and 7q
Summary
In this study we present a comprehensive 5,000-rad radiation hybrid (RH) map of a 40 cM region on equine chromosome 4 (ECA4) that contains quantitative trait loci for equine osteochondrosis. We mapped 29 gene-associated sequence tagged site (STS) markers using primers designed from equine expressed sequence tags (ESTs) or BAC clones in the ECA4q12-q22 region. Three blocks of conserved synteny were identified showing two chromosomal breakpoints in the segment of ECA4q12-q22.
Markers from other segments of HSA7q mapped to ECA13p and ECA4p.
Furthermore, a region of HSA7p was homologous to ECA13p. Therefore, we have improved resolution of the human-equine comparative map, which allows the identification of candidate genes underlying traits of interest.
Keywords Equus caballus, comparative map, ECA4, FISH, radiation hybrid mapping.
Introduction
Osteochondrosis (OC) is an inherited developmental orthopaedic disorder which is observed in young horses at moderate to high prevalences (Stock et al. 2005, Winter et al. 1996, Philipsson et al. 1993). A genetic test for equine OC would be of major interest in horse breeding. Recently, quantitative trait loci (QTL) for OC in Hanoverian warmblood horses were mapped within the 50-90 cM region of the ECA4 linkage map (Böneker et al. 2006). Homology between ECA4q and HSA7q has been shown previously (Milenkovic et al. 2002, Chowdhary et al. 2003, Perrocheau et al. 2006, Swinburne et al. 2006). Our objective in this study was to refine the human-equine comparative map of ECA4q12-q22 where the OC QTL has been mapped.
A detailed map of this region will be instrumental in our search for candidate genes affecting this economically important equine condition.
Material and methods
All microsatellites of the RH04b and RH04c groups of the Chowdhary et al.
(2003) map were compared to the human genome, Build 35.1 using BLAST (http://www.ncbi.nlm.nih.gov/genome/seq/HsBlast.html). UMNe054 at 29.63 Mb, COR089 at 28.82 Mb and LEX061 at 8.49 Mb showed significant matches on HSA7.
In addition, Swinburne et al. (2006) found three microsatellites of the RH04b and RH04c groups that were syntenic to regions on HSA7: LEX050 at 16.73 Mb, ASB29 at 92.23 Mb, and AHT006 (alias HMB6) at 89.76 Mb. Based on these BLAST hits, genes from the corresponding HSA7p and 7q region were chosen for inclusion in the study.
BAC library screening/sequence analysis: The equine CHORI 241 BAC library was screened for clones containing sorting nexin 13 (SNX13), IGF-II mRNA-binding protein 3 (IMP-3), 3-hydroxyisobutyrate dehydrogenase (HIBADH) and parathyroid hormone-responsive B1 (B1), all from HSA7p. High density BAC colony filters from the CHORI 241 library were probed according to CHORI protocols (http://bacpac.chori.org) with heterologous 32P-labelled inserts of the human cDNA IMAGE clones (IMAGp998G0610038 for SNX13, IMAGp998N209570 for IMP-3, IRALp962G2123 for HIBADH and IMAGp998J0238 for B1) provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). Additionally, a BAC containing LSM8 homolog (U6 small nuclear RNA associated) located on HSA7q was used to confirm synteny between ECA4q22 and HSA7q. Four BAC end sequences (CH241-465E23, CH241-229G5, CH241-169D19 and CH241-423M6) gave significant BLAST hits for the selected genes (SNX13, IMP-3, HIBADH, and B1 respectively) (Supplemental Table 1). In order to confirm the presence of LSM8 in CH241-102B13, a sequence primer was derived from an equine expressed sequence tag (accession no. CX599677) corresponding to human exons 2, 3, and 4 of LSM8 (BLAST E-value 1e-51).
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Chromosomal location: The equine BAC clones containing SNX13, HIBADH, IMP-3, B1, and LSM8 were localized on equine GTG-banded metaphase chromosomes (from 30 to 35 spreads) by fluorescent in situ hybridization (FISH) as described elsewhere (Böneker et al. 2005, Müller et al. 2005). The international horse chromosome banding standard (ISCNH 1997) was used as reference. The FISH assignments included: SNX13 to ECA4q14, IMP-3 to ECA4q21.1-q21.3, HIBADH to ECA4q21.1-q21.3, B1 to ECA4q21.3 and LSM8 to ECA4q22.
Radiation hybrid mapping: A total of 55 markers were typed on the Texas A&M University equine RH5,000 panel (Chowdhary et al. 2003). Twenty-four horse ESTs and 18 horse BAC end sequences (BESs) matched to 42 human genes or open reading frames located from 0.81 to 40.67 Mb on HSA7p, and from 86.47 to 122.12 Mb on HSA7q (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) (Supplemental Table 1). Genes indicated by (N) in Supplemental Table 1 were not identified in the equine BAC clones, but based on comparative data were considered to be in close proximity. Additionally, we chose primer sequences from one gene (COL1A2) previously mapped by Chowdhary et al. (2003), and twelve microsatellites (UMNe054, COR089, ASB22, LEX033, HTG007, AHT013, LEX050, LEX061, ASB29, HMB6, HMS19, HTG009) already mapped on the equine-human comparative RH5,000
map representing RH04b and RH04c groups (Chowdhary et al. 2003). All markers were first mapped by two-point analysis of the RHMAPPER-1.22 (Slonim et al. 1997) (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) to determine which chromosome they mapped to. The ECA4 RH5,000 map was constructed using the RHMAP3.0 software package (Boehnke et al. 1991, Lange et al. 1995). Two-point analysis (RH2PT) revealed one linkage group consisting of 42 linked markers at a LOD score threshold of 8.0. The order of the five genes already mapped by FISH (SNX13, IMP-3, HIBADH, B1 and LSM8) was fixed. After construction of a framework map (ADDMIN = 2.0) using a stepwise ordering under the equal retention model (Bishop & Crockford 1992, Boehnke et al. 1991), the comprehensive map was constructed by adding the remaining markers at their most probably position using RHMAXLIK (ADDMIN = 0.0).
Results
Based on these analyses, we mapped a total of 29 newly developed markers to ECA4q12-q22 (Fig. 1). The mean retention frequency was 0.20 with a minimum of 0.13 (HTG009) and a maximum of 0.34 (C7orf23). The entire RH5,000 map spans 496.0 cR and corresponds to approximately 45.4 cM on the equine linkage map. Our map has an average ratio of 10.9 cR5,000 per cM and a mean marker density of 0.9 markers per cM but the ratio was not constant for all of the marker intervals.
When aligned with the homologous regions of HSA7 (NCBI map viewer, Build 35.1), the RH5,000 data for ECA4q12-q22 displayed three conserved blocks. Two blocks showed synteny with 86.5 Mb - 95.4 Mb and 10.8 Mb - 36.3 Mb regions of HSA7p, and the third block corresponded to 107.8 Mb - 122.12 Mb (NCBI map viewer, Build 35.1) region of HSA7q. The breakpoints on ECA4q12-q22 were located between SLC25A13 (80.3 cR) and LEX061 (109.6 cR) and between AOAH (366.8 cR) and DNAJB (377.7 cR) (Fig. 1).
Thirteen markers showed no linkage to the ECA4q map. According to RHMAPPER-1.22 two-point software, three markers corresponding to 38.1 - 40.7 Mb of HSA7p and four markers corresponding to 102.3 - 107.2 Mb of HSA7q were assigned to the proximal region of ECA4p. Mapping of MGC11257, KDELR2 and DKFZp434J1 revealed human-equine homologous segments between HSA7p and ECA13p as well as synteny between HSA7q and ECA13p based on results for TRRAP, SMURF1 and AZGP1 (Table 1).
Discussion
When we compared the comprehensive RH5,000 map with the previously published equine ECA4 RH5,000 map of Chowdhary et al. (2003), we found differences in marker order and arrangement. First, the linkage group between UMNe054 and COL1A2 was inverted in our map. Microsatellites HMB6 and ASB029 were centromeric to COL1A2 in our map. Microsatellite HTG007 was mapped distal to UMNe054 in this RH5,000 map in agreement with Swinburne et al. (2006). These results show that RH04b group delineated by UMNe054 and COL1A2 was inverted. Microsatellites HTG009 and HMS19 were inverted in comparison to the map of Chowdhary et al.
(2003). A discrepancy between our mapping results and the comparative map of
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Milenkovic et al. (2002) and Perrocheau et al. (2006) was observed for the LAMB1 gene which was assigned to ECA4q13 by FISH analysis. We mapped the LAMB1 gene to ECA4p near microsatellite HMS06 on the RH5,000 map of Chowdhary et al.
(2003). When the microsatellite marker order and arrangement in the present RH5,000
map was compared to the sex-averaged linkage map of Swinburne et al. (2006), no discrepancies were found (Fig. 2). In comparison to the male linkage map of Penedo et al. (2005), the order of microsatellites COR089 and ASB22 was inverted in our map. Furthermore, using the equine RH5,000 panel, we were able to define the locations of four microsatellites (LEX050 on 155.5 cR, AHT013 on 176.3 cR, HTG009 on 417.5 cR and HMS19 on 428.6 cR) which were not resolved in previous studies.
Acknowledgements
This study was supported by grants from the German Research Council, DFG, Bonn (DI 333/12-1). We are grateful to H. Klippert-Hasberg for her technical support during the work in the laboratory. The authors thank C. Wittwer for the STS primer sequences for mapping the CPVL gene.
References
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Supplemental Table 1 Information for 42 sequence tagged site markers derived from horse BAC end sequences or ESTs. Genes indicated by (N) were not identified in the equine BAC clone, but are in close proximity.
Gene (forward in the first line, reverse in the second line)
PCR
CX596863 16.41 1e-51 TAGAAAACAAAGGGGCAAGG
CAGGAAGAGCCACATGAAAG 506
CT006390 117.90 1e-21 CCATGATCTTCATTCCCTCTG AGACTCACTGCTGGGAGCAC
98
AOAH 36.33 - 6.54
BI961651 36.33 4e-33 GCTGCTGTTGGCAGATGTC TTGAGGGACAATTGGAGAGG
AJ5841573 12.70 1e-08 TCGACAGCAGTTTGTAGCAG
TCACTTCCACTCAGGAGGAG 495
AZGP1 99.21 - 99.22
BM780848 99.21 9e-56 CCCAGGAAAAGAGAGGACAC
TGGGCCAGACGATTATTACTG 503
AM072941 33.37 1e-122 AGCCAAGCTACATTCTGCTG ATCACTTGAACGTGGAGGAG
40.54 4e-28 CTGTTCATCCTCGGGAGTAG TTTGGTTGATTTAAGTTTCAGG
496
C7orf23 86.47 - 86.49
CX598762 86.47 3e-21 TGTGACAAGGATACAACTGGAG
ACCCCTAAAGGGAAACTGTTC 320
CADPS2 121.55 - 122.12
AJ542841 121.65 6e-67 TGCTCATTTCATGTGCTTCC
CGTGCATAACCGTAGAGCTG 335
CAPZA2 116.10 - 116.15
CX596422 116.15 0.0 CTTTTGATATTCGCCCAGTTC AGCATTAGACCTTGGCCTTG
252
CPVL 28.81 - 28.96
DN511041 28.81 4e-61 TGACCAGAGTTACTACGGGAAG TAATTATTCATGATTTCAGTTAACCAC
CX597697 6.44 2e-13 GTGCTCTCCAAGGTCCAAAG
CTACCGTCCTGCTCTTACGG 266
DNAJB9 107.80 - 107.81
CX600182 107.81 3e-77 TGATACCACCAATCAGCACAC GGCAAATTCAATCTTTTCTTGG
442
FLJ22313 35.45 - 35.51
CX597132 35.45 2e-87 ATGATGGGCAAAATGCAAAC TTAATCCAGGCCTTTGAAGC
580
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117 Supplemental Table 1 continued
Gene (forward in the first line, reverse in the second line)
PCR
30.64 4e-09 TTGGGTCTCTGGCTTACACC CTACCTGCTCCCTGGACATC
AJ885834 113.66 2e-168 TTGACGGAGTAAAAGGGATTG TGAATGCTCAAAATGCAAGC
463
GARS 30.41 - 30.45
CX594065 30.43 1e-71 GGCAGAAATCGAGCACTTTG TGGGCTTTTGCTGAATACAAG 99
HBP1 106.40 - 106.44
CX596143 106.44 1e-76 GAGTTGGCCTTTCCTTGACC TGCAAGTGTTGCACTGTCAC
AM072947 27.41 6e-19 GAGCTGGAAGAGGGAGTTTC CCAGGCATTTGAAGACAGAG
AM072950 23.28 7e-17 ACCAGCGTGTCTTCATTTTC
AGAAATTCACACCAGGTTGC 198
ING3 120.18 -
120.21 CD464595 120.20 1e-76 AAGAGGGACGAAGAACATCAAG TGACGAACTGGCATTCTGTG 158
CT008293 32.69 0.0 ATGACAAGGGAATTCAGTGC GCCTAGATACGCTAACCGAAC
119.82 3e-06 TTGTCCTTCAAAATGACTCCTC TGGCTGTATGTGTGTGCAAG
159
KDELR2 6.28 - 6.30
CX605749 6.28 4e-134 TCAGTCTGCCAGCGTAAGTG
AAGGCAAGATGCATTAAACAG 205
AJ543064 36.14 6e-113 TTGGAGTAACTTTCCCTTGC
TTTGAAAAACAGTCTGCGTTC 504
LAMB1 107.16 - 107.24
DN8725154 107.16 3e-53 GCACCCCACAGCAGTTACAG ATCTGGGCCTTCTCTGCTTC
AJ5839913 117.43 4e-08 TCCACAAAGAGACTCACAACAG AGCCATGATTTTAATTTGGTTC
532
MDFIC 114.16 - 114.25
CX594432 114.25 5e-14 TATATGGCCATCACGACTGC
ACAAAGCTTGCATTTTGACC 403
MGC11257 0.81 - 0.95
CX600886 0.81 6e-38 TCTCCACCTTGCTGGCTTAC TAGGAGAGCAGCTGGAGCAC
172
Supplemental Table 1 continued (forward in the first line, reverse in the second line)
PCR
CX605596 102.33 6e-48 TGGATAAAGACCCACCTTGC TTCCATAGGAAGTCACAAATCC
508
PHF14 10.79 - 10.98
CX594630 10.85 5e-66 AGAACCCGAGGACGAAAAC GCAAGTTGCACATTCCGTTC
AJ543269 10.99 1e-15 TCACCATTTGAGGGAGAGAG CCTTAGCAAACCCTCAAAGC 377
SCAP2 26.48 - 26.68
CD528759 26.48 0.0 TTTGCCAACTGATGACCAAC GCAAATAATCAAGCAAAATGC
AJ543018 95.55 2e-14 TTTTAGGGGTCAGCAGCTTG TCTCCCTTTCCCCATCTTTC
525
SMURF1 98.27 - 98.39
CX599658 98.27 3e-11 TTGAGTCCATTCAAAAAGTCC CAGGAAGGTGGCTTTAGAGG 401
AM072955 17.75 4e-21 TCCATTATTATCCTGGTGGAAG GTGATCCCAAACACAACATTC 365
AJ885367 104.40 8e-14 ACACGGCATTAAGACCAACC GTGCTCAGAAACCACCACAG
526
STEAP1 89.43 - 89.44
CX593678 86.43 2e-74 TCTGTGCTTTTGCACTTGAAC TGATGGAAACCATCGGTAAG
250
TES 115.44 - 115.49
CX597165 115.48 1e-103 AAGTGCCACGAATTGTCTCC TTTTCGGCCAATTTGTTCTC 200
TRGV10 38.11 - 38.11
CD535783 38.11 2e-25 TCACTGCTGGAAGCATTCAC CCACCATCCAGCACAGTAG 450
CR955442 98.14 1e-33 AGAGCCTCTGGCACATAAGAC CCACCCATCACACAAGAGG
AJ618811 38.63 2e-13 CCCACATTTACCTCATCAGC AGTTTTGAGTTCCAGTTTCCAG
391
1NCBI map viewer Build 35.1
2Accession number of BAC end or horse EST matching to HSA 7
3Four pairs of primers failed to produce a product suitable for RH mapping and had to be replaced by adequate ones from the other end sequence of the same BAC clone. Accession number of BAC end sequence used for STS primer design
4For this gene, a canine EST was used to design STS primers.
5This EST gave a positive BLAST hit using the nucleotide-nucleotide BLAST.
6The sequence primer LSM8_exon4: 5’-AGG TAG TTT TTC CTC AGT GTG C-3’ was designed using the primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). A sequence corresponding to exon 3 and intron 3 of the human LSM8 gene (accession no. AM072952) was obtained.
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Table 1 Two-point RH mapping results (LOD score ≥ 12) of genes that were not mapped to equine chromosome 4q14-q22
Gene Gene location
(Mb) on HSA7
ECA1 Closest marker
Distance (cR)2
Retention3
TRGV10 38.11 - 38.11 4p AHT084 30.65 29.3 VPS41 38.54 - 38.72 4p TCRG 0.0 29.3 C7orf10 39.95 - 40.67 4p AHT084 33.97 28.3 NAPE-PLD 102.33 - 102.38 4p NV029 0.0 21.7 SRPK2 104.35 - 104.62 4p AHT084 19.0 22.8 HBP1 106.40 - 106.44 4p HMS06 1.71 28.3 LAMB1 107.16 - 107.24 4p HMS06 7.47 23.9
MGC11257 0.81 - 0.95 13p COR069 13.12 33.7
KDELR2 6.28 - 6.30 13p VHL161 2.53 38.0
DKFZp434J1015 6.43 - 6.44 13p VHL161 2.53 38.0
TRRAP 98.12 - 98.26 13p VHL161 3.98 34.8 SMURF1 98.27 - 98.39 13p VHL161 2.84 39.1 AZGP1 99.21 - 99.22 13p VHL161 5.98 39.1
1Equus caballus autosome.
2Distance to the closest linked marker on the 5,000-rad TAMU equine RH panel (Chowdhary et al. 2003).
3Retention frequency.
Figure 1 Comparison of the ECA4q12-q22 map with the equine cytogenetic, HSA7 (NCBI map viewer, Build 35.1) and equine RH5,000 (Chowdhary et al. 2003) maps.
Vertical lines in the linkage map indicate two markers located at the same position.
The five genes mapped in this study by FISH are in boldface in the cytogenetic map.
High-resolution comparative RH map of ECA4q12-q22
Figure 2 RH5,000 map of ECA4 aligned with linkage maps of Penedo et al. (2005) and Swinburne et al. (2006). Vertical lines in the linkage map indicate two markers located at the same position.
121