INSTITUT PERTANIAN BOGOR

DEPARTMENT OF SILVICULTURE

MSc. Tropical Silviculture

(Utilization of Forest Genetics Resources)

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

A Presentation Based on DNA Fingerprinting in Plants (Principles, Methods and Applications) By

PHILIP WORLANYO DUGBLEY

(P050128041)
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

TOPICS UNDER DISSCUSSION

The AFLP Technique: Principle, Advantages, and Limitations
Some Variants of Amplified fragment length

polymorphism (AFLP)
Sequence-Specific Amplification Polymorphism Analysis (S-SAP) Selective Amplification of Microsatellite Polymorphic Loci (SAMPL) Microsatellite-AFLP Methylation-Sensitive Amplified Polymorphisms (M-SAP)
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

TOPICS UNDER DISSCUSSION

AFLP-Based Expression Profiling Single-Strand Conformation Polymorphism Analysis Miscellaneous Techniques Mini-satellite Variant Repeat Mapping Two-Dimensional DNA Typing Methods Single-Nucleotide Polymorphisms (SNP)
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants The AFLP Technique

INTRODUCTION

Amplified fragment length polymorphism (AFLP) Defined

AFLP is a technology which was introduced by Vos et al (1995) and represents an ingenious combination of RFLP

analysis and PCR. AFLP technology is applicable to all

organisms without previous sequence information, and generally results in highly informative fingerprints. It rapidly

became one of the most popular and powerful approaches to

detect DNA polymorphisms.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants The AFLP Technique

INTRODUCTION

Typically, two successive PCRs are performed on the restricted template, using specifically designed primers that allow only a subset of the restriction fragments to be amplified. To achieve this, the 5-portions of the primers are made complementary to the adapters, whereas the 3-ends extend by a few, arbitrarily chosen

nucleotides (so-called selective bases or selective nucleotides)

into the restriction fragment
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

INTRODUCTION.

The AFLP technique is based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases. In this method, the exact matching of the 3-end of a primer is essential for amplification. Therefore, only those restriction fragments

are amplified in which the 3-primer extensions match the sequences

flanking the restriction sites
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

INTRODUCTION.

Polymorphisms between two or more genotypes may arise from three sources: Sequence variation in one or both restriction sites flanking a particular fragment (such as in RFLPs) Insertions or deletions within an amplified fragment (such as in RFLPs). Differences in the nucleotide sequences immediately adjacent to the restriction sites. AFLPs therefore detect higher levels of polymorphism than RFLPs. AFLP marker bands are mainly dominant, but codominant inheritance can sometimes be evaluated.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

PROCEDURES IN AFLP METHOD

Digestion
Adaptor Ligation (PO-4)

Amplification (PCR)
Electrophoresis
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Digestion
Two different restriction endonucleases are used in digestion. One is 4base cutter (MseI) and the other one is 6-base cutter (EcoRI).

MseI EcoRI

5TTAA3 5GAATTC3

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Adaptor Ligation
- Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments.

- One adaptor will complement to the Msel cut end, the other will complement to the EcoRI cut end.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

AMPLIFICATION (PCR)
DNA fragments with MseI-EcoRI ends will be selected as DNA
template for amplication. Two PCR primers complementary to the two adaptors are used in

amplification.
The PCR primers are labeled with radioactive or fluorescence dye for detection of DNA bands on gels.
De-naturation at 95 oC Primer Binding at 55 oC DNA Synthesis at 72 oC
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

ELECTROPHORESIS
- Polyacrylamide/agarose
gel is used for separating DNA bands. - Normally, 30-100 DNA bands can be detected by

AFLP on polycrylamide gel.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

About 30 - 40 bands visible

Example of AFLP gel from individuals sampled from a tropical tree species

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

CHARACTERISTICS OF AFLP
Dominant marker. DNA variation is detected by presence/absence of DNA bands

due to:
presence/absence of restriction sites additional bases (insertion) between two restriction sites are very large

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

ADVANTAGES OF AFLP
The main advantages of AFLP are;
No need for known sequences in the genome

Many polymorphisms compared to other Mtds.

By changing the selective nucleotides a

1 2 3 4

different part of the genome can be analysed. Whole genome analysis is (theoretically) possible.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

LIMITATIONS OF AFLP
The main disadvantages are:
Complex procedure Marker is dominant (i.e. heterozygotes are scored as homozygotes) The requirement for both good quality and medium quantities DNA (to ensure complete restriction), and of DNA compared to e.g. microsatellite analysis. Can be tedious to score.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants Miscellaneous AFLP Variants

Sequence-Specific Amplification Polymorphism (S-SAP)

One important modification of the basic AFLP technology

introduced by Waugh et al. which became known as S-SAP analysis. The initial steps of DNA digestion and adapter ligation are identical to the standard of AFLP . However, only one restriction site-specific AFLP primer is employed in the final amplification step, whereas the second primer is complementary to a defined DNA sequence
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

STILL ON S-SAP
The concept of combining gene-specific primers with AFLP primers is not restricted to transposons, but is potentially applicable for targeting and mapping any gene, gene family member, or other DNA sequence of interest. The only requirement is that, sufficient sequence information about primer design is needed.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

S-SAP Continuous
Thus, an S-SAP variant coined gene-anchored amplification
polymorphism (GAAP) was designed to amplify DNA sequences in the immediate vicinity of mtDNA coding regions.

Related approaches were developed for other transposons, and

several acronyms describe the different variants of the technique.
For example; Casa et al. (2000) designed a modification of the S-SAP technique that combines an MseI or BfaI-specific AFLP primer with a primer specific for the maize MITE family Heartbreaker (Hbr). The technique was called Hbr display
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Selective Amplification of Microsatellite Polymorphic Loci (SAMPL)

The SAMPL technique was introduced by Morgante and Vogel. It combines the high and controllable multiplexing rate of

the AFLP technique with the high levels of microsatellite

polymorphism by using AFLP-type primers together with compound microsatellite primers.

As in the case of S-SAP, the first step of the SAMPL

procedure is more or less identical to a standard AFLP analysis.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Selective Amplification of Microsatellite Polymorphic Loci (SAMPL)

DNA is digested with one rare and one frequently cutting restriction enzyme, and suitable adapters are ligated to the

resulting restriction fragments. A preamplification step is then

performed with two primers that are complementary to the adapters, plus one specific base extending from the 3-end into

the ligated genomic DNA fragment.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Selective Amplification of Microsatellite Polymorphic Loci (SAMPL)

In the second (selective) amplification, a single AFLP primer (targeting one of the restriction sites at the fragment ends) is combined with a microsatellite-specific primer, annealing to a compound dinucleotide repeat, which can be anywhere internal to the restriction fragment. Compound microsatellites consist of

two or more different simple sequence motifs directly adjacent to

each other, and are common elements of plant genomes.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Selective Amplification of Microsatellite Polymorphic Loci (SAMPL)

SAMPL primers anneal to the junction site of the two microsatellite motifs, providing a perfect 5-anchor in both directions.
(A motif is a distinctive sequence on a protein or DNA, having a threedimensional structure and allows binding interactions to occur)

TA and GC repeats must be of limited length to prevent selfannealing. SAMPL primers are optimally designed to have similar annealing temperatures as their corresponding AFLP primers.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

SAMPL CONTINUEOUS
Theoretically, it would be sufficient to digest
the DNA with a single restriction enzyme.

However, using two enzymes provides more

flexibility because both AFLP and SAMPL can then be applied to the same batch of preamplification products.
A(CA)7(TA)2T T(CT)7(AT)3A

SAMPL banding patterns from chickpea

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Microsatellite-AFLP
Whereas compound microsatellite primers are employed in
SAMPL, 5-anchored microsatellite primers are used in conjunction with AFLP primers in a method coined microsatellite-

AFLP (MFLP) by Yang et al.

Genomic DNA from various plant species was digested with MseI, followed by ligation of an MseI adapter and two rounds of

PCR. Selective PCR was performed with an Mse+3 primer and

one of a set of 74 33P-labeled, anchored microsatellite primers.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Microsatellite-AFLP
Each primer combination revealed complex banding patterns
comprising 70 to 150 bands on sequencing gels. As is the case with Selective Amplification of Microsatellite Polymorphic Loci

(SAMPL), Anchored Microsatellite Primed -PCR (AMP-PCR),

and Random Amplified Microsatellite Polymorphism (RAMP) markers, a subset of the polymorphisms obtained with MFLP are

assumed to be codominant, because they arise from variations in

the number of tandem repeats of the amplified microsatellite.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Methylation-Sensitive Amplified Polymorphisms (M-SAP)

Still another modification of the AFLP technique was developed for monitoring the state of genomic DNA methylation in fungi and plants. This type of approach incorporates the use of methylation sensitive restriction enzymes.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Methylation-Sensitive Amplified Polymorphisms

In M-SAP, template DNA is usually digested with one rare cutter such as EcoRI, and one of a pair of iso-schizomers in place of the

frequent cutter MseI.

Isoschizomers are restriction enzymes that cleave at the same target DNA sequence but display differential sensitivity to the

presence of 5-methylcytosine or 6-methyladenine in their

recognition sequence.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Methylation-Sensitive Amplified Polymorphisms

Pairs of isoschizomers have long been used in conjunction with

traditional RFLP analysis and Southern blot hybridization to compare the state of DNA methylation in various tissues or plant species of

interest
Genomic DNA samples from representatives of three major fungal taxa were digested with EcoRI MspI or HpaII. After ligation of

appropriate adapters, preamplification and selective amplification

steps were performed.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

AFLP-BASED EXPRESSION PROFILING

AFLP-based transcript profiling method allows genome-wide expression analysis in any species without the need for prior sequence knowledge. The general strategy to generate an AFLP-based expression profile of a given tissue involves the isolation of total mRNA, reverse transcription of mRNA into a population of cDNA molecules; restriction, ligation, preamplification, and amplification of double-stranded cDNA by a slightly modified AFLP procedure.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

AFLP-BASED EXPRESSION PROFILING.....

The resulting cDNA-AFLP fragments are separated on highresolution gels. The potential of the AFLP technique for generating mRNA

fingerprints was first recognized in the mid-nineties.

Despite the recent development of high-throughput full-genome

expression screens cDNA-AFLP still remains a useful technique

for several reasons.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

AFLP-BASED EXPRESSION PROFILING.....

First, all developmental stages, tissue types, or environmental
conditions under investigation can be compared in the same experiment, and the kinetics of the expression of defined mRNAs can be monitored

directly
Secondly, fragments of interest can easily be recovered from the gel for further analysis.

Third, cDNA-AFLP is inexpensive, does not require sophisticated

equipment, and can be performed in most laboratories.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants Miscellaneous AFLP Variants

Single-Strand Conformation Polymorphism Analysis

Principle: single strand DNA tend to fold into complex structure

which determines the mobility of the DNA strand in non denaturating gel. The S-SCP technology relies on the observation that the mobility of a single-stranded DNA (ssDNA) molecule on a non-denaturing polyacrylamide gel is a function of both its size and its three dimensional conformation. Because the latter (3D) depends on the precise base composition and the DNA fragments of identical size.
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Single-Strand Conformation Polymorphism Analysis

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Miscellaneous AFLP Variants

Suazo and Hall suggested a highly simplified AFLP protocol that involves; 1. The digestion of DNA and ligation of the adapters in a single reaction; 2. The use of a single, rare-cutting restriction enzyme (EcoRI) instead of two enzymes, the use of one adapter and one type of primer;

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Miscellaneous AFLP Variants

3. Amplification in one step instead of two; 4. Separation of fragments on agarose gels; and 5. Staining with ethidium bromide. Distinct, reproducible, and polymorphic DNA profiles similar of

RAPD patterns were obtained from honey bee (Apis mellifera)

DNA from Europe and Africa.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Minisatellite Variant Repeat Mapping (MVR)

A DNA profiling strategy was invented by Jeffreys and colleagues which relies on the internal heterogeneity of human

minisatellite repeats rather than length variation

Its principle is based on the observation that certain minisatellite arrays consist of heterogeneous basic units. These

units are distinguishable from each other by, for example, the
presence or absence of a restriction site.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Minisatellite Variant Repeat Mapping (MVR)

The earliest version of MVR mapping made use of a strategy reminiscent of the chemical DNA sequencing procedure.

A DNA fragment comprising the human minisatellite MS32 locus was PCR-amplified from genomic DNA, end-labeled, and then

partially digested with HinfI (which cuts once in each 29-bp basic unit)
or HaeIII.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Minisatellite Variant Repeat Mapping (MVR)

Finally, electrophoresis and autoradiography produced a continuous HinfI ladder reflecting the basic repeat length on one hand, and a

discontinuous HaeIII ladder on the other, from which the relative

positions of the variant units could be deduced.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Limitations of Minisatellite Variant Repeat Mapping (MVR)

Although MVR mapping procedures provide unprecedented levels of discriminatory capacity, the method is not generally

useful for DNA profiling in plants.

First, establishing MVR (i.e., cloning and sequencing of a minisatellite, design of suitable primers, etc.) is laborious.

Second, highly efficient individual-specific discrimination is

seldom needed in studies on plants.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Two-Dimensional DNA Typing Methods

In this method, genomic DNA is first digested by a rare-cutting

restriction enzyme such as NotI or AccIII Depending on genome size and complexity, several thousand

restriction fragments are generated, which are labeled by filling the

ends 32P-dNTPs. The size of the labeled fragments is reduced by a second restriction using a six-base cutter such as EcoRV. The resulting EcoRV fragments are separated on an agarose gel.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Two-Dimensional DNA Typing Methods

electrophoresis is carried out in

Two-dimension

polyacrylamide gels, and radioactive spots are visualized by autoradiography. Although somewhat cumbersome, the technique provides a high level of sensitivity, and the genome is broadly represented. Allows one to monitor differences between closely related genomes.

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Single-Nucleotide Polymorphisms (SNPs)

Since the late 1990s, single-nucleotide polymorphisms (SNPs)

have become increasingly popular as a molecular marker system. This type of sequence variation is characterized by a single base substitution at a particular position, a type of polymorphism that is also recognized by the RFLP technique if the SNP occurs in a restriction enzyme recognition site.

Cytosine Thymine Adenine Guanine

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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques

Single-Nucleotide Polymorphisms (SNPs)

To screen for SNPs, genomic DNA from several related test

organisms is amplified by PCR with either a specific pair of primers flanking a known sequence or by arbitrary priming. Single base

substitutions can be recognized by their impact on the mobility of

ssDNA molecules in SSCP gels PCR fragments that are polymorphic among the test organisms are then sequenced, and the SNP is localized. Most SNPs are biallelic markers and therefore highly useful for chipbased microarray technology.
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Introduction to Tropical Forest Genetics| AFLP Analysis

and Its Variants

Miscellaneous Techniques Individual 1

Single-Nucleotide Polymorphisms (SNPs)

A C G T G T C G G T C T T A A A A C G T G T C C G T C T T A A A

Individual 2

A C G T G T C G G T C T T A A A
A C G T G T C G G T C T T A A A

A C G T G T C C G T C T T A A A

Individual 3

A C G T G T C C T A C T T A A A
The position of the SNP is indicated by the box. Individual 1 is heterozygous, while individuals 2 and 3 are homozygous.
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Terima Kasih!
THANK YOU!!
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