Development of purification protocols for plant VLP (virus-like particle) isolation

Retrotransposons are mobile genetic elements which, with the use of reverse transcriptase and an RNA intermediate, take themselves out of one part of the genome and insert themselves into another part, in a copy and paste mechanism. These retrotransposons are believed to have played a significant role in the structure, size, and evolution of plant genomes. Studying plants with both large and small genomes will provide a way of developing a better understanding of the mechanisms that control plant genome size. The Liliaceae family is of particular interest as they contain some of the largest plant genomes ever recorded for an angiosperm, and they also contain species with much smaller genomes. In other words, there is great genome size variation within the Liliaceae family. The basis of the study relies greatly on next-generation sequencing (NGS) as this will allow us to characterize and study large plant genomes as well as the transposable elements they contain. For this study, the Ion Torrent Personal Genome Machine (PGM) was utilized. This NGS platform employs semiconductor sequencing technology. VLPs (virus like particles) from Lilium pardalinum, Prosartes smithii, Clintonia, Scoliopus, all of which are from the Liliaceae family as well as smilax from the sister family Smilacaceae were first isolated from fresh tissue. RNA from these VLP preps were extracted and converted to cDNA, which was then sequenced on the PGM in order to identify which transposable elements were present in the VLP pool. Identifying, characterizing, and comparing the specific active retrotransposons between larger and smaller plant genomes, through NGS and bioinformatics, will allow us to get a better understanding of how and why some genomes have expanded. Were these larger genomes a result of very “aggressive” retroelements or was it that the plant’s “defense system” was deficient and not able to fight against the retroelements? In other words, it would allow us to draw conclusions about the roles these retrotransposons play in the expansion of plant genomes. Once the sequencing results were generated, the fragments were assembled using MIRA, a de-novo assembler and DNA Star (manufacturer) SeqManNGen. Ribosomal RNA, chloroplast, and mitochondrial sequences were used as “contaminates” and any matches were removed. Following the de novo assembly with contaminates, the baittemplated assembly was also performed. This is also de novo assembly, however, conserved RT domains were used as “bait” and any fragments that matched to the bait were then highlighted. Based on the Ion Torrent Personal Genome Machine sequencing results and the bioinformatics, both through de-novo assembly and bait-templated assembly, show that no transposable element had been isolated or sequenced. The only sequenced results were ribosomal rRNA.