Development of microdissection and chromosome specific genomic library in Lilium tigrinum

Abstract

Techniques for microdissection and microcloning were established using chromosome 1 of triploid Lilium tigrinum. Chromosome 1 was dissected from a membrane slide using a microbeam system. Digestion with proteinase K was done before PCR amplification for more than 24 hours. The dissected chromosomes were then amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and linker adaptor-mediated PCR (LA-PCR). Successful PCR amplification relied on critical concentrations of both MgCl2 and Taq polymerase. The optimum concentration of MgCl2 and Taq polymerase was 2.5 mM and 0.5 U, respectively. Amplification of the dissected chromosome using DOP-PCR had more sensitivity dependent upon PCR factors, but LA-PCR was more dependent on the linker ligation. Amplified DNA products ranged from 100 to 2500 bp both for DOP- and LA-PCR. Evaluated clones only ranged from 100 to 1700 bp for DOP-PCR and 100 to 900 bp for LA-PCR. Based on the sequence results, most of the sequences from the DOP-PCR and LA-PCR showed no significant similarity with known data in NCBI database. However, about 2% of the sequence data was partially matched with plant microsatellites with low similarity. The results derived from the microdissection of a large genome organism such as Lilium showed informative and useful for the development of microsatellite repeat markers. Sequence data from the chromosome specific DNA library was considered for the development of microsatellite markers.