molecular markers for molecular in crop improvmen

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Molecular Markers



Understanding “Genome”

Genome = Total genetic materials of an organism

MitochondriaGenome

ChloroplastGenome

NuclearGenome

Chromosomes

Genes + Non-Genes



Purine

Pyrimidine



Genetic Variation in Inter-Genic Region

AC

AC

AC

AC

AC

Insertion

Deletion

Simple Sequence Repeat (SSR)

TransposonGene interuption

Chromosome DNA

Base substitution



GA

GA

GA

GA

GA

Simple Sequence Repeat (SSR)

A

B

A B

SSR

A (GA)10

B (GA)5



Transposon-induced mutation



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



RFLP“R estriction Fragment Length Polymorphism”

Molecular Marker



Molecular Marker Restriction Enzyme



A

B

C

D

E

Molecular Marker

(at b→e)



RFLP procedure

1. DNA isolation

2. Restriction enzyme digestion & agarose

electrophoresis&southern blot3. RFLP probe libraries

4. Probe labeling & hybridization

5. Detection

Molecular Marker



DNA isolation

1. High quality; clean, no shear DNA

2. High quantity of DNA required

Molecular Marker

M l l M k



1. DNA isolation




5. Detection

RFLP procedure

Molecular Marker

M l l M k



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



RFLP method

Molecular Marker

M l la Ma k



RFLP procedure

1. DNA isolation




5. Detection

Molecular Marker

Molecular Marker



RFLP probe libraries

1. Genomic libraries

2. cDNA libraries

Molecular Marker

RFLP b d l f G iMolecular Marker



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



2000 bp

500 bp

Molecular Marker

Molecular Marker



Plasmid(e.g., pUC18)

Molecular Marker

Molecular Marker



Molecular Marker

RFLP b d l t f DNAMolecular Marker



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



RFLP procedure

1. DNA isolation




5. Detection

a a



Molecular Marker



denature

A A

A A

U U

U U

denature

U U

U U

U U

Hybridization Detection

A A



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



PrePre--hybridization and Hybridizationhybridization and Hybridization

Molecular Marker



RFLP procedure

Detection1. Color detection

2. Chemiluminesence

Molecular Marker



DetectionDetection

Molecular Marker



Applications

1. DNA Fingerprinting

2. QTL mapping

Molecular Marker



Polymerase Chain ReactionPolymerase Chain Reaction ““PCR PCR ””

PCR PCR --based Markersbased Markers



T f l f PCR



Temperature profile for PCR

Annealing temperature affects to non specific products



Annealing temperature affects to non-specific products

Molecular Marker



Random AmplifiedRandom Amplified PolymorphicPolymorphic DNAsDNAs ““RAPDRAPD””

Molecular Marker



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



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



RAPD PrimerRAPD Primer -- OperonOperon KitKit

Molecular Marker



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



ApplicationsApplications Molecular Marker



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



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



Genetic Diversity of ShrimpGenetic Diversity of Shrimp

Molecular Marker



Gene Mapping in RICEGene Mapping in RICE

Molecular Marker



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



Limitations of RAPDLimitations of RAPD

@@ Dominant MarkersDominant Markers

@@ Reproducibility ProblemsReproducibility Problems

@@ Problems of InterpretationProblems of Interpretation

SCAR (Sequence Characterized Amplified Region) Molecular Marker



flanking region I flanking region II

specific primers weredesigned

corresponding to

flanking sequence of

interesting regionPCR analysis

Molecular Marker



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



• 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.



5’---------------GAATTC-----------------------TTAA----------------3’

3’---------------CTTAAG-----------------------AATT ----------------5’

Molecular Marker



+ 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



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



RE G SN

RE G SN

RE G SN

RE G SN

RE = Restriction Enzyme

G = Genome Size

SN = Selective Nucleotide Primer

Molecular Marker



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



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



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



• 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



K D M L 1 0

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0 0 0 1 0 0 0 0 0 9 0 0 0 0 0 0 1 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0 1 0 9 0 0 0 0 1 0 0 9 1 0 0 0 1 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 1 1 9 0 0 0 1 0 0 0 9 0 0 0 0 1 0 0 0 1 0 0 1 0 0

1 1 1 1 0 0 1 1 1 9 1 1 1 1 1 1 0 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 0 1 0 0 0 1 1 1 9 1 1 1 1 1 1 0 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 0 1 0 1 0 1 1 1 9 1 1 1 1 1 1 0 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 0 0 0 0 0 1 1 1 9 1 1 1 1 1 1 0 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0 1 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 0 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 9 9 1 1 1 0 1 1 1 0 1 1 1 1 1

1 0 1 0 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 0 1 0 0 0 1 1 1 1 1 1 1 1 0 1 0 1 1 1 1 1 0 1 1 1 0 0 0 1 1 1

1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 1 1 0 0 1 1 1 1 1 1

0 1 0 1 1 1 0 1 0 0 0 0 0 1 0 1 1 0 0 0 1 1 0 0 0 0 1 0 0 1 0 1

0 1 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0

1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1

1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 0 0 0 1 1 1 1 1 1

0 1 0 1 1 1 9 0 0 1 0 0 0 0 0 1 1 0 1 0 0 1 1 0 0 1 0 0 0 1 0 1

0 0 1 0 0 0 9 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0

0 1 0 1 1 1 0 1 0 1 0 0 0 0 0 0 1 0 1 0 1 1 1 0 0 0 0 0 0 0 0 0

Phylogenetictree of Thai Rice Varietiys Molecular Marker



หอมบางขาวพวงสพรรณบร 90

ฟาม ยหอมอนคาRD7

สพรรณบร 1หอมเชยงใหมหอมนางมนหอมจนหอมใบหอมศรนวลRD9

RD25

RD8

RD21

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



Molecular Marker



Microsatellite Markers

microsatellite DNA

Molecular Marker



microsatellite DNA

NNNNNAGTCAGTCAGTCNNNNN

(AGTC) 3

core sequences

Satellite DNA markers

Molecular Marker



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



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



Types of Microsatellites:

Molecular Marker



yp

RFLP based : Genomic DNA digestion

Southern blotHybridization

Detection

PCR based : PCR reactionElectrophoresis

Detection

RFLP based for microsatellite studies Molecular Marker



fl ki i IIflanking region I

Schematic of SSR assay(:SSLP Single Strand Length Polymorphism) PCR based for microsatellite studies

Molecular Marker



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



• 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



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



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



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



SSCP analysis Molecular Marker

Single Strand Conformation Polymorphism



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



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



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



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



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



• 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



Repeat length

changes



• 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



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



@ Polymorphic :High PIC (Polymorphic Information content) values

@ Co-dominant and dominant inheritance

@ Occurs thoughout the genome

@ Easy, fast and cheap to detect

@ Reproducible / transferable



Thank you!



SNP detection



Guidelines for the design and use of primersGuidelines for the design and use of primers

• Primer length



• 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



- 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



• Specificity

- depends on primer length



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)

molecular markers for molecular in crop improvmen

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