Journal of Heredity 2003:94(2)
© 2003 The American Genetic Association 94:161-169
Brief Communication |
Mapping of Rabbit Microsatellite Markers Using Chromosome-Specific Libraries
From the Department of Laboratory Animal Science (Korstanje, Gillissen, van Zutphen, and van Lith), the Department of Clinical Sciences of Companion Animals (Versteeg and van Oost), the Department of Horse Sciences and Herd Health (van Oost), and the Department of Cell Biology and Histology (Bosma), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80166, 3508 TD, Utrecht, the Netherlands; and the Laboratoire Mixte INRACEA de Radiobiologie et d'Etude de Genome, Institut National de la Recherche Agronomique, 78 352 Jouy en Josas Cedex, France (Rogel-Gaillard).
Address correspondence to R. Korstanje at the Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, or e-mail: rkorstan{at}jax.org.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResults and DiscussionReferences |
|---|
Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.
Although the rabbit is used both as an animal model for human diseases and for the production of meat, wool, and fur, the genetic map of this species is not as extensively developed as those of most other laboratory, companion, or production animal species. To enable the identification and localization of quantitative trait loci that are involved in the pathogenesis of complex diseases or in production traits, a dense genetic map containing DNA markers is needed.
In the past 5 years, a number of rabbit microsatellite markers have been developed (Korstanje et al. 2001c; Mougel et al. 1997; Rico et al. 1994; Surridge et al. 1997; van Haeringen et al. 1997). Some of these markers have been used for linkage studies using an F2 intercross population (Korstanje et al. 2001c). Sat13, an anonymous microsatellite marker, is linked to markers on chromosome 1 and the microsatellite located in the metallothionein 1 (MT1 ) gene to linkage group VI (LG VI). The microsatellites in the peripheral myelin protein 2 (PMP2 ) and ileal lipid-binding protein (FABP6 ) genes and the anonymous microsatellite Sol33 were found to be linked, thus forming a new linkage group (LG XI). For another nine polymorphic markers, the anonymous microsatellites Sat8 and Sol44 and the microsatellites located in the genes for the beta chain of the T-cell antigen receptor (TCRB ), MHC class II DP alpha-1 (RLADPA1 ), alpha-like globin cluster (HBA ), wey acid protein (WAP ), erythroid cell-specific 15-lipoxygenase (ALOX15 ), cytochrome P450IIC4 (CYP2C4 ), and beta-casein (CSN2 ), no linkage was found in the analysis of the F2 intercross. Thus, it is obvious that for establishing additional linkages more markers are needed. However, the development of microsatellite markers using conventional approaches can be time consuming. The use of microsatellite-enriched chromosome-specific DNA libraries is a more direct approach; we have recently demonstrated this by generating and mapping rabbit chromosome 1 microsatellite markers (Korstanje et al. 2001b).
Recently, five of the above-mentioned markers have been physically mapped in the rabbit (Zijlstra C et al., in press). PMP2 was mapped to 3q14–15, TCRB to 7p23, ALOX15 to 19q12, MT1 to 5q12, and Sol33 to 3q11. In the present study, chromosome-specific libraries were constructed for these chromosomes, and libraries were constructed for chromosomes 6 and 12. These two chromosomes were chosen to establish linkage with a microsatellite marker in the HBA gene cluster and a microsatellite marker in the MHC-related gene RLADPA1, which were genotyped in the F2 intercross population. These genes have already been physically mapped to 6q12 (Xu and Hardison 1991) and 12q11 (Rogel-Gaillard et al. 2001), respectively. Subsequently, these libraries were used to develop microsatellite markers. Polymorphic markers were used to genotype the F2 intercross and to construct linkage maps.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
Chromosome Isolation
The isolation of chromosomes 3, 5, 6, 7, 12, and 19 was done at the Department of Pathology (Cambridge, U.K.) by bivariate flowcytometry as described previously (Korstanje et al. 1999). Briefly, for flow sorting, primary cultures were established from lung fibroblasts from a male AX/JU rabbit according to standard protocols. AX/JU is an inbred rabbit strain, maintained at the Department of Laboratory Animal Science (Utrecht, the Netherlands). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum; to arrest cells in metaphase, colcemid (0.1 µg/ml) was added for 6 h. Cells were subsequently treated as previously described (Rabitts et al. 1995). Chromosome suspensions were spun, and the supernatant containing the chromosomes was stained for two hours with Hoechst 33258 at 2 µg/ml, chromomycin A3 at 40µg/ml, and magnesium sulfate at 2.5 mM. Fifteen min before analysis, sodium sulfite and sodium citrate were added at 25 mM and 10 mM, respectively. Bivariate flow analysis was performed with a FACStar plus flowcytometer (Becton Dickinson) equipped with two lasers (Spectra Physics). The lasers were set to excite Hoechst 33258 and chromomycin A3 separately, which allowed bivariate analysis of the chromosomes by size and base-pair composition. The identification of the chromosomal fraction was performed as previously described (Korstanje et al. 1999).
Construction of Microsatellite-Enriched Libraries of Chromosomes
The enrichment of the isolated chromosomes and the construction of the libraries was done as previously described (Korstanje et al. 2001b). After transformation of the library into E. coli, single colonies were picked and suspended into 100 µl of LB with Zeocin (50 µg/ml). After a 4 h incubation at 37°C, 5 µl of culture were lysed in 95 µl of water. Five µl of this dilution were used in a PCR reaction using M13 primers. Each reaction was performed in a 30 µl volume containing 1.5 µl (10 µM) of each primer, 6 µl PCR buffer (HT Biotechnology), and 1.5 units of Taq polymerase (HT Biotechnology). PCR products were blotted on Hybond N+ and hybridized with a 32P-labeled [CA]22 oligonucleotide probe according to standard procedures.
Sequencing of Rabbit Microsatellite Sequences
All colonies showing a strong hybridization signal at high stringency wash (6x SSC + 0.1% SDS at 65°C) were sequenced. After a PCR with the M13 forward and reverse primers, the PCR product was used for cycle sequencing, with the M13 forward or M13 reverse primers and the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), according to the standard protocol. The purified extension products were then analyzed on the ABI Model 310 DNA sequencer. All sequences were checked with use the BLAST module for uniqueness with respect to each other and to sequences deposited in the GenBank (Altschul et al. 1990).
Genotyping and Linkage Analysis
Unique sequences were used to generate primer pairs by analysis with the Lasergene computer program (DNAstar, Madison, WI, USA). The primer pairs were optimized and tested on a panel of different rabbit breeds. The panel consisted of (partially) inbred strains (AX/JU, IIIVO/JU, OS/J, WH/J, and X/J), random-bred strains (New Zealand White, Californian, ELCO, and Watanabe), and wild rabbits as previously described (van Lith et al. 1996). PCR was carried out in 10 µl reaction volumes containing 200 ng DNA, 720 nM forward primer, 720 nM reverse primer, 1 x PCR buffer (HT Biotechnology, Cambridge, U.K.), 0.2 mM dNTP (HT Biotechnology), 1–5 mM MgCl2, and 0.3 U Supertaq (HT Biotechnology). After an initial 5 min denaturation at 94°C, 30 cycles were performed as follows: 30 s denaturation at 94°C; 1 min annealing at 55, 60, 65, or 70°C; and 2 min extension at 72°C. A final elongation was carried out for 10 min at 72°C. Products were separated in a 6% denaturing acrylamide gel together with an allele sizing marker (10-base DNA ladder; Allele Sizing Set; Boehringer Mannheim Gmbh, Mannheim, Germany) and detected by autoradiography.
Most markers that were polymorphic between AX/JU and IIIVO/JU (with the exception of 5L1C11 ) were used for linkage analysis. Like the AX/JU strain, the IIIVO/JU is an inbred strain maintained at the Department of Laboratory Animal Science (Utrecht, the Netherlands). The AX/JU and IIIVO/JU strains were crossed, and the F1 hybrids were intercrossed to produce an F2 population. A total of 138 intercross progeny were available. The F2-intercross animals have previously been phenotyped for a number of biochemical markers (e.g., Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP; Korstanje et al. 2001a) and genotyped for microsatellite markers located in genes (e.g., MT1, PMP2, FABP6, and HBA ) and for anonymous microsatellite markers (e.g., Sol33; Korstanje et al. 2001c). In addition, we found a polymorphism in the podocalyxin-like gene (PODXL ), which was amplified with two primers (5'-GTGGCCACGTGTTAATCTATCTTG-3' and 5'-ACTCCAGCCAGGGTCACATTTTA-3') and had three different alleles in the test panel (van Lith et al. 1996): 246 bp for CAL, NZW, IIIVO, wild, ELCO, OS, and WH; 248 bp for AX, ELCO, and WH; and 254 bp for wild. For genetic analysis of the (IIIVO/JUxAX/JU) F2 intercross and mapping of the markers, we used the MAPMAKER/EXP computer package (Lander et al. 1987). For the establishment of linkage groups, we used a critical minimal LOD score of 3.0. Kosambi's mapping function was used for the construction of the map.
|
Results and Discussion |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
All colonies from chromosome-specific libraries of chromosomes 3, 5, 6, 7, 12, and 19 that hybridized to a radioactive-labeled [CA]22 probe were sequenced. On average, 20% of the clones had a hybridization signal. The lowest number was obtained with the library for chromosome 7 (15%); the highest, with the library for chromosome 12 (27%). After sequencing, 57% of the positive clones showed a microsatellite for which primers could be designed to amplify the microsatellite. The least were obtained for chromosome 3 (28%); the most, for chromosome 7 (71%). The remaining 43% showed CA repeats at the ends of the sequences, so no primers could be designed, or showed a CA-rich sequence with no clear microsatellite. Table 1 shows the results for all the primer sets that gave the expected amplification product. In our test panel (van Lith et al. 1996), the number of alleles varied between one and six (Table 1). Markers that were polymorphic between AX/JU and IIIVO/JU were used to genotype the F2 intercross progeny.
|
Chromosome 3
Two markers (D3Utr2 and D3Utr3 ) from the chromosome 3 library were linked to LG XI. We had previously assigned Sat3, Sol33, and D0Utr6 (D3Utr1 ) to this linkage group (Korstanje et al. 2001c). The recent physical mapping of Sat3 and Sol33 to chromosome 3 not only confirms the chromosomal assignment, but also makes it possible to determine the right orientation of the linkage group.
Chromosome 5
Three markers from the chromosome 5 library (D5Utr2, D5Utr3, and D5Utr4 ) linked to LG VI. The physical mapping of MT1 to chromosome 5 confirms the chromosomal assignment. Only one marker in this linkage group is physically mapped and makes determination of the orientation of the linkage group impossible. However, considering the homology between rabbit 5q and human 16q, we would expect HP, which is on one end of the linkage group, to be located on the q-arm of chromosome 5. This makes the orientation shown in Figure 1 the most likely one. In a previous study, we analyzed the atherosclerotic plaque formation in the aorta with our linkage data and found a correlation with LG VI with a peak of LOD 5.5 at the Est-2 locus (Korstanje et al. 2001c). We analyzed the data again with our new chromosome 5 (LG VI) linkage map. The correlation between atherosclerotic plaque formation in male rabbits and chromosome 5 was confirmed (results not shown).
|
Chromosome 6
Four markers from the chromosome 6 library (D6Utr2, D6Utr3, D6Utr4, and D6Utr5 ) are linked to D6Utr1 (in the HBA gene). As for chromosome 5, there is only one physically mapped marker in the linkage group. However, the physical and genetic location of this marker makes the orientation shown in Figure 1 the most likely one.
Chromosome 7
Five markers from the chromosome 7 library (D7Utr2, D7Utr3, D7Utr4, D7Utr5, and D7Utr6 ) could be linked to the physically mapped D7Utr1 (TCRB ). Again, the physical and genetic location of this marker determines the orientation of the linkage group with respect to the chromosome.
Chromosome 12
One marker from the chromosome 12 library (D12Utr2 ) linked to D12Utr1 (RLADPA1 ). The other marker from this library (DOUtr16) that was genotyped showed no linkage with any of these markers. The orientation of the linkage group is impossible to determine at this point.
Chromosome 19
Three markers from the chromosome 19 library (D19Utr2, D19Utr3, and D19Utr4 ) were linked to D19Utr1 (ALOX15 ) and the previously described biochemical marker Es-3 (Korstanje et al. 2001a). The physical mapping of D19Utr1 to the proximal part of the chromosome makes it likely that the orientation of the linkage group is as shown in Figure 1.
Conclusion
In conclusion, two previously reported linkage groups could be mapped to individual rabbit chromosomes (LG XI to chromosome 3 and LG VI to chromosome 5). Four new linkage groups were constructed, and these could be assigned to chromosomes 6, 7, 12, and 19. The previously found correlation between atherosclerotic plaque formation and chromosome 5 markers was confirmed.
|
Footnotes |
|---|
Corresponding Editor: Hector Seuánez
Received July 18, 2002
Accepted November 15, 2002
|
References |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
-
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic local alignment search tool. J Mol Biol. 215:403-410.[CrossRef][ISI][Medline]
Korstanje R, den Bieman M, Campos R, Estevez PJ, Lankhorst A, van der Loo W, van Zutphen LFM, van Lith HA, Ferrand N, 2001a. Genetic analysis and mapping of biochemical markers in an F2 intercross of two inbred strains of the rabbit (Oryctolagus cuniculus). Biochem Genet. 39:169-178.[Medline]
Korstanje R, Gillissen GF, den Bieman MG, Versteeg SA, van Oost B, Fox RR, van Lith HA, van Zutphen LFM, 2001b. Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library. Anim Genet. 32:308-312.[CrossRef][ISI][Medline]
Korstanje R, Gillissen GF, Kodde LP, den Bieman M, Lankhorst A, van Zutphen LFM, van Lith HA, 2001c. Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit. Physiol Genomics. 6:11-18.
Korstanje R, O'Brien PC, Yang F, Rens W, Bosma AA, van Lith HA, van Zutphen LFM, Ferguson-Smith MA, 1999. Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting. Cytogenet Cell Genet. 86:317-322.[Medline]
Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L, 1987. Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics. 1:174-181.[CrossRef][Medline]
Mougel F, Mounolou J-C, Monnerot M, 1997. Nine polymorphic microsatellite loci in the rabbit, Oryctolagus cuniculus. Anim Genet. 28:58-59.[CrossRef][ISI][Medline]
Rabitts P, Impey H, Heppell-Parton A, Langford C, Tease C, Lowe N, Bailey D, Ferguson-Smith MA, Carter N, 1995. Chromosome specific paints from a high resolution flow karyotype of the mouse. Nat Genet. 9:369-375.[CrossRef][ISI][Medline]
Rico C, Rico I, Webb N, Smith S, Bell A, Hewitt G, 1994. Four polymorphic microsatellite loci for the European wild rabbit, Oryctolagus cuniculus. Anim Genet. 25:367.[ISI][Medline]
Rogel-Gaillard C, Piumi F, Billault A, Bourgeaux N, Save J-C, Urien C, Salmon J, Chardon P, 2001. Construction of a rabbit bacterial artificial chromosome (BAC) library: application to the mapping of the major histocompatibility complex to position 12q1.1. Mamm Genome. 12:253-255.[CrossRef][Medline]
Surridge AK, Bell DJ, Hewitt GM, 1997. Polymorphic microsatellite loci in the European rabbit (Oryctolagus cuniculus) are also amplified in other lagomorph species. Anim Genet. 28:302-305.[CrossRef][ISI][Medline]
Van Haeringen WA, den Bieman M, van Zutphen LFM, van Lith HA, 1997. Polymorphic microsatellite DNA markers in the rabbit (Oryctolagus cuniculus). J Exp Anim Sci. 38:49-57.
Van Lith HA, den Bieman M, Lankhorst A, van Haeringen WA, Kuiper MTR, van Zutphen LFM, 1996. Development of a genetic map of the rabbit genome using DNA markers. In: Harmonization of laboratory animal husbandry. Proceedings of the sixth symposium of the Federation of European Laboratory Animal Science Associations. Basel, Switzerland, June 19–21, 1996. London: The Royal Society of Medicine Press Limited; pp. 38–40.
Xu J, Hardison RC, 1991. Localization of the 
-like globin gene cluster to region q12 of the rabbit chromosome 6 by in situ hybridisation. Genomics. 9:362-365.[CrossRef][ISI][Medline]
Zijlstra C, de Haan NA, Korstanje R, Rogel-Gaillard C, Piumi F, van Lith HA, van Zutphen LFM, Bosma AA, in press. Fourteen chromosomal localizations and an update o65f the cytogenetic map of the rabbit. Cytogenet Genome Res.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Li, X. Chen, Z. Fang, J. Shi, and H. Z Sheng Rabbits generated from fibroblasts through nuclear transfer. Reproduction, June 1, 2006; 131(6): 1085 - 1090. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||