A High Resolution Physical and RH Map of Pig Chromosome 6Q1.2 And

A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1 Flávia Martins-Wess, Denis Milan, Cord Drögemüller, Rodja Voβ-Nemitz, Bertram Brenig, Annie Robic, Martine Yerle, Tosso Leeb

To cite this version:

Flávia Martins-Wess, Denis Milan, Cord Drögemüller, Rodja Voβ-Nemitz, Bertram Brenig, et al.. A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1. BMC Genomics, BioMed Central, 2003, 4, pp.435-444. 10.1186/1471-2164-4- 20. hal-02680244

HAL Id: hal-02680244 https://hal.inrae.fr/hal-02680244 Submitted on 31 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. BMC Genomics BioMed Central

Research article Open Access A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1 Flávia Martins-Wess1, Denis Milan*2, Cord Drögemüller1, Rodja Voβ- Nemitz1, Bertram Brenig3, Annie Robic2, Martine Yerle2 and Tosso Leeb*1

Address: 1Institute of Animal Breeding and Genetics, School of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany, 2Institut National de la Recherche Agronomique (INRA), Laboratoire de Génétique Cellulaire, BP27, 31326 Castanet Tolosan Cedex, France and 3Institute of Veterinary Medicine, University of Göttingen, Groner Landstr. 2, 37073 Göttingen, Germany Email: Flávia Martins-Wess - [email protected]; Denis Milan* - [email protected]; Cord Drögemüller - [email protected]; Rodja Voβ-Nemitz - [email protected]; Bertram Brenig - [email protected]; Annie Robic - [email protected]; Martine Yerle - [email protected]; Tosso Leeb* - [email protected] * Corresponding authors

Published: 13 May 2003 Received: 7 March 2003 Accepted: 13 May 2003 BMC Genomics 2003, 4:20 This article is available from: http://www.biomedcentral.com/1471-2164/4/20 © 2003 Martins-Wess et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

Abstract Background: The generation of BAC/PAC contigs in targeted genome regions is a powerful method to establish high-resolution physical maps. In domestic animal species the generation of such contigs is typically initiated with the screening of libraries with probes derived from human genes that are expected to be located in the region of interest by comparative mapping. However, in many instances the available gene-derived probes are too far apart to allow the cloning of BAC/ PAC contigs larger than a few hundred kb. High resolution physical mapping allows to estimate the sizes of gaps and to control the orientation of the individual sub-contigs, which helps to avoid errors during the assembly of smaller contigs into final Mb-sized contigs. The recently constructed porcine IMNpRH2 panel allowed us to use this approach for the construction of high-resolution physical maps of SSC 6q1.2. Results: Two sequence-ready BAC/PAC contigs of the gene-rich region on porcine chromosome 6q1.2 (SSC 6q1.2) containing the RYRl gene were constructed. The two contigs spanned about 1.2 Mb and 2.0 Mb respectively. The construction of these contigs was monitored by the results provided by the mapping of 15 markers on the IMpRH7000rad and 35 markers on the IMNpRH212000rad radiation hybrid panels. Analyses on the IMpRH panel allowed us to globally link and orientate preliminary smaller contigs, whereas analyses on the high resolution IMNpRH2 panel allowed us to finally identify the order of genes and markers.

Conclusions: A framework map of 523 cR12000 was established covering the whole studied region. The order of markers on the framework 1000:1 RH map was found totally consistent with the data deduced from the contig map. The kb/cR ratio was very constant in the whole region, with an average value of 6.6 kb/cR. We estimate that the size of the remaining gap between the two contigs is of about 300 kb. The integrated physical and RH map of the investigated region on SSC 6q1.2 was used for a comparative analysis with respect to the syntenic regions on HSA 19q13.1 and MMU 7 and revealed a perfectly conserved gene order across the entire studied interval.

Page 1 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

Background of the existing 1.2 Mb contig [8] to 2.0 Mb and the gener- Comparative genome analysis increases the knowledge of ation of a new 1.2 Mb contig (Fig. 1). End sequences from genome evolution and is especially important in livestock all clones of the contig were generated and submitted to species where the currently available sequence informa- the EMBL database under accessions AJ514457- tion is very limited as compared to the vast amount of in- AJ514832. In total 292 end sequences from SSC 6q1.2 formation available from the human and mouse with an average read length of 708 bp totaling 207 kb of genomes. Radiation hybrid mapping is seen as an efficient genomic survey sequences were generated. Thus, the BAC/ technique for the generation of high-resolution gene PAC end sequences cover approximately 6 % of the stud- maps in different species and RH maps can be integrated ied genomic region. The end sequences contain an average in comparative mapping approaches to reveal the degree GC content of 47 % exceeding the value of 41 % that is of synteny conservation between species [1]. generally accepted as the average GC content in mammalian genomes [9]. The GC content analysis further con- Two RH panels have been reported for the pig: the 7 000 firms that SSC 6q1.2 is indeed closely related to HSA rad IMpRH panel [2] that provides medium-resolution 19q13.1, which has a GC content of 46 % in the corre- global mapping information, and the 12 000 rad sponding 4 Mb region. An analysis of repetitive elements IMNpRH2 [3], that can be used to construct high-resolu- revealed that 39.8 % of the end sequences consisted of re- tion local RH maps. Panels developed after a high level of petitive DNA. Of the 39.8 % repetitive DNA, 20.5 % were cell irradiation (10 000 to 50 000 rads) are very useful for SINE, 13.3 % were LINE, 2.4 % were of retroviral origin high resolution regional mapping studies but they require (LTRs), and 2.0 % represented DNA transposons. The pre- a characterization with a very large number of markers to dominance of SINEs is another typical hallmark of GC- be useful for genome-wide mapping studies [4]. rich and gene-rich genome segments [10]. The analysis of the end sequences also revealed three dinucleotide and The porcine RYRl gene region on SSC 6q1.2 is of special one tetranucleotide microsatellite (AJ514594, AJ514613, interest due to its economical importance. The porcine AJ514706, AJ514795). stress syndrome (PSS), which in pigs is caused by a single RYRl point mutation, is known to be associated with pos- The availability of the end sequences allowed the contin- itive characteristics like increased muscling and increased uous verification of the contig assembly by comparative lean meat content. Until now, it is not clear whether the mapping. In BLAST searches against the human draft ge- RYRl mutation is also responsible for the positive carcass nome sequence, approximately 15 % of the BAC/PAC end traits in stress susceptible pigs or whether these complex sequences showed significant (E < 10-5) matches to HSA growth traits are influenced by other closely linked genes 19q13.1, which allowed the precise comparative mapping on SSC 6q1.2 [5–7]. Furthermore, this genomic region is of 27 % of the tested BAC/PAC clones. Of the investigated also of special interest as it represents a GC-rich genomic clones, 73 % had no match in the human genome se- region with a very high gene content. To investigate this quence, 23 % had matches with one end sequence, and 4 genomic region we have previously reported the construc- % had matches with both end sequences. tion and analysis of a 1.2 Mb BAC/PAC contig [8]. Physical mapping and comparative analysis In the present study, we report the construction of high- During the contig construction many gene-specific STSs resolution framework and comprehensive RH maps of the were used, which allowed the unequivocal assignment of RYRl gene region on the porcine chromosome 6q1.2 us- genes to individual clones. Further genes were localized ing the porcine IMpRH and IMNpRH2 panels as well as by hybridization of heterologous cDNA probes to the in- the comparison of the RH maps to an extended clone- dividual BAC/PAC clones and BLAST analysis of the clone based physical map of this region. end sequences. Using these approaches, 33 genes in total were localized. Furthermore, the microsatellite SW193 Results and Discussion was also localized by STS content analysis thus anchor in Construction of the BAC and PAC contig and analysis of the physical clone-based map to the linkage map of this end sequences region [11]. We previously reported the construction of a 1.2 Mb BAC/ PAC contig on SSC 6q1.2 [8]. To extend the existing contig The gene assignments were compared with human and the porcine TAIGP714 PAC and RPCI-44 BAC libraries mouse maps and a comparative map for SSC 6q1.2, HSA were screened with new probes either derived from end 19q13.1 and MMU 7 was developed (Fig. 2). The gene or- fragments of previously isolated porcine genomic clones der in this region of the pig genome corresponds exactly or from human HSA 19q13.1 genes. Assembly of all 171 to the gene order of the NCBI HSA 19 map (http:// isolated BAC and PAC clones according to STS content, in- www.ncbi.nlm.nih.gov build 31). The gene order of MMU sert sizes and fingerprinting data resulted in the expansion 7 (http://www.ncbi.nlm.nih.gov MGSCv3) also

Page 2 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20 sizes of their respectivegenomi sizes of arede associated withgenes that are atthetop,markers lines of Physical map the isolatedBAC/PAC contigs. Figure 1

2.0 MbContig 32_N2_Sp6 BAC 32N2-155kb BAC 113 E2 BAC 62D12 BAC 153A8

50 kb (ITZ014)

283_N2_T7 62_D12_Sp6 = negativePCR = Sp6-end = T7-end = possiblydeleted = tilingpath = chimeric BAC 117 B1 E09 A492-120kb

BAC 237P18-210kb

BAC 283N2 J09_0028_T7 BAC 467E2

E08 A518-90kb

J09 0028-80kb

(ITZ005) B09_A138_Sp6 BAC 509I23 BAC 446I5 BAC 289B2 BAC 382M14

L23 0093 E08_A518_T7 N01 0093 B09 A138-160kb C05 A355

C12 A165-85kb B12 A178

263_A8_Sp6

c targets. BACs and PACs are indicated below th below indicated are c targets.BACs andPACs

475_K6_Sp6 BAC 263A8-125kb (ITZ006) B09_A138_T7 BAC 437D8-150kb BAC 475K6-140kb BAC 479 C14 - 150 kb BAC 336B4-150kb

BAC 482 M15 - 145 kb BAC 480 K14 - 120 kb BAC 466 G17 - 145 kb BAC 303 C23 - 145 kb G22_0081_T7

G22 0081-50kb

263_A8_T7

C09_A65_Sp6 (ITZ007) C09_A65_Sp6

NEUD4

F10_A504_Sp6 G22_0081_Sp6

1.2 MbContig F10 A504-70kb PPP1R14A

J16 0065-65kb E02_A455_T7 A03 0039 C09 A065

E02 A455-90kb I17 0037-135kb SPINT2 B16 0068-105kb

M24 0041-115 kb

H01 A166 F10_A504_T7

F04 A54

G23 0023 O12 0045 A03_0039_T7

KCNK6

G13_0089_T7 (ITZ012)

D03_A69_Sp6 M08_0006_Sp6

G13 0089-85kb E09_A169_T7

E02_A455_Sp6

C09_A65_T7 PSMD8

E09 A169-110 kb B06 0030-70kb I17_0037_T7

M08 0006-115 kb

RYR103/104

STS markers are represented byvertical STS markers G13_0089_Sp6 K13 0049-65kb RYR115/116 I16 0016-60kb

D03 A69-250kb

F10 A391-105kb

B08 A475-100kb BAC_187_C16_Sp6

RYR101/102

B10 A143-195kb M08_0006_T7

M08_0041_T7

RYR111/112

MAP4K1 G09_A181_T7

BAC 187C16-170kb N01_0050_T7 E03 A447-100kb M08 0041-150kb noted in bold. The physical sizes covered by thedifferenthybri sizes bold.The physical noted in K13 0061-120kb BAC 98B18-170kb N01 0050-90kb C02 A428-65kb B01A145

BAC 204H11 -170kb BAC 151G11 -170kb

G09A181 - 150 kb

BAC_162_H7_Sp6

A01_A465_T7

E03_A477_Sp6

PolR2I BAC_235_B21_Sp6

E03 A477

BAC 166D13-170kb C02_A428_T7 BAC 187M23-170kb ACTN4 235 C16

BAC 168H21-100kb A01A465 - 130 kb BAC 162H7-80kb N01_0050_Sp6

K13_0061_Sp6 CAPN4

BAC_172_J1_T7

COX7A1 M08_0041_Sp6 LGALS7

BAC 143J1-155kb

BAC 235B21-200kb

204_H11_T7 LGALS4

ECH1

B01 A302-110 kb BAC 172J1-150kb

BAC_162_H7_T7

HNRPL

B11_A416_T7

SIRT2

N23_0035_Sp6

(SIRT2) H13_0041_T7 216_K1_Sp6 (ITZ013) 216_K1_Sp6

H13 0041-65kb

A09 0094-70kb

NFKBIB

H07 A117 -80kb 166_D13_T7 F01 A466-70kb B11 A416-105kb 187_M23_Sp6 N23 0035-110 kb

I11 0051-165kb H04 A462-100kb

B01_A302_Sp6

MRPS12 H13_0041_Sp6 C09 A329-95kb

D01 A356-80kb

BAC_172_J1_Sp6

e markers with their corre with e markers N23_0035_T7

H04 A308-100kb

F06 A258-115 kb I11_0051_Sp6 O06 0032-75kb D08_A256_T7 BAC 216K1 E11 0019-(100kb) D08 A256-150kb

H06 0005-160kb

BAC_311_B11_T7 F06_A258_T7

L10 0005-65kb

BAC_299_D1_T7

BAC_338_B22_T7 (ITZ008) BAC_338_B22_T7

PAK4 (ITZ004) BAC 311 B11 -175kb

BAC 351N10-105kb BAC 387P21-(130kb) 444_G5_Sp6 BAC 338B22-115 kb

BAC 432 M14 - 135 kb BAC 299D1-(95kb)

dotted lines, cDNA hybridization pr dotted lines,cDNAhybridization BAC_528_O23_Sp6

BAC 477B7-135kb

BAC 120J12-150kb BAC 7E12

H06_0005_T7

GMFG 99_O22_T7

444 G5-150kb 216_K1_T7

521_D11_Sp6

BAC 528O23-145kb

BAC_338_B22_Sp6

BAC_299_D1_Sp6

F04_A442_Sp6

(FLJ10211) BAC_311_B11_Sp6

A07 A479-(70kb) 99 O22-170kb

430 D23-155kb BAC_477_B7_T7

F04 A442-70kb LOC163128 DKFZp434H247 (LOC126263) DKFZp434H247

521 D11 RPS16

SUPT5H

444_G5_T7 , ITZ009) , SUPT5H ( BAC_528_O23_T7

347 N19-150kb H11 A476-110 kb

C07_A24_T7

) DLL3 ( F04_A442_T7 G22 0050-60kb 463 N1-130kb C07 A24-90kb

A09 A121-140kb

sponding clone names, names, clone sponding 266 F8-170kb 99_O22_Sp6 319 D5-180kb

449 E24-160kb H11_A476_Sp6

(ITZ003)

F12 A542-90kb C10 A285-80kb

G22_0050_T7 521_D11_T7 F03 A7-225kb A08 A400-120kb J08 0040-115 kb 438 C20-190kb A05 0031-160kb 392 C20-170kb 424 G14-170kb 256 N2-180kb 345 L23-155kb 443 E11 -170kb 421 J2-160kb 419 C3-(190kb) BAC 308C15 BAC 125H13 BAC 354M10 BAC 22M8

443 J11

BAC 399L3 409_F21_T7

G11_A143_Sp6 L14 0086-100kb B03_A349_T7

419_C3_T7 (ITZ002) 419_C3_T7 D03_A224_T7 (ITZ010) D03_A224_T7

G04 A365-85kb B03 A349-85kb DYRK1B E16 0085-(85kb) J06 0031-110 kb J05 0024-140kb F04 A228-75kb D03 A224-75kb

H01 A361 G12 A82 FBL (LOC148002) FBL G11 A143-130kb 409 F21-165kb 445 F24-150kb A05 0046-90kb C03 A9-140kb 378 C18-185kb

dization probes depend on the intron theintron depend on dization probes D03_A224_Sp6

515 K5-170kb

A05_0046_T7 409_F21_Sp6 obeshorizontal arerepresentedas C07 A556 positions, and insert sizes. andinsert positions, 496 I3-(170kb) 306 I8-170kb

G04 A249-165kb SW193 N17 0085 F04 A399 H11 A80

O11 0075-80kb L08_0078_T7 (ITZ011) L08_0078_T7

L08 0078-70kb

A08_A240_T7 C07_A172_T7 A08 A240 - 115 kb C07 A172-90kb

G17 0095-90kb

PRX_F/R

SEI1_F/R

RBT1_F/R

C07_A172_Sp6

BLVRP_F/R H08 0005-75kb (ITZ015) A08_A240_Sp6

N06 0056 - 110 kb H08_0005_Sp6 (ITZ016) H08_0005_Sp6

C08 0070 SPTBN4

B05 A193 C08_0070_Sp6

Page 3 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

SSC6 HSA19 MMU7

0 kb 36.9 Mb CLIPR-59 22.6 Mb DKFZp434J046 OVOL3 POLR2I Capns1 CKAP1 200 kb CLIPR-59 37.1 Mb CAPNS1 22.4 Mb DKFZp434J046 COX7A1 POLR2I CAPNS1 COX7A1 400 kb 38.6 Mb 22.2 Mb

600 kb 38.8 Mb NYD-SP11 22.0 Mb

800 kb 39.0 Mb 21.8 Mb

NEUD4 PPP1R14A 1000 kb 39.2 Mb SPINT2 21.6 Mb Neud4 IMUP KCNK6 Ppp1r14a PSMD8 Spint2 RASGRP4 Psmd8 1200 kb 39.4 Mb RYR1 21.4 Mb

MAP4K1 Ryr1 0 kb M9 Map4k1 39.6 Mb ACTN4 21.2 Mb Actn4 LGALS7 Lgals7 LGALS4 Lgals4 200 kb ECH1 Ech1 39.8 Mb HNRPL 21.0 Mb Hnrpl Sirt2 NEUD4 SIRT2 NFKBIB Nfkbib PPP1R14A Mrps12 400 kb SPINT2 SARS2 MRPS12 KCNK6 40.0 Mb 20.8 Mb PSMD8 PAK4 RYR1 IL29 Gmfg 600 kb MAP4K1 40.2 Mb 20.6 Mb Rps16 ACTN4 GMFG Supt5h FLJ10211 LGALS7 800 kb Dyrk1b LGALS4 RPS16 Fbl ECH1 40.4 Mb SUPT5H 20.4 Mb HNRPL DLL3 SIRT2 Psmc4 1000 kb NFKBIB PP13 MRPS12 40.6 Mb 20.2 Mb PAK4 PPL13 GMFG CLC 1200 kb FLJ10211 LOC163128 DYRK1B LOC126263 40.8 Mb FBL 20.0 Mb FCGBP RPS16 SUPT5H PSMC4 1400 kb DLL3 41.0 Mb 19.8 Mb Akt2 DYRK1B Prx FBL MAP3K10 RBT1 1600 kb AKT2 41.2 Mb 19.6 Mb Spnb4 SW193 PLD3 1800 kb PRX SEI1 PRX 41.4 Mb RBT1 SEI1 BLVRP RBT1 SPTBN4 2000 kb BLVRP SPTBN4 41.6 Mb

Figure 2 Comparative maps of Sus scrofa chromosome 6q1.2, Homo sapiens chromosome 19q13 and Mus musculus chromosome 7. Comparative maps of Sus scrofa chromosome 6q1.2 (results from this study), Homo sapiens chromosome 19q13 (http://www.ncbi.nlm.nih.gov build 31), and Mus musculus chromosome 7 (http://www.ncbi.nlm.nih.gov MGSCv3). Gene order is perfectly conserved between the three species, however the gene order is inverted in the mouse with respect to the other two species. In the human map all known genes without hypothetical gene predictions are listed, while in the murine map only those genes are listed that have also been mapped in the pig. In the porcine map the position of the microsatellite SW193 is also indicated.

Page 4 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

corresponds exactly to the gene order of SSC 6 and HSA 19 tional markers at their most likely location, without alter- but the orientation is inverted. The perfect synteny conser- ing the multipoint distance between framework markers vation between mouse and the two other species can only (Fig. 3). be observed since the latest update of the mouse maps as in the previous mouse genome assembly a major rear- As shown in figure 3, the gene orders on the RH and phys- rangement of the gene order in this genome region was ical maps are generally in good agreement. This agree- observed [8]. ment is perfect between the physical map and the 1000:1 framework RH map produced on IMNpRH2 panel. It Whereas the gene order is perfectly conserved between hu- demonstrates that at the 50–100 kb scale, fully accurate man, mouse and pig, the physical distances between maps can be produced on this panel provided that 1000:1 genes vary somewhat between the three species. Within framework maps are drawn. the investigated region the gene-poor stretch between COX7A1 and NEUD4 accounts for the biggest part of Some minor discrepancies can be found when compre- these size deviations. The cloned region has a very uneven hensive maps are drawn. For instance, the location of gene density. At the top and at the bottom of the map (Fig. SPTBN4 on the IMNpRH2 map seems incorrect. However 2) genes are clustered extremely dense with very short a difference of log likelihood of only 1.57 is found be- intergenic regions, while in the middle of the map, between the maps constructed under the most likely order tween the COX7A1 and the NEUD4 gene the gene content and the expected order. We thus think that our RH data do is actually very low. not sufficiently support the hypothesis of a very small re- arrangement of this region. It should be pointed out, that RH mapping even if additional markers are added at their most likely In this study, we were able to build two comprehensive location on this kind of comprehensive map, their map- RH maps for SSC 6q1.2. On the 7000 rad IMpRH panel 15 ping does not affect the distance calculated between STS markers were genotyped, while on the 12 000 rad framework markers. IMNpRH2 35 STS markers were analyzed. Retention fre- quencies of markers ranged from 18.1 % to 32.8 % (aver- We also compared the resolution of both panels on the age 22.9 %) on the IMpRH panel and from 27.8 % to 44.3 framework map established between COX7A1 and % with an average retention frequency of 37.5 % on the BLVRP. On IMpRH the distance is 146 cR7000, whereas the porcine IMNpRH2 panel. same fragment is 438 cR12000 long on IMNpRH2. In this region the ratio between the resolutions is thus 3.01, During the building of these two contigs, we simultane- which is slightly higher than the value of 2.77 observed in ously analyzed data obtained on both IMpRH and the PRKAG3-RN region [3] and of 2.43 observed in a QTL IMNpRH2 panels using the Carthagene program. Inter- region close to the centromere of SSC 7 [12]. In the gene mediate rough analyses of RH data allowed us to monitor rich region between RYRl and BLVRP, which is precisely the construction of the contig. In particular it allowed us mapped on the reported clone contig, a ratio of 6.6 kb / to orient a subcontig in the gene poor region from ITZ002 cR12000 (1370 kb / 207 cR12000) is observed on the to ITZ004 as well as to estimate the size of remainmg gaps. IMNpRH2 panel.

When the full RH data set was available for both panels, it The RH map allowed us to confirm the close link between appeared that at the scale of 10–100 kb, the degree of res- the two contigs we produced. The distance between the olution of the IMpRH panel is not high enough, and fur- extremity markers of the contigs (ITZ002 and ITZ014) was thermore the order of genes that could be determined on estimated at 43.2 cR12000. Considering a ratio of 6.6 kb/ this panel is very sensitive to some small genotyping er- cR12000 in this region, we can estimate that the physical rors. To produce a final reference map we thus computed distance between both contigs could be around 285 kb, a 1000:1 framework map using only the 35 vectors pro- which is roughly similar to the 360 kb distance that would duced on IMNpRH2 panel. The framework status of the be estimated from the human-pig comparative map. map was tested by calculation of likelihood of maps produced after all local permutations in a slipping window of Conclusion 6 markers, and by global local inversions. We confirmed The IMNpRH2 panel allowed a highly accurate resolution that no altemate order could be identified with a differ- of closely spaced markers and was very useful in evaluat- ence of log likelihood of less than 3 compared to the pro- ing the assembly of a clone contig. In most instances not posed order. The framework map contained 24 of the 35 only the order of markers but also the physical distances IMNpRH2 markers. Using this framework map compre- between markers could be very accurately estimated from hensive maps were produced on each panel. In order to the RH12000 map. During the contig building it helped us avoid inflation of the map size, we chose to project addi- to orientate small sub-contigs, which were originally un-

Page 5 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

Physical map IMpRH SSC 6q1.2 IMNpRH2

0 cR ITZ012 100 kb

ITZ013 ITZ012 CAPNS1 30 cR COX7A1 POLR2I

POLR2I CAPNS1 COX7A1 ITZ013 ITZ004 60 cR

90 cR ITZ003

ITZ004

120 cR

ITZ003 ITZ002

150 cR ITZ002

180 cR ITZ014

ITZ005 CAPNS1 0 cR COX7A1 ITZ014 ITZ004 210 cR ITZ005 ITZ006 30 cR ITZ007 ITZ005

ITZ006 240 cR RYR1_Promoter RYR1_Promoter 60 cR ITZ007 MAP4K1 ITZ001 ECH1 270 cR PAK4 ITZ001 SUPT5H LGALS4 90 cR RYR1_Promoter LGALS4 ECH1 RPS16 NFKBIB MAP4K1 ITZ010 300 cR

120 cR ITZ001 PRX ITZ008 LGALS4 SPTBN4 ECH1 NFKBIB 330 cR

150 cR BLVRP LOC163128 DKFZp434H247 360 cR ITZ008 RPS16 PAK4 ITZ009 DLL3 180 cR LOC163128 DKFZp434H247 ITZ010 RPS16 SUPT5H 390 cR ITZ009 DLL3

ITZ011 420 cR ITZ010 SW193 PRX SPTBN4 SEI1 450 cR RBT1 BLVRP ITZ011 / SW193 ITZ016 ITZ015 PRX SEI1 480 cR RBT1 BLVRP ITZ015 ITZ016 SPTBN4

510 cR

GRIK5

540 cR

Figure 3 Comparison of the clone-based physical map, RH7000 map and RH12000 mapofSSC 6q1.2. The RH maps repre- sent comprehensive maps, where the markers of the 1000:1 framework map are highlighted in bold. Note that there is perfect agreement in the order of framework markers between the IMNpRH2 RH12000 map and the clone-based physical map. For the non-framework markers, altemate positions are indicated by thin vertical lines.

Page 6 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

Table 1: Primer sequences for RH mapping

Primer Forward sequence (5 '-3') Reverse sequence (5 '-3') Tm (°C) PCR-Product (bp) Retention Retention IMpRH IMNpRH2

BLVRB GGT TAT GAG GTG ACA GTG CCA AGA GCA CGA TGA TGG 55 148 0.14 0.38 CAPNSl TGT ATT CCT GAA CGG GAG TGC AAG AGA GGG CTA ATG 54 272 0.31 0.38 COX7A1 CAC CTA CTG GAC GAA TCC AGG TCC CGA GGT ATT ACA G 59 388 0.29 0.38 DLL3 ACC CCA GAA TCC TCG TAC AGA GCA GGA CAT AGC ATC 55 395 0.39 ECHl ACC AGG AGG TCC TGC TTG ATG TAG TTG AGG CCC TCT G 58 136 0.18 0.34 GRIK5 CAA CTT CCA GGC CCT GTC TTG ATG AGC TCG CCA ACC 60 174 0.31 ITZ001 TAG CCT TTC CTG TGG AGG ACA CAA AAG CAC ACA CCG 56 423 0.19 0.32 ITZ002 AGC CTT CAC CCA GAA GTC GAT GCA CAA GGA GCT GAG 59 287 0.36 ITZ003 CAT AGG GCT TTT CAC CAG TTC TTG CCG AAT ATC AGA G 54 297 0.28 ITZ004 TTC ACT TTG GGT CTG CTG ACC TAC CCA CTG CTA TGC 55 311 0.24 0.26 ITZ005 TCC AGA CAA GTG AGA AAC AA TTA ATT TAT AAT GCC TGG TCA 55 320 0.24 0.37 ITZ006 ATC TGC GGA GAG GAA AAG TCG TGT TTG TTG GAA TGT C 54 201 0.35 ITZ007 GTG ACT TGT AGA CCA CAG CCT ACA GAG GGA GAA TCC 55 346 0.35 ITZ008 AGC TGA GAC CAA TGC CAT ATA ATT GGG AGT TCT CGC 53 122 0.36 ITZ009 CTG CTC CTC ATT CCC ATG CCG TCT TAT GCT TGA GTC 55 187 0.40 ITZ010 CAG ATT GGG ATG AAG CTC TTA TCA ATC CCA ACA CAC C 54 114 0.22 0.37 ITZ011 AGA CGG GAA ATT GAG ACC TCC CTG TGG CAG TAA ATG 58 345 0.37 ITZ012 GTG GGG CCC TAT AAA GAC CCG AGG GTC AAA TGT CTG 56 223 0.38 ITZ013 TTG GAG GGT TCA ACT ACG GTA AAC CCG TTC ACG TTG 57 197 0.38 ITZ014 TGC CTG TTC ACG AAC CAC TCC TTG TGT GGG CTA CAG 59 100 0.32 ITZ015 AAG AAA AGG AAA AGG TTT GG TGT TCT CGC AAA CAG TGA G 58 304 0.33 ITZ016 AGA CAG GCT CCG ATG AAG AGA AAG GCT TCC CTG CTG 59 209 0.35 LGALS4 GAT GTC GCC TTC CAC TTC TGA TGA CCA GCT CGA AGG 56 142 0.18 0.35 LOC 163128 TCC AAA GAG AAG GTG GTG ATG AGG GCA TAG GAG AGC 55 170 0.36 DKFZp434H247 TTC TGC AGC TTC TTC TGC ATC GAA GTC CTG TTG CTG 54 145 0.41 MAP4K1 CCT ACC CAC GCC TAT GC CCA GCC AGC AGG AAA GC 56 136 0.33 NFKBIB CTG CAC CTG GCA GCC ATC GCT GGA GCA GCA CGC AAG 60 154 0.32 PAK4 CAG CGA GTG TCC CAT GAG CAT GGG TCA GCA GGA TGG 58 ~1600 0.19 POLR21 ACC ACC CGT GCC AAA AG CCG CGC ACT GTG TGA CT 55 172 0.37 PPP1R14 GCT GAG CAA GCT GCA GTC GGT ACA GCT CCT CCA AGC 58 164 0.32 PRX GCC TCA GGT GAC CTT GTC CCC ACA TCC AGC TCA AGC 58 117 0.21 0.38 RBTl CCA TGG ATG AGA CTG AGC GGC ACA GAA GAG GTT GTG 56 218 0.33 RPS16 CGC TGA TCA TCA CGA TGG GCT TTT GGG CAA GGA ACG 56 294 0.24 0.41 RYR103/104 TTC GTT TCT GCT TCG CC CTC TCT CCT CCC ATT TC 48 162 0.20 0.34 (RYR_Promoter) SEIl GAG CTG GAT GAT GCT GAG GCT GTG ATG GAG CTT GAG 56 184 0.32 SPTBN4 ATA TCC TGC CCC AAG AAG GAG GAG GTC GAC GTT TTG 58 282 0.20 0.38 SUPT5H AGG AGC TTC CCC AGG AAG TGG GTG AGG ATC GGG AAG 58 121 0.18 SW193 TGC CAT CCT TTC TTT CAT TAC G TCA CTC TGA GGG GTC CTG AC 62 101 0.37

linked, and to estimate the size of remaining gaps. Com- DNA sequence analysis bining analyses on both IMpRH and IMNpRH2 panels End sequences of isolated BAC and PAC DNA were gener- provides both the possibility to detect significant linkage ated with a LICOR 4200L automated sequencer system. between relatively distant markers on the IMpRH panel as Further analyses were performed with the online tools of well as to determine the accurate gene order on the higher the European Bioinformatics Institute http:// resolving IMNpRH2 panel. www.ebi.ac.uk/, BLAST database searches in the GenBank database of the National Center for Biotechnology Infor- Methods mation NCBI and the RepeatMasker searching tool for re- DNA library screening and chromosome walking petitive elements (Smit, A.F.A. and Green, P. http:// Library screenings were done as described [8]. Briefly, the repeatmasker.genome.washington.edu/). Single copy se- TAIGP714 PAC library [13], http://www.rzpd.de was quences were used to design primer pairs for the chromo- screened by PCR of hierarchical DNA pools. The porcine some walking using the programs GeneFisher and genomic BAC library RPCI-44 was screened by radioactive Primer3 http://bibiserv.techfak.uni-bielefeld.de/cgi-bin/ hybridization according to the RPCI protocols http:// gf_submit?mode=START, http://www-ge- www.bacpac.chori.org. nome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.

Page 7 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

RH mapping Dmltpt(X,Y) and D2pt(X,Y) are the multipoint and two Prior to RH mapping of swine genomic inserts, each de- point distances between markers X and Y. signed primer pair was tested for correct localization on SSC 6 on a somatic cell hybrid panel [14]. PCR was per- List of abbreviations formed in a 22.5 µl reaction volume containing 25 ng of BAC bacterial artificial chromosome; cR centi Ray; HSA template DNA, 100 µM of each dNTP, 1.5 mM MgCl2, 10 human chromosome; PAC Pl derived artificial chromo- pmol of each primer and 1 U Taq polymerase in the reac- some; RH radiation hybrid; SSC porcine chromosome; tion buffer supplied by the manufacturer (QBiogene, Hei- STS sequence tagged site; MMU murine chromosome; IM- delberg, Germany). After a 5 min initial denaturation at pRH INRA Minnesota porcine Radiation Hybrid panel, 94°C, 38 cycles of 45 s at 94°C, 45 s at the annealing tem- IMNpRH2 INRA Minnesota Nevada porcine Radiation perature of the specific primer pair, and 45 s at 72°C were Hybrid panel 2 performed in a MJ Research thermocycler (Biozym, Hess. Oldendorf, Germany). Finally an extension step at 72°C Authors' contributions for 5 min was performed. Primer sequences and annealing FM constructed most of the BAC/PAC contig, did the gen- temperatures are given in table 1. PCR products were sep- otypings of the RH panels and drafted parts of the manu- arated on 1–2 % agarose gels. Gels were scored twice by script. DM analyzed the RH data and wrote the radiation independent investigators. hybrid mapping related parts of the manuscript. CD provided some initial help with the RH analysis. RV isolated To construct the RH map, 15 markers were mapped on the several BACs and PACs of the final contigs. BB provided IMpRH panel [2] and 35 on the IMNpRH2 panel [3] ac- the TAIGP714 PAC library. AR and MY provided the IM- cording to the INRA protocols http://www.toulouse.in- pRH and IMNpRH2 panels and helped with analysis of ra.fr/lgc/pig/RH/IMpRH.htm. After genotyping the RH data. TL conceptualized the investigation, coordinated IMpRH panel the chromosomal assignments were per- the planning of the experiments and finalized the manu- formed with software available on the INRA WWW server script. All authors have read and approved the final and submitted to the IMpRH database http://imprh.tou- manuscript. louse.inra.fr/. Acknowledgments Statistical analysis of RH results We thank H. Klippert, S. Neander, and D. Seinige for expert technical as- Vectors obtained on IMpRH and IMNpRH2 panels were sistance. We would also like to thank P. de Jong and his lab for providing analyzed with Carthagene software [15]. A framework the RPCI-44 library and excellent supplementary information. This study map was built using buildfw option, which constructs a was supported by a grant of the German Research Council DFG (Le 1032/ 7-1+2) to T.L. 1000:1 framework map by a stepwise locus adding strate- gy under the haploid model of fragment retention. The References framework map was tested using a flips algorithm, which 1. Goldammer T, Kata SR, Brunner RM, Dorroch U, Sanftleben H, checks all local permutations in a window of 6 markers, Schwerin M and Womack JE A comparative radiation hybrid and a greedy algorithm, which tries to improve the map map of bovine chromosome 18 and homologous chromo- somes in human and mice Proc Natl Acad Sci USA 2002, 99:2106- by inversion of parts of the reference map. When the most 2111 likely order did not fit the expected order based on the hu- 2. Yerle M, Pinton P, Robic A, Alfonso A, Palvadeau Y, Delcros C, Hawken R, Alexander L, Beattie LB, Milan D and Gellin J Construc- man-pig comparative map, the likelihood of the two pos- tion of a whole genome radiation hybrid panel for high-reso- sible orders were calculated to determine the strength of lution gene mapping in pigs Cytogenet Cell Genet 1998, 82:182-188 the indication of a possible modification of gene order be- 3. Yerle M, Pinton P, Delcros C, Amal N, Milan D and Robic A Gener- ation and characterization of a 12,000 rads radiation hybrid tween both species. The final framework map was recom- panel for fine mapping in pig Cytogenet Genome Res 2002, 97:219- puted under a diploid model. Additional markers were 228 4. Flaherty L and Herron B The new kid on the block – a whole ge- mapped relatively to the framework map at their most nome mouse radiation hybrid panel Mamm Genome 1998, likely location, projecting the markers on the map using 9:417-418 the following formula (using the diploid model). 5. Fujii J, Otsu K, Zorzato F, de Leon S, Khanna VK, Weiler JE, O'Brien PJ and MacLennan DH Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia Science 1991, 253:448-451   6. Zhao F, Ambady S, Ponce de Leon FA, Miller LM, Lunney JK, Grimm DptFwkM2 ()n , Loc() M= Loc() Fwk+  Dmltpt() Fwk,* Fwk +  DR, Schook LB and Louis CF Microsatellite markers from a nnn1 ()+ ()  DptFwk2 n ,MMDptMFwk2 , n+1  microdissected swine chromosome 6 genomic library Anim Genet 1999, 30:251-255 7. Bidanel JP, Milan D, lannuccelli N, Amigues Y, Boscher MY, Bourgeois where Loc (M) is the location on the framework map of F, Caritez JC, Gmand J, Le Roy P, Lagant H, Quintanilla R, Renard C, marker M mapped between the nth and n + 1th markers of Gellin J, Ollivier L and Chevalet C Detection of quantitative trait the framework (respectively named Fwk and Fwk ), loci for growth and fatness in pigs Genet Sel Evol 2001, 33:289- n n+1 309

Page 8 of 9 (page number not for citation purposes) BMC Genomics 2003, 4 http://www.biomedcentral.com/1471-2164/4/20

8. Martins-Wess F, Voβ-Nemitz R, Drögemüller C, Brenig B and Leeb T Construction of a 1.2-Mb BAC/PAC Contig of the Porcine GeneRYR1 Region on SSC 6q1.2 and Comparative Analysis with HSA 19q13.13 Genomics 2002, 80:416-422 9. The intemational human genome sequencing consortium Initial sequencing and analysis of the human genome Nature 2001, 409:860-921 10. Smit AFA Interspersed repeats and other mementos of trans- posable elements in mammalian gcnomcs Curr Opin GenetDev 1999, 9:657-663 11. Rohrer GA, Alexander LJ, Keele JW, Smith TP and Beattie CW A microsatellite linkage map of the porcine genome Genetics 1994, 136:231-245 12. Demeure 0, Renard C, Yerle M, Faraut T, Riquet J, Robic A, Schiex T, Rink A and Milan D Rearranged gene order between pig and human in a QTL region on SSC Mamm Genome 2003, 14:71-80 13. Al-Bayati H, Duscher S, Kollers S, Rettenberger G, Fries R and Brenig B Construction and characterization of a porcine Pl-derived artificial chromosome (PAC) library covering 3.2 genome equivalents and cytogenetical assignment of six type 1 and type 11 loci Mamm Genome 1999, 10:569-572 14. Yerle M, Echard G, Robic A, Mairal A, Dubut-Fontana C, Riquet J, Pin- ton P, Milan D, Lahbib-Mansais Y and Gellin J somatic cell hybrid panel for pig regional gene mapping characterized by molec- ular cytogenetics Cytogenet Cell Genet 1996, 73:194-202 15. Schiex T, Chabrier P, Bouchez M and Milan D Boosting EM for Ra- diation Hybrid and Genetic Mapping WABI'2001 (Workshop on Algorithms in Bioinformatics) 2001, LNCS 2149

Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright

Submit your manuscript here: BioMedcentral http://www.biomedcentral.com/info/publishing_adv.asp

Page 9 of 9 (page number not for citation purposes)