N. Sandal1, L. Krusell1, S. Radutoiu1, M. Olbryt1, A. Pedrosa2, S. Stracke3, M. Parniske3, A. Bachmair2, T. Ketelsen1, J. Stougaard1
laboratory of Gene Expression, Department of Molecular and Structural Biology,
University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark department of Cell Biology and Genetics, University of Vienna, Rennweg 14,
A-1030 Vienna, Austria
Sainsbury Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, UK
New possibilities for genetic studies in the Lotus japonicus model legume have recently opened as a result of the genome sequencing initiative and the EST sequencing programs started on L. japonicus (Cyranoski 2001; Asamizu et al. 2000). When combined with the diploid genetics of L. japonicus, a small genome size estimated to -432 Mb and ample seed production from large self-fertile flowers, these genome initiatives will enable genetic linkage analysis and physical mapping to be done effectively. In addition several L. japonicus mutant populations have been generated and many mutant classes identified (Schauser et al. 1998; Szczyglowski et al. 1998; Imaizumi-Anraku et al. 1997).
Gross phenotypes divide the mutants into four classes:
1. Non-nodulating mutants that are impaired in early rhizobial interaction, Nod factor perception or downstream signaling;
2. Ineffective nodulating mutants that are either arrested during nodule organogenesis or impaired in nodule function;
3. Mutants with either increased or decreased nodule numbers. Here, inactivation of the normal autoregulatory mechanism leads to an excess of root nodules, whereas reduced nodule numbers may be caused by a variety of mutations including leaky mutations; and
4. Mutants with delayed nodulation. With these mutants, the infection and nodule-developmental process can be genetically dissected.
Although both transposon tagging (Schauser et al. 1999) and T-DNA tagging (Webb et al. 2000) have been accomplished, most of the mutant populations were produced by EMS mutagenesis and, in order to molecularly characterize the mutants from these collections, a map-based cloning procedure needs to be established. For this purpose, a genetic linkage map of L. japonicus Gifu has been developed.
AFLP marker technology has proved to be reliable and effective for the generation of plant linkage maps. The AFLP technique combines restriction fragment analysis with PCR into a multi-locus DNA fingerprinting system that is independent of prior knowledge of genome sequence. DNA fragments amplified by PCR are resolved by electrophoresis in either gels or capillaries allowing the large numbers of fragments arising from complex genomes to be detected and analyzed. We have chosen to use AFLP for providing the backbone markers of the L. japonicus map and to supplement this analysis with markers generated with more time-consuming RFLP and gene specific PCR
technology. The resulting genetic linkage map was based on a highly polymorphic interspecific F2 mapping population established from a cross between L. filicaulis and L. japonicus ecotype Gifu.
A total of 518 anonymous AFLP markers, 3 anonymous RAPD markers, 38 gene specific markers and 6 recessive symbiotic mutant loci were mapped. This first generation map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes. The length of the linkage map is 358 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in sections of the map and linkage group I could only be assembled using color mapping with the assistance of localization of markers by FISH. In Lotus, two mapping populations are in use, one based on a highly polymorphic interspecific cross between L. japonicus ecotype Gifu and L. filicaulis, another originating from an ecotype cross between Gifu and Miyakojima (Kawaguchi et al. 2000). The possibilities for genetic analysis and map-based cloning using these two populations have been evaluated by mapping of three symbiotic loci, Ljsyml, Ljsym5 and Ljharl-3. A combination of marker analysis and BAC end sequencing has delimited Ljsyml within a 2 cM region, the Ljsym5 locus within a 1 cM region and Ljharl within 0.42 cM.
Although map-based cloning of these loci is progressing, cloning of tagged loci is still more effective. This technique will change in the future as map-based cloning will become faster and easier. Sequencing of the Lotus genome (ecotype Miyakojima) will provide a basis for identifying single nucleotide polymorphisms and short insertion/deletions between mapping partners. Lack of PCR markers is one of the main obstacles to fine mapping, so PCR markers based on nucleotide differences will greatly expedite map-based cloning. Effective use of this possibility requires additional sequencing of a mapping partner, but the potential resolution is very high. At present, the best partner for survey sequencing is the Gifu ecotype.
Asamizu E et al. (2000) DNA Res. 7, 127-130 Cyranoski D (2001) Nature 409, 272
Imaizumi-Anraku H et al. (1997) Plant Cell Physiol. 38, 871-881 Kawaguchi M (2000) J. Plant Res. 113, 507-509 Schauser L et al. (1999) Nature 402, 191-195 Schauser L et al. (1998) Mol. Gen. Genet. 259, 414-423 Szczyglowski K et al. (1998) Mol. Plant-Microbe Interact. 11, 684-697 Webb KJ et al. (2000) Mol. Plant-Microbe Interact. 13, 606-616
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