A deep-coverage BAC library for Ponkan mandarin and its further use in screening ethylene regulated genes

A deep-coverage bacterial artificial chromosome (BAC) library of Yanxiwanlu Ponkan a late maturing cultivar was constructed and evaluated Results showed that the library contained a total of 61 000 clones Restriction analysis indicated that the DNA insert sizes ranged from 30 to 170 kb with an average of 82 kb No empty clone was found Therefore the library should cover similar to 13 6-fold of the citrus genome Continuous sub-cultivation of 5 randomly chosen clones for up to 100 generations showed no detectable change in their restriction profiles To demonstrate the application of the library in discovery of genes 7 ethylene-regulated genes were screened by PCR amplification from hierarchically pooled library clones with gene-specific primers Five positive clones each representing a different gene were thus identified from 3840 clones Sequencing of the clones showed that all of them matched the expected genes indicating that the library was highly representative (C) 2010 Elsevier B V All rights reserved