The Ty3- gypsy Retrotransposons in Gossypium and their affect on Genome Size Variation

1. The Ty3-gypsy Retrotransposons in Gossypium and their affect on Genome Size Variation Ashley Davidson1, Jennifer S. Hawkins2, Ryan J. Percifield2, and Jonathan F. Wendel2. 1Norfolk State University Norfolk, VA; 2Department of Ecology, Evolution and Organismal Biology, Iowa State University Ames, IA • ABSTRACT • The Ty3-gypsy group retrotransposons from representative species of the A, D, and K genomes of Gossypium, as well as the outgroup, Gossypioides kirkii were studied in order to determine their occurrence and proliferation and its affect on genome size. In each genome these sequences were obtained through PCR amplified using degenerate primers. The PCR products were cloned and sequenced and the sequence data from each species was compiled to construct a phylogenetic tree. According to the phylogeny the gypsy elements in each genome appear to have recently proliferated. The data suggests that genome size variation resulted in part from the augmentation of Ty3-gypsy group retrotransposons. Genome size variation in Gossypium appears to be directly influenced by lineage-specific amplification of transposable elements. The knowledge obtained from this study will be the building blocks that will lead to the future understanding of genome organization and evolution in other diverse organisms. • INTRODUCTION • The cotton genus, Gossypium • 5-10 million years old • Comprised of ~50 different diploid species • 8 different genome groups (A through G, and K) • Each genome has 13 chromosomes, but differ in chromosome size (DNA content) • Genome size variation ranging from 885Mb (New World “D” genome) to 2572Mb (Australian “K” genome) • Transposable elements • Every living organism investigated contains transposable elements that accounts for a portion of the “junk DNA” in the genome • Each Gossypium genome is composed of 40-65% of transposable elements • Ty3-gypsy is a type of LTR-retrotransposable element commonly found in plants and previously shown to be present in Gossypium • Gossypioides kirkii was used as the outgroup because of its phylogenetic relationship to Gossypium. • METHODS • Polymerase Chain Reaction (PCR) • Performed on representative species from the K, D, A, genomes and the outgroup G. kirkii • Amplified an array of reverse transcriptase (RT) sequences using degenerate primers • Agarose Gel Electrophoresis • Large and small gels ranging from 1-2% to visualize and isolate PCR products • Cloning • Ligation and transformation to clone the PCR products for further analysis • X-gal and IPTG was used to identify bacteria containing a recombinant plasmid • White colonies were picked and placed in growth media in 96-well plates • Sequencing • 384 samples were sequenced • The data was used to construct a phylogenetic tree • SUMMARY AND DISCUSSION • The results imply that the genomes have expanded through retrotransposon proliferation. • There is greater sequence variation between genomes than within indicating recent lineage-specific transposition. • Clustering of the Ty3-gypsy group retrotransposons, limited sequence divergence within clades, and limited number of insertions or deletions shows that the gypsys are relatively new and have proliferated recently and have not had time to accumulate mutations or sequence divergence. • FUTRUE IMPACT • Further studies can be done on not only Ty-3 gypsy retrotransposable elements, but other transposable elements to enhance our understanding of genome evolution and development in other diverse organisms. RESULTS Table I Percent Identity among RT sequences The depiction of the percent range identities of not only the individual genomes, but also of the genomes as in comparison to one another. Figure 1. A chromatograph of a sequenced Ty3-gypsy group retrotransposon from the A- genome of Gossypium Figure 2. RT phylogeny of Ty3-gypsy group retrotransposons from Gossypium consisting of the A, K, D genomes and the outgroup G.kirkii. Green represents the A genome, blue the K genome, pink the D genome, and orange the G.kirkii genome ACKNOWLEDGEMENTS I would like to thank Jennifer Hawkins for all of the time and energy she put in teaching me new techniques, Dr. Jonathan Wendel for allowing me to intern in his lab, Ryan Percifield for his assistance in Jennifer’s absence, Dr. Max Rothschild for leading an excellent summer program, and everyone working in the Wendel lab.