Construction of a yeast artificial chromosome library of pepper (Capsicum annuum L.) and identification of clones from the Bs2 resistance locus

A yeast artificial chromosome (YAC) library was constructed using high-mole cular-weight DNA isolated from pepper (Capsicum annuum L.) leaf protoplasts . Insert DNA was prepared by partial digestion using EcoRI and subjected to electrophoretic fractionation before in-gel ligation to the pJS97/98 YAC v ector. Prior to transformation of yeast spheroplasts, ligation products wer e subjected to a second electrophoretic size selection. The library consist s of about 19 000 clones with an average insert size of 500 kb, thus repres enting approximately three haploid genome equivalents. Three PCR-based mark ers tightly linked to the pepper Bs2 resistance gene were used to assess th e utility of this library for positional cloning. Three YAC clones containi ng pepper genomic DNA from the Bs2 resistance locus were isolated from the library. The clones ranged in size from 270 kb to 1.2 Mb and should prove u seful for the cloning of the Bs2 gene.