Random PCR of micro-dissected chromosome amplified predominantly repeated DNA in Lilium tigrinum

Large genome size in plants is largely caused by presence of repetitive DNA, which makes it of great interest to study the chromosome specific markers and function of complex large plant genome. In order to investigate evolutionary patterns of repetitive DNA, random PCR was carried out for a comprehensive repeat characterization and amplification in microdissected and micro-cloned L. tigrinum. The isolated chromosome was amplified by using DOP-PCR (degenerate oligonucleotide primed PCR) and LA-PCR (linker adaptor-mediated PCR) by which 100 to 2500 bp smear fragments of DNA, and 300 to 2000 bp predominant fragments were obtained. To construct DNA library of chromosome 1, obtained PCR products were cloned in the plasmid vector. The size of cloned inserts varied in DOP-PCR from 100-1700 bp and100-900 bp in LA-PCR, with predominant fragment at 500 to 1000 bp. Based on the BLAST-X search, 28% were matched with protein coding genes in the public database, while 72% of the sequences didn't exhibit any significant similarity with sequences already present in the plant database making up to 72% of unique sequences in the huge lily genome. Two clones obtained from micro-dissected chromosome were successfully detected on the chromosome 1 of diploid and triploid L. tigrinum. Among 513 genomic DNA sequences searches, 405 SSRs with perfect repeats were clearly verified and categorized based on number of repeats and their motifs. Six types of repeats were identified, among them penta-nucleotide repeats occurred at the high frequency.