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Molecular Marker
Characterization of plant genotypes
Morphological markers
Physiological markers
Biochemical markers
Molecular markers
etc.
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.
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
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
Molecular markers
Protein-based marker: Isozyme / Allozyme
multiple molecular forms of an enzyme
similar / identical catalytic activity
enzyme assay on PAGE
DNA-based marker
Isozyme marker
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
DNA-based markers
Approach:
Hybridization
Polymerase Chain Reaction
Types: RFLP Minisatellite
RAPD SCAR SSR AFLP SNP etc
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
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
RFLPs
Polymorphism:
homologous pieces of different lengths
mutation on restriction sites
mutation between restriction sites
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
RFLPs
PCR-RFLPs
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
SSR or microsatellites
Easy to detect via PCR
High polymorphism
Co-dominant marker
Library screening or Database search
require for sequence identification
SSR or microsatellites
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
RAPDs
Polymorphisms:
base changes at annealing sites
insertion/deletion within amplified fragments
Results: presence or absence of the bands
Cannot distinguish homozygote / heterozygote
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
RAPDs
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
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
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
AFLPs
SNPs or SSCPs
Single Nucleotide Polymorphisms
Single-Stranded Conformation Polymorphisms
SNP: major genetic source of phenotypic variability
differentiate individuals within a species
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
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
SNPs or SSCPs
Many approaches for detection
PCR-RFLP
primer extension
allele specific oligonucleotide ligation
allele specific hybridization
sequencing
SNPs or SSCPs