Construction and characterisation of a yeast artificial chromosome library containing two haploid Beta vulgaris L. genome equivalents

Abstract

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded protoplasts of a sugar beet (Beta vulgaris L.) cell suspension line, Ar+. This library contains 15,000 clones with an average insert size of 140 kb. Based on a sugar beet haploid genome size of 1.1 x 10(3) Mb it should represent approximately two haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. High-density filters, each containing 864 YAC clones, have been screened in colony hybridisation experiments. In this way, sugar beet YAC clones have been identified containing chloroplast DNA, mitochondrial DNA and two satellite DNAs. Ten pools of DNA from 1500 individual YAC clones have also been prepared for rapid PCR screening of the library. Using this approach, in combination with colony hybridisation, we have been able to isolate sugar beet YAC clones containing a transcribed low-copy-number gene.