Toward positional cloning of everblooming gene (evb) in plants: A BAC library of Rosa chinensis cv. Old Blush
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A majority of commercial rose varieties bloom repeatedly throughout the year, as compared to most rose species, other woody ornamentals, and fruit crops that bloom only once a year. This recurrent flowering feature named everblooming (evb) is found in the rose species Rosa chinensis. Although several molecular maps have been developed for rose, little is known about the evb gene. The purpose of this study was to develop a large-insert bacterial artificial chromosome (BAC) library as a starting tool for molecular cloning and analysis of the evb gene by map-based cloning. To construct the large-insert BAC library, nuclear mega base-size DNA was isolated from the recurrent blooming diploid species, Rosa chinensis cv. Old Blush. The DNA was then partially digested with BamHI and separated on agarose gels by multi-phase pulsed- field gel electrophoresis. Fragments between 100 kb and 150 kb were cloned into the pECBACl BAC vector and the clones having rose DNA inserts were arrayed in 80 384-well microplates. The library contains 30,720 clones, has an average insert size of 108 kb and covers roughly 5.9x genome equivalents, with a >99% probability of isolating a single-copy clone from the library. The library has been transferred to membrane for screening with genes involved in vernalization and floral development cloned from other species and will be used in map-based cloning of the evb gene using a Rosa wichurana ('Basye's Thornless') 'Old Blush' backcross population.
