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

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

3. 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

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

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

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

9. 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

10. 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

11. 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

12. 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)
GenBank Database

14. 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)
Primer3

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

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

17. 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

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

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

20. 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

21. 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)

22. 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

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

24. References (Papers)
Akkaya, M. S., A. A. Bhagwat, and P. B. Cregan, 1992: Length polymorphism of simple sequence repeat DNA in soybean. Genetics 132,
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,
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

25. References (Websites)
Genome Database for Rosaceae (GDR)
Integrated DNA Technologies (IDT)
Primer3

26. THANK YOU