Construction and characterization of a Nicotiana plumbaginifolia genomic library in a yeast artificial chromosome

Abstract

Large-size DNA of Nicotiana plumbaginifolia was manipulated in solution, concentrated by 2-butanol and cloned into a yeast artificial chromosome, pYACCMC5. A YAC library consisting of 22 000 clones with an average insert size of 190 kb has been constructed. The sizes of YAC inserts in this library ranged from 80–660 kb, indicating that 2-butanol can be used for concentrating large-size DNA with minimum shearing damage. This library represents 84% of the genome of N. plumbaginifolia. Characterization of this library revealed that only 6% of clones in the library contained sequences derived from chloroplast DNA. Some YAC inserts contained relatively long stretches of DNA, 100–180 kb, uninterrupted with repetitive sequences. Furthermore, both ends of most DNA molecules cloned in pYACCMC5 can be reisolated efficiently as recombinant plasmids in Escherichia coli. Approximately one third of the subcloned end fragments contained low- or single-copy DNA sequences. Single-copy end fragments are valuable probes to identify overlapping YAC clones by chromosome walking. The method for concentrating large-size DNA and the vector for YAC cloning described here should be useful in genome research programs. The YAC library presented here will facilitate mapping and cloning of genes from the genome of N. plumbaginifolia, which has been used for over a decade as a model species in plant cell genetics.