amplified the resultant clones using the universal nested primers for separate exon
2 and exon 3 of the Aime-MHC-I genes (Table S4). The nested PCR fragments of
exons 2 and 3 were subjected to single-strand conformation polymorphism (SSCP)
analysis. We sequenced the positive clones which have different combinations of
exon 2 and exon 3 banding patterns. Each exon 2–intron 2–exon 3 haplotype was
confirmed by sequencing at least three independent clones. The PCR and SSCP
conditions were described in Zhu et al. [1].
Data analysis
We calculated haplotypic frequencies and tested deviations from
Hardy-Weinberg equilibrium for each locus using Genepop4.0 [4]. Observed (HO)
and expected (HE) heterozygosities were estimated in Arlequin3.1 [5].
All the sequences were edited and aligned using the DNASTAR software
package (DNASTAR, Inc.). We checked for evidence of recombination on the exon 2,
8
intron 2, and exon 3 fragments using the maximum likelihood-based GARD (genetic
algorithm for recombination detection.) [6] and chi-square-based MaxChi2 (modified
maximum chi-square) [7] methods. A GARD analysis was conducted through a
web-based routine (http://www.datamonkey.org/GARD/) while MaxChi2 were
implemented in RDP 3.44 [8].
We tested positive selection at the Aime-MHC-I genes using a Random Effects
Likelihood (REL) method implemented in the HyPhy Package
(http://www.datamonkey.org/) [9]. REL adopts a maximum likelihood method to
compute rates of nonsynonymous (dN) and synonymous (dS) substitutions for each
codon [10]. Empirical Bayes analysis was then used to determine whether there was
evidence of positive selection (dN > dS) or purifying selection (dN < dS) when Bayes
factors are larger than 50. Since recombination is known to seriously overestimate
positive selection [11, 12], we detected recombination events in our datasets using the
GARD method described above prior to implementing REL. The HyPhy Package can
detect evidence of selection in the presence of recombination.
The dS/dN ratios were also calculated using MEGA 4.0 [13] and standard errors
were estimated through 1000 bootstrap replicates. To test for signatures of balancing
selection at the three Aime-MHC-I genes, we used a Z-test as implemented in MEGA
4.0 to compare the rates of synonymous and nonsynonymous substitutions at all sites,
including antigen binding sites (ABSs) and non-ABSs. ABSs were identified by
comparison to human sequences [2].
The number of nucleotide substitutions in the intron 2 (d) and the number of
9
synonymous substitutions in the exons 2 and 3 (dS) were estimated using a Kimura-2p
method in K-estimator [14]. We plotted mean d and dS against nucleotide position
using a sliding window size of 15 base pairs and a step size of 3 base pairs.
References for Supplementary Information
1.
Zhu Y, Wan QH, Yu B, et al. BMC Evol Biol 2013; 13:227.
2.
Bjorkman PJ, Saper MA, Samraoui B, et al. Nat Immunol 1987; 329:512-518.
3.
Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual,
3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York).
4.
Rousset F. Mol Ecol Resour 2008; 8:103-106.
5.
Excoffier, Laval LG, Schneider S. Evol Bioinform Online 2005; 1:47-50.
6.
Pond SLK, Posada D, Gravenor MB, et al. Mol Biol Evol 2006; 23:1891-1901.
7.
Maynard-Smith J. J Mol Evol 1992; 34:126-129.
8.
Heath L, Van Der Walt E, Varsani A, et al. J Virol 2006; 80:11827-11832.
9.
Pond SLK, Frost SDW. Bioinformatics 2005; 21:2531-2533.
10.
Pond SLK, Frost SDW. Mol Biol Evol 2005; 22:1208-1222.
11.
Anisimova M, Nielsen R, Yang Z. Genetics 2003; 164:1229-1236.
12.
Richman AD, Herrera LG, Nash D, et al. Genet Res 2003; 82:89-100.
13.
Tamura K, Dudley J, Nei M, et al. Mol Biol Evol 2007; 24:1596-1599.
14.
Comeron JM. Bioinformatics 1999; 15:763-764.
10
Table S4 Locus-specific primers of the three polymorphic Aime-MHC-I genes. The
primer sequences were published by Zhu et al. [1].
Locus
Primer
Primer sequence (5’-3’)
name
Aime- C
Aime-I
Aime-L
Aime-C/I/
1611E21F a
GCTCTCCCCCACTCAGTA
1611E33R a
TGCAGGTCTAAGAGGGAGAGCGCT
128E22F a
CCTGCTCTCCCCAACGCG
128E32R a
GTCCGGGGTTTCTGAAGAAGAACG
1300E22F a
GGGAGAAGGGTCGGGCGGGAC
1300E31R a
GTCGGGGGTTTCTGAAGAAGAATC
C1E2B1 b
TCAGCCCCTCCGCGCCCGCAG
C1E2B2 b
GACCCGGGCCGCGTCGCTCAC
C1E3B1 b
TCGCCTCCTGTCGGGCGGGGCCAG
C1E3B2 b
AGCCAGCCCCAGCGGAGGGG
Size
Ta
(bp)
(C)
1255
60.0
1467
60.0
1354
60.0
312
66.3
349
65.6
LE2
Aime-C/I/
LE3
Notes: The primer pairs (a) were used to amplify the long fragment of exon 2–intron
2–exon 3, and the primer pairs (b) were then adopted for the subsequent nested PCR
to get separate exon 2 and exon 3 fragments for single-strand conformation
polymorphism analysis.
11