1. Molecular Marker Characterization of plant genotypes
Morphological markers
Physiological markers
Biochemical markers
Molecular markers
etc.

2. Widely-used markers To distinguish varieties / genotypes
by observation / measurement
Characteristics:
growth habit, fruit color, shape, etc.
resp rate, PS content, hormone balance, etc.
fruit size, plant height, sugar content, etc.

3. Molecular Marker Useful when other methods not available / possible
Very similar morphology / anatomy
Growth and development stages
Environmental factors
Analysis of banding patterns
Statistics for evaluation of polymorphism

4. Molecular marker Study and management of genetic resources
Identifying and distinguishing genotypes
Marker assisted selection (MAS)
Complementary tool for DUS studies of cultivars
Distinctiveness / Uniformity / Stability

5. Molecular markers Protein-based marker: Isozyme / Allozyme
multiple molecular forms of an enzyme
similar / identical catalytic activity
enzyme assay on PAGE
DNA-based marker

6. Isozyme marker

7. Isozyme marker Enzyme Isozyme locus # allozymes
Shikimate dehydrogenase Sdh-1 1
Phosphoglucose isomerase Pgi-1 2
Pgi-2 2
Aminonentidase Amp-1 3
Amp-2 4
Amp-3 3
Amp-4 4
Alcohol dehydrogenase Adh-1 2
Phosphogluconate dehydrogenase Pgd-1 2
Pgd-2 1
Glu oxaloacetate transaminase Got-1 1
Got-3 1

8. DNA-based markers Approach: Hybridization Polymerase Chain Reaction
Types: RFLP Minisatellite
RAPD SCAR SSR AFLP SNP etc

9. DNA-based markers Patterns of small DNA sequences
Constant landmarks in the genome
May or May not have biological function
Linked to conserved or variable regions

10. RFLPs Restriction Fragment Length Polymorphisms
Digestion with restriction enzymes
Size fractionation on agarose gel
Southern hybridization (genomic or cDNA probe)
Analysis of hybridized restriction fragments

11. RFLPs Polymorphism: homologous pieces of different lengths
mutation on restriction sites
mutation between restriction sites

12. RFLPs Several bands per lane Highly polymorphic in a population
at a locus – max 2 alleles in an individual
Co-dominant marker
Laborious / Time consuming
Usually use isotope

13. RFLPs

14. PCR-RFLPs

15. SSR or microsatellites
Simple Sequence Repeat
several bases per repeat
tandem repeats flanked by unique sequences
primer design based on flanking sequences
polymorphism: number of repeating units

16. SSR or microsatellites
Easy to detect via PCR
High polymorphism
Co-dominant marker
Library screening or Database search
require for sequence identification

17. SSR or microsatellites

18. RAPDs Random Amplified Polymorphic DNAs
PCR with 1 short primer (usu decamer)
low annealing temperature
primer annealing in inverted orientation
at optimal distances
amplified products analyzed on agarose gel

19. RAPDs Polymorphisms: base changes at annealing sites
insertion/deletion within amplified fragments
Results: presence or absence of the bands
Cannot distinguish homozygote / heterozygote

20. RAPDs Simple, fast, relatively inexpensive assay
Many loci to be identified in 1 rxn
Can be automated
Inconsistent results (short primer / low temp)
Less informative for mapping with dominant nature
different lengths not identifiable

21. RAPDs

22. AFLPs Amplified Fragment Length Polymorphisms
digestion with 2 enzymes (rare/frequent cutters)
eg EcoRI and MseI
ligation of synthetic adapters to RFs
pre-selective amplification
primers corresponding to adapter sequences

23. AFLPs Amplified Fragment Length Polymorphisms selective amplification
1-5 nt added to 3’ end of each primer
1 nt added to each primer
1/16 amplified
banding patterns analyzed by PAGE

24. AFLPs Many loci to be identified in 1 rxn
High efficiency in detecting polymorphic DNA
More consistent pattern than RAPDs
Dominant marker
Technically challenging / labor intensive

25. AFLPs

26. SNPs or SSCPs Single Nucleotide Polymorphisms
Single-Stranded Conformation Polymorphisms
SNP: major genetic source of phenotypic variability
differentiate individuals within a species

27. SNPs or SSCPs Mobility of ssDNA dependent of nt sequence
looping or compaction
Polymorphisms at a single locus
base change by point mutation or
small insertion / deletion

28. SNPs or SSCPs Specific primers to amplify target region
Asymmetric PCR (1 primer in excess)
Regular PCR (denaturing ds DNA)
ss PCR products analyzed by electrophoresis
Base change revealed by labeled nucleotides
in automated sequencer

29. SNPs or SSCPs Many approaches for detection PCR-RFLP primer extension
allele specific oligonucleotide ligation
allele specific hybridization
sequencing

30. SNPs or SSCPs