CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY CONTAINING LARGE ECORI AND HINDIII GENOMIC FRAGMENTS OF LETTUCE

Existing bacterial artificial chromosome (BAG) vectors were modified t o have unique EcoRI cloning sites. This provided an additional site fo r generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed followin g the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC libra ry of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each liga tion varied between 92.5 and 142 kb with a combined average insert siz e of 111 kb. The library was screened with markers linked to disease r esistance genes; this identified 134 BAC clones from four regions cont aining resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from p revious estimates of genome size. The lack of hybridization to chlorop last and mitochondrial sequences demonstrated that the library was pre dominantly composed of nuclear DNA. The unique EcoRI site in the BAC v ector should allow the integration of BAC cloning with other technolog ies that utilize EcoRI digestion, such as AFLP(TM) markers and RecA-as sisted restriction endonuclease (RARE) cleavage, to clone specific lar ge EcoRI fragments from genomic DNA.