1. Cloning genes involved in the flavonoid pathway of pink and white Hydrangea macrophylla Melissa DellaTorre, Bowdoin College Danial Hasani, mentor, NFU Dr. Khairy Soliman, mentor, AAMU Dr. Tongming Yin, mentor, NFU

2. Importance of Anthocyanins Attract pollinators Environmental Adaptation Biodiversity Pest protection UV protection Powerful antioxidants (Dyer et al., 2013; Miller et al., 2011; Seeram, 2008)

3. Flavonoid biosynthetic Pathway Determines color by producing anthocyanins Chalcone isomerase (CHI) Flavanone 3 beta-hydroxylase (F3H) Dihydroflavonol reductase (DFR) Anthocyanidin synthase (ANS) UDPG-flavonoid glucosyl transferase (UF3GT) (Miller et al., 2007)

4. Hydrangea macrophylla Factors affecting color: pH Sugar content Metal content Temperature Light (Tanaka et al., 2008)

5. Objective Determine which major flavonoid pathway genes are present in the pink and white hydrangea flower Help to discover which gene is responsible for the color change

6. Material Collected 5 samples from 5 different flowers for both white and pink hydrangeas

7. Methods 1. RNA extraction with Trizol Transferred the material to 1ml Trizol extraction buffer. Added chloroform, phenol:chloroform:isoamyl alcohol (25:24:1), isopropanol, and centrifuged the samples at 14,000rpm for 10 min at 6°Cin after each was added.

8. Methods 2. DNA Digestion Purpose: Purification of RNA extract 250bp 750bp 500bp White Molecular Molecular Pink Marker Marker

9. Methods 3. Gel Electrophoresis Purpose: To check quality and size of RNA 250bp 750bp 500bp White Molecular Molecular Pink Marker Marker

10. Methods 4. cDNA library construction Purpose: To turn RNA into complimentary DNA

11. Methods 5. Polymerase Chain Reaction (PCR) Purpose: Amplification of genes

12. Results and Discussion M DFR F3H CHI ANS UFGT Pink Hydrangea Gene Expression White Hydrangea Gene Expression 500bp 1000bp

13. Conclusion There are copy number differences in CHI for pink and white hydrangea flowers Ex Taq caused negative results for F3H and UFGT Too many cycles resulted in extra band for ANS

14. Future Research Quantitative PCR Gene Sequencing

15. Acknowledgments Dr. Khairy Soliman, mentor, AAMU Danial Hassani, mentor, NFU Dr. Tongming Yin, NFU Dr. Moss Dr. Wang National Science Foundation

16. References Bailey D. (1992) Hydrangeas. Introduction to Floriculture p365-383 Clegg M., Durbin M. (2000) Flower color variation: A model for the experimental study of evolution. PNAS 97(13):7016-7023 Dyer A., Boyd-Gerny S., McLoughlin S., Rosa M., Simonov V., Wong B. (2013) Parallel evolution of angiosperm color signals: common evolutionary pressures linked to hymenopteran vision 198:301-307 Miller R., Owens S., Rorslett B. (2011) Plants and Color: Flowers and Pollination. Optics and Laser Technology. 43(2):282-294 Seeram, Navindra P. (2008). Berry Fruits: Compositional Elements, Biochemical Activities, and the Impact of Their Intake on Human Health, Performance, and Disease. Journal of Agricultural and Food Chemistry 56 (3): 627–9 Toyama-Kato Y., Yoshida K., Fujimon E., Haraguchi H., Shimizu Y., Kondo T. (2003) Analysis of metal elements of hydrangea sepals at various growing stages by ICP-AES. Biochemical Engineering Journal. 14(3):237-241

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