Construction of a Radiation Hybrid Map of Chicken Chromosome 2 And

Construction of a radiation hybrid map of chicken chromosome 2 and alignment to the chicken draft sequence Sophie Leroux, Mélanie Dottax, Suzanne Bardes, Florence Vignoles, Katia Feve, Frédérique Pitel, Mireille Morisson, Alain Vignal

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Sophie Leroux, Mélanie Dottax, Suzanne Bardes, Florence Vignoles, Katia Feve, et al.. Construction of a radiation hybrid map of chicken chromosome 2 and alignment to the chicken draft sequence. BMC Genomics, BioMed Central, 2005, 6, pp.12. 10.1186/1471-2164-6-12. hal-02682366

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Research article Open Access Construction of a radiation hybrid map of chicken chromosome 2 and alignment to the chicken draft sequence Sophie Leroux*, Mélanie Dottax, Suzanne Bardes, Florence Vignoles, Katia Fève, Frédérique Pitel, Mireille Morisson and Alain Vignal

Address: Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, 31326, France Email: Sophie Leroux* - [email protected]; Mélanie Dottax - [email protected]; Suzanne Bardes - [email protected]; Florence Vignoles - [email protected]; Katia Fève - [email protected]; Frédérique Pitel - [email protected]; Mireille Morisson - [email protected]; Alain Vignal - [email protected] * Corresponding author

Published: 04 February 2005 Received: 09 September 2004 Accepted: 04 February 2005 BMC Genomics 2005, 6:12 doi:10.1186/1471-2164-6-12 This article is available from: http://www.biomedcentral.com/1471-2164/6/12 © 2005 Leroux et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances. Results: Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR6000 long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths. Conclusion: We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate.

Background genome analyses. This will help to detect sequences con- Chicken is a model organism in various fields of biology, served between species, which should correspond to such as embryology or immunology. It is also the only unknown exons and to regulatory or other functional bird species for which the genome has been studied in regions. Such analyses will therefore be essential for the detail and a lot is expected from its use in comparative annotation of other genomes, including that of human

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[1,2]. Chicken is also actually the only major agricultural mapping. One hundred and seventy eight out of 219 species for which a draft assembly of the genome markers (81 %) amplified successfully. This high success sequence is available http://www.ensembl.org/ in primer design using EST is comparable to what was Gallus_gallus/[3,4]. Thanks to a significantly lower rate of achieved in other studies using the Iccare web server interspersed repetitive elements and to the use of a highly [16,19,20]. inbred bird for sequencing, this draft is probably more accurate than the first one published for human three GGA2 RH map (figure 1) years ago [5,6]. Nevertheless, previous comparisons of RH Altogether, genotyping data were obtained for a total of mapping data with the sequence data showed that some 270 markers. Two-point analysis with CarthaGene using a sequence segments are in wrong positions of the genome LOD threshold of 4 enabled the construction of 5 GGA2 assembly [7]. Therefore the integration of all available linkage groups containing a total of 198 markers, includ- chicken mapping resources will be essential for improving ing all the microsatellite and gene markers from the the quality of the assembly, building a more reliable and genetic map. Out of the remaining markers, two informative resource. In addition to the genetic and BAC (LOC51059 and ALDH5A1) should have been integrated contig maps that have already been used, the RH map will into the GGA2 RH map, given their location within the thus provide an independent source of data to assist the sequence assembly. One explanation, concerning the lack chicken genome sequence assembly process towards a fin- of these 2 markers, could be that the retention frequency, ished quality sequence. that is unusually low for these two markers and indicative of PCR problems, did not allow attaining a LOD score RH panels are available for several domestic animals: cow value significant for linkage. The other 70 markers map [8], pig [9], horse [10], dog [11] and cat [12]. The success- either to other chromosomes or to unknown regions. ful production of a RH panel in chicken is quite recent They correspond to the external boundaries of the regions [13] and therefore only a limited number of chicken chro- of conserved synteny with human, from which EST were mosomes have been studied to date, namely: GGA4 [14], chosen for marker development. GGA5 [15], GGA7 [16], GGA14 [7], and GGA15 [17]. The final RH map of chicken chromosome 2 is 2794 We present here the radiation hybrid map of chicken cR6000 long and comprises 86 markers. A one-to-one com- chromosome 2, built by using markers chosen from the parison with the genetic map shows a good overall agree- GGA2 genetic map and a substantial number of addi- ment, with a few improvements over the genetic map; for tional markers developed from chicken EST data. Markers example markers ADL114 and MCW310 are mapped with from the genetic map are essential to anchor the RH map a higher precision than previously on the GGA2 linkage onto GGA2. Markers developed from EST data were cho- group. At the end of the chromosome (position 2794 cR), sen on the basis of existing comparative mapping infor- the 3 markers (MCW157, MCW189, and MCW0073 / mation data, indicating conservation of synteny with HSF1) were in the same order in the linkage group, but its HSA1, HSA3, HSA6, HSA7, HSA8, HSA9, HSA10, HSA18 orientation is different from the genetic map. These mark- and HSA22. They were used primarily to saturate the map ers are mapped on the comprehensive RH map (less sig- in markers and also to increase the precision of compara- nificant than the framework one), so the orientation given tive maps. The GGA2 RH map obtained was aligned with in the genetic map may be the right one. the genomic sequence assembly to detect eventual discordances. The corresponding comprehensive map comprises a total of 198 markers (figure 1). Results and discussion Development of EST markers Comparative maps (figure 2) Eighty eight microsatellite markers and 10 genes were As a result of our map construction strategy, based in great selected from available data in the literature or the data- part on the development of EST markers, we found a few bases. In addition to this, 219 markers were developed observations worthy of comment. from EST data to saturate the map. At the time this study was initiated, comparative mapping data suggested con- Based on the information that the microsatellite servation of synteny of GGA2 with 9 human chromo- MCW0189, identified as being in the LIMK2 gene, somes and therefore genes were selected from regions of mapped to the GGA2 genetic linkage group[21], 5 chicken HSA1 (9 markers), HSA3 (28 markers), HSA6 (22 mark- EST markers were developed, corresponding to 8 Mb of ers), HSA7 (10 markers), HSA8 (84 markers), HSA9 (19 HSA22q12.2 containing the homologous gene. Recently, markers), HSA10 (7 markers), HSA18 (35 markers), Jennen et al indicated that the mapping of LIMK2 was HSA22 (5 markers). Primers were developed using the erroneous and that it was located on the GGA15 RH map Iccare web server [18] with the constraints specific to RH [17]. We confirm here this result, as all of our five markers

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RH (cR) DRAFT SEQUENCE (Mb) ADL228 MCW205 ABR0336 ADL228 0.41 MCW205 1.28 CENTG3 CSPG5 0,0 MCW082 MGC4607 MGC4607 3.51 33,0 ACVR2B MCW082 4.86 58,1 PAXIP1L ACVR2B 5.20 SHH EN2 PAXIP1L 7.10 106,4 ADL270 SHH 7.51 138,2 LEI234 ADL270 9.16 159,9 KIF5B LEI234 WAC 171,9 LMU013 10.72 MCW184 KIF5B 13.57 GAD2 MCW220 187,0 ADL190 LMU013 14.46 ADL152 WAC 14.69 GCT025 SPAG6 217,7 ADL190 14.70 ROS0355 251,9 GCT027 MCW184 14.73 LEI117 MCW220 14.77 271,5 MRC1 GAD2 15.59 ADL152 16.11 297,6 ITGA8 SPAG6 17.01 LEI117 17.35 GCT027 17.36 18.75 MRC1 SCAP2 ITGA8 LEI086 ADL309 19.73 MCW206 ADL176 NPY ADL185 480,5 THRB RARB ADL309 30.17 520,3 MCW063 ADL185 30.25 MCW206 30.49 557,0 GCT052 NPY 30.65 SCAP2 31.59 LEI086 31.67 605,2 RBMS3 ADL176 36.24 629,6 CGI THRB 36.37 ADL120 RARB 36.95 ROS0018 644,2 FLJ14566 MCW063 37.05 658,4 LOC131034 GCT052 37.51 RBMS3 38.71 682,0 SACMIL CGI 39.96 FLJ14566 40.24 725,3 FBXL2 ROS0018 40.39 ADL120 40.55 LOC131034 41.01 778,7 LEI089 SACMIL 41.92 FBXL2 43.70 ADL257 ADL257 45.34 ITGA9 LEI089 46.36 ROS0105 ITGA9 47.95 MCW062 54.85 MCW062 TGFBR1 55.811 TGFBR1 ROS0105 55.816 ATP9B ATP9B 56.53 MRS2L 57.66 VMP 57.75 948,9 VMP PRL 58.38 MRS2L E2F3 59.23 PRL KIF13A 60.24 995,9 GMPR 61.31 1018,1 E2F3 ADL235 61.67 CAP2 61.76 FLJ20330 1035,8 KIF13A FLJ20330 61.94 MAK ADL235 GMPR MCW039 62.94 CAP2 1060,8 MAK 62.97 MCW039 1098,5 BMP6 BMP6 64.12 F13A1 64.84 F13A1 ADL196 66.07 ADL373 NQO2 66.12 1160,3 ADL196 ADL373 66.18 MKPX FOXC1 MKPX 66.20 FOXC1 66.74 NQO2 BCL2 OVY OVY 67.46 BCL2 67.68 MGC13269 1228,2 ADL300 ADL300 67.83 LEI096 MCW034 MGC13269 67.93 LEI195 69.28 LEI195 LEI096 69.48 1290,1 LEI248 MCW034 69.66 MCW077 GCT023 ADL181 LEI248 71.69 MCW077 77.37 1337,3 MCW173 ADL181 77.43 MCW291 GCT023 77.64 KIAA1695 MCW173 80.01 1387,5 KIAA1695 83.31 MCW153 FH MCW087 1414,5 MCW027 MCW153 84.22 MCW087 84.83 LIV NR4A3 1426,9 MCW009 FH 85.57 INVS 1444,6 TMEFF1 MCW027 85.97 MCW009 87.97 1475,0 C6orf32 LIV 88.45 TMEFF1 88.59 1506,9 NETO1 INVS 89.06 1546,9 CIS4 NR4A3 89.25 LEI127 C6orf32 90.06 1572,1 MCW257 NETO1 92.42 CIS4 93.41 MCW137 1589,7 CDH19 LEI127 93.53 1611,2 MCW288 MCW257 94.29 MCW088 CDH19 95.10 VAPA 1633,7 PTPN2 MCW288 95.89 TSG MCW088 96.09 ROS0023 FLJ23403 1661,8 LEI147 PTPN2 96.64 1688,6 ZFP161 MCW137 97.24 ADL236 LEI147 97.30 1715,5 ROCK1 FLJ23403 97.99 SUDD ZFP161 99.35 1751,5 GATA6 ADL236 100.54 ROS0023 100.74 1787,2 OSBPL1A ROCK1 101.37 GATA6 101.86 SUDD 102.22 1836,3 SS18 OSBPL1A 102.48 SS18 103.29 1864,4 CDH2 CDH2 104.01 MCW096 104.96 MCW185 MCW185 105.35 1912,0 MCW096 B4GALT6 105.35 STRIN 105.54 1970,6 B4GALT6 NOL4 106.33 MAPRE2 106.81 1982,3 STRIN PRKDC 107.11 CEBPD 2015,4 PRKDC MCM4 107.38 MCM4 LEI237 108.89 OPRK1 2031,3 MAPRE2 OPRK1 109.31 LEI237 RGS20 109.42 NOL4 2063,0 RGS20 MCW234 110.38 2086,0 PENK PENK 110.57 MCW234 SDCBP 111.32 2098,6 ADL114 ADL114 111.34 ASPH MCW264 112.31 LEI105 LEI355 2119,0 SDCBP LEI355 112.47 2138,4 MCW264 ROS0150 112.78 CYP7B1 113.93 PDE7A 2149,0 ROS0150 FLJ12987 115.16 CYP7B1 FLJ12987 NCOA2 115.92 EIF2C2 2168,5 EYA1 116.15 ADL146 TOP1MT NCOA2 2208,2 COPS5 TSG 116.45 GCT002 116.79 2260,9 EYA1 STAU2 117.41 STAU2 2283,2 GCT002 GDAP1 117.70 PI15 117.88 2299,7 GDAP1 PXMP3 118.98 PKIA 119.68 2310,1 PI15 IMPA1 120.83 HEY 2326,1 PXMP3 FLJ14007 120.84 FLJ14007 MCW310 121.82 E2F5 2349,4 PKIA E2F5 122.06 CPNE3 122.51 CPNE3 2373,0 IMPA1 RIPK2 123.62 NBS1 RIPK2 2402,5 MCW310 NBS1 123.71 MCW056 MCW051 123.75 MCW166 2448,5 CALB1 MCW056 124.15 CCNE2 MCW166 124.40 2473,4 MCW314 MCW314 124.91 2505,2 UQCRB1 CCNE2 125.71 UQCRB1 126.14 2529,3 MCW245 MCW245 126.46 MATN2 TIEG 127.86 2545,5 FZD6 128.10 2560,8 NCALD DPYS 128.76 OXR1 OXR1 129.93 2569,8 TIEG ANGPT1 130.12 LEI070 TRHR 2578,4 FZD6 EIF3S6 130.60 KCNV1 TRHR 130.91 2591,5 DPYS EBAG9 131.03 TRPS1 KCNV1 131.20 LEI141 RAD21 EXT1 2623,2 ANGPT1 LEI070 132.66 2632,9 EIF3S6 NOV 132.70 EXT1 133.35 LEI104 HAS2 2652,0 EBAG9 LEI141 133.39 RAD21 133.70 2691,7 LEI228 TRPS1 134.43 2723,3 NOV LEI228 134.46 DDEF1 MCW324 TOP1MT 135.53 EIF2C2 TOP1MT 2793,8 MYC LEI031 136.30 ADL146 EIF2C2 138.25 MCW189 MCW157 KCNQ3 WISP1 LEI031 ADL146 138.48 LEI104 139.66 WISP1 141.83 HSF1 KCNQ3 142.34 MCW324 143.06 DDEF1 143.19 MYC 144.05 HSA2 146.43 147.37 UNKNOWN HSF1 147.39 MCW157 OTHER CHROMOSOME NO HIT

RadiationFigure 1 hybrid map of chicken chromosome 2 and comparison the draft sequence assembly Radiation hybrid map of chicken chromosome 2 and comparison the draft sequence assembly. The GGA2 RH map (left) is 2794 cR long and is aligned to the genome sequence assembly (right). The limits between the 5 framework groups of the RH map are indicated by double slashes. Markers present only in the comprehensive map are indicated with their most likely position and the confidence interval, to the left of the map. The coloured framed boxes indicate the results from the BLAST analysis, for the markers that were not found on the chromosome 2 sequence assembly http://www.ensembl.org/ Gallus_gallus/.

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HUMAN GENE CHICKEN GENE ORDER ORDER

7q36.2_PAXIP1L ACVR2B PAXIP1L 10p13_ITGA8 KIF5B 10p11.22_KIF5B ITGA8 3p24.2_THRB 3p24.1_RBMS3 THRB 3p23_FBXL2 RBMS3 3p22.3_ACVR2B CGI 3p22.1_FLJ14566 FLJ14566 3p21.32_SACM1L LOC131034 3q22.1_CGI SACM1L 3q22.1_Loc131034 FBXL2 6p24.3_BMP6 VMP 6p22.3_GMPR PRL 6p22.3_KIF13A E2F3 6p22.3_E2F3 KIF13A 6p22.3_PRL GMPR 6p22.3_VMP BMP6 6p22.1_C6orf32 KIAA1695 9q31.1_TMEFF1 TMEFF1 C6orf32 18p11.31_ZFP161 NETO1 18p11.21_PTPN2 CIS4 18q11.1_ROCK1 CDH19 18q11.2_GATA6 PTPN2 18q11.22_OSBPL1A ZFP161 18q11.2_SS18 ROCK1 18q11.2_CDH2 GATA6 18q12.1_B4GALT6 OSBPL1A 18q12.1_STRIN SS18 18q12.1_MAPRE2 CDH2 18q12.2_KIAA1695 18q22.1_CDH19 B4GALT6 18q22.2_CIS4 STRIN 18q22.3_NETO1 PRKDC MAPRE2 RGS20 8q11.21_PRKDC PENK 8q11.23_RGS20 SDCBP 8q12.1_PENK FLJ12987 8q12.1_SDCBP COPS5 8q13.1_COPS5 EYA1 8q13.2_FLJ12987 GDAP1 8q13.3_EYA1 PI15 8q21.11_GDAP1 PXMP3 8q21.11_PI15 PKIA 8q21.11_PXMP3 IMPA1 8q21.12_PKIA UQCRB 8q21.13_IMPA1 MATN2 8q22.1_UQCRB NCALD 8q22.1_MATN2 TIEG 8q22.3_NCALD FZD6 8q22.3_TIEG DPYS 8q22.3_FZD6 ANGPT1 8q22.3_DPYS EIF3S6 8q23.1_ANGPT1 EBAG9 8q23.1_EIF3S6 NOV 8q23.2_EBAG9 MYC 8q24.12_NOV 8q24.21_MYC

FigureComparison 2 of gene orders between the GGA2 RH map and the homologous human regions Comparison of gene orders between the GGA2 RH map and the homologous human regions. The GGA2 RH map (this study) is compared to the order of homologous genes in human (left) Each colour (plain boxes) corresponds to a human chromosome. Arrows on the right indicate the gaps between the 5 framework maps.

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mapped to the GGA15 RH linkage group (data not only a few local inversions that can be put on the account shown). The MCW0189 marker remains on the GGA2 RH of genotyping errors or other artefacts that may remain map, at the position corresponding to where the former despite all precautions taken, such as the double genotyp- LIMK2 would have been expected. The result of the BLAST ing process. search of MCW0189 in the genome assembly indicated a homology with a sequence fragment of unknown loca- In the upper part of the chromosome, several markers tion. No similarity with a LIMK sequence could be found absent in the sequence assembly could be localized on the in this fragment. RH map: sequences corresponding to CENTG3 and CSPG5 genes are aligned with "Unknown" sequence (not A recently published paper describing the mapping of assigned to known chromosomes) and ABR0336 is found IL1B on GGA2 suggested a conservation of synteny with on the GGA27 sequence. The RH two point LOD scores HSA2q [22]. The IL1B primers we tested failed to amplify between CENTG3, CSPG5, ABR0336 and their neighbours correctly, but other chicken EST markers located near on GGA2 (MCW082, ACVR2B or PAXIP1L) are higher IL1B, whose position is 113.3 Mb on HSA2q, had been than 6. In addition, the genetic map confirms the localiza- used in another mapping project in our laboratory [16]. tion of ABR0336 on chicken chromosome 2. In the same Of these, ACTR, positioned at 111.9 Mb and BIN1 at region of the RH map, MCW071 (a microsatellite in the 125.1 Mb on HSA2, were mapped to GGA7. If IL1B, EN2 gene) belongs to a genomic region with no sequence located between these two markers in human, is indeed available (no blast hit); the genetic map confirms the RH on GGA2, the fragment of conserved synteny must be very position of this marker. These results suggest possible small. The sequence of IL1B in the chicken genome assem- improvements to be made in the sequence assembly for bly is in a fraction of unknown location. this region of chromosome 2.

Previous data suggested that the RYR2 gene, mapped on On the sequence assembly, the TSG gene, at position HSA1, was located on GGA2 [23]. We have therefore 116.4 Mb, is close to EYA1 and STAU2, whereas on the RH tested 9 chicken EST markers orthologous to HSA1 genes, map TSG maps at 1660 cR, close to PTPN2 and LEI0147, but only one (FH) could be localized on the GGA2 RH located at positions 96.4 and 97.3 Mb respectively. At the map. As this localisation could be questioned, the identity same position as TSG on the RH map, the VAPA gene cor- of the fragment was confirmed after sequencing of the responds to a sequence fragment of unknown location. PCR product. Moreover, the RH map in this region is in agreement with comparative mapping, so we suspect there may be prob- As already observed for other chicken chromosomes lems in the sequence assembly in the TSG region. [7,15-17,24-26], the GGA2 regions investigated here, corresponding to regions of human chromosomes 3, 6, 7, 8, Around position 2100 cR, two local inversions between 9, 10 and 18 showed a high number of intra-chromo- the RH map and the sequence orders are observed. The somal rearrangements within the regions of conserved first concerns PRKDC and MAPRE2, and the second synteny. SDCBP and ADL114. In both cases, EST markers mapped close to these genes are missing in the GGA2 sequence Alignment of the RH map to the genomic sequence (figure assembly: CEBPD near PRKDC and ASPH near SDCBP are 1) in the fraction of unassigned sequence. However, in the A first draft chicken genome assembly was recently depos- case of the PRKDC-MAPRE2 region, the sequence assem- ited into public databases by a team led by R. Wilson and bly is in agreement with the gene order on human chro- W. Warren, from the Washington University School of mosome 8. The order of the markers in the RH map is Medicine in St. Louis (1st March, 2004, http:// supported in both cases by a difference of LOD greater www.ensembl.org/Gallus_gallus/). The sequence cover- than 8, when compared to the alternate order. age is 6.6X. For GGA2, 147.590.765 bp were sequenced, 166 known genes were identified, and a total of 1432 In the lower part of the chromosome, several markers genes were defined altogether when including results localized on the RH map are absent from the GGA2 from prediction programs. sequence assembly: the HEY gene at position 2330 cR is localized on GGA26 in the assembly, whereas the two- For each gene or microsatellite marker from our GGA2 RH point LOD scores with its neighbours on the GGA2 RH map, we compared the fragment sequence with the map are trust-worthy (11.5 with IMPA1, 8.8 with PKIA). chicken genome sequence assembly by using the BLAST The MATN2 and NCALD genes are in the unknown frac- algorithm [27]. The position for each marker in the tion of genomic sequence, whereas the 2-point LOD sequence is indicated in figure 1. The agreement between scores with their neighbours, around 2550 cR, are higher the RH map and the sequence order is almost perfect, with than 6. At the end of the chromosome (position 2690 cR),

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we observed an inversion between the RH map and the lished chicken genetic map. In addition, 11 other micros- sequence concerning the NOV gene and the microsatellite atellite markers and 10 genes were selected from other LEI228. The order in the RH map is supported by a differ- published data [28]. Primer information for these markers ence of LOD greater than 4 when compared to the order can be found in the ARKdb farm animal database http:// in the sequence assembly. www.thearkdb.org/browser?species=chicken and the ChickAce database https://acedb.asg.wur.nl/. Altogether, out of 198 markers localized on GGA2 through RH mapping, only one, labelled "no hit" in figure Primers for 219 EST were designed using the Iccare web 1, could not be found by BLAST in the chicken genome server [18], http://genopole.toulouse.inra.fr/bioinfo/ sequence; 10 were assigned to existing genomic sequence Iccare/. All publicly available chicken EST (> 420,000) of unknown location; 2 were assigned to a wrong chromo- were collected in a local database and compared to the some and 4 were not localized at the same position when human genome sequence. EST selection targeted towards both maps were compared. With less than 10% of markers GGA2 was based on available comparative mapping data either missing in the sequence or for which the two maps with the human genome. Long introns, whose positions disagree, these results confirm the high quality of the could be predicted in chicken on the basis of their posi- genome assembly, with only few finishing improvements tion in the human genes, were avoided and primers were to be made in the near future. chosen in the most divergent regions of the chicken/ human sequence alignments, to avoid cross-amplification The GGA2 RH map is available on the ChickRH web of the hamster DNA present in the hybrids. Information server http://chickrh.toulouse.inra.fr/ also used for RH on primers for EST markers is given in the additional genotyping data collection. table.

Conclusions PCR conditions We have built a high resolution radiation hybrid map of PCR were carried out in 15 µl reactions containing 25 ng chicken chromosome 2 using the chickRH6 panel. Our hybrid DNA, 2 mM MgCl2 (Life technologies: Carlsblad, goal was to provide jointly a source of potential polymor- CA, USA), 0.3 U Taq DNA polymerase (Life technologies), phic markers and of candidate genes for QTL mapping on 1X buffer (Life technologies), 200 µM of each dNTPs (Life this chromosome. In the course of our work, the first draft technologies), 0.25 µM of each primer, and 1X loading chicken genome assembly was released and we aligned it buffer (350 mM sucrose and 0.2 mM cresol). After a 5 min to our GGA2 RH map. Although the sequence assembly is denaturation step, 30 to 35 cycles (depending on the globally in good agreement with our data, a limited primers) of 30 sec at 94°C, 30 sec at annealing tempera- number of discrepancies and the mapping of sequence ture, 30 sec at 72°C, were performed, followed by a final fragments of unknown location or of markers not present elongation step of 5 min at 72°C. Chicken DNA was used in the genomic sequence, show that RH mapping it still as positive control; hamster DNA and TE were used as neg- useful. ative controls.

Future developments of the chicken RH map will now be PCR amplification results were scored on 2% agarose gels. based on the genomic sequence, using it for choosing STS Each marker was genotyped twice. markers in selected regions to develop RH framework maps and thereby detect eventual problems in the Map construction genomic sequence assembly. This is clearly needed in the The CarthaGene program [29] was used to build the RH regions for which the genetic map is still not complete, map for chromosome 2 http://www.inra.fr/bia/T/ such as some microchromosomes, but also for parts of CarthaGene/. First, linkage groups were constituted by macrochromosomes, as shown in this study. using a two-point LOD threshold of 4, after which a 1000:1 framework map (a map whose likelihood is at Methods least 1000 fold higher than the next possible highest like- Radiation hybrids lihood using the same markers in alternate orders) for The generation of the 6000 rads chicken RH panel has each group was built under a haploid model. Then, the already been described [13]. This panel, named framework maps obtained were aligned on the genetic ChickRH6, consists of a total of 90 hybrids. The mean map and the orientation and distance between each group retention frequency of markers is 21.9%. were estimated using the "printbestmap" option in the Carthagene program. Finally, after this final validation, all Gene selection and primers design distances between markers were re-evaluated under a dip- Seventy-seven microsatellite markers well distributed loid model. along chicken chromosome 2 were selected from the pub-

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Markers not included in the framework map, but display- 4. Burt DW: The chicken genome and the developmental biologist. Mech Dev 2004, 121:1129-1135. ing two point Lod scores higher than 4 with framework 5. McPherson JD, Marra M, Hillier L, Waterston RH, Chinwalla A, Wallis markers, were mapped relative to the framework map by J, Sekhon M, Wylie K, Mardis ER, Wilson RK, Fulton R, Kucaba TA, using the CarthaGene "buildfw" option which calculates Wagner-McPherson C, Barbazuk WB, Gregory SG, Humphray SJ, French L, Evans RS, Bethel G, Whittaker A, Holden JL, McCann OT, the most likely position of a marker and gives a confi- Dunham A, Soderlund C, Scott CE, Bentley DR, Schuler G, Chen HC, dence interval. Jang W, Green ED, Idol JR, Maduro VV, Montgomery KT, Lee E, Miller A, Emerling S, Kucherlapati, Gibbs R, Scherer S, Gorrell JH, Sodergren E, Clerc-Blankenburg K, Tabor P, Naylor S, Garcia D, de Jong PJ, Cat- The map was drawn using the MapChart software [30]. anese JJ, Nowak N, Osoegawa K, Qin S, Rowen L, Madan A, Dors M, Hood L, Trask B, Friedman C, Massa H, Cheung VG, Kirsch IR, Reid T, Yonescu R, Weissenbach J, Bruls T, Heilig R, Branscomb E, Olsen Maps comparison A, Doggett N, Cheng JF, Hawkins T, Myers RM, Shang J, Ramirez L, Data on the location of the chicken genes in the sequence Schmutz J, Velasquez O, Dixon K, Stone NE, Cox DR, Haussler D, assembly was obtained by BLASTN searches using the Kent WJ, Furey T, Rogic S, Kennedy S, Jones S, Rosenthal A, Wen G, Schilhabel M, Gloeckner G, Nyakatura G, Siebert R, Schlegelberger B, Ensembl http://www.ensembl.org/ browser. Korenberg J, Chen XN, Fujiyama A, Hattori M, Toyoda A, Yada T, Park HS, Sakaki Y, Shimizu N, Asakawa S, Kawasaki K, Sasaki T, Shin- tani A, Shimizu A, Shibuya K, Kudoh J, Minoshima S, Ramser J, Seranski Data on human gene order were obtained from the Iccare P, Hoff C, Poustka A, Reinhardt R, Lehrach H: A physical map of web server. the human genome. Nature 2001, 409:934-941. 6. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Authors' contributions Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng SL, MD, SB, FV and KF carried out the molecular study. SL XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor built the RH map, and drafted the manuscript with FP. Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, MM made the ChickRH6 panel. AV coordinated the Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern project and finalized the manuscript. A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Additional material Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nussk- Additional File 1 ern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang Description of the markers. The GCTXXXX markers were developed A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, Yao A, Ye J, using the Iccare web server. The EXTXXXX markers were chosen from Zhan M, Zhang W, Zhang H, Zhao Q, Zheng L, Zhong F, Zhong W, published information. Zhu S, Zhao S, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik Click here for file A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, [http://www.biomedcentral.com/content/supplementary/1471- Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, 2164-6-12-S1.xls] Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nel- son K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rod- Acknowledgements riguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, We wish to thank Denis Milan for helpful discussions over the use of Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn- Carthagene. 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Schmid M, Nanda I, Guttenbach M, Steinlein C, Hoehn M, Schartl M, Haaf T, Weigend S, Fries R, Buerstedde JM, Wimmers K, Burt DW, Smith J, A'Hara S, Law A, Griffin DK, Bumstead N, Kaufman J, Thom- son PA, Burke T, Groenen MA, Crooijmans RP, Vignal A, Fillon V, Morisson M, Pitel F, Tixier-Boichard M, Ladjali-Mohammedi K, Hillel J, Maki-Tanila A, Cheng HH, Delany ME, Burnside J, Mizuno S: First Publish with BioMed Central and every report on chicken genes and chromosomes 2000. Cytogenet Cell Genet 2000, 90:169-218. scientist can read your work free of charge 24. Crooijmans RP, Dijkhof RJ, Veenendaal T, van der Poel JJ, Nicholls "BioMed Central will be the most significant development for RD, Bovenhuis H, Groenen MA: The gene orders on human disseminating the results of biomedical research in our lifetime." chromosome 15 and chicken chromosome 10 reveal multi- ple inter- and intrachromosomal rearrangements. Mol Biol Sir Paul Nurse, Cancer Research UK Evol 2001, 18:2102-2109. Your research papers will be: 25. Jennen DG, Crooijmans RP, Kamps B, Acar R, Veenendaal A, Van Der Poel JJ, Groenen MA: A comparative map of chicken chromo- available free of charge to the entire biomedical community some 24 and human chromosome 11. Anim Genet 2002, peer reviewed and published immediately upon acceptance 33:205-210. 26. Smith J, Paton IR, Murray F, Crooijmans RP, Groenen MA, Burt DW: cited in PubMed and archived on PubMed Central Comparative mapping of human Chromosome 19 with the yours — you keep the copyright chicken shows conserved synteny and gives an insight into chromosomal evolution. Mamm Genome 2002, 13:310-315. Submit your manuscript here: BioMedcentral http://www.biomedcentral.com/info/publishing_adv.asp

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