a genetic linkage map of the rat derived from recombinant inbred strains

Mammalian Genome 7, 117-127 (1996).

�9 Springer-Verlag New York Inc. 1996

A genetic linkage map of the rat derived from recombinant inbred strains

M. Pravenec, 1 D. Gauguier, 2'3 J.-J. Sehott, 2'4 J. Buard, z'4 V. K[en, s V. Bfl~i, 5 C. Szpirer, 6 J. Szpirer, n J.-M. Wang, 7 H. Huang, 7 E. St.Lezin, 7 M.A. Spence, s P. Flodman, s M. Printz, 9 G.M. Lathrop, 3'1~ G. Vergnaud, 2'4 T.W. Kurtz 7

lInstitute of Physiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic 2Laboratoire de Genetique Moleculaire, Centre d'Etudes du Bouchet, Vert le Petit, France 3Wellcome Trust Centre for Human Genetics, Oxford, UK 4Laboratoire de Genetique des Especes, Institut de Biologic, Nantes, France 5Department of Biology, 1st Medical Faculty, Charles University, Prague, Czech Republic 6Department of Molecular Biology, Universite Libre de Bruxelles, Rhode-St-Genese, Belgium 7Department of Laboratory Medicine, University of California, San Francisco, California, USA 8Department of Pediatrics, University of California, Irvine, California, USA 9Department of Pharmacology, University of California, San Diego, California, USA t~ U358, Hopital St. Louis, Paris, France

Received: 21 June 1995/Accepted: 11 September 1995

Abstract. We have constructed a genetic linkage map in the rat by analyzing the strain distribution patterns of 500 genetic markers in a large set of recombinant inbred strains derived from the spontaneously hypertensive rat and the Brown-Norway rat (HXB and BXH recombinant inbred strains). 454 of the markers could be assigned to specific chromosomes, and the amount of genome covered by the mapped markers was estimated to be 1151 centimorgans. By including a variety of morphologic, biochemical, immunogenetic, and molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH recombinant inbred strains.

Introduction

the amount of genotype data available in these strains is quite limited (Hedrich 1992).

In the current study, we have constructed a linkage map in the rat by determining the strain distribution patterns of a total of 500 markers in the HXB and BXH recombinant inbred strains. The HXB and BXH strains represent the largest set of rat RI strains in the world and were derived from the spontaneously hypertensive rat (SHR) and Brown-Norway rat (BN.Lx), progenitors for genetic studies of cardiovascular phenotypes including blood pressure and cardiac mass, and for genetic studies of limb morphology and development (Pravenec et al. 1989, 1990, 1995; K~en et al. 1990, 1993). By including a variety of morphologic, biochemical, immunogenetic, and DNA molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH RI strains.

The rat provides a number of important models of human disease and is widely used for experiments in molecular biology, physiology, biochemistry, and pharmacology. The rat is also being increasingly used for genetic studies; as a consequence, there is growing interest in the development of a genetic linkage map of the rat (Jacob et al. 1995; Yamada et al. 1994). With its convenient size and well-characterized biochemical and physiologic back- ground, the rat is particularly well suited for studies of mechanisms whereby quantitative trait loci (QTL) influence complex phenotypes. Recombinant inbred (RI) strains have proven to be ex- tremely useful for gene mapping in the mouse (Taylor 1978; Jef- freys et al. 1987; Taylor and Reifsnyder 1993) and may offer certain advantages over classical crosses for genome scanning and mechanistic studies of complex traits such as blood pressure or addictive behaviors (Pravenec et al. 1989, 1995; Gora-Maslak et al. 1991; Plomin et al. 1991; Crabbe et al. 1994). For example, in a given set of RI strains, the strain distribution patterns of multiple genetic markers need only be determined once, and, therefore, investigators can concentrate on phenotyping without having to worry about running the thousands of genotyping reactions typi- cally required in studies of large segregating populations. In the rat, however, only a few sets of RI strains have been derived, and

Correspondence to: M. Pravenec

Materials and methods

Derivation of recombinant inbred strains. The RI strains were derived from spontaneously hypertensive rats (SHR/Ola) and normotensive Brown-Norway rats (BN.Lx/Cub; Pravenec et al. 1989). The BN.Lx progenitor is a BN congenic strain that carries a segment of Chromosome (Chr) 8 from the polydactylous PD strain (Kfen 1975), and as a consequence the RI strains exhibit polymorphisms on Cbr 8 that are not always observed when comparing other strains of SHR and BN rats. A total of 36 RI strains were originally derived from crosses of female SHR and male BN.Lx rats (HXB strains, n = 26) or female BN.Lx rats and male SHR (BXH strains, n = 10). Of the 36 original strains, 32 surviving strains are still available (22 HXB strains and 10 BXH strains) and are currently beyond F35; the four extinct HXB strains were beyond the F2o generation when breeding stopped. For derivation of the genetic map, DNA samples were available from all 36 of the original strains.

Genetic markers and nomenclature. We analyzed the RI strain distribution patterns (SDPs) for a total of 500 biochemical, morphologic, immunogenetic, and molecular genetic markers. Locus names and abbrevia- tions are as specified by Jacob and associates (1995) with the exception of more than 200 new markers that are described below. Typing of the RI strains for some of the biochemical, morphologic, immunogenetic, and molecular markers has been previously described: Alpl, B, C, Es2, Es3, Es4, Es6, Es8, EslO, Esl4, Esl5, Esl6, Esl8, Fh, 14, lgk@, lgh@, Lx, Msl5-1 (DXUc~sfl) to Ms15-6 (D4Ucsfl), Ren, Rtla, Rt2, RtS, RtS, and Svpl (Pravenec et al. 1990); KIklrs (a kallikrein-related sequence;

118 M. Pravenec et al.: Gene map of rat RI strains

Pravenec et al. 1991); Hspal (Hamet et al. 1992); Cac5-1 (D5Ucsfl) to Cac5-9 (D5Ucsf2) and Agt (Kurtz et al. 1991); Inha, Cryga, and Lca (Pravenec et al. 1994); 116 and R133 (IZffen et al. 1993); and Mgdl (Bender et al. 1994). The esterase 13 (Esl3) isozymes were detected according to Kendall (1983), and the aconitase 1 (Acol) and glyoxalase 1 (Goxl) isozymes according to Cramer and colleagues (1986). The Rt6 alloantigen was detected by flow cytometric analysis with biotinylated P4/16 (anti- Rt6.1) or GY/12 (anti-Rt6.2) monoclonal antibodies (Butcher 1987).

The molecular markers used in the current study consisted primarily of microsatellite polymorpbisms typable by polymerase chain reaction (PCR) analysis and microsatellite/minisatellite markers typable by Southern blot/ DNA fingerprint analysis. The PCR primers for typing the microsatellite markers of Jacob and coworkers (1995) were obtained from Research Genetics (Huntsville, Ala). PCR primers for typing other microsatellites were synthesized in the UCSF Biomolecular Resource Center from previously published sequences of Serikawa and associates (1992): A2m, Abpa, Acaa, Adrb2, Alb, Ampp, Apeh, Apoc3, Cat, Chrm3, Ckb, Cpb, Cryga, Cyp2b2, D1Pasl, D6Cep8, Dcpl, Eno2, Fga, Fgg, Grl, lgf2, lghe, 116, lnha, KIkl, Lsn, Lsn2, Mbpa, Mdh2, Mtlpa, Myc, Mycs, Myh3, Myl2g, Mylclv, Npy, P9ka, Prph, Pklr, Ppy, Pthlh, Rbp2, Scn2a, SIc2al, Spr, Sst, Syb2, Tat, Thyl, Tnfa, Tpml, To, l, Ttr; Goldmuntz and colleagues (1993): D1N64, Pthrl ; Zha and coworkers (1993): Atp l a l , D2N35, D2N91; Rem- mers and associates (1993): D13N2, Trneglr; Kobayashi and colleagues (1992): Ncam; Deng and Rapp (1992): Nprl; Cicila and coworkers (1993): Cp450as, P450cl la; Deng and associates (1994): Drdla, Edn3, Ednra, Ednrb, Prl; and Kershaw and colleagues (1995): Pgml.

Primers amplifying a 3' portion of the rat Jun gene were designed from the Genbank sequence (accession number X17215); upstream primer, 5'- aat gtg ctg gag tgg gaa gg; downstream primer, 5'-tgg aaa atc ttc agt gtg cgg c. Restriction fragment length polymorphism between BN.Lx and SHR rats was detected by cutting the PCR products with the Hinfl enzyme: the SHR DNA contained a Hinfl restriction site, the BN.Lx DNA did not.

Primers amplifying a CA repeat microsatellite polymorphism in the 3' portion of the rat Bcl2 gene were designed from the Genebank sequence (accession number L14680); upstream primer, 5'-atg aaa agg nc act aaa gc; downstream primer, 5'-ata gct gat ttg acc art tgc c.

The Slc9a3 gene (sodium-hydrogen exchanger 3) was previously assigned to Chr 1 by somatic cell hybrid analysis (Szpirer et al. 1994a), and in the RI strains was detected on Southern blot analysis by cDNA probing of TaqI-digested genomic DNA, which yielded a 2.7-kb fragment in the SHR strain and a 2.9-kb fragment in the BN.Lx strain.

Multiple DNA polymorphisms were detected by Southern blot analysis of anonymous mini- and microsatellite loci with probes for synthetic tandem repeats (STRs) or natural tandem repeats (NTRs). These include over 200 new genetic markers as well as some markers that have been previously tested in the RI strains (Pravenec et al. 1990; Kurtz et al. 1991). These markers have been assigned new locus names in accordance with the rules of the rat nomenclature committee. The Msl5 loci, including D3Ucsf2 (Ms15-4), D4Ucsfl (Msl5-6), D19Ucsf2 (Ms15-2), D2OUcsfl (Ms15-3), DXUcsfl (Msl5-1), and DXUcsf2 (Ms15-5) loci, were marked with a probe corresponding to the consensus repeat sequence of the myo- globin 33.15 minisatellite. Southern blot analysis was performed by probing AluI-digested genomic DNA with alkaline phosphatase-labeled oligonucleotide aga ggt ggg cag gtg gag agg tgg gca ggt gg (Pravenec et al. 1990; Kurtz et al. 1991). For Southern blot analysis of the Ct8 loci including D2Ucsf2 (Ct8-2) and D18Ucsf2 (Ct8-1); the Gn5 loci including D2Ucsfl (Gtt5-1), D3Ucsf3 (Gtt5-4), D17Ucsfl (Gtt5-5), and Dl8Ucsfl (Gtt5-3); and the Gaca4 loci including D5Ucsfl (Gaca4-5), DVUcsfl (Gaca4-1), D8Ucsf2 (Gaca4-2), and Dl l Ucsfl (Gaca4-4), AluI-digested genomic DNA was probed with (ct)8, (gtt)5, and (gaca)4 oligonucleotides, respectively. The HinfI restriction enzyme and a (cac)5 oligonucleotide probe were used for detection of Cac5 loci including D3Ucsfl (Cac5-2), D4Ucsf2 (Cac5-5), D5Ucsf2 (Cac5-9), DVUcsf2 (Cac5-7), D8Ucsfl (Cac5-8), D15Ucsfl (Cac5-6), D16Ucsfl (Cac5-3), D17Ucsf2 (Cac5-4), and D19Ucsfl (Cac5-1). These oligonacleotides were end labeled with digoxegenin-labeled deoxyuridine triphosphate by a terminal transferase reaction, and after hybridization and blotting the hybrids were detected by enzyme immunoassay (Boehringer Mannheim) as previously described (Pravenec et al. 1991, 1992).

The new series of anonymous molecular markers not previously tested in the RI strains was derived by the use of other STRs. The STRs that mimic minisatellite structure can simultaneously detect many polymorphic minisatellites on Southern blots and can also be used to screen genomic libraries to identify new natural tandem repeats (Vergnaud et al. 1993). One set of 18 minisatellite markers was isolated as previously described

(Vergnaud et al. 1991a) by screening approximately 20,000 clones from a Sprague-Dawley rat genomic library (CLONTECH RL1032m) with the following STR probes: 14c3, 14c5, 14c14 (Vergnaud 1989); 16c2, 16c4, 16c17, 16c20 (Vergnaud et al. 1991b); 13cl, 14c31 (Mariat and Vergnaud 1992); and 14c16, 16c27 (Mariat et al. 1993). Two STR probes, 14c16 and 16c27, gave a polymorphic pattern of hybridization on Southern blots. A second set of 64 anonymous minisatellite markers was obtained by another procedure to be described in detail elsewhere (manuscript in preparation). A third set of markers (NTR markers) was isolated from a rat library of short DNA fragments after screening with STR probes. Sequence analysis of these clones provided both PCR typable markers and, after PCR am- plification and purification of the repeated element, probes for use on Southern blots.

The nomenclature for the new Southern blot markers is based on the official guidelines and reflects the pattern of hybridization of the probes on Southern blots. For example, D3Cebr8Os2 identifies a locus on Chr 3 marked by probe Cebr80, where s2 denotes the second system detected according to its position of migration. For PCR typable markers that identify anonymous repeat sequences, the NTR nomenclature was used. For example, D1Ntr8 identifies a locus on Chr 1 amplified by primers of the marker NTR8. When the repeated element was used as a probe on Southern blots, the polymorphic pattern of hybridization was reflected by the nomenclature described for Cebr probes (for example, D8Ntr44slO). South- ern blots and hybridizations of the minisatellite probes were performed as previously described (Vergnaud 1989).

Map construction. Genotype results were independently scored by two individuals and entered into the Map Manager computer program of Manly (version 2.5b3) (Manly 1993). Data entry was verified by two individuals. When a priori evidence of linkage was available, the RI strain linkage analysis was based on conventional binomial distribution statistics by use of a 95% confidence level (Silver 1985). In the absence of any a priori linkage data, we used the Map Manager routine for Bayesian analysis of RI strains (95% confidence level), a routine that is more stringent than the conventional analysis (Neumann 1990). Linkage groups were assigned to specific chromosomes on the basis of the results of somatic cell hybrid analysis, previously published chromosome locations of selected markers, or the chromosome locations of microsatellite markers reported by Jacob and associates (1995). The multiple opportunities for crossovers during the production of RI strains limit the usefulness of RI strains for establishing gene orders. Nevertheless, by choosing orders that minimized the number of doable crossovers, we were able to derive a map that was fundamentally similar to the F 2 map of Jacob and colleagues (1995). In most of the discrepant cases, RI strain orders could be readily adjusted to match those on the F z map without greatly increasing the number of double recombinants.

Results

In the 36 recombinant inbred strains for which DNA was available, we determined the strain distribution patterns (SDPs) o f 500 biochemical, morphologic, immunogenet ic , and molecular genetic markers. Of the 500 genetic markers tested, 454 could be assigned to specific chromosomes (Tables 1 and 2). The assigned markers fell into 41 linkage groups (each including at least two loci). This represents about twice the number of linkage groups as in the F 2 map of Jacob and coworkers (1995). Because of the multiple opportunities for recombinat ion during the development of RI strains and the limited power of RI strains for detecting linkages between distantly separated genes, the greater number o f linkage groups in the RI strains is to be expected. The l inked markers span approximately 1151 cM of the genome as calculated according to the method of Jacob and associates (1995). For the purpose of gene mapping with the RI strains, the amount of genome swept by all o f the assigned markers is est imated to be approximately 1746 cM, as calculated according to Neumann (1990). This figure tends to overest imate the true extent of genome coverage because the covered distance attributed to markers near the ends of the linkage groups will be inflated. Table 2 shows the linkage data for all chromosomes .

M. Pravenec et al.: Gene map of rat RI strains 119

Table 1. Distribution of markers on each chromosome.

Genetic Number of Number Number of Total Chromosome length linkage of anonymous number number (cM) groups genes markers of markers

1 82.3 3 11 20 31 2 83.0 1 7 27 34 3 113.6 3 5 29 34 4 107.7 2 11 24 35 5 39.9 3 5 16 21 6 56.6 3 3 18 21 7 117.9 2 5 38 43 8 81.5 1 11 23 34 9 23.4 3 3 14 17

10 72.6 4 6 24 30 11 20.0 2 1 12 13 12 26.6 1 3 16 19 13 73.4 1 6 14 20 14 33.8 1 2 11 13 15 11.4 2 1 6 7 16 21.0 2 1 13 14 17 53.7 1 3 8 11 18 49.0 2 3 18 21 19 51.9 2 14 9 23 20 15.1 1 3 5 8 X 17.0 1 1 4 5 Totals 1TST-.T- 4-'T ~ ~

Genetic length is determined by summing distances between markers. The number of linkage groups does not include assigned markers that are not linked to any other markers.

D i s c u s s i o n

We have generated a rat linkage map with 454 genetic markers in a large set o f recombinant inbred strains derived f rom the SHR and the BN.Lx rat. The map includes multiple molecular markers and many of the classic immunogenet ic and biochemical genetic markers previously applied in the rat. The RI strain linkage map is in reasonable accord with the F 2 map of Jacob and colleagues (1995) derived with 430 markers in the (BN.Lx x SHR) intercross (n = 46) and the composi te map assembled by Yamada and coworkers (1994) with 276 markers. It should be emphas ized that with the RI strain linkage map, as with linkage maps derived by evaluating relatively small numbers of meiotic events, the conf idence inter- vals for the map distances between individual loci are relatively large (Table 2). Thus, the current RI map should be regarded as a screening tool; additional fine genetic mapping will require the analysis o f larger segregating populations. Because we typed the RI strains for many of the markers of Jacob and associates (1995) and Yamada and colleagues (1994), the current map serves to integrate l inkage data f rom both of these groups.

In a comparison of the RI map with the F 2 map of Jacob and coworkers (1995), some gross discrepancies were observed in the genetic distances for several pairs o f closely l inked loci: in the F e map, several marker pairs were closely l inked on Chr 5 (D5Mit4 and D5Mit11), 10 (DlOMit3 and DlOMit4), and 18 (D18Mit4 and Grl), whereas these appeared to be only loosely linked in the RI strains. Gene orders on the RI map generally matched those on the F 2 map of Jacob and associates (1995) and, in a few discrepant cases, could be readily adjusted without greatly increasing the number of double recombinants.

The RI map is very similar to the map compi led by Yamada and colleagues (1994); however , there are several important discrepancies. On the Chr 8 map of Yamada and colleagues, the Es6 and Thyl loci are placed in separate l inkage groups, and the rela- t ionship be tween Es6 and Apoc3 is uncertain. In contrast, we have found a close linkage between the Es6, Apoc3, and Thyl loci; furthermore, these loci appear to be linked to Rbp2 and other genes located distally. The order of the Es6, Apoc3, Thyl and Ncam loci is identical to that of homologous loci on mouse Chr 9. The RI strain data are also consistent with the linkage results reported by Kobayashi and coworkers (1992). On the other hand, the linkage

Table 2. Genetic linkage map of the rat derived from RI strains?

Distance between loci 95% lower and

Locus (cM) upper limits (cM)

Chromosome 1 Slc9a3

9.5 3.3-28.9 D1Ntr8

2.5 0.5-8.8 D1Mit9

3.3 0.8-10.7 DICebr68sl

8.3 3.0-23.7 DIPasl

5.1 1.4-16.1 D1 Cebrpl31s2

2.8 0.5-10.5 Cyp2b2

10.5 3.8-30.8 Klkl

DICebr16s1 0.0 0.0-3.5

DICebrlO3sl 0.9 0.0-5.8

C 0.0 0.0-2.9

DIN64 5.3 1.5-17.1

Rt6 2.8 0.5-10.5

DICebr52s3 0.0 0.0-2,9

Sa 0.0 0.0-3.1

DICebrTs3 1.2 0.0-8.2

D 1 Ceb r21 s2 4.1 0.7-16.9

Mtlpa 0.0 0.0-3.4

Myl2 1.5 0.2-6.5

Lsn 3.3 0.8-10.7

Ig]'2 1.5 0.2-6.5

D 1Ceb r 10s3 5.8 1.8-17.0

DIMit27

D1 Cebr72sl 0.0 0.0-3.1

DICebrlOOsl 1.6 0.2-6.7

DICebrlOOs2 1.7 0.2-7.3

DICebr31sl 1.7 0.2-7.3

DICebr31s2 8.1 2.7-25.0

DIMitl4 0.8 0.0-5.4

DICebrl9s2

Jak2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pthrl

Chromosome 2 D2Mit4

3.3 0.8-10.7 D2Ucsf2

4.4 1.3-13.2 D2Mit6

5.8 1.8-17.0 D2Cebrp1104s1

0.8 0.0-5.2 D2Cebrpl lO4s2

6.5 2.0-20.1 D2Mitl 7

0.0 0.0-2.9


Table 2. Continued. Table 2. Continued.





D2Mitl8 Scn2a 0.9 0.0-5.6 7.5 2.5-22.0

Cpb D3CebrPlO38sl 2.9 0.5-11.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D2Mit7 D3Cebr2s4 9.5 3.3-28.9 0.8 0.0-5.2

D2Mit9 D3Cebr9sl 8.5 2.8-26.8 3.7 0.9-12.2

D2Cebr28s4 Cat 2.6 0.5-9.6 4.2 1.0-14.2

D2Cebrlls2 D3Mit15 3.5 0,8-11.2 2.9 0.5-11.0

Fga D3Mit16 0.0 0.0-2.9 1.6 0.2-6.7

Fgg D3Mit6 1.7 0.2-7,3 1.5 0.2~6.5

Pklr D3Mitl 7 0.0 0.0-3.1 1.5 0.2-6.5

Npr l D 3 Ucsf3 0.0 0.0-3.0 I0.0 3.7-28.8

D2CebrlO4sl D3Mit13 1.6 0.2-7.0 0.0 0.0-2.9

R802 D3Mit4 1.6 0.2-6.7 4.9 1.4-15.3

P9ka D3Cebr4s2 3,9 0.9-12.8 0.0 0.0-3.1

D2CebrlOs6 D3Cebr4s5 0.8 0.0-5.4 2.6 0.5-9.6

Atplal Goxl 2.4 0.4-8.5 4.6 1.1 16.0

D2Cebr42s3 D3Mit4 1.8 0.2-7.9 1.8 0.2-7.9

D2Cebr204s17 D3Mit3 1.8 0.2-7.9 0.7 0.0-4.6

D2Mitl4 D3Mit2 1.6 0.2-7.0 6.9 2.3-19.6

D2CebrlOs2 D3Cebr45s8 1.3 0.0-9.3 2.6 0.5-9.6

D2Ucsfl D3Cebr8Os2 1.3 0.0-9.3 1.7 0.2-7.3

D2Cebr4s8 D3Cebr8Os l 2.6 0.5-9.6 2.7 0.5-10,0

D2N91 Svpl 5.6 1.8-16.2 8.7 3.1-25.2

D2N35 Edn3 1.6 0.2-7.0

D2Cebrp133s9 Chromosome 4 4.7 1.3-14.5 Rt8

D2Cebrlls4 2.7 0.5-10.0

D2Mitl6

D2Cebrp476s2

D2Mit8

Chromosome 3 D3CebrP97s12

13.9 5.1-45.2 R63

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D3Cebr69sl

D4Cebr6s8 0.0 0.0-3.3

D4Cebr6sl6 0.0 0.0-3.1

116 0.0 0.0-2.9

D4Cebr88sl 0.0 0.0-4.8

D4Cebrp165sl 0.0 0.0-4.8

R133 3.6 0.9-11.7

D4 Ucsfl 0.0 0.0-3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D3Cebr204s4 D4Cebr28s5 0.8 0.0-5.2 0.0 0.0-3.1

D3Cebr26s l D4Cebrp149s8 1.6 0.2-7.0 3.7 0,9-12.2

D3Uesfl D4Mit2 0.0 0.0-3.3 0.0 0.0-2.9

D3Ucsf2 D4Mit9 1.7 0.2-7.6 6.3 1.9-18.9

D3Cebrp2OTs7 D4Cebr46s5 0.0 0.0-2.9 4.6 1.1-16.0

D3Cebr83s l D4Cebrp215s9 4.7 1.3-14.5 2.5 0.3-12.1

D3 Mit l O D4Cebrp14 5s3 7.5 2.5 22.0 1.4 0.0-9.9

D3Mit9 D4CebrplO16s14z 12.5 4.7-37.9


Table 2. Continued.

Locus

Distance between loci 95% lower and (cM) upper limits (cM)

Z6 0.5-9.6 Klklrs

1,6 0.2-6.7 ~ryl

1.6 0.2-6.7 D4Mit5

2.4 0.4-8.5 Upy

8.1 2.7-25.0 D4Mitl 1

3,1 0.6-11.6 lgk@

8.1 2.7-25.0 Spr

10.0 3.7-28.8 Ampp

8.3 3.0-23.7 D4Mitl 9

1.6 0.2-6.7 D4Cebr7sl 7

2.5 0.5-8.8 A2m

1.6 0.2-6.7 Eno2

2.7 0.5-10.0 D4Cebr7s7

6.3 1.9-18.9 D4Cebr9s4

1.7 0.2-7.6 D4Mit22

Table 2. Continued.



D5Cebr2s2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B

Chromosome 6 D6Mit5

2,5 0.5-8.8 D6Cep8

0.0 0.0~,.8 D6Cebr23s11

0.0 0.04.8 D6Cebrp424s2

1.7 0.2-7.6 D6Cebrp40s27

0.9 0.0-6.3 D6Cebr204s20

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D6Mit9 11.5 4.2-35.9

D6Mit4 0.8 0.0-5.2

D6Cebrp97s14 9.5 3.3-28.9

D6Cebr36sl 0.8 0.0-5.2

D6Mit8 2.4 0.4-8.5

D6Mit2

D6Cebr2s3 12.5 4.7-37.9 3.7 0.9-12.2

Pthlh D6Cebrp165s2 2.5 0.5-9.2 0.0 0.0-3.1

D4 Ucsf2 D6Cebrp91s2 2.8 0.5-10.5 0.8 0.0-5,2

D4Cebr204s3 5 D6Cebrp91s l 2.9 0.5-11.0 9.5 3.3-28.9

D4Cebrp l O16s8 D6Cebr82s I 0.8 0.0-5.2

Chromosome 5 lghe D5Ucsfl 2.8 0.5-10.5

6.3 1.9-18.9 lgh@ D5Mitl I 5.3 1.5-17.1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D6MitlO D5Mit2 3.6 0.9-11.7

3.5 0.8-11.2 Ckb D5Cebrp312s4

0.0 0.0-9.8 Chromosome 7 Aco l D7Cebr6s13

5.0 0.5-35.7 2,0 0.2-9.1 D5Cebr63s2 D7Cebr69s5

2.4 0.4-8.5 0~9 0.0-5.6 D5Cebr63sl D7Cebr205sl

0.8 0.0-4.8 0.9 0.0-5.6 D5MitlO D7Cebr6s5

0.8 0.0-4.8 0.9 0.0-6.3 D5Mitl D7Uesfl

3.6 0.% 11.7 0.0 0.0~. 1 D5Mit9 D7CebrplOO5sl

4.0 1.0-13.5 3.9 0.7-15.7 D5Ucsf2 D7Cebr46sl

0.0 0.0-3.3 4.2 1.0-14.2 D5CebrlOsl2 D7Ntrl l

1.6 0.2-6.7 0.0 0.0-3.1 D5Mit4 D7Cebr204s8

3.6 0.%11.7 0.0 0.0-3.0 D5Mit5 Prph

0.8 0.0-4.8 1.5 0.2-6.5 Jun D7Ucsf2

1.7 0.2-7.6 1.5 0.2-6.5 Pgml D7Cebr59s4

5.1 1.4-16.1 2.4 0.4-8.5 D5Mitl4 D7Cebr24s2

0.0 0.0-2.9 0.8 0.0-4.8 Slc2al D7Cebr24sl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 1.9-18.9

D5Cebr2sl D7Cebr74sl 0.7 0.0~..6 3.7 0.%12.2



Locus

Distance between loci (cM)

95% lower and upper limits (cM) Locus


D7Mit8 5.2 0.9-24.4 5.8 1.8-17.0 Lx

D7MitlO 0.0 0.0-6.0 4.7 1.3-14.5 Rt5

D7Mit2 0.7 0.0-4.6 0.0 0.0-3.6 Ncam

D7Mit11 3.3 0.8-10.7 1.9 0.2-8.2 Apoc3

Bzrp 0.7 0.0-4.6 0.8 0.0~-.8 DSMit3

D7Mit13 0.0 0.0-3.0 2.6 0.5-9.6 D8Mit4

Cypl lb l 0.9 0.0-5.6 0.8 0.0-5.2 Thyl

Cypl]b2 11.5 4.2-35.9 0.0 0.0-2.9 Rbp2

D7Mit14 3.3 0.8-10.7 1.7 0.2-7.6 D8Ntr44s10

D7Mit3 0.0 0.0-2,9 1.7 0.2-7.3 D8CebrlOs5

D7Cebrp187s3 0.0 0.0-3,4 0.0 0.0-3.1 D8Ucsfl

D7Cebr77s1 3.9 0.9-12.8 2.7 0.5-10.0 Mylclv

D7Mit4 1.6 0.2-6.7 2.9 0.5-11.0 Apeh

Myc 9.5 3.0-31.4 10.0 3.2-34.3 D8Ucsf2

D7Mit5 2.2 0.2-10.1 5.9 1.3-23.2 D8Cebr46s6

D7Cebrl4Cl6s3 3.7 0.9-12.2 3.5 0.6-13.8 D8Mitll

D7Cebr204sl I 0.7 0.0-4.6 0.0 0.0~4.1 Acaa

D7Cebrl4C16s2 0.7 0.0~-.6 3.5 0.6-13.8 D8Cebrp203sl

D7Cebr204sl2 3.7 0.9-12.2 3.3 0.8-10.7 D8Cebrp97s22

D7Mit6 0.0 0.0-3.3 1.8 0.2-7.9 D8Cebrl6s5

D7Cebr77s3 0.0 0.0-3.0 12.9 4.5~-3.0 D8CebrlO3s2

D7Cebr59s6 12.2 4.3-39.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D8Cebr46s2 D7Mit7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D8Mit6 13.9 5.145.2 D7Cebrp179s6

5.9 1.3-23.2 D7Cebrp179s7

0.0 0.0-4.8 DTMitl7

0.8 0.0-5.4 D7Cebr205s3

1.8 0.2 7.9 D7CebrlOsl

Chromosome 8 D8Cebr49s4

0.7 0.0~-.6 D8Cebr49s2

0.8 0.0-5.2 D8Cebr92sl

0.8 0.0-5.2 D8Cebr92s2

5.1 1.4-16.1 Tpml

3.5 0.8-11.2 D8Mitl2

4.4 1.3-13.2 Es6

0.8 0.0-5.0 D8Mit5

0.8 0.0-5.0 D8Cebr81sl

0.0 0.0-3.3 D8Cebrp97s13

0.8 0.0-5.4 D8Cebr81s4

Chromosome 9 D9Mitl

1.2 0.0-8.2 D9Cebrp4Os l 4

1.2 0.0-8.2 D9Cebr204s l

0.8 0.0-5.4 D9Cebr16C27s1

0.8 0.0-5.4 D9Cebr16C27s2

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Alp1 5.8 1.8-17.0

Inha 3.3 0.8-10.7

Cryga 4.9 1.4-15.3

D9Cebrp60s12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D9Mit6 1.5 0.2-6.5

D9Cebrl6s3 0.8 0.0-5.2

D9Cebr25sl 0.8 0.0-5.2

D9Cebr l 6s6 1.6 0.2-7.0

D9Cebr3s4 0.0 0.0-3.3

D9Cebr205s2 0.0 0.0-3.0


Table 2. Continued.



D9Cebr65sl 0.7 0.0~-.6

D9Cebr65s2

Table 2. Continued.

Chromosome 10 DlOMit4

0.9 0.0-6.3 DlOCebrplOI6s2

0.9 0.0-6.3 DlOCebr4s9

0.8 0.0-5.4 DlOCebr4s7

4,0 1.0-13.5 D l OCebrp97s5

3.9 0.9-12,8 Myh3

4.6 1.3-13,9 DlOMit2

0.9 0.0-6.1 DlOCebr44s3

1.9 0.2-8.2 Syb2

0.0 0.0-2.9 abpa

6.9 2.3-19.6 DlOCebrplO16s5

1.5 0.2 6.5 DlONtr44s7

0.0 0.0-5.0 DlOCebr23s8

7.1 1.6-31.6 DlOMit7

1.7 0.2-7.3 PPy

0.0 0.0-3,0 Slc4a

9.1 3.2-26.9 Dcpl

DlOCebrp207sl 3.6 0.9-11.7

DlOCebr45s7 6.0 1.9-17.9

DlOMit5 2.4 0,4-8.5

DlOCebrp312s3 1.7 0.2-7.3

D l ONtr32 1.9 0.2-8.2

DlOCebr27s2 0.8 0,0 5.2

D l OMit6

D l OCebrp40s28 8.1 2.1-31.7

DlOCebr204s21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Es13 2.2 0.0-19.6

D10Cebr39s1 1.7 0.2-7.3

DlOCebr39s2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DlOMit3

Chromosome 11 D l l Ucsfl

0,0 0.0-2.9 Sst

3.3 0.8-10.7 DllCebrl ls6

0.0 0.0-2.9 D1 ICebrlO5sl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DI lMitl 1.7 0.2-7.3

DI 1Cebrl5sl 0.7 0.04.6



Dl lCebr87sl 0.7 0.0-4.6

D11Cebr87s2 1.5 0.2-6.5

D l l Mit2 5.6 1.8-16.2

D l l Mit4 2.7 0.5-10.0

D11Cebr204s16 0.9 0.0-5.8

D1 lCebr77s5 2.9 0.5-11.0

D11Cebr77s6

Chromosome 12 D12Cebr4s3

0.8 0.0-5.2 Dl2Mit8

0.0 0.0-3.3 D12Cebrp454sl

3,9 0.9-12.8 D12Cebrp97s9

0.0 0.0-3.1 D12Cebrp97s4

1,7 0.2-7.3 Dl2Ntr2

1.2 0.0-8.2 Dl2Cebr21s8

1.2 0.0-8.2 Dl2Mit6

0.0 0.0-2.9 D12Mit5

4.6 1.3-13.9 D12Mitl

2.5 0.5-8.8 Pail

1.6 0.2-6.7 Lsn2

0.7 0.0-4.6 Mdh2

0.0 0.0-2.9 Dl2Mit7

0.8 0.0-5.4 D12Mit3

0.0 0.0-3.1 Dl2Cebr4s4

3.7 0.9-12.2 D12Cebrls1

3.9 0.9-12.8 D12Cebr6s4

D12Mit4

Chromosome 13 D13Mit4

7.5 2.5-22.0 D13Mit5

1.5 0.2-6.5 D13Mit3

6.7 1.5-28.2 Mgdl

2.8 0.3-14.0 Trneglr

7.1 2.4-20.7 Fh

7.8 2.6-23.4 D13Cebr28s8

9.5 3.3-28.9 D13N2

2.4 0.4-8.5 D13Mit2

1.7 0.2-7.6 D13Cebr2s5

5.1 1.4-16.1 Lca

4.6 1.3-13.9 D13Cebr5s3

0,8 0.0~.8



Locus

Distance between loci (cM)

95% lower and upper limits (cM)

D13Cebr5s4 0.8 0.0-4.8

Ren 3.1 0.6-11.6

D13Cebrp149sl I 4.2 1.0-14.2

D13Cebr9s3 0.8 0.0-5.2

D13Cebr9s2 1.7 0.2-7.6

Bcl2 0.0 0.0-3.4

D13Cebr7sl5 5.3 1.7-17.1

D13Mitl

Chromosome 14 D14Cebrp136s2

0.8 0.0-5.2 D14Cebrp 36 7s6

2.7 0.5-10.0 Dl4Mit9

1.7 0.2-7.6 Dl4Cebr7sl4

0.0 0.0-3.1 D14Mit8

0.7 0.0~.6 Dl4Mit4

1.5 0.2-6.5 D14Mit3

2.6 0.3-13.0 Dl4Cebrp312s2

1.9 0.0-15.3 H

8.3 2.5-28.9 Alb

6.0 1.9-17.9 DI4Mitl

4.9 1.4-15.3 D14Mit5

2.7 0.5-10.0 Dl4Cebr8sl

Chromosome 15 Ednrb

4.0 1.0-13.5 D15Ucsfl

0.9 0.0-5.8 D15Cebr204s39

6.5 2.O-21.0 D15Cebr7s13

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D15Cebr7s11 0.0 0.0-3.1

D15Cebr79sl

Dl5Mit3

Chromosome 16 Dl6Mit2

2.4 0.4-8.5 D16CebrplO38s2

0.8 0.0-5.2 Dl6Ucsfl

0.0 0.0-3.1 D16CebrlOs10

0.0 0.0-3.1 Dl6Cebr204sl3

3.6 0.4-20.1 D16Cebr204s40

1.7 0.0-13.8 D16Cebrp215s4

2.6 0.3-13.0 Dl6Mit5

0.0 0.0-2.9 Mbpa

3.3 0.8-10.7 D16Mitl

Locus


4.4 1.3-13.2 D16Mit3

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D16Cebr48sl 0.7 0.0-4.6

D16Cebr50s2 1.5 0.2-6.5

D16Cebr5Osl

Chromosome 17 Dl7Ucsfl

2.7 0.5-10.0 DI7Ue~f2

0.8 0.0-5.0 Drdla

1.5 0.2-6.5 D 17Mit 7

13.9 5.1-45.2 D l 7Cebrp203s2

0.8 0.0-5.2 DI 7Mit2

3.3 0.6-13.0 Prl

6.8 1.8-24.0 D17Mit3

12.2 4.3-39.1 Chrm3

2.6 0.5-9.6 D17Mit5

9.1 3.2-26.9 D17Mit6


4.9 1.4-I5.3 D18Mit7

3.3 0.8-10.7 D18Mit2

3.5 0.8-11.2 Ttr

3.6 0.9-11.7 Dl8Mit3

6.5 2.0-20.1 D18Cebrl9sl

3.7 0.9-12.2 Grl

10.0 3.7-28.8 Adrb2

0.8 0.0-4.8 Dl8Mit8

2.6 0.5-9.6 Dl8MitlO

D18Uesfl 3.5 0.6-13.8

Dl8Mit9 1.7 0.2-7.3

D18Ucsf2 0.9 0.0-5.6

D18Cebrp205sl 0.8 0.0-5.0

D18Cebrp187s6 0.0 0.0-3.1

D18Cebr6sl4 0.0 0.O-3.1

D18Cebr69s2 1.6 0.2-6.7

Dl8Cebr51sl 1.6 0.2-6.7

Dl8Cebr51s2

Dl8Mit4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D18Cebrp6Osl 1

Chromosome 19 Dl9Cebrpl5Osl

0.0 0.0-2.9


Table 2. Continued.



Agt 2.9 0.3-15.1

DI 9Cebr204s28 7.7 1.7-36.0

Dl9Mit7 4.7 1.3-14.5

Tat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Esl4 0.0 0.0-2.9

Esl5 0.0 0.0-2.9

Esl6 0.0 0.0-2.9

Esl8 6.9 2.3-19.6

Es2 0.0 0.0,2.9

Es4 0.0 0.0-2.9

Es8 0.0 0.0-2.9

EslO 0.7 0.0~,.6

Es3 3.5 0.8 11.2

Hmox 1.6 0.2-6.7

D 19Mit2 9.1 3.2-26.9

Rt2 3.7 0.9-12.2

Dl9Ucsfl 5.1 1.4-16.1

Edn ra 1.7 0.2-7.6

Dl9Ucsf2 0.8 0.04.8

D19Cebr204s23 3.5 0.8-11.2

D19Cebr204s27

D19Cebrp97slO


0.0 0.0-3.5 D2OUcsfl

13.9 5.145.2 Hspal

0.0 0.0-2.9 Rtla

0.0 0.0-2.9 Tn)h

1.2 0.0-8.2 D2OCebr4sl

0.0 0.04.8 D20Cebr32s3

0.0 0.0-3.1 D2OCebrp215s7

Chromosome X DXMit5

11.9 4.5-35.1 Mycs

0.0 0.0-2.9 DXUcsfl

1.6 0.2-6.7 DXCebr7sl6

3.5 0.8-11.2 D X U c ~

Dashed lines separate groups of markers on each chromosome that could not be linked in the analysis of the recombinant inbred strains; cM, centimorgans; the most likely distances between loci and the 95% confidence limits for the linkage distances were calculated according to Silver (1985).

between the Apoc3-Thyl-Ncam group and the Rbp2 locus in the RI strains should be considered provisional as the number of recombinants was near the limit allowed for declaring linkage. We have also detected close linkages between a number of other genes that appear to be unlinked in previously published maps of Serikawa and associates (1992) and Yamada and colleagues (1994). For example, we have found that the Cryga and lnha loci are closely linked on Chr 9, and these genes are also known to be closely linked on mouse Chr 1 and human Chr 2q (Pravenec et al. 1994). We have also found close linkages between the Mdh2 and Lsn2 loci on Chr 12, the Lca and Ren loci on Chr 13, and the Ttr and Grl loci on Chr 18, although no such linkages were found by Yamada and colleagues (1994). The RI strain data and the results compiled by van Zutphen and den Bieman (1984) indicate that the Es2 and Es3 genes are very tightly linked on Chr 19, whereas the map of Yamada and coworkers (1994) suggests the possibility of much looser linkage.

In the current study, we have mapped a number of new loci including the genes for Bcl2, Esl3, Jak2, Jun, and Slc9a3 and more than 200 new anonymous markers. The Jun gene was also assigned to 5q31-33 by in situ hybridization (Szpirer et al. 1994b), and the Slc9a3 gene was recently assigned to Chr 1 by somatic cell hybrid analysis (Szpirer et al. 1994a). However, in the RI strain analysis, positioning of Slc9a3 was difficult, and its location relative to the other markers on Chr 1 is unclear and likely to change. The new anonymous loci consisted of a variety of minisatellite markers that yield mono-locus or multi-locus patterns on Southern blot analysis with natural or tandem repeat sequences. Although these newly mapped anonymous markers are not particularly convenient for use in genetic studies in segregating populations, they represent a very useful addition to the cumulative marker set being established in the RI strains. The new minisatellite markers and the PCR typable microsatellite markers were found to be dispersed throughout the rat genome and appeared to be distributed equally among chromosomes according to chromosome size, with the pos- sible exception of the X Chr, for which a relative deficiency of repetitive sequence markers has also been noted by Dietrich and associates (1994) in the mouse and Jacob and colleagues (1995) in the rat. The repetitive sequence markers appeared to be stably inherited, as we observed only two mutant bands in one of the RI strains associated with the (ct)8 and (gtt)5 synthetic repeat probes in the HXB7 strain. Although the BN.Lx rat served as one of the progenitors for the RI strains, we were unable to map the brown (B) locus on Chr 5 because of difficulty in scoring coat color phenotypes in some RI strains. A similar problem has been en- countered by Hedrich (1992) in the LXB RI strains.

Although the statistical characteristics of quantitative trait loci (QTL) mapping studies in RI strains remain to be clearly defined (Belknap 1992; Neumann 1992), it appears that RI strains can provide an important screening tool for genetic studies of complex phenotypes. For example, several groups have successfully used RI strains derived from the mouse to map QTLs related to complex behaviors and to audiogenic seizures (Gora-Maslak et al. 1991; Neumann and Collins 1991; Plomin et al. 1991). Because the HXB and BXH RI strains were derived from a hypertensive progenitor and a normotensive progenitor, the current set of RI strains should be particularly useful for genome scanning studies of QTLs reg- ulating blood pressure and other cardiovascular phenotypes. In addition, the BN.Lx progenitor exhibits an abnormality in limb morphology (polydactyly-luxate syndrome) (K~en 1975) that is variably expressed in the different RI strains. Accordingly, the current set of RI strains should also be useful for mapping genes involved in limb morphology and development.

Because complex phenotypes such as blood pressure or behavior often exhibit substantial variability even among animals within a single, highly inbred strain, there can be considerable uncertainty as to the phenotype values assigned to individual animals in F2 or backcross populations. With the availability of multiple genetic


markers and increasingly dense genetic maps, the importance of rigorous phenotyping will begin to attract greater attention. From this perspective, one of the experimental advantages of recombinant inbred strains is that one can estimate the phenotype associated with a given genotype by averaging the phenotype results of multiple animals within a strain. Moreover, in contrast to the finite life spans of individual genotypes represented in a segregating population, the genotypes represented among recombinant inbred strains are perpetually available and can be repeatedly studied. This enables one to use RI strains for detailed mechanistic studies of QTL action. These features, together with the cumulative nature of RI strain marker sets and the convenient size of the rat as an experimental animal, should facilitate the use of combined genetic and physiologic approaches in the study of complex phenotypes. The RI strain linkage map and the F 2 map recently derived by Jacob and coworkers (1995) should help to accelerate the estab- lishment of the rat as a key organism in mammalian genetics research.

Note:

The SDPs will be made available on the Ratmap World Wide Web site (http://ratmap.gen.gu.se). The RI strains may be obtained on a collaborative basis by contacting Dr. Pravenec.

Acknowledgments. We thank Alena Musilov~i for technical assistance in the breeding of the RI strains, A. Panczak for providing the strain distribution pattern of the Goxl alleles, G. Butcher for providing the strain distribution pattern of the Rt6 alleles, J. Orlowski and A. Wilks for the gift of the S1c9a3 and Jak2 cDNA probes. This work was supported by grants from the Ministry of Health of the Czech Republic to V. K~en, V. Bila, and M. Pravenec; the US-Czechoslovak Science and Technology Program to V. K~en, M. Printz, M. Pravenec, and T. Kurtz, a grant from the PECO Program of the European Commission (EURHYPGEN Project) to M. Pravenec and V. Kfen; grants from the National Heart, Lung, and Blood Institute to T. Kurtz and M. Printz; grants from the FRSM, the CGER- ASLK, and the Association contre le Cancer to C. Szpirer and J. Szpirer.

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a genetic linkage map of the rat derived from recombinant inbred strains

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