Molecular Marker

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