1. Introduction The genus Gossypium (Cotton) is believed to have originated between five to fifteen million years ago. Evidence indicates that 2 genome groups, known as the A- & D-genome, developed in different geographic regions before combining about one to two million years ago to form the modern polyploid form of cotton. The modern polyploid form of cotton (Gossypium hirsutum) feeds a $12 billion international industry. A comparison of the A- & D-genomes contained in polyploid cotton reveals a duplication of genes coding for the anthocyanin pathway. The anthocyanin pathway is a linear pathway found among many of the higher land plants. The flavonoid metabolites produced by the genes of the pathway are known to protect the plant from microorganisms, herbivores and ultra-violet light. Also, these metabolites produce pigments in various tissues of the plant, which aides in the identification of phenotype (visible) changes. Based on our knowledge, we are trying to understand how the anthocyanin pathway has been affected by combining the A- and D-genomes to form the AD-genome of polyploid Gossypium. Materials and methods Identified genes present in the anthocyanin pathway under investigation, and found their DNA sequences by performing BLAST searches on the NCBI (National Center for Biotechnology Information) website. Created phylogenetic trees of the DNA sequences to make sure that they were the only genes responsible for the anthocyanin pathway. Ordered primers for the genes CHS, CHI, F3H and UF3GT. Used primers and PCR protocol to amplify a specific gene for both the A- and D- genomes. Each gene was then transformed into bacteria cells and the cells grew into colonies on petri dishes overnight. Colonies that appeared to contain our genes were tested using gel electrophoresis to confirm that the bacteria contained our gene. The colonies containing our gene were saved, cloned and sequenced to see if our bacteria contain our gene of interest. Acknowledgments We would like to thank the NSF Plant Genomics Research Program (grant # DBI-0638418), especially Adah Leshem-Ackerman and Jonathan Wendel for the opportunity to participate in the program. We are very grateful to Lex Flagel for his mentorship, patience, and willingness to set aside his other projects to guide us through this project. Thank you, also, to the other members of the Wendel Lab who were so supportive, helpful, and such graceous hosts: Armel Salmon, Dharminder Pathak, Lei Gong, Ying Bao, Corrinne Grover, Guanjing Hu and Kara Grupp. Results We were able to find DNA sequences of the following genes for the anthocyanin pathway: CHS (Chalcone Synthase), CHI (Chalcone Isomerase), F3H (Flavanone 3-Hydroxylase), DFR (Dihydroflavanol Reductase), ANS (Anthocyanidin Synthase), UF3GT (UDP glucose flavonoid 3-oxy- glucosyltransferase). References Winkel-Shirley, Brenda (2001) Flavonoid Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell ` Biology, and Biotechnology. Plant Physiology, Vol. 126 Rausher, Mark D., Richard E. Miller, and Peter Tiffin (1999) Patterns of Evolutionary Rate Variation Among Genes of the Anthocyanin Biosynthetic Pathway. Molecular Biology and Evolution, Vol. 16 Parks, C.R., W. L. Ezell, D.E. Williams, D.L. Dreyer (1975) The Application of Flavonoid Distribution to Taxonomic Problems in the Genus Gossypium. Bulletin of the Torrey Botanical Club Rausher, Mark, Yingqing Lu, and Kyle Meyer (2008) Variation in Constraint Versus Positive Selection as an Explanation for Evolutionary Rate Variation Among Anthocyanin Genes Journal of Molecular Evolution, Vol. 67 Koes, Ronald, Walter Verweij, and Francesca Quattrocchio (2005) Flavonoids: A Colorful Model for the Regulation and Evolution of Biochemical Pathways. Trends in Plant Science, Vol. 10 Grotewold, Erich (2006) The Genetics and Biochemistry of Floral Pigments. The Annual Review of Plant Biology, Vol. 57 Jim Kollman 1, Spencer Mesick 1, Lex Flagel 1, and Jonathan F. Wendel 1 1 Department of Ecology, Evolution and Organismal Biology at Iowa State University, Ames, Iowa Figure 3. An overview of the protocols used to amplify and sequence a specific gene from the anthocyanin pathway. For further information Please contact Spencer Mesick, Jim Kollman, Lex Flagel or Jonathan Wendel through Wendel Lab, Department of Ecology, Evolution and Organismal Biology at Iowa State University, Ames, Iowa. It is our understanding that the anthocyanin pathway contains six main genes that can be found bolded in figure four. Out of the six genes we successfully identified and isolated all four genes (CHS, CHI, F3H, UF3GT) for which we designed primers. According to our phylogenetic trees, of these four isolated genes, only CHS and CHI appear to be single copy genes in Gossypium. Currently, all four isolated genes are waiting to be sequenced in order to confirm their identity and copy number. Evolution of duplicate pathways in polyploid Gossypium Conclusions Future goals It is our hope that other researchers will continue our project and soon find the answers to our important, and unanswered questions:  How many genes in the genome encode each step of the pathway?  Which copies of each gene are used at each step?  How many genes have been retained since polyploidy?  Have the duplicate copies diverged in their function? When did they diverge? Before or after polyploidy?  How are these genes expressed in different tissues and under different environmental conditions?  How have any of these changes affected metabolite levels and total fitness of the plant? Figure 5. Phylogenetic tree of the CHI gene. Figure 1. A-genome originated in Africa while the D-genome originated in Mexico. Figure 2. The A- and D-genome formed the AD-genome. Figure 6. Gel electrophoresis of PCR products for the F3H (lanes 2 & 3) and UF3GT (lanes 6 & 7) genes. Figure 7. Example X-gal/IPTG plate. Figure 8. Gel electrophoresis of plasmid screen for the F3H and UF3GT genes. We used ligation and transformation protocols to grow our PCR product in E. coli. Gel electrophoresis allowed us to isolate and identify our amplified PCR products for each gene. Figure 4. Anthocyanin pathway. From the phylogenetic trees that were made we interpreted CHS, CHI and DFR to be single copy genes for Gossypium. The F3H, ANS and UF3GT genes appear to have possible ancient and/or recent duplications for Gossypium. The plasmid screen helped us identify genes that had been properly transformed into the E. coli cells. According to our interpretation of our plasmid screens, we successfully isolated the following genes for both the A- and D-genome: CHS, CHI, F3H and UF3GT.