Table S3 Identification of the 37 single nucleotide polymorphisms (SNPs) according to CanFam3.1 and CanFam2.1, their location on the dog genome assembly CanFam2.1, primer pairs, amplicon size in base pairs (bp), annealing temperature (Ta) and method used for genotyping in 95 cases and 95 controls. The ten SNPs employed for validation in 843 German shepherd dogs are in bold.
CFA / SNP(CanFam2.1)
Gene / Location in the canine gene / Position on the dog genome (Mb) / Forward primer (5’ - 3’)
SNP motif
Reverse primer (5’ - 3’) / Product size (bp) / Ta
(°C) / Method (enzyme,
Tb in °C)
1 / NW_003726047:g.13548080C>T
(NW_876270:g.13559913C>T)
OSTF1 / intron 4 / 86.171 / AAGCAATAAGGCAACACCAC
TGGAACTGGA[C/T]ACTCTTGGCT
GCAGCATACCTCTACCATGC / 533 / 58 / RFLP (BciVI, 37)
1 / NW_003726047:g.29375641A>C
(NW_876270:g.29388222A>C)
CENPP / intron 5 / 101.999 / CTGGTCTGCCTTCTTCACTG
AGTTGATAGC[A/C]TTTCCTATTA
TCACTTATAGTCCCAACTACTCAGG / 519 / 58 / Taqman
1 / NW_003726047:g.40679980C>T
(NW_876270:g.40514469C>T)
MARK4 / intron 12 / 113.126 / AGAAACAGCATCTGCTCTGG
GGGCCCAGGA[C/T]GGCGTGTGAA
CCTCATCTGTCATCCTCCTG / 420 / 58 / RFLP (BtscI, 50)
1 / NW_003726047:g.47026853A>G
(NW_876270:g.46879282A>G)
TBCB / intron 2 / 119.491 / TCCCGGGATTTAAGTCTCTC
TCCCTCTCTG[A/G]GTCTCTGGGA
GTACGTGCATGTGTTTGTGG / 476 / 58 / RFLP (HinfI, 37)
3 / NW_003726054:g.37139132G>A
(NW_876307:g.37109176G>A)
PGM2 / intron 8 / 77.186 / GTTCAACACCCTTTGAGCAC
AAAAACAACC[G/A]ATGCATTGCT
ACCACTTTGAGCCTTGTTTG / 587 / 57 / RFLP / (SfaNI, 37)
4 / NW_003726055:g.5441210C>T
(NW_876310:g.5439118C>T)
TARBP1 / intron 33 / 8.439 / TTCCAGAGCCTCAGTGTTTC
CAGCAAAATA[C/T]ACCTCCCTCT
AAGCTGTATTGCGTCAGGTC / 553 / 58 / Taqman / -
4 / NW_003726056:g.8920678T>G
(NW_876311:g.9126752T>G)
MYPN / intron 3 / 22.620 / TTGAAAGGCATTTACATCAGTG
ACCTTAGGTA[T/G]TACCAAATAC
ATCAGCAATCCAATCTCAGC / 512 / 58 / RFLP / (RsaI, 37)
8 / BICF2P1184132
(MDGA2) - (RPS29) / intergenic / 28.909 / GAGCTCCAGCTCTCAAACCA
ACTGTGAGTT[A/G]AACATGTAAA
GCCACATCTGGGAAGAAGAG / 470 / 58 / Taqman / -
9 / BICF2S22937555
(FCN1) - (OLFM1) / intergenic / 54.312 / CCCACCACCATCTTGTCTTC
TTAGAAAGGT[G/C]ACTTTCCAGG
TCCCAGCTGGAGATCTCTGT / 450 / 60 / Taqman / -
9 / NW_003726076:g.3433414A>G
(NW_876333:g.3448481A>G)
C9H9orf116 / intron 2 / 54.550 / ATAGACAACCCTGCCACACC
GATTTCTGAC[A/G]TACAGGGCCA
TCCTGCCAAGCAAAAAGAGT / 450 / 58 / RFLP / (CviQI, 25)
16 / NW_003726087:g.10983842G>A
(NW_876261:g.10982539G>A)
(NRG1) - (WRN) / intergenic / 35.217 / TTCTGGGCCAGTCATGAGAT
GAAGAGAAAT[G/A]GAAGATGGAA
AGCAAGATGCCCATGAAAAT / 467 / 60 / Taqman / -
19 / NW_003726094:g.1930502T>C
(NW_876268:g.1932412T>C)
RAB3GAP1 / intron 15
(isoform 1) / 40.947 / ACAGGTAAGGATTTCCCAATG
TTCCCAATGA[T/C]AGTATCTAAA
TTCTTGCCACTCTCTTGTCC / 501 / 58 / Taqman / -
19 / NW_003726094:g.13718110T>G
(NW_876268:g.13721560T>G)
ACVR2A / intron 7 / 52.737 / GATTGGCTTATTTGCACGAG
TTAAGAATGA[T/G]AGTGTATTTT
TTTTCCCTTTTGTGGTTCTG / 576 / 58 / Taqman / -
19 / NW_003726094:g.17533130T>G
(NW_876268:g.17541151T>G)
FMNL2 / intron 2 / 56.556 / TCAGAATAGCTCACCCATCC
TGTGGCCAAA[T/G]ATCTCACCTC
CGTCGGAACTCACATAAACC / 534 / 58 / RFLP / (BglII, 37)
26 / NW_003726107:g.2648636G>A
(NW_876283:g.2648127G>A)
(ZNF70) - (VPREB1) / intergenic / 31.831 / ACCTGAAGCAACCCCAACTA
TCAGATCCTC[G/A]CAGTGACAGA
CTTTTCTATCCCCTGCTCCA / 321 / 60 / RFLP / (BtsI, 55)
26 / BICF2P844355
(VPREB1) - (PRODH) / intergenic / 32.016 / ATGTCCTGGCACACCAAAG GCTTACCTGC[G/A]TCCCTTCCCC
ACGTGGTAGCTGGAGGACAG / 434 / 62 / RFLP / (BtsCI, 50)
26 / NW_003726107:g.9637149A>C
(NW_876283:g.9641369A>C)
PRKG1 / intron 5 / 38.824 / CCAATGGAAGGCCATAACTC
GAAGGAGCAT[A/C]AAATGCAGAA
CTTGGTAGGCAATGTCAT / 524 / 58 / RFLP / (SfaNI, 37)
29 / NW_003726112:g.24955022A>G
(NW_876288:g.24954473A>G)
PEX2 / intron 1 / 27.954 / GAGTATTGTCAAGGGCCTGTC
AGTGTTTAGT[A/G]TAACAATGTT
ATCCCTCTTTGATGCATGTC / 589 / 58 / RFLP / (MaeIII, 55)
32 / NW_003726116:g.5225404C>T
(NW_876297:g.5189709C>T)
LOC100855862 (BMP3-like) / intron 2 / 8.190 / AAGGTGATTTTGGAGGGAAC
CATTTGCTTC[C/T]CTAATAAACA
TGTATGGCAGAGATGCACAC / 533 / 58 / Taqman / -
32 / NW_003726116:g.8405881G>A
(NW_876297:g.8367470G>A)
CDS1 / intron 10 / 11.367 / GCAGTGTTGAGACGGGTAAG
GAATATTACT[G/A]TCACAAACCA
GTTCTATTGGGCAGCATGTC / 563 / 60 / RFLP / (Tsp45I, 65)
32 / NW_003726116:g.10233722T>C
(NW_876297:g.10194392T>C)
PTPN13 / intron 6 / 13.194 / AGTGTTCTGCTGGAGGTTTG
TGATTATAAA[T/C]TTTCAAGGGC
TTGTTTGTTTTGCACAATGG / 502 / 58 / RFLP / (ApoI, 50)
32 / NW_003726116:g.11190288C>T
(NW_876297:g.11150740C>T)
IBSP / intron 3 / 14.151 / TCGAAGAGCCAAATTAGAGG
CAATTCTCAC[C/T]GAAAGTCTGC
TGACAGTGGCCTTATCACTG / 542 / 56 / RFLP / (HphI, 37)
32 / NW_003726116:g.11221524A>G
(NW_876297:g.11182056A>G)
MEPE / exon 4 / 14.182 / TCAAGAGCCAGCACCTAAAG
AGACAATGCT[A/G]TCAACAAATC
TGGGTGCTTTTGCTTTTAAC / 477 / 56 / RFLP / (HincII, 37)
33 / NW_003726117:g.973418T>G
(NW_876298:g.974981T>G)
EPHA3 / intron 5 / 3.975 / GCAGCTTCCATTACACTTGG CTCCCTGTGT[T/G]TCCTTCCAAT
TGTTCAAGGCCTCTGTTAGC / 504 / 58 / RFLP / (NIaIV, 37)
33 / NW_003726117:g.4052195T>C
(NW_876298:g.4022182T>C)
EPHA6 / intron 3 / 7.022 / TGGTTTTGCACTTAGTCTGAT
TAAAATCTGA[T/C]ATAGCCAAAG
AACTCCAGGGAGAGAACTGG / 507 / 58 / Taqman / -
33 / NW_003726117:g.7438639G>A
(NW_876298:g.7454694G>A)
ABI3BP / intron 3 / 10.455 / TCACCTCTTAGGGAAAGGAAG
AGTATGCAGC[G/A]TCCAAGTACT
TTCCCATGTGTAACCCTGTC / 502 / 58 / RFLP / (HgaI, 37)
33 / NW_003726117:g.7967386T>C
(NW_876298:g.7983324T>C)
PCNP / intron 2 / 10.983 / AGATGGGAGGGTTTTGTTTG
AAACACGTGA[T/C]GTCTTTAAAA
TGATTCGGTTCTCTGGTCTC / 577 / 58 / RFLP / (Tsp45I, 65)
33 / BICFPJ518789
(RG9MTD1) - (PCNP) / intergenic / 10.969 / GCATGCTCATTCACAGTTTG
GGAAGTAGTG[C/G]CTTTTCTTTT
GGAGGTTGGTAGCATGTTTG / 299 / 58 / RFLP / (CvikI-1, 37)
34 / NW_003726119:g.414871G>A
(NW_876300:g.421687G>A)
TRIO / intron 12 / 3.422 / AGGGGAAAGACAGAAGGAAG
AGGGCAAGGC[G/A]CTGTCCTTCC
AATGGAAGACGTTCTCATGC / 539 / 58 / RFLP / (HaeII, 37)
34 / NW_003726119:g.4846215A>G
(NW_876300:g.4850749A>G)
SEMA5A / intron 3 / 7.851 / TGGGCATCATCCTGATTTAG
AGTGGCAGAT[A/G]TGAGTCACAA
TTGAAGAACTTGTCCCAAGC / 517 / 58 / Taqman / -
34 / NW_003726119:g.11239992A>G
(NW_876300:g.11245718A>G)
SLC6A3 / intron 11 / 14.246 / GCCTCTACTGGAGGGTGTG
TCAGGATGGC[A/G]CCCCAGTTCC
AACTCTGCAGGTCCAGACAC / 577 / 58 / RFLP / (HhaI, 37)
34 / NW_003726120:g.11547417T>G
(NW_876301:g.1154628T>G)
FGF12 / intron 3 (isoform 1) / 27.034 / TCCTCAATCAAAATGAAATCAC
TAATGGTATT[T/G]CAGGAAGCTA
CAAGGTTATTCAGCCAGCAG / 536 / 58 / RFLP / (Hpy188III, 37)
34 / NW_003726120:g.23562075A>G
(NW_876301:g.23560347A>G)
PLD1 / intron 4 / 39.045 / CGGTCTTAAGGCATTTGTTG
TAAAAGTCAC[A/G]GCTTCTCATT
TAGGGCCAGAGAGGAGTAGG / 528 / 58 / RFLP / (AluI, 37)
35 / NW_003726121:g.690224T>C
(NW_876302:g.690336T>C)
LOC606970 (uncharacterized) / intron 1 / 3.690 / GTCTGTCGATGTACGCTGTG
GGGAACTTGC[T/C]GAAACAAGGC
GGTGGGACCTCACAGTTAAG / 509 / 58 / Taqman / -
35 / NW_003726121:g.16934280C>T
(NW_876302:g.16953392C>T)
TPMT / intron 7 / 19.953 / AAAGCTCAGTTGTGGCAAAG
GTGAATGAGA[C/T]GACATACAAG
TTCTGTGACACGGATAGCAG / 567 / 58 / RFLP / (BsmBI, 55)
35 / NW_003726121:g.19904761C>G
(NW_876302:g.19922509C>G)
(CDKAL1) - (SOX4) / intergenic / 22.923 / AAGAGCGGGGTCACACATA
GACAGGGGGA[C/G]GGGGTGCACA
GCTCCCTCGATGTGAGACTAA / 500 / 60 / Taqman / -
35 / NW_003726121:g.26217499G>A
(NW_876302:g.26236243G>A)
GABBR1 / intron 14 / 29.236 / GGAGCCGAATTAACCTCTTC
TCTGACCTCC[G/A]TCCCATGCTC
CATTTCGCTTCATGTCACAG / 504 / 58 / RFLP / (BccI, 37)
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Table S4SBE primers and SNP motifs for the tennucleotide polymorphisms (SNPs) genotyped in 843 German shepherd dogs
CFA / SNPGene / Chromosomal position (Mb) / SBE primer (5’ - 3’)
SBE orientation
SNP motif
3 / NW_003726054:g.37139132G>A
PGM2 / 77.186 / CGCTGAAAGAAAAAACAACC
G/A-forward
G>A
9 / BICF2S22937555
(FCN1) - (OLFM1) / 54.312 / GTATTTAGCCCCTGGAAAGT
G/C-reverse
C>G
26 / BICF2P844355
(VPREB1) - (PRODH) / 32.016 / GTGCCAGGGGAAGGGA
G/A-reverse
C>T
33 / NW_003726117:g.973418T>G
EPHA3 / 3.975 / GGGATTCCCTCCCTGTG
T/G-forward
T>G
33 / NW_003726117:g.4052195T>C
EPHA6 / 7.022 / TGCGGTGTCTAAAATCTGA
T/C-forward
T>G
33 / NW_003726117:g.7967386T>C
PCNP / 10.983 / CTGTACAACATCAAACACGTGA
T/C-forward
T>C
34 / NW_003726119:g.414871G>A
TRIO / 3.422 / CAGAGGGAGGAAGGACAG
G/A-reverse
C>T
34 / NW_003726119:g.4846215A>G
SEMA5A / 7.851 / AGGATATTTATTTTAGTGGCAGAT
A/G-forward
A>G
34 / NW_003726119:g.11239992A>G
SLC6A3 / 14.246 / CCCCTCATCAGGATGGC
A/G-forward
A>G
34 / NW_003726120:g.11547417T>G
FGF12 / 27.034 / AGGAACATTAGCTTCCTG
T/G-reverse
A>C
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Table S5 Odds ratios (OR) of SNPs flanking the highly associated SNPs on dog chromosomes 26 (BICF2P844355), 33 (g.7967386T>C) and 34 (g.414871G>A) with their 95% lower (CL) and upper confidence limits (CU), ²- and -log10P-values for genotypic, allelic and trend association considering 277 CHD-A German shepherd dogs as controls and 566 German shepherd dogs classified as CHD-C/D/E as cases
SNP / CFA / Position (Mb) / OR / OR-CL / OR-CU / Genotype / Allele / Trend² / -log10P-value / ² / -log10P-value / ² / -log10P-value
BICF2P754746 / 26 / 31.533 / 0.31 / 0.15 / 0.59 / 14.44 / 3.14 / 15.99 / 4.20 / 13.03 / 3.51
BICF2P270835 / 26 / 31.572 / 0.24 / 0.11 / 0.49 / 14.61 / 3.17 / 17.44 / 4.53 / 12.91 / 3.49
BICF2P1380063 / 26 / 32.243 / 0.56 / 0.40 / 0.80 / 15.91 / 3.46 / 10.90 / 3.02 / 6.11 / 1.87
BICF2G630247517 / 33 / 10.864 / 3.25 / 1.84 / 5.73 / 13.61 / 2.96 / 18.37 / 4.74 / 12.88 / 3.48
BICF2G630247561 / 33 / 10.977 / 2.45 / 1.98 / 3.02 / 61.35 / 13.32 / 70.03 / 16.23 / 60.99 / 14.24
BICF2G630247570 / 33 / 11.024 / 0.21 / 0.10 / 0.44 / 14.96 / 3.25 / 19.74 / 5.05 / 14.10 / 3.76
BICF2P1248243 / 34 / 3.405 / 0.15 / 0.10 / 0.23 / 89.31 / 19.39 / 102.66 / 23.40 / 83.59 / 19.22
BICF2P1063291 / 34 / 3.429 / 0.20 / 0.14 / 0.28 / 76.28 / 14.18 / 20.29 / 17.12 / 16.95 / 14.82
BICF2P1084628 / 34 / 3.894 / 2.87 / 1.64 / 5.00 / 13.52 / 2.94 / 15.53 / 4.09 / 10.46 / 2.91
BICF2S2399226 / 34 / 4.310 / 2.05 / 1.36 / 3.08 / 10.69 / 2.32 / 12.05 / 3.29 / 10.53 / 2.93
BICF2P1062716 / 34 / 14.202 / 0.19 / 0.10 / 0.34 / 33.20 / 7.21 / 36.40 / 8.79 / 30.98 / 7.59
BICF2P554459 / 34 / 14.652 / 3.66 / 1.65 / 8.14 / 15.44 / 3.35 / 11.54 / 3.17 / 10.94 / 3.03
TIGRP2P397016_RS9217137 / 34 / 27.082 / 0.27 / 0.15 / 0.47 / 20.17 / 4.38 / 23.49 / 5.90 / 23.49 / 4.56
BICF2S23732555 / 34 / 27.245 / 0.48 / 0.31 / 0.74 / 13.04 / 2.83 / 10.96 / 3.03 / 7.81 / 2.28
Table S6 Possible candidate genes for the CHD-QTL in German shepherd dogs
CFA / SNP-position / QTL-position (Mb) / Candidate genes and their positions (Mb) / Function for bone formation(Mb)
3 / 77.186 / 79.4-91.7 / phosphoglucomutase 2 (PGM2)
fibroblast growth factor receptor-like 1 (FGFRL1) / 77.2
94.3 / Endochondral bone formation, skeletal dysplasia
9 / 54.312 / 37.4-54.6 / collagen type XI alpha 1 (COL11A1) / 54.2 / Formation of cartilage collagen fibrils, differentiation and spatial organization of growth plate chondrocytes
26 / 32.016 / matrix metalloproteinase 11 (MMP11)
calcineurin binding protein 1 (CABIN1) / 31.7
31.4 / Extracellular matrix turnover
Expression of muscle genes
33 / 3.975 / 3.2-12.7 / ephrin type-A receptor 3 precursor (EPHA3) / 3.9
33 / 7.022 / 3.2-12.7 / ephrin type-A receptor 6 (EPHA6) / 7.0
33 / 10.983 / 3.2-12.7 / target of nesh-sh3 (ABI3BP)
SUMO1/sentrin specific peptidase 7 (SENP7)
pest-containing nuclear protein (PCNP) / 10.5
10.8
10.9
34 / 3.422 / 4.5 / triple functional domain (PTPRF interacting) (TRIO) / 3.4 / Chondrocyte proliferation and differentiation
34 / 7.851 / 4.5
34 / 14.246 / - / solute carrier family 6 (neurotransmitter transporter, dopamine), member 3(SLC6A3) / 14.2 / Locomotion impairment
34 / 27.034 / - / fibroblast growth factor 12 (FGF12) / 27.0 / Cell survival activities
The genes PGM2, COL11A1, MMP11, CABIN1, EPHA3, EPHA6, ABI3BP, SENP7, PCNP, TRIO, SLC6A and FGF12are connected through a joint network using GENEMANIA ( The same pathway share CABIN1, EPHA3, EPHA6 andTRIO. Joint co-expression has been indicated for all candidate genes in Table S3. Physical interactions were shown among EPHA3, EPHA6 andTRIO.
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