FIRST EUROPEAN FOOD CONGRESS 4-9, November, 2008 Ljubljana, Slovenia Prof.Dr.Fatih YILDIZ, (fatih@metu.edu.tr) and Maher DERNAIKA Middle East Technical University Department of Food Engineering and Biotechnology Ankara/Turkey

TITLE DNA Barcoding of Fruit and Vegetable Cultivars: Two examples, Apricots and Strawberries

Purpose To identify a unique DNA fingerprint in each of the Apricot and Strawberry subspecies (Cultivars) To differentiate among them at the molecular level by Simple Sequence Repeat (SSR) markers (Microsatellites) To be able to trace an unknown apricot or strawberry subspecies

Molecular Markers A readily detectable sequence of DNA or protein whose inheritance can be monitored

Molecular Markers Polymorphic Reproducible Display co-dominant inheritance Fast and inexpensive to detect

SSRs (Simple Sequence Repeats) tgtgtgtgtgtgtgtgtgtg (TG ) 10 Microsatellite (SSRs) are molecular markers of tandemly repeated di- tri- tetra-nucleotide sequences

Steps of the work Collecting Apricot and Strawberry Samples Isolating DNA from fruits and leaves Picking SSR Markers from Databases Designing Primers Applying PCR for the amplification of the regions flanking the repeats. Running Gel Electrophoresis Comparing SSRs of different Apricot and Strawberry cultivars. Pointing out unique DNA fingerprints

Samples collection Apricot and Strawberry Samples were collected from a Horticultural Research Center to assure the genetic purity of the Cultivars Fruits and Leaves are frozen at -20 0C for storing

DNA ISOLATION There are many protocols for extracting DNA. CTAB based protocol is used in our Laboratory for obtaining a pure DNA ready for PCR applications

SSRs from Databases DNA sequences of many species have been discovered and many DNA markers have been identified Publicly available through website databases Genome Database for Rosaceae (GDR) http://www.bioinfo.wsu.edu/gdr/ http://www.ncbi.nlm.nih.gov/ GenBank Database http://www.psc.edu/general/software/packages/genbank/genbank.html

Primer Design We design primers for the regions flanking the SSRs already taken from the GDR DataBase We design the primers depending on two programs: Integrated DNA Technologies (IDT) http://eu.idtdna.com Primer3 http://frodo.wi.mit.edu/

Primers designed for Apricots Forward GTCCCCTATTCGACAACC Reverse TGGCTACCAAAGAACACG Forward ATTCGGCACGAGGGATTTG Reverse AAGGCCACAGATAATGACCAG Forward AATTCGGCACGAGGCTG Reverse GATGGAGAACATTACATGGCTACC Forward ATTCGGCACGAGGCTTCAC Reverse CTTTCCTGTTTTCACTAGCTCCATC Forward AAGTCTCGTCAATAAAGGATTTG Reverse GGCCACAGATAATGACCAG

Primers designed for Strawberry Forward GGAGAGTGTTGAGTGTTTAG Reverse TTAAATCTCCATCCAAACATAC Forward CCCCTATTCGACAACCAATG Reverse GGCTACCAAAGAACACGAAC Forward ACGCTTGCTGATGGAGAACTAC Reverse TCCTCAACCGTGCAATCAAATC Forward GCACGAGGATTGTTTGAACC Reverse CTACAGGCAGACCAACACAC Forward TCGTCGAGTTCTACGCTTGCTG Reverse ACCGTGCAATCAAATCCCACTCTC

PCR and Gel Electrophoresis After designing the primers we run PCR to amplify the simple sequence repeats region Then, we run it on Gel Electrophoresis to monitor the differences in the migration distances of the DNA bands for all the cultivars for each SSR Marker

Primers-PCR-Gel (Strawberry) tgtgtgtgtgtgtgtgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgtg (TG) (TG) Forward primer 12 14 Reverse primer Subspecies A (Balcalı-4) Subspecies B (Tioga)

Primers-PCR-Gel (Apricot) acacacacacacacacacac acacacacacacacacacacacacac (AC) (AC) Forward primer 10 13 Reverse primer Subspecies A (Hacihaliloglu) Subspecies B (Kabaasi)

Comparing DNA bands (Strawberry) SSR 2 Is our Prospective Candidate Marker SSR 1 SSR 2 Subspecies A (Balcalı-4) Subspecies B (Tioga) Subspecies A (Balcalı-4) Subspecies B (Tioga) TG (14) TG (12) TG (12) TG (12) NOT Informative Informative

DNA Sequencing of SSR 2 DNA sequencing DNA extraction from Gel SSR 2 Subspecies A (Balcalı-4) Subspecies B (Tioga) TG (14) DNA sequencing TG (12)

Results We can compare and differentiate different cultivars of apricot and strawberry by identifying different repeats in the same marker Therefore, each subspecies will have a unique simple repeat that considered as a fingerprint

All Rosaceae family would be Identified & In the Future, All Rosaceae family would be Identified & a DNA fingerprint Database would be Publicized

References (Papers) Akkaya, M. S., A. A. Bhagwat, and P. B. Cregan, 1992: Length polymorphism of simple sequence repeat DNA in soybean. Genetics 132, 1131-1139 T-J. Kang, and M. W. Fawley,1997: Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas. Plant Molecular Biology 35: 943–948, 1997. N. DOGRAR, S. AKIN-YALIN, and M. S. AKKAYA, 2000: Discriminating durum wheat cultivars using highly polymorphic simple sequence repeat DNA markers. Plant Breeding 119, 360-362 Kevin M Folta, Margaret Staton, Philip J Stewart, Sook Jung, Dawn H Bies, Christopher Jesdurai, and Dorrie Main, 2005: Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria × ananassa). BMC Plant Biology, 5:12 G. CIPRIANI and R. TESTOLIN, 2004: Isolation and characterization of microsatellite loci in Fragaria. Molecular Ecology Notes, 4, 366–368 D . J . SARGENT, A . M. HADONOU, and D. W. SIMPSON, 2003: Development and characterization of polymorphic microsatellite markers from Fragaria viridis, a wild diploid strawberry. Molecular Ecology Notes 3, 550–552

References (Websites) Genome Database for Rosaceae (GDR) http://www.bioinfo.wsu.edu/gdr/ http://www.ncbi.nlm.nih.gov/ http://www.wikipedia.org/ Integrated DNA Technologies (IDT) http://eu.idtdna.com Primer3 http://frodo.wi.mit.edu/

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