Acknowledgements

This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-2)

3.6 References

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Table 1 Location of 16 positional candidate genes on HSA1, the Equine Gene name and their position on EquCab2 assembly

Gene Gene location (Mb)

on HSA1¹

Equine Gene name

Gene location (Mb) on ECA2² collagen, type IX, alpha 2 (COL9A2) 40.54 - 40.56 COL9A2 17.78-17.81 Smad nuclear interacting protein 1 (SNIP1) 37.77 - 37.79 LOC100054834 20.07-20.08 thyroid hormone receptor associated protein 3

(THRAP3)

36.46 - 36.54 LOC100055016 21.14-21.21

collagen, type VIII, alpha 2 (COL8A2) 36.33 - 36.33 LOC1000693339 21.29 - 21.29 neurochondrin (NCDN) 35.80 - 35.80 LOC100055370 21.73 - 21.74 CUB and Sushi multiple domains 2 (CSMD2) 33.75 - 34.40 LOC69927 22.96 - 23.56 polyhomeotic homolog 2 (PHC2) 33.56 - 33.61 PHC2 23.71 - 23.76 FLJ25476 protein (FLJ25476) 33.49 - 33.54 LOC100070027 23.79 - 23.81 brain-specific angiogenesis inhibitor 2 (BAI2) 31.97 - 32.00 BAI2 24.92 - 24.95

collagen, type XVI, alpha 1 (COL16A1) 31.89 - 31.94 COL16A2 24.97 - 25.02 lysosomal associated multispanning membrane

protein 5 (LAPTM5)

30.98 - 31.00 LOC100056331 25.72 - 25.75

matrilin 1, cartilage matrix protein (MATN1) 30.96 - 30.97 LOC100056375 25.76 - 25.77 mitochondrial trans-2-enoyl-CoA reductase

(MECR)

29.39 - 29.43 LOC100070598 27.01 - 27.04

phosphatase and actin regulator 4 (PHACTR4) 28.57 - 28.70 PHACTR4 27.54 - 27.60 ficolin 3 (FCN3) 27.57 - 27.57 FCN3 28.41 - 28.42 exostoses-like 1 (EXTL1) 26.22 - 26.24 LOC100071242 29.41 - 29.43

1 Homo sapiens, NCBI map viewer, build 36.3

2 Horse (Equus caballus)UCSC Genome Browser, build

Trend 0.22 0.34 0.19 0.04 0.21 0.01 0.28 0.04 0.28 0.03 0.13

Allele 0.20 0.30 0.17 0.04 0.16 0.01 0.24 0.05 0.24 0.04 0.12

Error probabilities Geno- type 0.02 0.04 0.02 0.11 0.03 0.04 0.04 0.04 0.04 0.03 0.03

Trend 1.52 0.90 1.74 4.44 1.56 6.61 1.17 4.10 1.17 4.82 2.31

Allele 1.62 1.06 1.92 4.31 1.93 6.33 1.40 3.98 1.40 4.21 2.38

χ2 -tests for associaton Geno- type 8.02 6.24 7.76 4.44 7.13 6.61 6.50 4.10 6.50 4.82 6.87

Controls 3.50 3.50 3.50 35.70 3.50 35.70 3.57 6.25 28.57 5.36 28.57

MAF (%) Affected 8.70 7.70 9.60 47.10 10.40 39.50 8.82 100.00 13.24 18.00 16.00

Controls 28 28 28 28 28 28 28 24 28 28 28

Horses Affected 74 45 52 52 24 24 34 31 34 25 25

Odds ratio 2.60 2.28 2.87 2.02 3.14 2.75 2.61 0 0.38 3.88 0.48

SNP AAWR02028119:g.2038T>C AAWR02028119:g.2038T>C AAWR02028119:g.2038T>C AAWR02028318:g.8256C>T AAWR02028119:g.2038T>C AAWR02028318:g.8256C>T AAWR02028119:g.2038T>C AAWR02028280:g.3080G>T1 AAWR02028343:g.10957A>G AAWR02028065:g.1247G>A AAWR02028343:g.10957A>G

Gene NCDN NCDN NCDN FCN3 NCDN FCN3 NCDN MECR EXTL1 SNIP1 EXTL1

Table 2 Number of affected and controls, Odds ratio, Minor allele frequency and significant results of the association analyses using χ2 -tests for SNPs on ECA2 associated with different phenotypes of OC in Hanoverian warmblood horses. Trait OC OCD OC-F OCD-F OC-T OCD-T 1 not in Hardy-Weinberg equilibrium

-0.5

Figure 1 Multipoint chromosome-wide Zmeans and LOD scores of the genomic region on ECA2 harbouring the Quantitative Trait Locus (QTL) for equine osteochondrosis (OC) in fetlock and/or hock joints. The maximum of the curve is marked by an arrow which shows the respective markers.

Figure 2 Multipoint chromosome-wide Zmeans and LOD scores of the genomic region on ECA2 harbouring the Quantitative Trait Locus (QTL) for equine osteochondrosis dissecans (OCD) in fetlock and/or hock joints. The maxima of the curves are marked by arrows which show the respective markers.

-1.0

Figure 3 Multipoint chromosome-wide Zmeans and LOD scores of the genomic region on ECA2 harbouring the Quantitative Trait Locus (QTL) for equine osteochondrosis in fetlock joints (OCF). The maxima of the curves are marked by arrows which show the respective markers.

Figure 4 Multipoint chromosome-wide Zmeans and LOD scores of the genomic region on ECA2 harbouring the Quantitative Trait Locus (QTL) for equine osteochondrosis dissecans in fetlock joints (OCDF). The maxima of the curves are marked by arrows which show the respective markers.

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0

0 10 20 30 40 50 60 70 80 90 100 110 120 Chromosome position in Mb

Zmean and LOD score

Zmean LOD score ABGe006 HMS54

Figure 5 Multipoint chromosome-wide Zmeans and LOD scores of the genomic region on ECA2 harbouring the Quantitative Trait Locus (QTL) for equine osteochondrosis in hock joints (OCH). The maxima of the curves are marked by arrows which show the respective markers.

Supplementary Table 1 Number of families analysed, their sizes and prevalences of osteochondrosis (OC), osteochondrosis dissecans (OCD), osteochondrosis in fetlock (OC-F) and hock (OC-H) joints and osteochondrosis dissecans in fetlock (OCD-F) and hock (OCD-H) joints by family and in total

Prevalences in % for

Half-sib family

Number of progeny

Male Female

OC OCD OC -F OCD-F OC-H OCD-H 1 4 1 3 100.0 100.0 50.0 50.0 75.5 75.0 2 9 4 5 44.4 33.3 11.1 0.0 33.3 33.3

3 7 5 2 71.4 42.9 71.4 42.9 28.6 14.3

4 15 9 6 53.3 33.3 26.7 6.7 26.7 26.7 5 8 1 7 100.0 25.0 75.0 12.5 37.5 12.5 6 4 2 2 100.0 25.0 50.0 25.0 50.0 0.0 7 5 3 2 100.0 40.0 80.0 40.0 20.0 0.0 8 6 2 4 100.0 66.7 83.3 50.0 66.7 33.3 9 20 12 8 75.0 60.0 50.0 20.0 50.0 45.0 10 8 4 4 62.5 25.0 50.0 12.5 12.5 12.5 11 5 3 2 40.0 20.0 20.0 0.0 20.0 20.0 12 5 2 3 100.0 80.0 100.0 80.0 0.0 0.0 13 5 3 2 40.0 40.0 40.0 40.0 0.0 0.0 14 3 1 2 100.0 66.7 100.0 66.7 0.0 0.0 Total 104 52 52 73.1 45.2 51.9 25.0 32.7 24.0

Supplementary Table 2 Characteristics of the microsatellites used in this study

Marker Acc. No Forward / reverse primer sequence (5’-3’)

COR041 AF108358 F: CAACTTGGGTTCTCGCTTAG

R: TCCGGAAAGTGCAAAGTTAG 60 1.73 226-244 7 62.3 56.6 ABGe004 AM900758 F: TGAAAATCTTTCATTGCCAGAG

R: TCTACCCTCCCCCTTTGC 60 3.11 238-264 8 55.5 66.9 ASB18 X93532 F: TGCAGACAAAGCTGGACACTC

R: CTGCTGAGAAAGCTTCTGC 60 5.25 190-206 7 64.0 49.9 ABGe109 AM946988 F: GGGTGGCTCCTTAGAGCTTC

R: CCCCTCCCTTGTTTATATGC 60 6.21 204-218 6 72.7 69.9 ABGe005 AM900759 F: GGGAGTGGTGAGGGATGTC

R: GCCCAAGCTCCTTAGCTTAAC 60 7.48 122-144 6 55.7 52.7 TKY384 AB048290 F: TGCAGCAAGAAACCTAAACA

R: CTTCAGTTGTAATCAGGCTC 55 9.00 103-131 11 64.8 64.6 HTG019 F: GTATGTGCTGTACCTTCTGC

R: ATGAGAAAGACGATAGATGATAT 57 9.49 143-153 3 44.1 32.3 ABGe143 FM165573 F: TTCTGCTTTGGGATGGTTTC

R: CCATTCTCTTCCCCAGAAAG 60 10.16 211-219 5 90.0 84.0 ABGe110 AM946989 F: CAAGTGCCCACTGACTGATG

R: TGTTGCTGTGAATGGGACAG 60 11.89 129-164 9 62.9 66.0 ABGe144 FM165574 F: CAAAAATGGCAAGATTTCATCC

R: TGCCCACTGACAGATGAATG 60 12.43 115-141 10 78.1 80.8 TKY615 AB103833 F: CCAGACCCACCCAAAAGATA

R: GGGCAAAGTGGTCTGAGAAG 60 14.06 243-255 8 82.0 79.0 ABGe111 AM946990 F: CCATGTTCACGTCCATTTTG

R: CTAGGGGGTTCAAGGCACTC 60 15.65 145-161 7 81.7 73.2 ABGe060 AM940021 F: GCGACAGCTTCCACTTTG

R: CAGAGATGAGTCCTGCTTGG 58 16.54 180-188 4 46.0 37.2 UMNe323 AY391338 F: GATCCTGCAGGAAAGCATGT

R: CCGCTCGGAATATTTCATTG 55 19.17 166-212 12 75.8 70.1 COR037 AF101406 F: GAGCAATTTCTGGGTCTGAG

R: CACCCCTCTCTTTGTTTGTC 58 21.60 238-244 4 63.1 52.2 COR090 AF154943 F: GGTTTGTCTCTTTGAGGTGTG

R: TGCTCATATCTTCACCCTGC 58 22.45 91-101 5 61.2 57.0 TKY003 AB048314 F: GGTTCACACAGGAGTCAGGGA

R: CCTTCTGGTTTGCCTCGTCTC 60 22.73 162-174 6 59.3 56.3 AHT035 AJ271520 F: TGACTTAGAGCTTTTGCTCCC

R: CCAGAAGTCCAGGCATTTGT 60 23.34 125-143 8 70.5 69.2 VIAS-H17 L23550 F: AATTAACCACTATGCCACAGGG

R: GTGTGGACCTAGCACCACTC 60 23.64 139-147 5 62.0 54.4 UMNe513 AY464512 F: AGAATCTCACTTAGCTCCGCC

R: TGCTGTTCTTTACAGCGTGG 60 24.65 232-254 9 52.9 50.8

Table S2 continued

ABGe062 AM940023 F: TTCAACGTGCAGAGTGAAAG

R: AACGCTCAGCCCAAGGAG 52 26.39 176-200 6 55.9 53.1 ABGe006 AM900760 F: CTGAAACCAGCCAGGAAAAG

R: TCTCCTAGCCGGGAGAAAAC 58 26.89 175-209 9 61.4 74.0 ABGe063 AM940024 F: TAGCCTTTTCTTTGGTGTGC

R: GCTCAAAAATTTGATCAACGAC 58 27.87 117-143 6 64.8 59.3 HMS054 AY768954 F: AATGAACGGAGAAGCAGAAAGGC

R: ACTGCGGTGCAGGTTGGAGG 58 28.45 112-122 4 52.4 49.5 ABGe007 AM900761 F: CCCACATATTGTAGATTAGTGATTCTC

R: AACAGTGTTAAACCATAGCAAGG 59 29.47 227-253 6 67.5 71.9 ASB017 X93531 F: GAGGGCGGTACCTTTGTACC

R: ACCAGTCAGGATCTCCACCG 60 30.60 89-115 12 81.0 75.6 AHT067 AJ507684 F: CGGAGTTGGTATTTAACACACG

R: ACGCACCAACTTCTGAAAGG 60 30.94 234-252 5 75.1 71.2 ABGe008 AM900762 F: GTGGGACTAAGCTGGGGTTC

R: CTACTTCCCTGCGTCCTGTC 61 31.17 149-165 8 66.7 68.7 UM007 AF195128 F: GGGAATAGAGAAAGGTGAAG

R: TTAGAGTTCCTGCTCCTCC 60 31.90 119-157 10 71.3 68.6 ABGe009 AM900763 F: AGAGCTGCACTGTCAACTGC

R: CACATGACCTCATTGGTTCC 61 31.99 234-242 5 53.7 56.1 AHT073 AJ507690 F: AACAGGACTGCAGAGACTCCA

R: CTCCCTCACTAAACTGTGACCC 60 32.16 145-157 7 86.5 76.1 UCD380 U67403 F: GTGGGATGGGGTAGGAAC

R: ACTGGGAAGTGCTGCAAA 58 32.50 115-135 4 59.7 45.7 HMS051 AY775553 F: GCTCCATGTTTCCTGACAGG

R: GAAAAGAAAGTGTCAGACTGCC 58 32.97 164-178 4 47.4 39.2 ABGe113 AM946992 F: CTCCCTGCTTTTCCTGTCTC

R: GGATCCTTTGTCTCCTGCTG 60 33.05 156-171 7 67.9 69.2 ABGe342 FM179616 F: ACCCACTGTGAAACCCTTTG

R: TGTGGAGAATGGAGGAGACC 60 34.42 220-237 7 79.0 77.2 ABGe343 FM179617 F: CGGGAGAAAGTCAGGAAGC

R: AAGGGTGTGAATCCCCTTG 58 35.23 76-94 7 70.6 67.9 ABGe064 AM940025 F: CTCTGGCCAGGCTTGCTC

R: TCAAATCTTGGATCTGAGTGC 58 36.88 106-114 4 27.7 24.5 ABGe344 FM179618 F: GCACAGGAAGACCACACAAG

R: TGAGCAGGAGAGGACTAGGG 60 36.89 164-178 7 73.3 68.1 TKY474 AB103692 F: TGTCCTACTTCCCAGCTACG

R: CCTGCCTTTGCAGTTCAGTG 60 38.50 153-159 4 38.2 30.7 TKY784 AB104002 F: GATCAGTACTTTGCAAATGGATAAC

R: GTAACTCCAAGGCTACGTTC 60 38.79 200-214 7 74.5 69.1

Table S2 continued

TKY340 AB044841 F: CAGGCTTTGGGTTTCATTATG

R: TCAGTCTCCATTATAGGAGG 58 39.26 132-154 5 66.0 60.2 ABGe065 AM940026 F: ATGCTCGTTTCACAAAGAGG

R: TCTTTTTGTGCAGGGTGTG 56 41.11 232-312 16 71.4 68.6 ABGe345 FM179619 F: GGCCTTCTTGACCTAACTGC

R: CCATCTACCATCCGGTCTTC 58 42.56 239-265 11 78.9 71.6 TKY2643 AB216586 F: TCTAATTGCAGCCTCCTTTG

R: TGCTCCCATTTCTCTTATGC 56 43.13 270-284 6 42.2 38.5 ABGe346 FM179620 F: CCAGCACATTCTGAGGAACC

R: GCTTCAAGAAGGGGTTCTG 58 46.26 125-149 10 65.8 62.8 ABGe347 FM179621 F: ACTACTAGGGTGCATTGTTTTTAGG

R: GTGAACATGTTGCCCCTCTG 62 49.29 257-269 6 67.9 60.8 ABGe348 FM179622 F: GGCGCTCTCAAATCATGC

R: ATGGGTGGTTCCAGAAACTG 60 50.57 172-184 7 74.1 68.9 ABGe349 FM179623 F: CCAGGAATATGAAACCACAGG

R: GGTCCCTCACTCCTTCAATG 60 52.42 247-263 7 72.8 68.6 COR049 AF108366 F: TGACATTCACAATGACCAGC

R: CGGCCTGCACTATAGACTTC 58 55.12 194-196 2 3.5 3.4 TKY352 AB044852 F: TCTGCTAAGTTCAATGGGTC

R: TTCTTACTTAACAACCATCG 58 55.37 77-95 5 64.4 58.2 AHT012 ECA001445 F: ACCCAAAGTCATGGGAATCA

R: TTGTTGCCGACAACATGC 60 59.08 102-114 6 63.9 60.2 ABGe066 AM940027 F: GAGAAGCTGTTGCTAAAGTGC

R: CCGGGATTTAGTACTGCAAG 58 59.16 94-100 4 54.3 51.2 TKY605 AB103823 F: AGTGCCATCTTGAATTGCTG

R: CGATGAAATTGAGGATTTATGGA 60 60.64 157-165 4 54.7 40.4 HMS16 U89806 F: AGAGTAATCAATGGATGAGTGGAC

R: TGTTGTCGCAAATGGCAGGCATC 60 65.14 142-154 6 37.3 45.1 TKY358 AB044858 F: GAAGCAGTGCCTCTTATGTG

R: CAGAACAGTCAGGACTTGAC 60 70.51 154-164 5 61.3 54.5 A-14 Y10239 F: CAGCTGGGTGACACAGAGAG

R: GTCATCACTACTCCCTACAC 60 74.47 208-236 9 76.7 65.5 ASB13 X93527 F: CTCTGAAAGAGCAGGATTGG

R: GTCTTCTAAGTGGTAAGAGCC 60 75.64 122-132 5 67.8 61.6

100G3_T7

MS AJ542943 F: GGGTGAACAGTAGGGGAAAC

R: CTGTTGTAGAGAGGGGGCTC 65 86.46 185-197 5 70.9 63.4 UMNe076 AF191706 F: CCCTCAGGTTGAGGACTCAG

R: AGGTGACAACCTGGATTTGC 60 87.06 100-104 3 47.2 39.9 TKY798 AB104016 F: GAGCAGAAGGTACGAGAAGA

R: AACTTAACCAGGCTGTTCTG 52 93.95 237-247 6 58.5 63.2

Table S2 continued

COR026 AF101395 F: GGCGTCCAACGTAAAGTAGA

R: CCTCTTCGGAAACTCTGACA 55 117.18 228-230 2 26.8 22.9 COR043 AF108360 F: ACGCTCATTTCATAGGCTCTGAC

R: GCAGGAGTGGTGTTTGTTTCAC 55 117.54 132-134 2 10.9 9.8

Ta: Annealing temperature

Mb: Positions on the horse genome assembly EquCab2 in megabases bp: Basepairs

HET: Observed heterozygosity PIC: Polymorphism information content

Markers in bold indicate microsatellites which were used in the previous whole genome scan for osteochondrosis in Hanoverian warmblood horses (Dierks et al. 2007)

Permutation sequences were built with all variations of di-, tri- and tetra-repeat motifs with a minimum length of 15 repeats and a maximum length of 30 repeats in the identified QTL region. These sequences were aligned with the horse genome assembly (Broad Institute, version EquCab2, 2007) using the SSAHA2 package (Sequence Search and Alignment by Hashing Algorithm combined with the cross-match sequence alignment program developed by Phil Green at the University of Washington, version 1.0.1, The Wellcome Trust Sanger Institute, UK, 2007).

Alignment results that obtained a maximum score per length (100% identity) were used for primer design. For this purpose flanking sequences were taken from EquCab2 and equine PCR primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) after masking repetitive elements with the RepeatMasker (http://www.repeatmasker.org/). The marker positions on ECA5 on EquCab2 were verified using the Basic Local Alignment Search Tool (BLASTall version 2.2.17) of NCBI.

The average PIC and HET of the 29 newly developed microsatellites was 62.2% and 67.5% with a minimum of 21.7% and 25.1%.

RFLP (enzyme recognition sequence, number of cutting sites in PCR product BstUI, CGCG, 1 PspOMI, GGGCCC, 1 BsoBI, CYCGRG, 1

SNP ID AAWR02027999:g.2814 G>A AAWR02027999:g.5421 T>C AAWR02027999:g.8263 G>A AAWR02028065:g.328 T>C AAWR02028065:g.1247 G>A AAWR02028065:g.1259 G>A AAWR02028065:g.1404 G>C AAWR02028065:g.1487 C>T AAWR02028098:g.8027 T>C AAWR02028098:g.8077 G>A AAWR02028098:g.8135 G>T

HO 43.0 35.7 50.0 11.5 22.6 56.9 31.7 29.4 - 24.0 34.7

PIC 30.9 30.4 37.3 10.3 18.0 34.9 25.0 23.9 - 18.9 30.2

Corres- ponding location in human gene Intron 4 Exon 12 Exon 18 Exon 3 Intron 3 Intron 3 Intron 3 Exon 4 Intron 94 Intron 9 Intron 9

Ta 60 60 60 60 60 60

Bp 510 595 679 552 511 581

Primer forward (5’-3’), primer reverse (5’-3’) GTGAGGAGAGGAGCATGTGG CAAACAGAACCCCTCTCTGC CTCACATTGAGGGCTTCGAG CCAGAGCAGGGAAATGACTC ATGACCAGACCCACTTCCTG GCGACTGTCCAAATGATGAG GGGATCGAGACCCTCAGAAC AGTAGACACGAGGGGTAGGG GACTCCAGCTCCCATGACTC GCTCAATGCGCTTGTTGTTC AGCTGTGGGTCACAGAAACC CAAGGACTTCGAGCCATTTC

Supplementary Table 3 Primer sequences, product size, annealing temperature and the corresponding location in the human gene, polymorphism information content, heterozygosity and for 65 SNPs (43 SNPs genotyped in this study), one indel mutation, RFLP information about nine intragenic single nucleotide polymorphisms Gene symbol COL9A2 SNIP1 THRAP3

RFLP (enzyme recognition sequence, number of cutting sites in PCR product

SNP ID AAWR02028098:g.8198 A>G AAWR02028104:g.4121 A>G AAWR02028119:g.1809 T>C AAWR02028119:g.1839 T>A AAWR02028119:g.1890 T>G AAWR02028119:g.1923 T>G AAWR02028119:g.2038 T>C AAWR02028119:g.2092 T>C AAWR02028168:g.9615 C>T AAWR02028168:g.9790 T>C AAWR02028168:g.10120 T>C AAWR02028168:g.10510 C>A

HO 17.2 34.6 12.6 - - 24.5 33.0 8.0 - - 46.0 43.1

PIC 14.5 30.5 12.6 - - 20.3 30.6 8.0 - - 34.8 32.9

Corres- ponding location in human gene Intron 9 Exon 2,utr Intron 5 Intron 54 Intron 54 Intron 5 Intron 5 Intron 5 Intron 134 Intron 134 Exon 14 Intron 14

Ta 58 60 60 60

Bp 596 555 574 572

Primer forward (5’-3’), primer reverse (5’-3’) CTGAGAAGAAAGGTGCCAAC CAGGAGCTGATGAATTTTCG CCTGTCCAGCCAGACAGTTC GGGTGTGGAAGGAACAGATG CTTACCCGGCACCTCCTTAG CTGGGTAGTTGGGTGACAGG CCTCCACTGCTCTTCTCTTTC GCTCAAAAGCCAGTGACTCC

Supplementary Table 3 continued Gene symbol COL8A2 NCDN CSMD2

RFLP (enzyme recognition sequence, number of cutting sites in PCR product XbaI, TCTAGA, 1 HpyCH4III, ACNGT, 2

SNP ID AAWR02028175:g.63095 T>A AAWR02028177:g.20061 G>A AAWR02028177:g.20112 C>T AAWR02028177:g.20238 C>T AAWR02028177:g.20455 C>T AAWR02028177:g.20465 G>A AAWR02028222:g.11368 T>C AAWR02028222:g.60042 T>C AAWR02028224:g.8093 T>C AAWR02028224:g.9690 T>G AAWR02028224:g.9696 C>G AAWR02028224:g.9716 A>C

HO 35.7 - 52.5 31.3 - - 50.0 51.5 57.7 55.5 53.9 53.9

PIC 36.3 - 37.2 29.0 - - 33.7 32.6 36.9 37.1 37.2 37.2

Corres- ponding location in human gene Intron 6 Intron 54 Exon 5 Exon 5 Intron 44 Intron 44 Intron 4 Intron 6 Intron 68 Intron 71 Intron 71 Intron 71

Ta 58 60 58 60 60 60

Bp 504 586 400 578 563 805

Primer forward (5’-3’), primer reverse (5’-3’) CGAGACAGACAGAGGAGAGC CTGGGAGCCTTAGGAAGAAC CAAGCCTTCTCTTCCCTGTG CAGTGGGCACCTAGAAAAGG TGAGGGATTAAACTCCAAAGG TCCAAATGCATGAAAGATAAA GACTCAGCTCCCACCTTCTG ACTCAAGCTCAGGGTGTGGT CACAGAGCAGGCATGTGG AGAGCCAGTGACAAGGAAGG GGGACATTGGTATTGGCATC CATCTTGCCATACCCTGGAG

Supplementary Table 3 continued Gene symbol PHC2 FLJ25476 BAI2 COL16A1

RFLP (enzyme recognition sequence, number of cutting sites in PCR product HpyCH4III, ACNGT, 2

SNP ID AAWR02028224:g.9722 A>G AAWR02028249:g.33564 C>T AAWR02028249:g.33767 C>T AAWR02028249:g.47110 C>T AAWR02028249:g.51137 C>T AAWR02028249:g.51242 A>G AAWR02028249:g.51267 A>T AAWR02028249:g.51625 T>C AAWR02028249:g.53623 A>G AAWR02028249:g.53628 G>C AAWR02028249:g.53660 G>A AAWR02028249:g.53851 C>T

HO 53.9 47.8 29.0 37.7 21.4 21.4 37.9 39.0 - - - -

PIC 37.2 37.5 25.7 37.3 17.3 17.3 32.9 32.8 - - - -

Corres- ponding location in human gene Intron 71 Exon 8, utr Exon 8, utr Intron 1 Intron 4 Intron 4 Intron 4 Exon 5 Intron 74 Intron 74 Intron 74 Intron 74

Ta 60 58 58

Bp 529 530 1042

Primer forward (5’-3’), primer reverse (5’-3’) ATCGAGTCAGCACCCATCAG CAGGGTTTGCCTGTTTTGAC AAGTCGTCTGTCTGCCTGAG ACAAGTGGGAGCGACAAAC AGACCACGACTGTGAGCAAG TAGTTGGGCTGTACCCTGTG CCTTTCGTCAGGCCACTG TCTTCAGAGTGCCCCTCTTC

Supplementary Table 3 continued Gene symbol LAPTM5 MATN1

RFLP (enzyme recognition sequence, number of cutting sites in PCR product HphI, GGTGA, 1

SNP ID AAWR02028249:g.53883 C>T AAWR02028249:g.53930 C>T AAWR02028249:g.54034 C>T AAWR02028280:g.30776 Indel C>G AAWR02028280:g.30801 G>T AAWR02028280:g.30817 C>T AAWR02028294:g.37096 G>A AAWR02028294:g.37121 A>C AAWR02028294:g.37122 A>C AAWR02028294:g.37146 C>T AAWR02028294:g.37334 C>T AAWR02028294:g.38018 C>A

HO 35.6- - - 65.6 11.0 47.3 - - 16.5 - 18.5 27.2

PIC 30.8 - - 52.5 13.2 33.3 - - 15.3 - 17.9 25.6

Corres- ponding location in human gene Intron 7 Intron 74 Intron 74 Intron 6 Intron 6 Intron 6 Intron 54 Intron 54 Intron 5 Intron 54 Intron 5 Intron 4

Ta 60 60 60

Bp 555 551 574

Primer forward (5’-3’), primer reverse (5’-3’) CTGTGAGGGAAGCAGGAGAG CCATTTTCATGGACTTCACTTC CATTTACCTCGAGGGTTTGC GGGGGAAGTAAAGGCTGTTT GCTCTTTCACGTGGTGTTTG AACTGGCGCAGTTAGACAGG

Supplementary Table 3 continued Gene symbol MECR PHACTR4

RFLP (enzyme recognition sequence, number of cutting sites in PCR product EcoRI, GAATTC, 1 FauI, CCCGC, 3

SNP ID AAWR02028294:g.38022 G>C AAWR02028294:g.38119 G>C AAWR02028318:g.8256 C>T AAWR01015756:g.9740 A>G AAWR01015756:g.9875 A>G AAWR02028343:g.10957 A>G AAWR02028343:g.11029 T>C

HO 27.2 27.2 51.5 46.2 - 20.8 35.3

PIC 25.6 25.6 37.4 36.4 - 19.3 30.2

Corres- ponding location in human gene Intron 44 Intron 44 Intron 4 Exon 6 Intron 64 Intron 9 Intron 9

Ta 60 60 60 60

Bp 574 558 580 600

Primer forward (5’-3’), primer reverse (5’-3’) GCTCTTTCACGTGGTGTTTG AACTGGCGCAGTTAGACAGG CTTTGCCTTTGGGATTTCAG GAAAGGGAGATGGACTGAGG CAATCTCCTCCAGGACTTGC CCAAGGAGGTGGAAGGACTC CAGAGACCCCTTCTTTGGAC CACACAGCCCCCTACAGAAC

Supplementary Table 3 continued Gene symbol PHACTR4 FCN3 EXTL1 1 Product size (basepairs) 2 Annealing temperature (°C) 3 Due to low heterozygosity (<5%) the SNP was not genotyped 4 Polymorphism was in same linkage phase like another one

SupplementaryTable 4 Multipoint non-parametric chromosome-wide test statistics (Zmean and LOD score) with their chromosome-wide significant error probabilities (PZ, PL) and their map positions (POS) on the horse genome assembly 2.0 for OC and OCD in fetlock and/or hock joints in Hanoverian warmblood horses

POS 21.15 THRAP3_In9c 2.37 0.009 2.14 0.0009*

21.15 THRAP3_In9b 2.37 0.009 2.14 0.0009*

21.15 THRAP3_In9a 2.37 0.009 2.14 0.0009*

24 21.29 COL8A2_SNP1 2.42 0.008 2.18 0.0008*

Supplementary Table 4 continued

Supplementary Table 4 continued

POS in cM¹

POS in Mb²

Marker OC OCD

Zmean PZ LOD

score PL Zmean PZ LOD score PL

29.47 ABGe007 2.07 0.02 0.81 0.03 1.98 0.02 0.97 0.02 42.7 30.60 ASB17 1.85 0.03 0.64 0.04

43 30.94 AHT067 1.77 0.04 0.58 0.05 p_Z:error probability for Zmean

p_L: error probability for LOD score ECA: Equus caballus autosome Mb: Mega base

1Map position in the genetic map of Swinburne et al. (2006)

2Position on equCab2 assembly found by BLAST

Genome-wide significant after Bonferroni’s correction using P genome-wide (1-P chromosome-wide)^1/r with r being the length of equine chromosome 2 (120.86 Mb) divided by the total equine genome length (2680 Mb). *: P<0.05, **: P<0.01, ***: P<0.001

SupplementaryTable 5 Multipoint non-parametric chromosome-wide test statistics (Zmean and LOD score) with their chromosome-wide significant error probabilities (PZ, PL) and their map positions (POS) on the horse genome assembly 2.0 for OC and OCD in fetlock joints in Hanoverian warmblood horses

POS

SupplementaryTable 5 continued 23.80 FLJ25476_Ex5b 3.84 0.00006*** 3.40 0.00004*** 2.34 0.010 1.64 0.003 23.80 FLJ25476_Ex5a 3.84 0.00006*** 3.40 0.00004*** 2.34 0.010 1.64 0.003 33.5 24.66 UMNe513 3.82 0.00007** 3.43 0.00004*** 2.41 0.008 1.69 0.003 34.2 24.92 BAI2_SNP1 3.82 0.00007** 3.43 0.00004*** 2.43 0.007 1.70 0.003 34.29 24.97 COL16A1_SNP1 3.81 0.00007** 3.43 0.00004*** 2.43 0.007 1.70 0.003 25.02 COL16A1_In68 3.81 0.00007** 3.42 0.00004*** 2.43 0.007 1.70 0.003 34.25 25.02 COL16A1_SNP2 3.81 0.00007** 3.42 0.00004*** 2.43 0.007 1.70 0.003 36 25.75 LAPTM5_SNP1 3.77 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 36.01 25.75 LAPTM5_SNP2 3.77 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 36.05 25.76 MATN1_SNP1 3.76 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 36.06 25.77 MATN1_SNP2 3.76 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 36.08 25.77 MATN1_SNP4 3.76 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 36.09 25.77 MATN1_SNP5 3.76 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003 25.77 MATN1_In7 3.76 0.00008** 3.39 0.00004*** 2.43 0.007 1.70 0.003

SupplementaryTable 5 continued

POS in cM¹

POS in Mb²

Marker OC-F OCD-F

Zmean PZ LOD

score PL Zmean PZ LOD score PL

27.56 PHACTR4_In4 3.35 0.0004** 2.99 0.00010** 2.39 0.008 1.67 0.003 27.88 ABGe063 3.63 0.00014** 3.18 0.00006*** 2.43 0.007 1.70 0.003 28.41 FCN3_In4 3.63 0.00014** 3.18 0.00006*** 2.43 0.007 1.70 0.003 28.42 FCN3_Ex6 3.63 0.00014** 3.18 0.00006*** 2.43 0.007 1.70 0.003 41.7 28.46 HMS54 3.63 0.00014** 3.18 0.00006*** 2.43 0.007 1.70 0.003 29.41 EXTL1_In9a 3.60 0.0002** 3.16 0.00007** 2.38 0.009 1.68 0.003 29.41 EXTL1_In9b 3.60 0.0002** 3.16 0.00007** 2.38 0.009 1.68 0.003 29.47 ABGe007 3.59 0.0002** 3.14 0.00007** 2.36 0.009 1.67 0.003 42.7 30.60 ASB17 3.35 0.0004** 2.83 0.0002** 1.87 0.03 1.32 0.007 43.0 30.94 AHT067 3.26 0.0005* 2.66 0.0002** 1.72 0.04 1.11 0.012

31.17 ABGe008 0.09 0.5 0.00 0.5

43.41 31.91 UM007 1.65 0.05 0.62 0.05 pZ:error probability for Zmean

pL: error probability for LOD score ECA: Equus caballus autosome Mb: Mega base

1Map position in the genetic map of Swinburne et al. (2006)

2Position on equCab2 assembly found by BLAST

Genome-wide significant after Bonferroni’s correction using P genome-wide (1-P chromosome-wide)^1/r with r being the length of equine chromosome 2 (120.86 Mb) divided by the total equine genome length (2680 Mb). *: P<0.05, **: P<0.01, ***: P<0.001

SupplementaryTable 6 Multipoint non-parametric chromosome-wide test statistics (Zmean and LOD score) with their chromosome-wide significant error probabilities (PZ, PL) and their map positions (POS) on the horse genome assembly 2.0 for OC in hock joints in Hanoverian warmblood horses

POS

SupplementaryTable 6 continued p_Z:error probability for Zmean

p_L: error probability for LOD score ECA: Equus caballus autosome Mb: Mega base

1Map position in the genetic map of Swinburne et al. (2006)

2Position on equCab2 assembly found by BLAST

Genome-wide significant after Bonferroni’s correction using P genome-wide (1-P chromosome-wide)^1/r with r being the length of equine chromosome 2 (120.86 Mb) divided by the total equine genome length (2680 Mb). *: P<0.05, **: P<0.01, ***: P<0.001

Supplementary Figure 1 Haploview

21.73 Mb

23.35 Mb 23.80 Mb

25.02 Mb

25.76 Mb

27.03 Mb 27.56 Mb

28.41 Mb

29.41 Mb

Supplementary Figure 1 Linkage disequilibrium and positions of 35 SNPs (call rate

>50% of 43 genotyped SNPs) on ECA2. LD coefficients (r²) between the SNP pairs are indicated and the positions on ECA2 are pictured

Gene symbol

SNP ID SNP name in haploview

AAWR02027999:g.2814 G>A COL9A2_SNP1 AAWR02027999:g.5421 T>C COL9A2_SNP2 COL9A2

AAWR02027999:g.8263 G>A COL9A2_SNP3 AAWR02028065:g.328 T>C SNIP1_Ex3 AAWR02028065:g.1247 G>A SNIP1_In3a AAWR02028065:g.1259 G>A SNIP1_In3b AAWR02028065:g.1404 G>C SNIP1_In3c SNIP1

AAWR02028065:g.1487 C>T SNIP1_Ex4 AAWR02028098:g.8027 T>C

AAWR02028098:g.8077 G>A THRAP3_In9c AAWR02028098:g.8135 G>T THRAP3_In9b THRAP3

AAWR02028098:g.8198 A>G THRAP3_In9a AAWR02028119:g.1809 T>C NCDN_In5d AAWR02028119:g.1839 T>A

AAWR02028119:g.1890 T>G

AAWR02028119:g.1923 T>G NCDN_In5c AAWR02028119:g.2038 T>C NCDN_In5b NCDN

AAWR02028119:g.2092 T>C NCDN_In5a AAWR02028168:g.9615 C>T

AAWR02028168:g.9790 T>C

AAWR02028168:g.10120 T>C CSMD2_Ex14 CSMD2

AAWR02028168:g.10510 C>A CSMD2_In14 AAWR02028177:g.20061 G>A

AAWR02028177:g.20112 C>T FLJ25476_Ex5b AAWR02028177:g.20238 C>T FLJ25476_Ex5a AAWR02028177:g.20455 C>T

FLJ25476

AAWR02028177:g.20465 G>A

BAI2 AAWR02028222:g.11368 T>C BAI2_SNP1 AAWR02028222:g.60042 T>C

AAWR02028249:g.33564 C>T LAPTM5_SNP1 LAPTM5

AAWR02028249:g.33767 C>T LAPTM5_SNP2 AAWR02028249:g.47110 C>T

AAWR02028249:g.51137 C>T AAWR02028249:g.51242 A>G AAWR02028249:g.51267 A>T

AAWR02028249:g.51625 T>C MATN1_SNP5 MATN1

AAWR02028249:g.53623 A>G

Supplementary Figure 1 continued

Gene symbol

SNP ID SNP name in haploview

AAWR02028249:g.53628 G>C AAWR02028249:g.53660 G>A AAWR02028249:g.53851 C>T

AAWR02028249:g.53883 C>T MATN1_In7 AAWR02028249:g.53930 C>T

MATN1

AAWR02028249:g.54036 C>T AAWR02028280:g.30776 Indel C>G

AAWR02028280:g.30801 G>T MECR_In6b MECR

AAWR02028280:g.30817 C>T MECR_In6c AAWR02028294:g.37096 G>A

AAWR02028294:g.37121 A>C

AAWR02028294:g.37122 A>C PHACTR4_In5b AAWR02028294:g.37146 C>T

AAWR02028294:g.37334 C>T PHACTR4_In5a AAWR02028294:g.38018 C>A

AAWR02028294:g.38022 G>C PHACTR4

AAWR02028294:g.38119 G>C PHACTR4_In4 AAWR02028318:g.8256 C>T FCN3_In4 AAWR02028318:g.9740 A>G FCN3_Ex6 FCN3

AAWR02028318:g.9875 A>G

AAWR02028343:g.10957 A>G EXTL1_In9a EXTL1

AAWR02028343:g.11029 T>C EXTL1_In9b

CHAPTER 4

Fine mapping of quantitative trait loci on equine chromosome 4 responsible for osteochondrosis in Hanoverian warmblood horses

Claudia Dierks, Karina Komm, Virginie Lampe and Ottmar Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Germany

4 Fine mapping of quantitative trait loci on equine chromosome 4

Im Dokument Fine mapping of quantitative trait loci (QTL) for osteochondrosis in Hanoverian warmblood horses (Seite 69-103)