Annotation Gene identification

Figure 1. Strategy of the genome analysis of Lotus japonicus MG-20.

3.2. Genome sequencing. We have constructed a genomic library of L. japonicus in a transformation competent artificial chromosome (TAC) vector as a source of genome sequencing clones. High molecular weight DNA from L. japonicus accession MG-20 was partially digested with Mbol or Hindlll and ligated with pYLTAC7. The average insert sizes were 87 kb, 96 kb, 105 kb and 106 kb for four independent preparations, a total of which is 7.7 haploid genome equivalent. The TAC libraries thus generated were arrayed in 93 384-well microtiter plates, and 48 DNA pools each containing 384 clones were subjected to PCR screening.

Seed clones for sequencing were selected by high throughput PCR screening using primer sets designed on the basis of ESTs and cDNA markers of L. japonicus and other legumes (Figure 1). The nucleotide sequence of each selected clone was determined according to the bridging shotgun method with an approximate redundancy of 5. The finished sequences were subjected to gene assignment by similarity search and computer prediction. At present, a total of 688 seed clones have been selected. Ninety-four of them are ready for sequencing, 5 are being sequenced, 96 are in the finishing stage, and 44 are being annotated.

3.3. Linkage mapping. Mapping of the seed clones on the basis of genome sequence information was also performed. First of all, simple sequence repeats (SSR) were searched in the sequence of each sequenced clone (Figure 1). Primers were designed to amplify identified SSR regions and the polymorphism in the parents of the mapping family (accessions MG-20 and B-129) was tested. When the polymorphism was observed, linkage analysis was performed using the mapping population of 127 F2 plants.

If no SSR was found, single nucleotide polymorphism (SNP) was searched by comparing the sequences of the Miyakojima clones and those of the corresponding chromosomal regions of Gifu. dCAPS marker was generated at the position where the SNP was detected, then the linkage analysis was carried out using F2 mapping population. According to this procedure, a total of 57 clones have been mapped on the 6 linkage groups so far (Figure 2).

.TM0002

.TM0002

JTM0027 TM0023 „TM0016 ^-TM0032 _^TM0036

-TM0014

Figure 2. Positions of the sequenced clones on the linkage map.

i—TM0080

I—TM0260

-TM0014

Figure 2. Positions of the sequenced clones on the linkage map.

4. References

The Arabidopsis Genome Initiative (2000) Nature 408, 796-815

Liu et al (1999) Proc. Natl. Acad. Sci. USA 96, 6535-6540

5. Acknowledgements

We thank Drs T. Aoki, M. Kawaguchi, M. Parniske, J. Stougaard, and T. Uchiumi for providing information for selection of seed clones and for valuable discussions. This work was supported by the Kazusa DNA Research Institute Foundation.

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