Construction of a BAC library for the investigation of the S locus in Buckwheat

Mio Nagano¹, Jotaro Aii², Clayton Campbell³, Taiji Adachi⁴ and Shinji Kawasaki⁵

¹ Department of Biology, Kochi University, 2-5-1 Akebono-cho, Kochi, 780-8520, Japan. ² Plant resources and Cell engineering Laboratory, Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Niigata, 956-8603, Japan ³ Kade Research Ltd. 135, 13 Street, Morden, Manitoba, R6M 1E9, Canada ⁴ Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka, 599-8531, Japan ⁵ National Institute of Agrobiological Sciences (NIAS), 2-1-2, Kannon-dai, Tsukuba, Ibaraki, 305-8602, Japan

Mio Nagano, Jotaro Aii, Clayton Campbell, Taiji Adachi, and Shinji Kawasaki. 2005. Construction of a BAC library for the investigation of the S locus in Buckwheat. Fagopyum 22: 13-20.

Abstract: A bacterial artificial chromosome (BAC) library was constructed from a homostylous self compatible wild buckwheat species, Fagopyrum homotropicum, for analysis of the heterostylous self-incompatibility system in common cultivated buckwheat. F. homotropicum was used for fine mapping of the super-gene S-locus, which regulates the style morphology and incompatibility reaction. A protoplast method was applied to prepare intact genome DNA without any contaminants such as polyphenols, polysaccharides, and inclusion bodies found in buckwheat leaves. A library consisting of 24,096 clones was constructed, and transferred to 7 high-density microplate-size membranes for distribution. As the average insert size was 97.4 kb, the library coverage was estimated to be 1.9 haploid genome equivalents based on a genomic size of 1,080 M bp. Screening of the BAC filters with single and low-copy molecular markers close to the S-locus successfully identified two positive BAC clones per probe, proving that the library was representative in this region. This library, the first construction of a BAC library in the genus Fagopyrum, will be useful, not only for positional cloning of the distylous S-locus super-gene region, but also for analyses of other traits in cultivated and wild buckwheat species.

Key words:BAC library, buckwheat, heterostylous, positional cloning, self-incompatibility