molecular markers for molecular in crop improvmen
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8/2/2019 Molecular Markers for Molecular in Crop Improvmen
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Molecular Markers
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Understanding “Genome”
Genome = Total genetic materials of an organism
MitochondriaGenome
ChloroplastGenome
NuclearGenome
Chromosomes
Genes + Non-Genes
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Purine
Pyrimidine
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Genetic Variation in Inter-Genic Region
AC
AC
AC
AC
AC
Insertion
Deletion
Simple Sequence Repeat (SSR)
TransposonGene interuption
Chromosome DNA
Base substitution
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GA
GA
GA
GA
GA
Simple Sequence Repeat (SSR)
A
B
A B
SSR
A (GA)10
B (GA)5
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Transposon-induced mutation
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Two main types of base subst.:
1) Transition (purine—>purine or pyrimidine—>pyrimidine)
Ex.- A—>G or T—>C
2) Transversion (purine—>pyrimidine or pyrimidine—>purine)Transitions are more common.
Base substitutions
A
B
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RFLP“R estriction Fragment Length Polymorphism”
Molecular Marker
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Molecular Marker Restriction Enzyme
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A
B
C
D
E
Molecular Marker
(at b→e)
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RFLP procedure
1. DNA isolation
2. Restriction enzyme digestion & agarose
electrophoresis&southern blot3. RFLP probe libraries
4. Probe labeling & hybridization
5. Detection
Molecular Marker
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DNA isolation
1. High quality; clean, no shear DNA
2. High quantity of DNA required
Molecular Marker
M l l M k
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1. DNA isolation
2. Restriction enzyme digestion & agarose
electrophoresis&southern blot3. RFLP probe libraries
4. Probe labeling & hybridization
5. Detection
RFLP procedure
Molecular Marker
M l l M k
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RFLPRFLP
Genomic DNA
digestion withdigestion witha restriction enzymea restriction enzyme
Restriction fragments
Restriction fragments were separated according to size
R estriction F ragment L ength P olymorphism Molecular Marker
M l l M k
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RFLP method
Molecular Marker
M l la Ma k
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RFLP procedure
1. DNA isolation
2. Restriction enzyme digestion & agarose
electrophoresis&southern blot3. RFLP probe libraries
4. Probe labeling & hybridization
5. Detection
Molecular Marker
Molecular Marker
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RFLP probe libraries
1. Genomic libraries
2. cDNA libraries
Molecular Marker
RFLP b d l f G iMolecular Marker
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DNA Motif % Methylation
C-G 82C-A 19
C-T 19
C-C 7
C-A-G > 80
C-T-G > 80
C-A-T < 4
Pst I
Methylation analysis of plant DNA (Gruenbaum et al. 1981).
RFLP probe development from Genomics
methylation-sensitive enzyme“more variation”
Molecular Marker
Molecular Marker
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2000 bp
500 bp
Molecular Marker
Molecular Marker
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Plasmid(e.g., pUC18)
Molecular Marker
Molecular Marker
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Molecular Marker
RFLP b d l t f DNAMolecular Marker
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Short, single- or low-copy
genomic DNA or cDNAclones are typically used asRFLP probes.
RFLP probe development from cDNA
mRNA
cDNA
RT-PCR
Cloning
(pUC18, pBluescript SK)
Molecular Marker
Molecular Marker
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RFLP procedure
1. DNA isolation
2. Restriction enzyme digestion & agarose
electrophoresis&southern blot3. RFLP probe libraries
4. Probe labeling & hybridization
5. Detection
a a
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Molecular Marker
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denature
A A
A A
U U
U U
denature
U U
U U
U U
Hybridization Detection
A A
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Alkaline phosphatase (AP)
5’ bromo-4-chloro-3-indolyl pho
Nitro blue tetrazoliumBlue color
Horseraddish peroxidase (HRP)
Hydroperoxide4-chloro-1-napthol
Violet color
Chemiluminesent
AP: substituted dioxitane
HRD: luminol
light
Molecular Marker
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PrePre--hybridization and Hybridizationhybridization and Hybridization
Molecular Marker
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RFLP procedure
Detection1. Color detection
2. Chemiluminesence
Molecular Marker
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DetectionDetection
Molecular Marker
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Applications
1. DNA Fingerprinting
2. QTL mapping
Molecular Marker
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Polymerase Chain ReactionPolymerase Chain Reaction ““PCR PCR ””
PCR PCR --based Markersbased Markers
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T f l f PCR
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Temperature profile for PCR
Annealing temperature affects to non specific products
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Annealing temperature affects to non-specific products
Molecular Marker
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Random AmplifiedRandom Amplified PolymorphicPolymorphic DNAsDNAs ““RAPDRAPD””
Molecular Marker
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Characters of Characters of RAPDsRAPDs
@@ PCR PCR --Based MethodBased Method
@@ SingleSingle AbitraryAbitrary Primer (8Primer (8--10 base pairs)10 base pairs)
@@ Amplify Anonymous Stretches of DNAAmplify Anonymous Stretches of DNA
@@ Dominant MarkerDominant Marker
@@ Number of fragment amplified / primer (6Number of fragment amplified / primer (6 -- 12)12)
template sequence Molecular Marker
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unamplified DNA
primer extension
primer extension
cycle 1cycle 1 denaturation and anneal primer
= RAPD primer
danaturation and anneal primercycle 2cycle 2
primer extension
cycle 3cycle 3
Molecular Marker
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RAPD PrimerRAPD Primer -- OperonOperon KitKit
Molecular Marker
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Amplification ConditionsAmplification Conditions
@@ TemperatureTemperature
denaturedenature 9494--9595 oocc
annealingannealing 3535--3636 oocc
extensionextension 7272 oocc
@@ TimeTime
30 sec30 sec--1 min1 min
30 sec30 sec--1 min1 min
1 min1 min -- 2 min2 min
@@ CycleCycle
3535--40 cycles40 cycles
Molecular Marker
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ApplicationsApplications Molecular Marker
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λ H
i n d
I I I K i 3
K i 1 1
K i 2 0
K i 2 1
K i 2 4
K i 2 5
K i 2 7
K i 2 8
K i 3 2
K i 3 6
K i 3 7
K i 4 0
K i 4 3
K i 4 4
λ
H i n d
I I I
Kbp
23.19.5
6.64.32.32.0
0.6
Kbp
23.19.56.64.32.3
2.0
0.6
OPA16
OPB01
Genetic Diversity in MaizeGenetic Diversity in Maize
Applicationspp
Molecular Marker
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4 4 8
4 4 9
4 5 0
4 5 1
4 5 2
4 5 3
4 5 4
4 5 5
M 4 5 6
4 5 7
4 5 8
4 5 9
4 6 0
4 6 1
4 6 2
DNA Fingerprinting in RICE (KDML105)DNA Fingerprinting in RICE (KDML105)
Molecular Marker
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Genetic Diversity of ShrimpGenetic Diversity of Shrimp
Molecular Marker
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Gene Mapping in RICEGene Mapping in RICE
Molecular Marker
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Advantages of RAPDAdvantages of RAPD
@@ FastFast
@@ SimpleSimple
@@ InexpensiveInexpensive
@@ No need to know the information about target DNANo need to know the information about target DNA
Molecular Marker
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Limitations of RAPDLimitations of RAPD
@@ Dominant MarkersDominant Markers
@@ Reproducibility ProblemsReproducibility Problems
@@ Problems of InterpretationProblems of Interpretation
SCAR (Sequence Characterized Amplified Region) Molecular Marker
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flanking region I flanking region II
specific primers weredesigned
corresponding to
flanking sequence of
interesting regionPCR analysis
Molecular Marker
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AFLP is a DNA fingerprinting technique which detects DNA
restriction fragments by means of PCR amplification.
AAmplifiedmplified FFragmentragment LLengthength PPolymorphism (olymorphism (AFLPAFLP))
Defined
Molecular Marker
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• AFLPs are differences in restriction fragment lengths caused by
- SNPs (Single Nucleotide Polymorphisms) or
- INDELs (Insertions / Deletions)
that create or abolish restriction endonuclease recognition sites.
AFLP assays are performed by selectively amplifying a pool of
restriction fragments using PCR.
• The key to the AFLP method is the design and combination of primersfor
selectively amplifying restriction fragments. The selective nucleotidesanneal to precise, randomly chosen sequences immediately internal toa restriction
endonuclease recognition site.
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5’---------------GAATTC-----------------------TTAA----------------3’
3’---------------CTTAAG-----------------------AATT ----------------5’
Molecular Marker
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+ EcoRI
MseI
Primer+3 5’--------------AAC
GAC ------------ 5’ Primer+3
4.5 % denaturing PAGE
AATTC-------------------------T G------------------------AAT
EcoR I adapter
TTAA
Mse I adaptor
TA
AATTCT ---------------------CTTATTAAGA---------------------GAAT
Primer+1 5’ --------------A
C------------- 5’ Primer+1
AATTCTTG-------------------GACTTATTAAGAAC-------------------CTGAAT
PCR I
PCR II
Number of band producing by selection of AFLP Molecular Marker
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If b is the number of nucleotides in the recognition site of a restriction
endonuclease, the G-C content of the target genome is 50%, and theprobability
of observing each nucleotide motif is equal in the target genome, thenthepredicted number of DNA fragments amplified by AFLP primers with
n selective nucleotides from completely digested DNA is
(N = Genome size, bp.)
Molecular Marker
Genome size of Arabidopsis is 1x108 bp
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RE G SN
RE G SN
RE G SN
RE G SN
RE = Restriction Enzyme
G = Genome Size
SN = Selective Nucleotide Primer
Molecular Marker
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RE G SN
RE G SN
RE G SN
RE G SN
RE = Restriction Enzyme
G = Genome Size
SN = Selective Nucleotide PrimerFurther Reading
The Effects of Nuclear DNA Content (C-value) on the Quality and Utility of
AFLP Fingerprints
Annals of Botany 95: 237–246, 2005
(FAY et al, 2005)
2
Advantages
Molecular Marker
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Generation of many informative bands per reaction.
No sequence information is required.
High producibility
PCR - based technique
Random distribution throughout the genome
Wide range of applications
DisadvantagesDisadvantages Molecular Marker
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Dominant marker
Band profiles can not be interpreted in terms of loci and alleles
Similar sized fragments may not be homologous
Purified, high molecular weight DNA required
(typically 0.2 to 2.5 µg per individual)
Expensive and Technically demanding
Applications Molecular Marker
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• Genetic studies,
• Biodiversity studies
• The analysis of germplasm collections
• The identification of closely-linked DNA markers
• The construction of genetic maps and physical maps
• Map- based gene cloning
AFLP Fingerprint of Domestic Thai Rice VarietiesAFLP Fingerprint of Domestic Thai Rice Varieties Molecular Marker
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Data scored from AFLP Gel Molecular Marker
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Phylogenetictree of Thai Rice Varietiys Molecular Marker
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หอมบางขาวพวงสพรรณบร 90
ฟาม ยหอมอนคาRD7
สพรรณบร 1หอมเชยงใหมหอมนางมนหอมจนหอมใบหอมศรนวลRD9
RD25
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เหลองประทวหอมท งดาวหอมประทปขาวตาแห งIR36
หอมเมองเพชรหอมขม นพวงมาลยหอมนวลสพรรณบร 2RD5
ชยนาท 1หอมดอกกาพล
กล นหอมหอมดาวหอมทองหอมสรนทรหอมอดมหอมแดงน อยขาวทยRD23
RD1
เหลองราน าดอกไม แพรหอมหอมมะยมหอมแมจ
RD11หมอครวหอมนายพลประทม 60KDML 105
หอมศรนวลหอมเกษตรRD27
หอมโพRD4
สพรรณบร 60RD15
เลศพลาลยร อยเอด 1
ยโสธร 1ศรสะเกษ 1อบล 1สรนทร 4มหาสารคาม 1
Automated AFLP
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Molecular Marker
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Microsatellite Markers
microsatellite DNA
Molecular Marker
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microsatellite DNA
NNNNNAGTCAGTCAGTCNNNNN
(AGTC) 3
core sequences
Satellite DNA markers
Molecular Marker
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Satellite DNA markers
Minisatellites : size length 200 bp - 10 kbp
core sequences > 20 bpabundant at centomeres and
telomeres
Microsatellites: size length 100 - 1000 bpcore sequences 1- 6 bp
distribute throughout the genome
Type of repeats Molecular Marker
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D i T r i T e t r a H e x a P e n t a
(AT)n (GGT)n (GAAT)n (GACCAT)n (CCATCAT)n(TCC) n (GATG)n (GAGAAA)n
(ATT)n (CAAA)n (CATCAG)n D e c a
(TCC)n (GACA)n (CGCCAT)n (CCATCATCAT)n(CAA)n (CAGG)n (TTTCTC)n
(CATT)n (CGTCAT)n
(CAGC)n (CATCTT)n(CACT)n
(GGCT)n
(GACC)n
Molecular Marker
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Types of Microsatellites:
Molecular Marker
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yp
RFLP based : Genomic DNA digestion
Southern blotHybridization
Detection
PCR based : PCR reactionElectrophoresis
Detection
RFLP based for microsatellite studies Molecular Marker
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fl ki i IIflanking region I
Schematic of SSR assay(:SSLP Single Strand Length Polymorphism) PCR based for microsatellite studies
Molecular Marker
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Repetitive sequence
primer Iprimer II
plant A
plant B
microsatellite
plant A
plant B
flanking region IIflanking region I
specific primers were designed corresponding to
flanking sequence of microsatellite
PCR analysis and analyze on 6 %denaturingpolyacrylamide gel with silver staining
A B
(Forward primer) (Reverse primer)
Advantages of microsatellite markers
Molecular Marker
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• Give high polymorphism
• Distributed throughout the genome• Mendelian inheritance
• Population genetic, Genetic diversity,
Genome mapping, etc.
Allele standard 427 bp
311 bp
427 bp
311 bp
Molecular Marker
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h1h2h3h4
i1i1
i2
RM225
A l l e l e s t a n
d a r
d
i2
i3 i4i5i6 i7 i8
i3 i8
i1
RM261
A l l e l e s t a n
d a r d
g1g2
g3
g4
RM17
A l l e l e s t a n
d a r d
h1
h2h3
i1 i2 i3
RM258
A l l e l e s t a n d a
r d
g1g2
h1
h2
RM201
A l l e l e s t a
n d a r d
h1h2
i1 i2 i3
i4 i5 i6 i7
i1
RM3
A l l e l e s t a n d a r d
i4 j1
i5
j1 j2
RM00
A l l e l e s t a n
d a r d
j1 j2
j3
k1
k2
l1
RM248
A l l e l e s t a n
d a r d
g1
g2
g3
g4
g5g6h1h2h3h4
i1
RM257
A l l e l e s t a n
d a r d
249 bp
200 bp
151 bp
140 bp
118 bp
100 bp
82 bp
60 bp
311 bp
249 bp
200 bp
151 bp
140 bp
118 bp
100 bp
82 bp
60 bp
Inter-MITE polymorphisms (IMP): a high throughput transposon-based genome mapping andfingerprinting approach
Theor Appl Genet (2001) 102:773–781 © Springer-Verlag 2001
R.-Y. Chang · L.S. O’Donoughue · T.E. Bureau
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Miniature inverted-repeat transposable elements (MITEs)represent a large superfamily of transposons (Retroelements)
that are moderately to highly repetitive and frequently associated with genes.
inter-simple sequence repeats (ISSR)
Lin et al 2005
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Using IRRR to detect the genetic diversity in 57 rice accessions and 65
barley lines introduced from more than 10 countries
http://www.regional.org.au/au/allelopathy/2005/1/4/2541_linwx.htm
ISSR
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SSCP analysis Molecular Marker
Single Strand Conformation Polymorphism
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Homozygote A Heterozygote Homozygote B
Denature with Heat and Formamide
Native Gel Electrophoresis
b r a s s i a n a
g r a n d i s 1
g r a n d i s 2
ro p i q u a n
r o p h y l l a
E. camaldulensis
e r e t i c o n i s
e l l i a l a
a l i g n a
Molecular Marker
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C 2
C 3
C 4
C 6
C 8
C 9
C 1 0
C 1
3
C 1 5
C 1 7
C 1 8
C 1 9
E .
b
Kb
0.798 bp
E . g
E . g
E . p
r
E . u
r
E .
t e
E . p
E . s a
Amplfied genomic DNA of extract from eight Eucalyptus species and with 12 clones of
Eucalyptus camaldulensis using E-F primer pair were approximately 789 bp. All amplification
products were electrophored on 1% agarose gel and ethidium bromide stained. Molecular
weight ladder sizes are in Kb.
ATGPrimer EPrimer EPrimer E
Primer IPrimer DPrimer G
Molecular Marker
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C 2
C 3
C 4
C 6
C 8
C 9
C 1 0
C 1 3
C 1 5
C 1 7
C 1 8
E .
b r a s s i a n a
E . g r a n d i s 1
E . g r a n d i s 2
E . p r o p i q u a n
E . u r o p h y l l a E. camaldulensis
E .
t e r e t i c o n i
s
E . p e l l i a l a
E . s a l i g n a
C 1 9
SSCP analysis from eight eucalyptus species of the amplified products withE-F CAD specific-primer combination were observed on 3.5 % polyacrylamide
and silver stained.
Exon1 Exon2 Exon3 Exon4 Exon55’
Primer FPrimer FPrimer F Primer C
TATA-box
Primer H
3’
Polymorphism Information Content (PIC) value
“Commonly used in genetics as a
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measure of polymorphism for a marker locus”
PIC = (1-ΣPi2) – (ΣPi2)2 + ΣPi4
= H - (ΣPi2)2 + ΣPi4
Heterozygosity is defined as the probability that a random
individual chosen from the population is heterozygous at a locus
and is given in a randomly mating population by
Heterozygosity = (1-ΣPi2)
(Botstein et al., 198
RM106 Pi Pi2 Pi4
1 22 41 0.5365854 0.2879239 0.0829001
2 19 41 0.4634146 0.2147531 0.0461189
RM106
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E Pi2 1 0.502677
H (1 - E Pi2) 0.497323
(E Pi2)2 0.2526841
E Pi4 0.1290191
PIC 0.3736579
RM17 Pi Pi2 Pi4
1 2 43 0.046511628 0.002163332 4.68E-06
2 17 43 0.395348837 0.156300703 0.02442991
3 13 43 0.302325581 0.091400757 0.008354098
4 2 43 0.046511628 0.002163332 4.68E-06
5 9 43 0.209302326 0.043807463 0.001919094
E Pi2 1 0.295835587
H (1 - E Pi2) 0.704164413
(E Pi2)2 0.087518694
E Pi4 0.034712462
PIC 0.651358181
Comparison of markers
RFLP RAPD SSLP AFLP
Molecular Marker
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• Technique Restriction
endonuclease digestion
DNA amplification
with random primers
PCR of simple
sequence repeat
regions
PCR of a subset of
restriction fragments
from extended adapter
primers
• Nature of
polymorphism
Single base changes,
insertions, deletions
Single base changes,
insertions, deletions
Repeat length
changes
Single base changes,
insertions, deletions
• Abundance in
the genome
High Very high Medium High
• Level of
polymorphism
Medium Medium High Medium
• Dominance Codominant Dominant Codominant Mixed
• DNA amt.
require
2-10 ug 10-50 ng 50-100 ng 1-2 ug
Molecular Marker Technology
Molecular Marker
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Type I : Known function, Unique position
- ESTs (Expressed Sequence T ags)
Developed Marker
- RFLP (Restriction Fragment Length Polymorphism)- SCAR (Sequence Characterized Amplified Region)- SSR (Simple Sequence Repeats)- STS (Sequence T ag Site)
Type II : No function, Unique position Developed Marker
Type III : No function, Variable position Generic Marker- RAPD (Random Amplified Polymorphic DNA)- AFLP (Amplified Fragment Length Polymorphism)
- SNPs (Single Nucleotide Polymorphism)
Desirable properties of molecular markers for diversity
Molecular Marker
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@ Polymorphic :High PIC (Polymorphic Information content) values
@ Co-dominant and dominant inheritance
@ Occurs thoughout the genome
@ Easy, fast and cheap to detect
@ Reproducible / transferable
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Thank you!
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SNP detection
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Guidelines for the design and use of primersGuidelines for the design and use of primers
• Primer length
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• Primer length- 18-30 nucleotides
- avoid long run on single base
(4 or more G’s or C’s in a row)
• Melting Temperature (Tm)
- optimum in the range 52-58 C- avoid Tm above 65 C
- Tm = 2(A+T) + 4(G+C) ---> 18-30 bases
• GC Content
- provide the strength of annealing
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- provide the strength of annealing- should be between 45-60 % GC
• 3’-end sequence- essential for the control of mis-priming
- G or C is desirable at the 3’ end
• Dimers and false priming cause misleading results
- should not form hairpins
- no primer dimer formation
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• Specificity
- depends on primer length
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depends on primer length
• Degenerate primers
- based on the amino acid sequence
of conserved regions
• Complementary primer sequences- no intra-primer homology beyond 3 base pairs
- inter-primer homology (interfere with hybridization)- primer dimer (homology at 3’ end)
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