welcome

9/22/2012

Mithilesh Singh, M Sc.(GPB)

Mithilesh Kumar
Singh
9/22/2012

Mithilesh Singh, M Sc.(GPB)

Sr. M sc.(GPB)
PALB 1205 2

What is marker ?
3

Characters
Inheritance followed

Different classes of
markers

Morphological
Biochemical markers
Mithilesh Singh, M Sc.(GPB)

9/22/2012

Markers at DNA level

came to the rescue

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9/22/2012

Abundant

Highly polymorphic

No environmental influence

No influence of ontogeny of individual

Co-dominant or dominant

Mithilesh Singh, M Sc.(GPB)

9/22/2012

Changing relative
importance of different
molecular markers

Nature Reviews Genetics

Volume 5 2004- Page 63
6

Mithilesh Singh, M Sc.(GPB)

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Codominant Biallelic Single locus

P1 F1 P2

RFLP

Polymorphic
SSR
Polyallelic
RAPD
DNA markers
Biallelic

ISSR

Dominant

Multi loci
Monomorphic
7

Mithilesh Singh, M Sc.(GPB)

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AFLP

Single locus

Multi loci

Mithilesh Singh, M Sc.(GPB)

9/22/2012

Comparison of DNA markers for their efficiency

Markers

RFLP

Minis
atellit
es

RAPD

SSR

ISSR

SSCP

CAPS

SCAR

AFLP

Abundance
Level of
polymorphism
Locus specificity
Codominance of
alleles
Reproducibility
Labour-intensity
Technical
demands
Operational costs
Development
costs
Quantity of DNA
required
Amenability to
automation
9

High

Medium

Mithilesh Singh, M Sc.(GPB)

Low

9/22/2012

Yes

No

What is AFLP ?

Amplified fragment length polymorphism

(AFLP) are generated by complete
restriction endonuclease digestion of
genomic DNA followed by selective
reamplification and electrophoresis of a
subset of fragments and resulting in
unique, reproducible fingerprint for each
individual.
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Contd.
11

PCR based ( Vos et al., 1995)

RFLP + PCR

Robust

Faster, less labour intensive

more information.
DNAs of any origin or
complexity.

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Steps in AFLP:
12

Digestion

Adaptor Ligation

Amplification

Electrophoresis

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Step I. Restriction Digestion

Rare cutter (EcoRI)

Frequent cutter (Mse I)

Genomic DNA

Restriction fragment

confirmation

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13

Rationale.
(i) The frequent cutter generate small DNA fragments.

(ii)

fragments to be amplified is reduced.

(iii)label one strand of the ds PCR products,

prevents the occurrence of 'doublets' on the gels
Unequal mobility of the two strands of the amplified fragments.

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14

Step II. Ligation of Adapters

Adapter (ECORI)

Restriction Fragment

Adapter
(Mse i)

Restriction fragments with adapters are called

as Tagged Restriction Fragments (TRF)

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15

Types of TRFs

Restriction Fragment

Adapter

Adapter

Adapter

Restriction Fragment

Restriction Fragment

Adapter

Adapter

Adapter

confirmation

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16

Contd.
17

Two different adaptors.

target sites for primer

annealing

core sequence and enzymespecific sequence

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EcoRI-adapter
5-CTCGTAGACTGCGTACC-3
3-CATCTGACGCATGGTTAA-5

MseI-adapter
5-GACGATGAGTCCTGAG-3
3-TACTCAGGACTCAT-5

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18

Restriction-ligation
19

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EcoRI PRE-SELECTIVE PRIMER

GTAGACTGCGTACC

AATT

CA

CA

AT

GAGTCCTGAGTA

MseI PRE-SELECTIVE PRIMER

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20

EcoRI SELECTIVE PRIMER (labeled)

FAM

GTAGACTGCGTACC

AATT

CACT

GTAGACTGCGTACC

AATT

CA

SELECTIVE PRIMER
CA

AT

GAGTCCTGAGTA

GACA

AT

GAGTCCTGAGTA

MseI SELECTIVE PRIMER

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21

Selective PCR product

contains many unlabeled
fragments that will not be
visible on ABI

EcoRI: 6bp cutter --> one cut every 4096 bp

MseI: 4bp cutter -->

one cut every 256 bp

MseI
MseI
EcoRI

MseI

MseI
MseI
MseI

MseI
MseI

MseI
EcoRI

MseI
MseI

MseI

MseI

MseI

MseI

MseI

MseI

MseI

MseI

MseI

MseI
EcoRI
MseI

EcoRI

MseI

EcoRI
MseI

MseI
MseI
EcoRI

MseI

MseI

MseI

MseI

MseI

MseI

MseI
MseI
MseI

22

MseI

Mithilesh Singh, M Sc.(GPB)

MseI

9/22/2012MseI

PRIMERS
23

AFLP reactions generally employed two oligonucleotide

primers:
corresponding to the EcoRI-ends
corresponding to the MseI-ends.
AFLP primers consist of three parts:
5 part corresponding to the adapter,
restriction site sequence and
3 selective nucleotides.
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Contd.
24

EcoRI
MseI

CORE
ENZ
EXT
5-GACTGCGTACC AATTC NNN-3
5-GATGAGTCCTGAG TAA

NNN-3

The PCR primers are labelled.

Good PCR primers

design
Different adaptor require different design of primers and
also different stringent PCR conditions.
Design of primer is dectated by design of adaptor.

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9/22/2012

Contd.
25

All primers start with a 5' guanine (G) residue.

A 5'

G-residue in the unlabeled primer is crucial to prevent

the phenomenon of double bands.
Incomplete addition of an extra nucleotide to the
synthetised strands.

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26

AMPLIFICATION
Fragments with MseI-EcoRI ends
are preferentially amplified.
1. lower annealing temperature.
2. MseI-MseI fragments have an
inverted repeat.
Stem-loop structure
compete with primer annealing.
Mithilesh Singh, M Sc.(GPB)

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A two-step amplification
strategy (complex genome)

27

Preamplification

Selective-amplification

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28

Advantages

1. background 'smears reduced.

2. less bands
3. unlimited template DNA .

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Electrophoresis
29

Polyacrylamide gel.

50-100 DNA bands.

Mithilesh Singh, M Sc.(GPB)

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Selective Bases

30

DNA bands detected is high.

Selective bases at the 3-end of

primers.

Prime DNA synthesis from a

subset of the restriction sites

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Contd.
31

1 additional selective
folds.

3 additional selective bases

4,096 folds.
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16

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32

Reliability of no. of
selective bases

A EcoRI primer with +A

four different MseI primers:

+C
+CT
+CTC
+CTCA.

Each additional selective nucleotide

generate a fingerprint which is
subset of the preceding fingerprints,
Mithilesh Singh, M Sc.(GPB)

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Result and conclusion

33

Appearance of bands that do not occur

in the preceding fingerprints.

Mismatch fragments are amplified

Selectivity is incomplete.
Selective primers up to 3 selective bases
are acceptable

Mithilesh Singh, M Sc.(GPB)

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selectivity of AFLP primers.

AFLP fingerprints are shown
of yeast DNA using primer
combinations with one
selective base for the coRI
primer and one (lanes A), two
(lanes B), three (lanes C) and
four (lane D) selective
nucleotides respectively for
the MseI primer. The primer
combinations used are
EcoRI+A/MseI+CTCA (panel
I), EcoRI+AlMse-I+CTGC
(panel H) and
EcoRI+T/MseI+CTCA (panel
III). The molecular weight size
range of the fingerprints is
from 40 to 370 nucleotides.
34

Mithilesh Singh, M Sc.(GPB)

9/22/2012

Choosing selective primer combinations

EcoR1-AGT
EcoR1-AGC

Use few of these

(expensive),
but allows use of multiple colors
(multiplex run on ABI)

Use many of these to

get enough markers
(cheap)
And use these to
optimize
number of bands

MseI-CGT
MseI-CGA
MseI-CGC
MseI-CGG
etc.
MseI-CGTG
MseI-CG

An additional
nucleotide reduces
number of peaks 4-fold
One less nucleotide
increases number
of peaks 4-fold

The number of fragments detected in a

single reaction can be 'tuned' by selection
of specific primer sets
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35

36

A few population genetic

programs for AFLP
RAPDFst: Fst (Lynch and Milligam, 1994)
analyses
MVSP, NTSYS: Jaccard coeficient, Nei and Li
(1979)
Arlequin, TFPGA: Amova
Genalex: st, analog of Fst, Amova
Structure, BAPS: inference of population
structure.
Hickory: Bayesian estimation of F statistics for
dominant markers.
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37

Advantages of AFLP

Only small amounts of DNA.

Two primers- reproducible results.
Many restriction fragment subsets can be
amplified by changing the nucleotide extensions
on the adaptor sequences.
Hundreds of markers can be generated reliably.

Mithilesh Singh, M Sc.(GPB)

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Contd.
38

High resolution due to stringent PCR conditions.

variety of genomic DNA samples.
No prior knowledge of the genomic sequence

Mithilesh Singh, M Sc.(GPB)

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disadvantages
39

Markers are dominant

Can be tedious to score

Size homoplasy

Reproducibility?
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40

Reproducibility
High reproducibility has generally been
reported
However, DNA quality is crucial component
(use same DNA extraction protocol for all
samples!)

Assess quality of data by repeating several

samples from scratch i.e. starting with DNA
extraction
Mithilesh Singh, M Sc.(GPB)

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41

Number of bands in AFLP

1.
Genome size
profile
2.
selective nucleotides
is determined
by
3. Dilution of PCR product

Why optimize number of bands?

Size homoplasy !!!!!

Difficult to score

Mithilesh Singh, M Sc.(GPB)

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Insensitivity to template
concentration
fingerprint patterns are very similar
using different template quantities
(25 ng to 25 pg).

42

Mithilesh Singh, M Sc.(GPB)

9/22/2012

Equal intensity from variable

template concentration
Labeled primer used limited quantity
Primer is exhausted.
Amplification stops.
further thermo cycling does not affect the band pattern
This characteristic is elegantly utilized in the AFLP
protocol, which uses an excess of PCR cycles.

43

Mithilesh Singh, M Sc.(GPB)

9/22/2012

Why MseI ? Why not TaqI ?

Eukaryotic DNAs
Msel
TaqI

AT-rich
TTAA
TCGA
Why EcoRI ?

Reliable (low cost).

Six-cutter enzyme.
Complete restriction of the DNA.

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Mithilesh Singh, M Sc.(GPB)

44

Application of AFLP
in crop improvement

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45

AFLP applications
Linkage mapping
Parentage analysis
Measuring genetic diversity
Population genetics
Development of single locus markers
from AFLP for MAS
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46

Case study

Relationship between hybrid

performance and AFLP based genetic
distance in highland maize inbred
lines
Legesse et al., 2007
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Dendrogram derived from UPGMA analysis based on 32 maize

inbred lines

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Conclusion
Significant positive correlation manifested
between GDs and hybrid performance for most of
the traits in inbred line x tester combinations
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Conclusion
Far from being almost forgotten AFLP is a
highly useful technique and if fostered by
parallel development of new analysis
methods will continue to be at the forefront
in answering important scientific questions.

s
Mithilesh Singh, M Sc.(GPB)

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54

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a

9/22/2012

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in

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Mithilesh Singh, M Sc.(GPB)

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