A NEW METHOD IN OPTICAL MAPPING VISUALIZES THE STRUCTURE OF THE S LOCUS REGION OF Brassica campestris L.

¹ Natl. Food Res. Inst., Tsukuba 305-8642, Japan

² Grad. Sch. Sci. Tech., Niigata U., Niigata 950-2181, Japan

³ Hokuriku Natl. Agr. Exp. Stn., Joetsu 943-0193, Japan

⁴ Grad. Sch. Agr. Sci., Tohoku U., Sendai 981-8555, Japan

⁵ Fac. Agr., Iwate U., Morioka 020-8550, Japan

⁶ Dept. Biol. Sci., Nara Inst. Sci. Tech., Ikoma 630-0101, Japan

⁷ Res. Inst. Seed Production, Sendai 989-3204, Japan

⁸ Fac. Eng., Grad. Sch. Osaka U., Suita 565-0871, Japan

DNA combing in conjunction with fluorescence in situ hybridization (FISH) enables high-resolution visual mapping of the multiple gene cluster on the large DNA fragment. In Brassica species, self-incompatibility is regulated by a single S locus with multiple alleles. The S locus, spanning several hundreds kilobases, may contain several genes, including SLG and SRK. We have cloned a 76 kb fragment, containing both SLG⁹ and SRK⁹ genes into a P1-derived artificial chromosome (PAC) vector (E89 clone, Suzuki et al. 1997). In order to visualize directly DNA structure of the S locus on the E89 clone, we demonstrated hybridization of digoxigenin-labeled SLG⁹ and SRK⁹ probes to the E89 clone using the DNA molecular combing technique. A droplet of hybridized DNA was spotted onto an APS-coated glass slide. The probe DNA sequences on the combed molecules were immunolocalized by mean of fluorescent labeled anti-digoxigenin antibodies. Two fluorescent signals were localized in linear at the center and about 20 kb inside from one end of the molecule. Moreover, two small fluorescent signals, which correspond to homologous sequences between cloning vectors of the E89, SLG⁹ and SRK⁹ were visualized at both the ends of the combed molecule. The resulting visual map is consistent with the physical map. The direct visual mapping system by DNA combing and FISH has great possibility to be a powerful tool for analysis of DNA sequences.