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Supplementary materials
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Supplementary Figures
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Supplementary Figure 1 Phylogenetic tree of 22 species, based on FTO precursor
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sequences.
Supplementary Figure 2 Linkage disequilibrium (LD) structure of 18 SNPs in the
FTO region.
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Supplementary Tables
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Supplementary Table 1 Primers used in this study
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Supplementary Table 2 cDNA database sequences used for phylogenetic analysis
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Supplementary Table 3 Genetic variations identified in 8,005 bp of chicken FTO
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Supplementary Table 4 Association of M2, and M4 to M15, with chicken growth,
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body composition and fatness traits
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17
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2
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Supplementary Figure 1 Phylogenetic tree of 22 species, based on FTO precursor
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sequences. This tree was constructed using the UPGMA method of the MEGA 4.0.1
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program using 1000 bootstrap replications. Numbers above or below branches
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indicate the bootstrap values (percentage of 1000 replicates). Number scale in the
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lower left indicates genetic distance. I to VI represent six distinct phylogenetic
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groups.
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Ⅰ
Ⅱ
Ⅲ
Ⅳ
Ⅴ
Ⅵ
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3
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Supplementary Figure 2 LD structure among 18 SNPs in the FTO region. Location
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of each SNP is indicated at the top of the diagram. LD is presented as a 2×2 matrix in
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which red represents very high LD (r2) and white indicates absence of correlation
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between SNPs. The r2 values were calculated with Haploview 3.2. Three SNPs (M4,
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M15 and M17) that did not follow Hardy-Weinberg equilibrium were excluded from
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LD analysis.
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4
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Supplementary Table 2 Primers used in this study
Name
Forward (5’-3’)
Reverse (5’-3’)
P-M
tccaagctaattttcagaaagt
attttgtagatgaacaattac
P-Race1
aatggagcttatgacgagcct
aagcagtggtaacaacgcagagt
P-Race2
aagcagtggtaacaacgcagagt
ccctcagtaggccaaggaact
P-Race2N aagcagtggtaacaacgcagagt
taagtgcatcctggctttcca
FTO-Q1
gaacatggcagtgagttggcatc
ggtggagctgaaccgaggtgaa
P-actin
ctcccccatgccatcctccgtctg
gctgtggccatctcctgctc
P1
gtcctacaaggttggctttactcg
tgctacagggactcggaaatga
P2
ttcattcatacgcccacttc
catgtcaaatcctagaccct
P3
aatgccttgttgttcttgttt
ttggaagtactagaagtcaactgaa
P4
ttaattactagaactacaagcaaag
atctgagcaaattctacttatgt
P5
aatggagcttatgacgagcct
atgaggcatggcatagcacat
P6
tgaaaacaaaagcagcagaat
tttcatggcaatagaagtatc
P7
gtctggttggtaaactaggggaaac
gcccatcacctcctgacactg
P8
ggaggcacaatacaaacagga
agcattccacctcacaaagga
P9
atacattggaaaccggcaaac
gactcaggaacgaaggtggaa
P10
gctttcactttcaatttcat
catcctcctccacatccttt
P11
gccagtcagaagcagcggagag
actgtgcacctcttgggctgaac
PGC1α-Q
gactcaggtgtcaatggaagtg
atcagaacaagccctgtggt
STAT3-Q
ggaggcatttgggaagta
atgggcaggtcaatggta
PPARα-Q
acggagttccaatcgc
aacccttacaaccttcacaa
gcactgcccatccctctaa
cggctgccacgaaacaa
AMPK-Q
IGF1R-Q
gtacttcagtgcttcggatgtg
cttcttcagagttggaggtgct
GHSR-Q
tgggcgtcgagcatgaga
acagcaccgtgaggcaga
Leptin-R-Q
taccgtgcctactgctga
aactggcgttgttattgc
HL-Q
ttggcttacatttgctcacc
gctcctaggctgtacccaatta
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4
Annealing
Temp (°C)
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60
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60
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60
59
56
60
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60
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60
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59
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Supplementary Table 2 cDNA database sequences used for phylogenetic analysis
Protein ID2
No. Species
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Gallus gallus1
NP_001172076
2
Anas platyrhynchos1
ADH51649.1
3
Anser anser1
ADH51652.1
4
Homo sapiens
NP_001073901.1
5
Macaca mulatta
XP_001092038.1
6
Pan troglodytes
XP_510968.2
7
Oryctolagus cuniculus
ACJ53748.1
8
Equus caballus
XP_001492838.2
9
Bos Taurus
NP_001091611.1
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Ovis aries
NP_001098401.1
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Rattus norvegicus
NP_001034802.1
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Sus scrofa
NP_001106162.1
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Mus musculus
NP_036066.2
14
Monodelphis domestica
XP_001372283.1
15
Ornithorhynchus anatinus XP_001520255
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Xenopus laevis
NP_001087481.1
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Danio rerio
AAH92844.1
18
Saccoglossus kowalevskii
XP_002738337.1
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Micromonas sp.
ACO64022.1
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Micromonas pusilla
ABY26524.1
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Ostreococcus tauri
XP_003082290.1
22 Ostreococcus lucimarinus XP_001420808.1
Sequences generated in the current study. 2 NCBI accession numbers for FTO
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1
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peptides of each species.
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6
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Supplementary Table 3 Genetic variations identified in 8,005 bp of chicken FTO
Name SNPs1
Location
Note2
M1
M2
M3
M4
M5
M6
M7
M8
G4938404T
C4938390T
C4938374T
A4938324G
T4938170A
A4938150C
G4938090A
G4938082A
5’flanking
5’flanking
5’flanking
5’flanking
5’flanking
5’flanking
5’UTR
5’UTR
Sequencing
Sequencing
Sequencing
Sequencing
Sequencing
Sequencing
Sequencing
Sequencing
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
C4937926T
T4937918C
C4937901T
G4910140A
G4854795A
C4848865G
T4848695G
G4677213A
A4675075G
T4674902G
intron1
intron1
intron1
intron1
intron5
intron6
intron6
exon9
3’UTR
3’UTR
Sequencing
Sequencing
Sequencing
RFLP (BclI)
RFLP (TaqI)
RFLP (Bstz17I)
RFLP (PmlI)
Ala467Val; RFLP (BbvI)
RFLP (TaiI)
RFLP (Csp6I)
M19
M20
M21
M22
M23
M24
M25
M26
M27
M28
GC4938293/-G4938095T4886652A
G4886441A
A4910324G
G4910130A
G4910120A
G4910112T
C4910085T
A4886281G
5’flanking
5’flanking
exon3
Leu158Ile
exon3
Gly228Glu
intron1
intron1
intron1
intron1
intron1
intron3
M29
M30
M31
M32
M33
M34
M35
M36
G4879926C
G4879857T
C4879769T
T4879757C
C4879747T
G4879729C
C4856349T
G4856346A
intron3
intron3
intron3
intron3
exon4
exon4
intron4
intron4
6
Ser258Leu
Thr264Ser
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M37
A4856092G
intron5
M38
M39
M40
M41
M42
M43
M44
M45
M46
G4856080A
G4856031A
T4855970C
A4854827G
A4854823C
G4854790T
G4854688A
C4854674A
G4854656A
intron5
intron5
intron5
intron5
intron5
intron5
intron5
intron5
intron5
M47
M48
M49
M50
M51
M52
M53
M54
M55
M56
T4854641A
G4854628C
A4854598G
G4848431A
A4848417G
T4677457C
G4677441C
C4677400G
C4677368T
C4677049T
intron5
intron5
intron5
intron7
intron7
intron8
intron8
intron8
intron8
exon9
M57
M58
M59
M60
M61
M62
M63
M64
M65
M66
C4677027T
C4675250T
C4675228T
G4675138A
A4675098G
T4675093C
G4675010A
T4674975G
4675072T
GTAGAC/-
exon9
3’UTR
3’UTR
3’UTR
3’UTR
3’UTR
3’UTR
3’UTR
3’UTR
3’UTR
M67
G4674913T
3’UTR
Synonymous
Synonymous
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released in May 2006 (http://genome.ucsc.edu/cgi-bin/hgBlat).
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genotyped by sequencing and further used for association analysis. M12 to M18 were
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genotyped by PCR-RFLP and further used for association analysis; the restriction
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enzyme for each SNP is indicated in brackets. M21 and M22, and M33 and M34 are
The location of each SNP is designated according to the Chicken Blat search
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2
M1 to M11 were
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non-synonymous SNPs causing the indicated amino acid changes, whereas M56 and
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M57 are synonymous SNPs.
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Supplementary Table 4 Association of M2, and M4 to M15, with chicken growth, body composition and fatness traits (P values and
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LSM±SEM)
SN
Ps
Traits
P value
Least squares mean ± Standard error
M2
LMF (%)
0.024
64.23 ± 0.53a (395) / CC
59.23 ± 2.19b (31) / CT
(0)/ TT
M2
CSALMF (μm2)
0.045
19675 ± 307.6a (170) / CC
17491 ± 1091b (17) / CT
(0)/ TT
M4
LMCPC (%)
0.001
20.76 ± 0.07aA (168) / AA
20.06 ± 0.16bB (24) / AG
20.6 ± 0.22aAB (13) / GG
M4
BMCFC (%)
0.013
1.04 ± 0.03A (168) / AA
1.05 ± 0.08A (24) / AG
0.71 ± 0.11B (13) / GG
M5
WLR (%)
0.009
27.66 ± 2.46aA (19) / AA
20.72 ± 1.47bAB (38) AT
17.29 ± 2.1bB (19) / TT
M6
WLR (%)
0.024
24.27 ± 1.67aA (38) / AA
19.11 ± 2.03abAB (26) / AC
14.28 ± 2.9bB (12) / CC
M7
BW7 (g)
0.017
58.93 ± 0.43ab (322) / AA
61.93 ± 1.31ac (40) / AG
63.66 ± 2.47a (7) / GG
M7
BW14 (g)
0.010
122.1 ± 0.90b (351) / AA
128.0 ± 2.66a (48) / AG
134.2 ± 5.33a (7) / GG
M7
BMCPC (%)
0.040
24.84 ± 0.08a (167) / AA
24.94 ± 0.24a (23) / AG
25.95 ± 0.44b (4) / GG
M8
SL63 (mm)
0.016
80.29 ± 1.43A (29) / AA
75.35 ± 1.08B (73) / AG
77.14 ± 1.39AB (38) / GG
M8
WLR (%)
0.043
16.2 ± 2.26b (17) / AA
20.55 ± 1.63ab (33) / AG
25.40 ± 2.42a (25) / GG
M9
HW (mm)
0.035
30.1 ± 0.12b (189) / CC
30.53 ± 30.53a (155) / CT
30.19 ± 0.18ab (69) / TT
M9
WLR (%)
0.018
24.32 ± 1.74aA (36) / CC
18.58 ± 1.96bAB (26) / CT
14.00 ± 2.73bB (13) / TT
M10
WLR (%)
0.044
16.03 ± 2.46b (18) / CC
20.56 ± 1.41ab (36) / CT
25.03 ± 2.25a (25) / TT
M11
HW (mm)
0.027
66.3 ± 0.45bB (172) / CC
67.62 ± 0.49aA (159) / CT
66.31 ± 0.69abAB (69) / TT
M11
WLR (%)
0.010
24.62 ± 1.69aA (35) / CC
18.55 ± 1.84bAB (27) / CT
14.09 ± 2.66bB (14) / TT
M12
BW14 (g)
0.031
125.2 ± 1.33A (197) / AA
121.0 ± 1.16B (208) / AG
121.0 ± 1.95AB (75) / GG
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M12
BW28 (g)
0.028
313.8 ± 4.07A (195) / AA
301.0 ± 3.56B (207) / AG
306.2 ± 6.04AB (72) / GG
M12
ADG0-4 (g/d)
0.028
10.14 ± 0.15A (195) / AA
9.68 ± 0.13B (207) / AG
9.87 ± 0.22AB (72) / GG
M13
DW (g)
0.008
1374.68 ± 15.42aA (117) / AA
1321.45 ± 9.84abAB (216) / GA
1345.39 ± 14.54bB (139) / GG
M14
WW (g)
0.033
62.89 ± 0.92b (70) / GG
65.31 ± 0.52a (199) / GC
65.74 ± 0.58a (205) / CC
M14
SL42 (mm)
0.031
61.68 ± 0.64a (67) / GG
60.67 ± 0.35ab (194) / GC
59.7 ± 0.39b (203) / CC
M14
SL56 (mm)
0.009
73.31 ± 0.7aA (69) / GG
72.33 ± 0.39aAB (196) / GC
70.93 ± 0.43bB (203) CC
M14
LMSS (%)
0.027
65.42 ± 3.95b (70) / GG
75.27 ± 2.22a (200) / GC
69.35 ± 2.49ab (205) / CC
M15
HW (mm)
0.028
29.87 ± 0.21a (46) / GG
30.37 ± 0.1b (275) / GT
30.09 ± 0.14ab (156) / TT
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BW7, BW14, BW28 = body weight at 7, 14, 28 days of age, respectively; ADG0-4 = average daily gain from 0 to 4 weeks; LMF = leg muscle
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flesh color; CSALMF = cross sectional area in leg muscle fiber; LMCPC = crude protein content of leg muscle; BMCPC = crude fat content of
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breast muscle; SL42, SL56, SL63 = shank length at 42, 56, 63 days, respectively; WLR = water loss ratio of breast muscle; HW = head width;
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DW = dressed weight; WW = wing weight; LMSS = leg muscle shear stress. Statistical analysis was performed using GLM by SAS 8.0, and
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data are shown as least square mean ± S. E. M.
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different (P < 0.01), and those not sharing a small letter were significantly different (P < 0.05). Numbers in brackets indicate the number of birds
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in that SNP genotype group. The SNP genotype is indicated after the sample size.
Means not sharing a superscript capital letter in the same column were highly significantly
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