Construction of Chromosome #1 Specific Library of Lilium tigrinum

Metaphase chromosome #1 of Lilium tigrinum was microdissected and microcloned. DOP-PCR and linker-mediated PCR were used to amplify microdissected L. tigrinum chr. #1. DOP-PCR and linker-mediated PCR yielded products ranging in size from 100 to 3000 bp, with predominant fragments at 300 to 2000 bp. When two methods were compared, linker-mediated PCR gave rise to a little bigger fragment. Southern hybridization with DIG-labeled genomic DNA confirmed that the products from both methods were amplified from the L. tigrinum genome. Cloning using size-fractionated DNA gave recombinant clones larger than 500 bp. DOP-PCR gave larger recombinant clones than linker-mediated PCR. The insert size was approximately from 300 to 2000 bp. DOP-PCR produced DNA fragments ranging in size from 400 to 2000 bp, and linker-mediated PCR from 300 to 1200 bp, with predominant fragment at 500 to 1000 bp. When randomly selected 30 clones were sequenced, only two clones from DOP-PCR showed homology with Lilium henryi or L. philadelphicum and others were identified as unknown unique sequences. On the other hand, 8 out of 15 clones from linker-mediated PCR showed homology with microsatellite region of other plant species. Based on the results obtained in this experiment, DOP-PCR was more suitable for finding unknown unique sequences than linker-mediated PCR.