4 Fine mapping of quantitative trait loci on equine chromosome
4.3 Materials and methods
Pedigree structure and phenotypic traits
For the multipoint linkage analyses, we employed 14 paternal half-sib families consisting of 104 foals, 99 of their mares and eight stallions (Supplementary Table 1). These Hanoverian families were identical with the previously analysed data set employed by Dierks et al. (2007). Screening for polymorphic SNPs was performed in
these eight unrelated stallions. The 104 foals were also genotyped for association analyses.
We obtained digital radiographs of metacarpo- and metatarsophalangeal (fetlock) joints of all limbs and tibiotarsal (hock) joints of both hind limbs. Diagnosis of osteochondrosis was done following the recording and evaluation scheme developed for warmblood horses (Kroll et al. 2001). The sagittal ridge of the 3rd metacarpal and metatarsal bone of fetlock joints, the intermediate ridge of the distal tibia and the lateral trochlea of the talus were considered as predilection sites for OC in these joints. Horses exhibiting irregular bone trabeculation with variable radiolucency, irregular bone margin, new bone formation or osteochondral fragments at these predilection sites were regarded as affected by osteochondrosis. Two categories of radiographic changes were distinguished in the analysis, firstly animals with all types of radiographic signs of osteochondrosis including osteochondral fragments (OC) and secondly horses with changes which were only characterised by osteochondral fragments visible as isolated radiodense bodies (“joint mice”, “chips”) in the joint space of the predilection sites of fetlock and/or hock joints (OCD). Animals without any signs of radiographic changes in the regarded joints were classified as free from OC and only these horses were used as controls.
Development of microsatellites and single nucleotide polymorphisms (SNPs)
For the in silico development of new microsatellite markers, we build permutation sequences with di-, tri- and tetranucleotides with at least 15 and up to 30 repeats.
Those generated repeat motifs were aligned with the horse genome assembly (UCSC Genome Bioinformatics, version EquCab2, 2008) 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. Their positions were determined by alignments of the flanking sequences on the second horse genome assembly EquCab2 (http://www.broad.mit.edu/ftp/pub/assemblies/mammals/horse/Equus2/) after
mas-king repetitive elements with the RepeatMasker (http://www.repeatmasker.org/). In addition to this, eight microsatellites were chosen published by Ellegren et al. (1992) and Tozaki et al. (2007). All 74 microsatellites used in this study and their characteristics were shown (Supplementary Table 2).
A total of 70 SNPs and two Indel mutations were identified within 19 PCR products in the regarded region (Supplementary Table 3). For non-parametric multipoint linkage and association analyses 26 SNPs were selected for genotyping due to their heterozygosity and linkage phase with other adjacent SNPs in the eight stallions. We preferentially chose SNPs with a high number of heterozygous animals or which showed all three possible genotypes. Some SNPs were intergenic because SNPs from the SNP table were chosen due to their position on EquCab2 with the objective of a uniform coverage of the genome-wide significant OC QTL.
For the development of 18 SNPs, we used the SNP table from Broad institute (http://www.broad.mit.edu/ftp/distribution/horse_snp_release).
15 SNPs (14 intragenic) could be developed using equine BAC clones within six positional candidate genes. These were the sorting nexin 13 (SNX13) gene, the insulin-like growth factor 2 mRNA binding protein 3 (IBF2BP3) gene, the 3-hydroxyisobutyrate dehydrogenase (HIBADH) gene, the Bardet-Biedl syndrome 9 (BBS9) gene, the KIAA0895 protein (KIAA0895) and the acyloxyacyl hydrolase (AOAH) gene. BAC DNA was prepared from positive BAC clones using the Quiagen plasmid midi kit (Quiagen, Hilden, Germany). BAC DNA end sequences were obtained using the ThermoSequenase kit (AmershamBiesciences, Freiburg, Germany) and a LI-COR 4300 automated sequencer (LI-COR, Lincoln, NE, USA).
Only BAC clones with significant BLAST hits on ECA4 were selected. Equine PCR primers for SNP identification were designed using the PRIMER3 software after masking repetitive elements with the RepeatMasker. These primers yielded PCR products within a size range of 466 to 623 bp.
Genotyping of microsatellites
F PTC 100™ or PTC 200™ (MJ Research, Watertown, MA, USA) thermo cyclers and a general PCR program with variable annealing temperature (Ta) were used for the
PCR amplification of the microsatellites. The reaction started at 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 sec, optimum annealing temperature (Ta) for 1 min, 72 °C for 30 sec, and at 4 °C for 10 min. All PCR reactions were performed in 12 µl reaction volumes using 10 ng DNA, 1.2 µl 10x incubation buffer containing 15 mM MgCl2, 0.5 µl DMSO, 0.3 µl each dNTP (100 µM each) and 0.1 µl Taq Polymerase (5 U/µl, Qbiogene, Heidelberg, Germany). The amount of primers was between 2.0 and 15.0 pmol in the multiplex reactions. All forward primers were end labelled fluorescently with IRD700 or IRD800. Most of the primer pairs were pooled into PCR multiplex groups. For the analysis of the marker genotypes, the PCR products were size-fractionated by gel electrophoresis on an automated sequencer (LI-COR, Lincoln, NE, USA) using 6% polyacrylamide denaturing gels (Rotiphorese Gel40, Carl Roth, Karlsruhe).
Genotyping of SNPs
The PCR reactions were performed in a total volume of 30 µl containing 10 ng of genomic DNA as template, 10 pmol of each primer and 1 U Taq polymerase (MP Biomedicals, Eschwege, Germany). Thermocycling was carried out under the following conditions: initial denaturation at 94°C for 4 min was followed by 35 cycles of 94°C for 30 s, 58°C or 60°C for 1 min, 72°C for 1 min and a final cooling at 4°C for 10 min.
The amplicons were sequenced on an automated capillary sequencer, MegaBACE 1000 (GE Healthcare, Freiburg, Germany). The sequencing reaction was carried out using the DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare).
Amplification started with an initial denaturation at 94°C for 1.5 min, followed by 34 cycles of 20 sec denaturing at 94°C, 15 sec annealing at 50°C and 2 min elongation at 60°C. Finally, the reaction was cooled down to 4°C for 10 min. The reaction product was cleaned up using a Sephadex G50 filtration (GE Healthcare). Sequence data was analysed with the Sequencher 4.7 program (GeneCodes, Ann Arbor, MI, USA). In the first step the sequence analysis was performed for the PCR products of eight unrelated Hanoverian warmblood stallions. If a heterozygous SNP was found
for at least one stallion, all progeny and the corresponding mares of the respective families were analyzed.
For eight of the identified SNPs a RFLP assay was designed because the respective SNP affected the recognition site of a restriction enzyme. The PCR products for six SNPs also contained at least one additional recognition site for this enzyme as an internal control (Supplementary Table 4). Out of the eight SNPs, two revealed only one cutting site in the respective PCR product. For one SNP we used a Real Time (RT) PCR TaqMan® SNP Genotyping Assay (Applied Biosystems, Darmstadt, Germany) with allele specific primers. These primers were labelled with different fluorescence dyes. For genotyping of SNP alleles the 7300 RT-PCR system (Applied Biosystems) detects the different wavelengths of emitted light from the employed dyes.
Statistical analyses
Multipoint non-parametric linkage analyses were performed using the Merlin software (multipoint engine for rapid likelihood inference, version 1.1.2) (Abecasis et al. 2002) for 14 paternal half-sib groups including 74 microsatellite markers and 26 SNPs on ECA4. 33 of these microsatellites were already included in the previously performed whole genome scan by Dierks et al. (2007). The Zmean and LOD score test statistics were used to test for the proportion of alleles shared by affected individuals identical by descent (IBD) for the considered marker loci (Kong and Cox 1997). The maximum (minimum) achievable Zmean was 12.94 (–2.81) for LDA and thus high enough to reach a genome-wide significance level of 5%. The corresponding maximum (minimum) value for LOD scores was 8.47 (–0.43). In the case of no linkage, Zmean approaches the minimum achievable value due to an equal distribution of alleles among affected relatives. A significant chromosome-wide co-segregation of a marker allele with the phenotypic expression of OC or OCD in the examined population was assumed for p-values < 0.05. Genome-wide error probabilities were obtained by applying a Bonferroni correction: Pgenome-wide = 1 - (1 – Pchromosome-wide)1/r, where r = length of ECA4 (108.57 Mb) divided by the total equine genome length in Mb. The length of the horse genome was assumed to be 2680 Mb.
In addition, the data was evaluated using the ALLELE procedure of the software package SAS/Genetics (Statistical Analysis System, Version 9.2, SAS Institute Inc., Cary, NC, USA, 2008) to estimate the observed heterozygosity (HO), the polymorphism information content (PIC) and to test for Hardy-Weinberg equilibrium.
Association was tested using the CASECONTROL procedure of SAS and χ2-tests were employed for the genotypic and allelic distributions and the allelic trend among affected horses and controls.
4.4 Results
The non-parametric multipoint linkage analyses for ECA4 including 74 microsatellites and 26 SNPs showed chromosome-wide significant Zmeans and LOD scores for OC in fetlock and/or hock joints at 4.92 to 39.76 Mb reaching a genome-wide significance level for both test statistics between 7.98 and 16.51 Mb, at 27.15 and at 28.79 Mb (Supplementary Table 5).
The highest Zmeans and LOD scores were 4.08 and 3.85 with corresponding genome-wide error probabilities of p=0.0005 and p=0.0002 for the microsatellite TKY3171 at 9.80 Mb (Figure 1). For fetlock OC, chromosome-wide significant error probabilities were reached for both test statistics in the region from 7.42 to 13.10 Mb, at 27.15 Mb and at 28.79 Mb and furthermore, between 56.15 and 59.84 Mb. The highest Zmean was 2.42 and the highest LOD score was 1.27 with chromosome-wide error probabilities of p=0.008 for both test statistics for the marker ABGe067 at 8.75 Mb (Figure 2). Chromosome-wide significant error probabilities were also reached for the trait hock OC from 3.62 to 5.30 Mb and from 6.21 to 6.24 Mb (Supplementary Table 6). The highest Zmean was 1.89 and the highest LOD score was 1.26 with chromosome-wide error probabilities of p=0.03 and p=0.008 for the microsatellite ABGe131 (Figure 3).
The mean distance among the all used markers on ECA4 was 1.05 Mb. The 74 microsatellite markers had on average 6.6 alleles, a mean observed heterozygosity (HET) of 63.2% and polymorphism information content (PIC) of 58.4%.
The SNPs were tested for Hardy-Weinberg equilibrium and for strong LD among alleles. SNP BIEC2-845191 was not in Hardy-Weinberg equilibrium. The r2-values indicating relationship of SNPs, showed by tagging with threshold r2≥0.8 that all
SNPs had linkage disequilibrium below the r2 value. A casecontrol analysis based on χ2-tests for genotypes, alleles and trend of alleles revealed significant associations for genotype and/or allele test statistics and showed a total of three significant SNPs (BIEC2-893170 at 13.10 Mb, AAW02031763:g.79876T>C at 14.08 Mb and BIEC2-851132 at 15.99 Mb). One SNP (BIEC2-893170) was highly associated for each trait of OC (Table 1).
4.5 Discussion
This study is an important step towards identifying genes responsible for equine OC on ECA4. The genome-wide significant interval spanned 8.53 Mb for OC in fetlock and/or hock joints and this genomic region appears most promising for discovering associated genes. The three significantly associated SNPs were found in this genome-wide significant QTL. Two SNPs were located in intergenic regions, one SNP (AAW02031763:g.79876T>C) was found in intron 2 of the HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1). This gene is involved in encoding a cytoplasmic phosphoprotein that regulates cell proliferation. HECW1 acts as a transducer molecule for developmental processes, including segmentation and neuroblast specification. Genes close to the two associated intergenic SNPs are GLI-Kruppel family member 3 (GLI3), receptor (G protein-coupled) activity modifying protein 3 (RAMP3) and ETS domain protein Elk-1-like (LOC647102). No function is known for LOC647102. GLI3 is a candidate gene for different kinds of polydactyly (Radhakrishna et al. 1999). Wang et al. (2000) demonstrated that cAMP-dependent protein kinase dependent processing of vertebrate GLI3 in developing limb generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. They concluded that mutations of the GLI3 gene produce a range of limb patterning malformations. Previous expression analyses showed no significant difference in expression of GLI3 in affected joints compared with unaffected cartilage (Semevolos et al. 2005). RAMP3 interacts with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells (Aiyar et al. 2001). Calcitonin is involved in control of bone metabolism and therefore seems to be a suitable functional candidate gene for osteochondrosis.
Those results have to be verified on the basis of more samples of affected and unaffected horses. In addition, we have to develop more SNPs in the region of the QTL. The Illumina Equine 50K BeadChip might be helpful to find more associated SNPs in the regarded region.
4.6 Acknowledgements
This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-2).
4.7 References
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metacarpo- and metatarsophalangeal joints of horses. Journal of the American Veterinary Medical Association 203: 101-4.
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Philipsson, J., Andreasson, E., Sandgren, B., Dalin, G., and J. Carlsten, 1993 Osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints in Standardbred trotters. II. Heritability. Equine veterinary journal Supplement 16: 38-41.
Radhakrishna, U., Bornholdt, D., Scott, H. S., Patel, U. C., Rossier, C. et al., 1999 The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; no phenotype prediction from the position of GLI3 mutations. American Journal of Human Genetics. 65: 645-655.
Semevolos, S., Strassheim, L., Haupt, J., and A. Nixon, 2005 Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis.
Journal of Orthopaedic Research 23: 1152-1159.
Stock, K. F., Hamann, H., and O. Distl, 2005 Prevalence of osseous fragments in distal and proximal interphalangeal, metacarpo- and metatarsophalangeal and tarsocrural joints of Hanoverian Warmblood horses. Journal of Veterinary Medicine A 52: 388-94.
Swinburne, J. E., Boursnell, M., Hill, G., Pettitt, L., Allen, T. et al., 2006 Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families. Genomics 87: 1-29.
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Wang, B., Fallon, J. F., and P. A. Beachy, 2000 Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100: 423-434.
Wittwer, C., Hamann, H., Rosenberger, E., and O. Distl, 2006 Prevalence of osteochondrosis in the limb joints of South German Coldblood horses. Journal of Veterinary Medicine A 53: 531-9.
Wittwer, C., Löhring, K., Drögemüller, C., Hamann, H., Rosenberger E. et al., 2007 Mapping quantitative trait loci for osteochondrosis in fetlock and hock joints and palmar/plantar osseus fragments in fetlock joints of South German Coldblood horses. Animal Genetics 38: 350-7.
Ytrehus, B., Carlson, C. S., and S. Ekman, 2007 Etiology and pathogenesis of osteochondrosis. Veterinary Pathology 44: 429-448.
0,0
Figure 1 Multipoint chromosome-wide Zmean and LOD score on ECA4 harbouring a quantitative trait locus (QTL) for equine osteochondrosis (OC) in fetlock and/or hock joints. The maximum of the curve is marked by an arrow and shows the position of the genotyped microsatellite.
Figure 2 Multipoint chromosome-wide Zmean and LOD score on ECA4 harbouring a quantitative trait locus (QTL) for equine osteochondrosis in fetlock joints (OC-F). The proximal and distal maxima of the curves are marked with arrows and show the positions of the genotyped microsatellite makers.
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5
0 10 20 30 40 50 60 70 80 90 100 110 120
Position in Mb
Zmean and LOD score
Zmean LOD score ABGe131
Figure 3 Multipoint chromosome-wide Zmean and LOD score on ECA4 harbouring a quantitative trait locus (QTL) for equine osteochondrosis in hock joints (OC-H). The maximum of the curve is marked with an arrow and shows the position of the genotyped microsatellite marker.
Table 1 Association analysis using χ2-tests for 23 Single Nucleotide Polymorphisms (SNPs) on ECA4 for the traits Osteochondrosis in fetlock and/or hock joints (OC), Osteochondrosis dissecans in fetlock and/or hock joints (OCD), Osteochondrosis in fetlock joints (OC-F), Osteochondrosis dissencans in fetlock joints (OCD-F), Osteochondrosis in hock joints (OC-H) and Osteochondrosis dissecans in hock joints (OCD-H).
Horses Minor allele frequencies (%)
χ2-tests for
association Error probabilities Trait/SNP
affected controls affected controls Odds
ratio
Geno-type Allele Trend Geno-
type Allele Trend OC
BIEC2-893170 75 26 25.33 50.00 0.33 12.80 10.86 10.00 0.00166 0.00099 0.00156
BIEC2-851132 74 28 18.92 32.14 0.48 7.56 4.07 4.11 0.02288 0.04370 0.04260
OCD
BIEC2-893170 46 26 27.17 50.00 0.36 8.14 7.57 6.76 0.01709 0.00594 0.00932 OC-F
BIEC2-893170 53 26 20.75 50.00 0.25 19.06 14.11 13.53 0.00007 0.00017 0.00023 BIEC2-85113 52 28 16.35 32.14 0.39 8.37 5.32 5.25 0.01524 0.02114 0.02196
OCD-F
BIEC2-893170 25 26 22.00 50.00 0.26 9.67 8.65 7.79 0.00795 0.00328 0.00524 AAWR02031763 1 25 26 36.00 32.69 3.75 8.07 10.01 7.98 0.01771 0.00156 0.00472
OC-H
BIEC2-893170 34 26 29.41 50.00 0.42 4.37 5.28 4.32 0.11234 0.02153 0.03777 OCD-H
BIEC2-893170 25 26 30.00 50.00 0.43 3.50 4.24 3.46 0.17399 0.03945 0.06279
1 AAWR02031763:g.79876T>C
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 Marker name, accession number, primer sequences, annealing temperature, position, allele size, number of alleles, HET and PIC values of the microsatellites used in this study
Marker Acc. No Forward/Reversed Primer Sequence Ta (°C) Mb
ABGe127 AM989453 F: CTCCCAGGACTAGGATTAAGAG
R: CAGGTCTCTGCCTGTAGAAAG 58 0.20
121-139 5 75.24 70.76
TKY0810 AB104028 F: CCACCTACGATGGGTTCAAT
R: CCAAGAGCACTGATACAGAG 58 1.18
251-269 8 53.99 54.11
ABGe128 AM989454 F: AAGAGAACAGTGACCGTTTCAG
R: TGACGTAAAGAATGGGTGATTC 58 2.05
178-182 3 59.33 51.95
ABGe129 AM989455 F: GAGAAAGCAAGGTGGATGTG
R: CCCATCGTATATGTATGTCCAC 58 2.34
76-106 5 64.56 55.27
AHT043 AJ271528 F: ACACAAGTGACAGGAGCGTG
R: TGGAAGCATGCAAGAGGTC 60 2.92
156-190 12 81.99 76.43
ABGe130 AM989456 F: CCATAATGACTTGTTCACTTGC
R: GAAGCTTCTTCCCCACTGAG 58 3.62
109-127 8 60.48 53.93
NVHEQ029 AF056393 F: GAGATTTTGCCCCAAAGGTTA
R: CTCTTCTTTCTTCCCCAGGTCT 56 4.53
92-100 5 61.14 57.64
ABGe131 AM989457 F: CTGAAAATCAGGACCGTGAC
R: CAAAGGTTCACCCAAGATTC 58 4.92
136-160 10 89.95 84.03
TKY1923 AB215866 F: AAAGCTCAAAGCTGGATGC
R: CTACAATGCATCTTCCCACAC 52 5.30
268-288 6 75.59 70.41
ABGe132 AM989458 F: AGAATGGACCCAAACCTACC
R: ACACTGCCTGTTTCTGGTTC 58 5.94
187-203 4 63.94 55.10
TKY0797 AB104015 F: CACCCCAATCGATGTCGAAG
R: CCATGCTGTGGTGGCATC 58 6.21
128-158 4 61.50 53.75
AHT084 AJ507701 F: TGGCAATCTGCAGGGAAC
R: GATCTTGTGATTGTGTGTGTG 60 6.34
82-192 15 74.76 72.45
ABGe133 AM989459 F: ATCCCTTACAGAGCCCAATG
R: TGCTGTTCTCTGGGAAGAAG 58 6.77
211-231 3 40.48 36.16
UMNe587 AY735285 F: TTGGTCAGAATAGGTAGTTATG
R: AGATGGATATGTACATGGATAC 60 6.90
242-274 11 77.04 69.99
HMS006 X74635 F: GAAGCTGCCAGTATTCAACCATTG
R: CTCCATCTTGTGAAGTGTAACTCA 60 7.23
157-167 6 77.40 67.63
ABGe134 AM989460 F: TGAAAAGACCAAACATGCTG
R: TGGTTCCATCTTGTTAACTCC 58 7.42
208-222 6 61.24 57.01
Supplementary Table 2 continued
Marker Acc. No Forward/Reversed Primer Sequence Ta (°C) Mb
TKY2285 AB216228 F: ACCAGCAGAGCATTCTTCAC
R: ATCCCAATGTGTGACCTCTC 62 7.98
191-213 8 76.42 76.16
ABGe067 AM940028 F: CCACTGGGGTGTAAATCTGA
R: CTGCGTGGAAGCTGTTTTAT 58 8.75
136-144 8 76.42 76.16
TKY3171 AB217114 F: CAGACCTTTCCTTCCAGCTC
R: CTAAGAGTGCCACCCCTATCT 58 9.80 93-97 3 26.42 21.77
ABGe068 AM940029 F: AATAAGCGAGCATTAGCACAA
R: AGCACTGTGCATTTTGGACT 58 10.89
238-252 6 76.42 71.95
ABGe069 AM940030 F: CATGGCAACGACAATACAAA
R: TGGATTTACAGTGCAAGCAG 56 11.48
158-208 14 69.34 69.71
ABGe070 AM940031 F: AAGTGCCTTCTCAGGAGGAT
R: GCCCTGCCTTAGCAGACT 58 12.59
135-145 6 58.29 55.49
UMNe224 AF536301 F: ATGCTTAGCAAGGCCGTG
R: TCCAAAGATAGCGGCAGTG 60 13.35
149-152 3 65.26 52.96
ABGe071 AM940032 F: AATAGTCATGGCGTGTCAGG
R: GCATCTGGTGCTTTTTGTCT 58 14.38
133-155 5 56.81 53.19
TKY942 AB104160 F: TTGTGCAGCTGTGGCTTAG
R: ATAGGTGAGGGGCTGTGAG 58 15.67
88-104 7 61.32 55.21
UMNe404 AF536301 F: TTGGAACTTTTAGCAAAGAACC
R: GATCCATTCCCACATATGGC 60 16.51
162-174 4 32.14 28.29
ABGe072 AM940033 F: TGTGACAAATGCCATGAGAG
R: CTGTTCGCTCTGAAACAGGT 58 18.15
187-190 4 62.74 51.88
ABGe073 AM940034 F: AGTCTTGCCCTTGCCTTTT
R: AGGCAGAGCAAAAGGATCA 60 18.61
255-295 9 70.42 63.77
UMNe063 AB104160 F: GGATTTTCTTCTTTTGAATGGC
R: TTTACAATAGCCAAGATGCGG 57 19.57
128-144 5 38.10 35.68
ABGe074 AM940035 F: GGTCAGAGATGAGCAGAAATG
R: TGAATCTATAGGAGGCTGTTCG 62 20.09
93-121 8 38.50 36.40
ABGe075 AM940036 F: CGGGTGGAAAAGAGAAGG
R: CACACCAGAAGTCCTGTAGTCA 58 21.53
98-114 7 64.79 60.09
ABGe076 AM940037 F: CACAATGTCAAGCAGGTTCA
R: ATCTTTGTCAGGGAGGGTTG 58 23.00
272-278 2 13.15 12.24
ASB003 AB104160 F: AATTCATCTCAGTGCTCTACCAGC
R: TTCATTTTCTACATGCACTACAGC 60 23.51
196-208 6 58.37 55.12
Supplementary Table 2 continued
Marker Acc. No Forward/Reversed Primer Sequence Ta (°C) Mb
ABGe077 AM940038 F: TGACTCCAGCTTGAGGAGAC
R: GGAAGCTGAGGATGACAAATC 58 24.34
224-242 8 45.07 44.22
ABGe078 AM940039 F: ATGTTGGGCACAGTTACCAC
R: AACTCAATGAAACAAACCAAGC 60 27.15
280-286 4 42.45 36.02
ABGe079 AM940040 F: AGGAGGATGTTTGCCTCTTAC
R: TGAACCAGGAAAGGTAGCAG 58 28.38
186-194 4 51.17 46.79
TKY772 AB103990 F: CAGCTTTCATTGTCTGATGTCT
R: ACTGGTCAATAGGCTTGTGG 60 28.79
222-236 6 45.97 44.62
TKY0337 AB103990 F: ACTCAAGAGGTCAATCAGAGG
R: CTCTTCCACTCTGCATTCTG 60 29.88
222-242 7 73.71 67.45
ABGe080 AM940041 F: GCAAACTGAGTGGTCCTTTG
R: CCAGGAAAATCTGGTTAGCA 54 31.27
212-266 10 72.77 69.16
HTG1 AF169162 F: TTGCCTAGAATTGCTGAACC
R: TGGCCATCATTTATACATACCA 52 32.34
266-284 6 73.33 66.29
COR057 AF108374 F: GGAGGAGAGGAAGAGAGTGG
R: ATCCAGGGCTCTCCATAGTC 58 32.74
235-243 5 69.81 63.55
ABGe081 AM940042 F: AGCCTCAGCTCAAGAAAACA
R: TGGCAGTAAGACCTTCCTTTT 58 33.52
186-198 5 60.09 50.28
ASB029 X93543 F: CTGGCCCATAAAAAAACACTG
R: TGTATGGTTGTCAGCTCAAACC 60 36.65
121-137 5 24.88 22.94
ABGe082 AM940043 F: TCAATGACAATCATCCTCCTG
R: CAGAGCAAGGGTGGAAATC 52 39.76 86-98 5 61.50 58.24
UMNe138 AF536259 F: AGCTGGAAAGGACCACTTT
R: AACCAACAGGGGGTTTCC 58 41.57
136-152 5 65.73 60.50
LEX061 AF075661 F: TCAGTGTTCCCATCTGTA
R: TGAAATCACACCTTTACTTTA 50 42.31
142-160 7 72.17 71.77
LEX050 AF075652 F: ATAGTCTGGGGTTAGGTAAGG
R: TCTAGCCCAATGTAAATGC 55 49.36
112-124 5 57.35 52.46
AHT013 F: CTTCCTCAGGTGCATAGGTTG
R: TCATTAAAATACAACCTGCCCC 60 50.31
136-142 4 54.29 49.75
ABGe083 AM940044 F: GAGGTCTTTATGTTGGCTTCC
R: AAGCCTATTGCTTTGGGATAG 58 51.20
90-108 6 55.40 51.08
ABGe084 AM940045 F: CCCACATAAAGAATGTGAAACA
R: CGCCTGAACATAGAATAACAAA 55 52.07
182-210 10 77.46 71.35
Supplementary Table 2 continued
Marker Acc. No Forward/Reversed Primer Sequence Ta (°C) Mb
ABGe085 AM940046 F: TTTATTGAAAGTCAGCCCTTG
R: TTTGAGCTCTGTGTCAGCAA 58 53.56
94-102 4 62.91 58.96
ABGe086 AM940047 F: ATCTCAACGTGGGATGTCTG
R: GCTTTGGAGTGCAAATTAGG 58 54.68
135-155 8 76.06 71.80
ABGe087 AM940048 F: GCTGTCTGGGTCTGAATCC
R: AGATAGCAGACTGGGGAAGG 58 56.15
139-155 7 69.95 65.39
TKY1451 AB215394 F: CTGAGATTAACGGCCCAGTA
R: TCAGTCATGTATTCCTGTGCAT 58 57.31
190-208 9 79.72 77.38
ABGe088 AM940049 F: GATATCCTTTCAGGCAGCAG
R: TGTTTCTGTTTGGTTTGTGG 58 58.12
226-246 10 74.18 70.69
TKY830 AB104048 F: ATTGGAATGTCAGGTGTAGC
R: AGGCAGGCCAGTTTGATTG 55 58.77
140-144 3 67.61 59.00
LEX033 AF075635 F: TTTAATCAAAGGATTCAGTTG
R: TTTCTCTTCAGGTGTCCTC 55 59.50
186-204 9 76.70 71.34
ASB022 X93536 F: AGGAATGTGAAATACAGGAGG
R: TTTGTGGTCTTCCGTGCACC 62 59.51
149-167 8 70.00 68.89
COR089 AF154942 F: CCTGCCATAAATTTGTTTCC
R: TCCCTACCTCATCTCCACAC 58 59.84
276-298 10 75.71 69.45
ABGe089 AM940050 F: GGACAAAAGTGCTTTGCCTA
R: CAGAGCATGTGACTGTGGAG 58 61.40
128-142 3 23.94 21.31
ABGe090 AM940051 F: GTGATGAAGGGGTTGAAGAG
R: AGCAGTTCTTACCTCCTGGAC 58 61.91
245-263 5 61.97 58.55
ABGe091 AM940052 F: AAACAAAAGCTGCATGTTGA
R: CTGAATTGTATTGGGGGAGA 62 62.37
130-142 6 65.88 60.36
TKY1817 AB215760 F: TGTTCCCAGAAGGAAAATTG
R: AAAAAGCTCAACCTCTGTGG 58 63.32
229-267 10 86.38 79.39
ABGe059 AM919498 F: AGTTGCCTCTGGTCTTGCAG
R: GCTGGCAGAATGTCTGTTTTC 60 63.98
192-212 8 78.87 69.81
HTG007 AF169291 F: CCTGAAGCAGAACATCCCTCCTTG
R: ATAAAGTGTCTGGGCAGAGCTGCT 55 64.17
118-128 5 62.38 54.60
TKY552 AB103770 F: CTAGAGGTGCCTTCCCAGAC
R: ACCACCAAGACGAAAGGTGA 60 65.15
124-132 5 69.52 66.85
TKY354 AB044854 F: AGTGAGGTCTTCCTTGACTG
R: TGTTAGATGGTGGTAAGTGC 60 69.24
140-174 12 67.62 63.29
Supplementary Table 2 continued
Marker Acc. No Forward/Reversed Primer Sequence Ta (°C) Mb
TKY720 AB103938 F: CAGGAGTATCCAGAATTGCAGA
R: CCAGCTGTGTGTAACGCAAT 60 69.96
235-255 6 73.11 67.26
HTG009 AF169293 F: TGTGGGAAGAGTGTCAATAGCTGT
R: AGGCATCTGGTTTGCTGCAATTTC 55 71.14
118-138 6 52.13 47.66
TKY661 AB103879 F: CGAGGTCTTGGAACCTATCC
R: TTCACTTCAGACAACTCTATTGAAGA 60 77.94
140-152 4 66.20 60.86
TKY363 AB044863 F: CTCAGACTAAGCGGTACTAG
R: ATGGATACATTCTGGGGAAC 55 96.44
164-176 7 78.40 71.31
AHT061 AJ507678 F: TCTGCATCCTCATGTTCAATG
R: TTGACTTATTTCACTCAGCCCA 60 97.24
222-232 6 68.10 63.21
TKY698 AB103916 F: TGTTGAGGCAAGGGTTCTTT
R: CTCCATTGCCCACTCCTTAG 60 97.58
238-258 9 68.08 65.06
SGCV23 U90601 F: GGCTTAAGATATGGGTGAGTAAGG
R: GCCCACCCTCTTACTTTTCTCAA 58 102.74
198-246 17 82.93 83.81
Ta: Annealing temperature in °C
Mb: Location on the horse genome assembly 2.0 in megabases bp: base pairs
HET: Observed heterozygosity
PIC Polymorphism information content
Markers in bold indicate those microsatellites which were used in the previous whole genome scan for osteochondrosis in Hanoverian warmblood horses (Dierks et al. 2007)
Location of the SNP intergenic intergenic intergenic Intron 1
Ta 60 58 60 58
Bp 612 567 466 585
Primer forward (5’-3’) Primer reverse (5’-3’) GTGATTGGAGGGGATCACAG GAGAAAGGCATTTCGCTCAG GGAATAAGGCAGGGATATTTCG AGGACCTCATGTTGGCTCAG TGAATGAAGTCCATTGTTGGAG GGGCACACATTTTTAGTGAGC TGGTAGCCAAACTGCATCAC TCAAACCCAATTTGGTGCTC
Primer forward (5’-3’) Primer reverse (5’-3’) GTGATTGGAGGGGATCACAG GAGAAAGGCATTTCGCTCAG GGAATAAGGCAGGGATATTTCG AGGACCTCATGTTGGCTCAG TGAATGAAGTCCATTGTTGGAG GGGCACACATTTTTAGTGAGC TGGTAGCCAAACTGCATCAC TCAAACCCAATTTGGTGCTC