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 once a year. This recurrent flowering feature of the commercial roses resulted from a flowering mutation named everblooming (evb). The mutation is recessive to once blooming and is found in the rose species Rosa chinensis. Although several molecular maps have been developed for rose, little is known about the evb gene, except for its classic genetics. 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 megabase-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. Size selected fragments estimated between 100 kb and 150 kb in size were cloned into the pECBAC1 BAC vector and the clones having rose DNA inserts were arrayed in 80 384-well microplates individually, with each clone being barcoded. 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 is now available to be screened with the genes cloned from other species that control vernalization and floral development and will be used in mapbased cloning of the evb gene using a Rosa wichuraiana (??????Basye??????s Thornless??????) x ??????Old Blush?????? backcross population.