5.4.10
PHYSICAL MAPPING OF THE Sw-5 RESISTANCE LOCUS IN LYCOPERSICON ESCULENTUM

R FOLKERTSMA1, M SPASSOVA2, M PRINS1, J HILLE2 and R GOLDBACH1

1Department of Virology, Wageningen Agricultural University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands; 2Department of Genetics, Free University, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.

Background and objectives
Tomato spotted wilt virus (TSWV), the type species of the genus Tospovirus, is one of the major crop-infecting viruses in the cultivation of many important crops worldwide [1]. Hardly any natural resistance genes, useful for breeding programmes, are available. Recently a resistance gene was found in tomato, referred to in the literature as Sw-5. The gene originates from Lycopersicon peruvianum and was introgressed into L. esculentum. The gene renders tomato plants highly resistant to the tospoviruses TSWV, GRSV and TCSV. The resistance reaction is thought to be a hypersensitive response. Genetic analyses revealed that the gene is located in the telomeric region of chromosome 9, tightly linked to the RFLP marker CT220 [2].

Materials and methods
In order to investigate the physical organization of the CT220 region, a BAC library was made in pBeloBAC1 1. High molecular-weight DNA was isolated from cultivar Stevens, homozygous for the Sw-5 locus.

Results and conclusion
Intact nuclei were isolated from seedlings and embedded in agarose plugs. The DNA was partially digested with HindIII and high molecular-weight DNA was isolated using pulsed field gel electrophoresis. The DNA was ligated into the BAC vector. At present the library contains 18,816 clones with an average insert size estimated to be 98.8 kb (n=50; standard deviation=24.5 kb), ranging from 152 to 30 kb. RFLP marker CT220 was converted into a CAPS marker and the library was screened using PCR. Left- and right-end markers of BAC clones were obtained using IPCR and plasmid rescue, respectively. Positive clones were identified, generating a contig around CT220 with an estimated size of approximately 120 kb. A physical map and further details of the analysis of the contig will be presented.

References
1. Goldbach R, Peters D, 1994. Seminars in Virology 5, 113-120.
2. Brommonschenkel SH, Tanksley SD, 1997. Molecular and General Genetics 256, 121-26.