Rashidah H. Farid, AAMU M.S. Student Dr. Tomgming Yin, Host Professor, NFU Danial Hassani, PhD Candidate Mentor, NFU

Research Topic Importance of Peach Flower Anthocyanin Pathway Literature Review Research Purpose Identified Problems Methods Results & Discussion Personal Goal: CTAB Protocol Methods Results China Experience Thank You

Peach blossoms are highly prized in Chinese culture More vitality than any other tree Peach rods to protect them from evils Identifiable by their bright pink flowers increase pollination

Anthocyanins are a group of reddish-blue, water- soluble pigments common in many flowers, fruits and vegetables. Possible health attributes, such as reduction of coronary heart diseases, antioxidant properties, and anti-cancer activities. Bright coloration essential to pollination. Figure 1. Genes involved in color expression in flowers.

Stanciu, Gabriela al et., 2010, Spectrophotometric study on stability of anthocyanins extracts from black grapes skins., Ovidius University Annals of Chemistry Volume 21, Number 1, pp , Varying factors effect the concentration of anthocyanins: pH, light exposure, temperature and oxygen. LIU Chang-ming,ZHANG Xian,XU Qing,XIAN Feng(College of Horticulture,Northwest A&F University,Yangling,Shaanxi ,China. AFLP Analysis on Dual Color Flower Gladiolus Mutants Induced by Radiation[J];Acta Botanica Boreali- Occidentalia Sinica; Dual color flower mutant and the control were related to the changes of genetic material, some of them may be related to the genes involved in flower color formation.

James T. Midcap, Extension Horticulturist, University of Georgia, Athens. Hydrangea Flower Color, In acidic soils, aluminum more available- binds to anthocyanin pigments producing blues In alkaline soils, erratic aluminum uptake so pigments are pink.

To determine whether there is a difference in anthocyanin pathway genes expression that distinguish red color from white color in peach flowers. Focused on six genes: ANS, CHS, CHI, DFR, F3H, UFGT

3RD PCR JUN 5TH RED FLOWER Primer Annealing Temperature Taq Enzyme Specificity Figure 2. Amplification failed. M

Re-configure the grouping of primers based on annealing temperatures Use a higher Specificity Taq Enzyme vs Master Mix Optimized the total cDNA to 50 ng per reaction

ANS-1F 5' GTGCTGTCACTTGGGTTGG 3'ANS-1R 5' TCTTCTCCTTTGGTGGCTC 3' ANS-2F 5' GCGTGCTGTCACTTGGGTT 3'ANS-2R 5' TCTTCTCCTTTGGTGGCTC 3' ANS-3F 5' GTTGAAGAAGGCAGCAGTG 3'ANS-3R 5' ACGCTTGTCCTCAGGGTAT 3' ANS-4F 5' AGCGACATTACCAATACAC 3'ANS-4R 5' CTGCTGCCTTCTTCAACTC 3' CHI-1F 5' CGTTTCCACCGTCAGTCAA 3'CHI-1R 5' GATCACCACGTTCCCAGCT 3' CHI-2F 5' AACGATACTGCCACTAACC 3'CHI-2R 5' TTCCCATTTCCTGCCTCAT 3' CHI-3F 5' AACGATACTGCCACTAACC 3'CHI-3R 5' CTACACTCTGCCTTGCTCC 3' CHI-4F 5' AACGATACTGCCACTAACC 3'CHI-4R 5' AAACGCCGTGCTTTCCTAT 3' CHS-1F 5' CCCAGTGACACCCACCTTG 3'CHS-1R 5' TTCTTCGTAGCCCTCTTCC 3' CHS-2F 5' CACCCACCTTGACAGTTTA 3'CHS-2R 5' CTTTCTTCGTAGCCCTCTT 3' DFR-1F 5' CGAAGAGCACCAGAAGTCA 3'DFR-1R 5' GGGAAGAACAAATGTAGCG 3' DFR-2F 5' TGACGAAACCGACTGGAGC 3'DFR-2R 5' ATCGAACTTTGTGGGAATA 3' DFR-3F 5' GAACCGTGAATGTCGAAGA 3'DFR-3R 5' GGGAAGAACAAATGTAGCG 3' F3'5'H-1F 5' TTCTCCAACCGTCCACCTA 3'F3'5'H-1R 5' CAACATCTCCGCCACCACA 3' F3'5'H-2F 5' TTCTCCAACCGTCCACCTA 3'F3'5'H-2R 5' ATCCCTTGTAAATCCATCC 3' F3'5'H-3F 5' CTCCAACCGTCCACCTAAT 3'F3'5'H-3R 5' ATCCCTTGTAAATCCATCC 3' F3H-1F 5' TCATCGTCTCCAGCCATTT 3'F3H-1R 5' CAGCATTCTTGAACCTCCC 3' F3H-2F 5' GTTCTTCGCTCTGCCCTCG 3'F3H-2R 5' CGCCTTTGTCAATGCCTCT 3' F3'H-1F 5' TCCCAGTTCCTGAAGACCC 3'F3'H-1R 5' CACTCCTGCCAACACCATC 3' F3'H-2F 5' CCAGTTCCTGAAGACCCAC 3'F3'H-2R 5' AACACTCCTGCCAACACCA 3' UFGT-1F 5' TATCATTCGGCAGTTTCGC 3'UFGT-1R 5' GGAGCTGTGAGCCCTATTT 3'

RT-PCR total volume of 50 µl consist of the following : 3 µl of cDNA (50 ng) Changed to: 1 µl of cDNA 3 µl of primer (1.5 µl F/R) Changed to: 2 µl of primer 30.7 µl of H 2 O Changed to: 34 µl of H 2 O 5 µl of 10x Ex Taq Buffer Changed to: La Taq Buffer 4 µl of dNTP Mix 4 µl of MgCl 2 (25mM) 0.3 µl Ex Taq Changed to: 0.3 µl La Taq Annealing Temp 58°C

WHITE JUN 7TH NEW cDNA EXACT TM FOR PRIMERS RED JUN 7TH NEW cDNA EXACT TM FOR PRIMERS M M

Figure? M

Objective: To extract total RNA from the Hydrangea using the RNA CTAB Protocol. Sample Collection: Collected hydrangea flowers ; transfer to - 80°C until RNA extraction. Petals were ground in liquid nitrogen in preparation for extraction.

0.5 g of Material add to 5ml CTAB extraction buffer water bath, plus 2% 2 - mercaptoethanol, heated two 65 ° C placed in 10ml centrifuge tubes; then incubated at 65 ° water bath 15min. Centrifuged at room temperature 12000rpm 10min. The supernatant is conserved and treated twice, with chloroform: isoamyl alcohol (24:1) solution. Centrifuged at room temperature 12000rpm 10min. Add 1/4 volume of 10M LiCl; 4 ° overnight- next day, 4 ° 12000rpm centrifuge 20min Discard supernatant, add 500ul SSTE dissolved about 10 minutes, transferred to 1.5ml tubes Add an equal volume of chloroform: isoamyl alcohol, vortex, mix, 4 ° 12000rpm centrifuge 10min; conserve the supernatant Adding twice the volume of ethanol in the supernatant, placed in -70 °, 30min to 2 hours 4 ° C, 12000rpm centrifuge 15-20min, the supernatant drained, dried at room temperature10min, adding appropriate amount of DEPC water (60ul) dissolved RNA, spare.

Sample IDUser name Date and Time Nucleic Acid Conc.UnitA260A280260/280260/230Sample Type RNA-CTAB #1Guest :15: ng/µl RNA sample 2Guest :20: ng/µl RNA PLGuest :48: ng/µl RNA PL 2Guest :50:3825.4ng/µl RNA PL 3Guest :51:4582.7ng/µl RNA PL 4Guest :52:53126ng/µl RNA PL 5Guest :54:1074ng/µl RNA

National Science Foundation Alabama A&M University/CREST Nanjing Forestry University Dr. K. Soliman, Advisor, AAMU Dr. Y. Wang, Co-Advisor, AAMU Ms. Lisa Gardner and Dr. Elica Moss