Molecular marker technology in studies on plant genetic diversity

PCR based techniques

PCR with arbitrary primers

PCR with Amplified fragment

length polymorp

hisms

Sequence-tagged sites

Microsatellites,

SCARs, CAPS, ISSRsBy

Chanakya Pachi

Molecular Markers

• The development of molecular techniques for genetic analysis has led to a great augmentation in our knowledge of crop genetics and our understanding of the structure and behavior of various crop genomes. • These molecular techniques, in particular the

applications of molecular markers, have been used to scrutinize DNA sequence and it’s variation(s) in and among the crop species and create new sources of genetic variation by introducing new and favorable traits from landraces and related crop species.

Markers can aid selection for target alleles that• Are not easily

assayed in individual plants,

• Minimize linkage drag around the target gene, and

• Reduce the number of generations required to recover a very high percentage of the recurrent parent genetic background

Introduction

• A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. • It can be described as a variation, which may arise

due to mutation or alteration in the genomic loci, that can be observed. • A genetic marker may be a short DNA sequence,

such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites.

Some commonl

y used types of genetic markers

RFLP (or Restriction fragment

length polymorphis

m) 

AFLP (or Amplified fragment

length polymorphis

m

RAPD (or Random

amplification of

polymorphic DNA)   

VNTR (or Variable number tandem repeat) 

Microsatellite polymorphis

m SNP (or Single

nucleotide polymorphis

m)

 STR (or Short tandem

repeat) 

SFP (or Single feature

polymorphism) 

DArT (or Diversity Arrays

Technology)

DNA-based technologies•PCR based techniques • PCR with arbitrary primers• Amplified fragment length polymorphisms (AFLPs)• Sequence-tagged sites• Microsatellites• SCARs• CAPS• ISSRs

PCR BASICS• The polymerase chain reaction• PCR is a rapid, inexpensive and simple

way of copying specific DNA fragments from minute quantities of source DNA material • It does not necessarily require the use of

radioisotopes or toxic chemicals• It involves preparation the sample DNA • A master mix with primers, • Followed by detecting reaction products

PCR procedures: steps• Denaturation: • DNA fragments are heated at high

temperatures, which reduce the DNA double helix to single strands. • These strands become accessible to

primers• Annealing: • The reaction mixture is cooled down.• Primers anneal to the complementary

regions in the DNA template strands, and double strands are formed again between primers and complementary sequences

• Extension: • The DNA polymerase synthesises a

complementary strand. • The enzyme reads the opposing strand

sequence and extends the primers by adding nucleotides in the order in which they can pair. • The whole process is repeated over and

over

• The DNA polymerase, known as 'Taq polymerase', is named after the hot-spring bacterium Thermus aquaticus from which it was originally isolated. • The enzyme can withstand the high temperatures

needed for DNA-strand separation, and can be left in the reaction tube. • The cycle of heating and cooling is repeated over and

over, stimulating the primers to bind to the original sequences and to newly synthesised sequences. • The enzyme will again extend primer sequences. • This cycling of temperatures results in copying and then

copying of copies, and so on, leading to an exponential increase in the number of copies of specific sequences. • Because the amount of DNA placed in the tube at the

beginning is very small, almost all the DNA at the end of the reaction cycles is copied sequences. The reaction products are separated by gel electrophoresis. • Depending on the quantity produced and the size of the

amplified fragment, the reaction products can be visualized directly by staining with ethidium bromide or a silver-staining protocol, or by means of radioisotopes and autoradiography

PCR procedures: cycle 1

PCR procedures: cycle 2

PCR procedures: cycle 3

CONDITIONSStep Reaction Temperature (°C) Time (Seconds)

1 Initial Denaturation 94 120

2 Cycle Denaturation 94 15

3 Cycle Annealing 58 30

4 Cycle Extension 72 80

5 Repeat from Step 2 to 4 30 Cycles

6 Final extension 72 600

7 Infinite Hold 4

PCR with arbitrary primers• MAAP (multiple arbitrary amplicon

profiling) is the acronym proposed to cover the three main technologies that fall in this category:• Random amplified polymorphic DNA

(RAPD) • DNA amplification fingerprinting

(DAF) • Arbitrarily primed polymerase chain

reaction (AP-PCR)

RAPD• Random Amplified Polymorphic DNA• It is a type of PCR reaction, but the segments

of DNA that gets amplified are random. • The researcher performing RAPD creates

several arbitrary, short primers (8–12 nucleotides)

• Then proceeds with the PCR using a large template of genomic DNA, to enable amplification of fragments.

• By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

• No knowledge of the DNA sequence for the targeted genome is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where.

• This makes the method popular for comparing the DNA of biological systems that have not had the privileged attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank).

• Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples.

• Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats.

• In recent years, RAPD has been used to characterize, and trace, the phylogeny of diverse plant and animal species.

How it Works?• Unlike traditional PCR analysis, RAPD does not

require any specific knowledge of the DNA sequence of the target organism: • The identical 1somer primers may or may not

amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. • For example, no fragment is produced if

primers annealed too far apart. • Therefore, if a mutation has occurred in the

template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel

Diagrammatic summary

• This picture shows an image of a very high quality RAPD gel. Both, presence and absence of most bands are very clear and the background is transparent.

• The researcher would have no doubts while selecting bands and collecting data from this gel.

• Consequently, the interpretation of results can be very confident.

Interpreting RAPD banding patterns

• DNA polymorphism among individuals can be because of:• Mismatches at the primer site• The appearance of a new primer site• The length of the amplified region between primer sites

• Because of the nature of RAPD markers, only the presence or absence of a particular band can be assessed. • Criteria for selecting scoring bands:

• Reproducibility—need to repeat experiments• Thickness• Size• Expected segregation observed in a mapping population

Examples

Bell pepper

Modern roseMeadow fescue

Amplified fragment length polymorphisms(AFLPs)

• AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering.

• Developed in the early 1990s by Keygene• AFLP uses restriction enzymes to digest genomic

DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments.

• A subset of the restriction fragments is then selected to be amplified.

• This selection is achieved by using primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments.

• The amplified fragments are separated and visualized on polyacrylamide gels, either through autoradiography or fluorescence methodologies, or via automated capillary sequencing instruments

Digestion

Amplification

Electrophores

isAFLP

The procedure of this technique is divided into three steps:• Digestion of total cellular DNA with one or more

restriction enzymes and ligation of restriction halfsite specific adaptors to all restriction fragments.• Selective amplification of some of these fragments

with two PCR primers that have corresponding adaptor and restriction site specific sequences.• Electrophoretic separation of amplicons on a gel

matrix, followed by visualisation of the band pattern.

Main features:• A combination of the RFLP and PCR technologies• Based on selective PCR amplification of restriction

fragments from digested DNA• Highly sensitive method for fingerprinting DNA of any

origin and complexity• Can be performed with total genomic DNA or with cDNA

('transcript profiling')• DNA is digested with two different restriction enzymes• Oligonucleotide adapters are ligated to the ends of the

DNA fragments• Specific subsets of DNA digestion products are amplified,

using combinations of selective primers• Polymorphism detection is possible with radioisotopes,

fluorescent dyes or silver staining

DNA digestion and ligation• One restriction enzyme is a frequent cutter (four-base recognition site, e.g. MseI)

• The second restriction enzyme is a rare cutter (six-base recognition site, e.g. EcoRI)

• Specific synthetic double-stranded adapters for each restriction site are ligated to the DNA fragments generated

Summarizing the technology

Interpreting AFLP bands• The AFLP technique detects polymorphisms arising from

changes (presence or size) in the restriction sites or adjacent to these• Different restriction enzymes can be used, and different

combinations of pre- and selective nucleotides willincrease the probability of finding useful polymorphisms• The more selective bases, the less polymorphism will be

detected • Bands are usually scored as either present or absent• Heterozygous versus homozygous bands may be

detected,based on the thickness of the signal, although this can betricky

Applications• Genetic diversity

assessment• Genetic distance

analysis• Genetic fingerprinting• Analysis of germplasm

collections• Genome mapping• Monitoring diagnostic

markers

Examples

• Limonium sp.• Stylosanthes sp.• Indian mustard

PCR-based technologies

Sequences-tagged sites

Microsatellites SCARs CAPS ISSRs

Unlike arbitrary primers, STS rely on some degree of sequence knowledge

Markers based on STS are codominant

They tend to be more reproducible becauselonger primer sequences are used

Require the same basic laboratory protocols and equipment as standard PCR

Sequence-tagged sites as markers

Microsatellites

• Microsatellites are also called simple sequence repeats (SSRs)• Microsatellites are short tandem repeats (1-

10 bp)• To be used as markers, their location in the

genome of interest must first be identified• Polymorphisms in the repeat region can be

detected by performing a PCR with primers designed from the DNA flanking region

Identifying microsatellite regions

Structure

Selecting primers

Methodology and visualisation• Methodology:• DNA extraction• PCR with primers specific for microsatellite flanking

regions• Separation of fragments

• Visualization:• By agarose gel electrophoresis, using ethidium

bromide staining and UV light, or • Acrylamide gels using silver staining or radioisotopes,

or • Through automated sequencers, using primers

prelabelled with fluorescence.• Data analysis

Staining with Ethidium bromide

Staining with Silver Nitrate

• Advantages:• Require very little and not necessarily high quality

DNA • Highly polymorphic • Evenly distributed throughout the genome • Simple interpretation of results • Easily automated, allowing multiplexing • Good analytical resolution and high

reproducibility

• Disadvantages: • Complex discovery procedure • Costly

Applications:

Individual genotyping

Germplasm evaluation

Genetic diversity

Genome mapping

Phylogenetic studies

Evolutionary studies

Sequence characterized amplified

regions (SCARs)

• SCARs take advantage of a band generated through a RAPD experiment

• They use 16-24 bp primers designed from the ends of cloned RAPD markers

• This technique converts a band—prone to difficulties in interpretation and/or reproducibility—into being a very reliable marker

Steps to obtain SCAR polymorphismsA potentially

interesting band is

identified in a RAPD gel

The band is cut out of the gelThe DNA fragment

is cloned in a vector and sequenced

Specific primers (16-24 bp long) for that DNA fragment are designed

Re-amplification of the template DNA

with the new primers will show a new and simpler PCR pattern

Diagram of the SCAR procedure

Advantages Disadvantages

Simpler patterns than RAPDs

Robust assay due to

the design of specific long

primers

Mendelian inheritance.

Sometimes convertible

to codominant markers

Require at least a small

degree of sequence knowledge

Require effort and

expense in designing

specific primers for each locus

Cleaved amplified polymorphicsequence (CAPS)

• This method is based on • The design of specific primers, • Amplification of DNA fragments, and • Generation of smaller, possibly variable,

fragments by means of a restriction enzyme• This technique aims to convert an

amplified band that does not show variation into a polymorphic one

Steps for generating CAPSA band, DNA, gene sequence

or other type of marker isidentified as important

Either the band is detected through PCR (and cut out of

the gel, and the fragment cloned and sequenced) or the fragment sequence is already

available

Specific primers are designed from the fragment

sequence

The newly designed primers are used to amplify the

template DNA

The PCR product is subjected to digestion by a

panel ofrestriction enzymes

Polymorphism may be identified with some of the

enzymes

Generating CAPS

Advantages

• Robust assay

because specific long

primers are designed

• Codominant markers

• Benefit from markers

that may have

already been

mapped

• Identify

polymorphisms in

markers that were

previously not

informative

Disadvantages

• Require at least a

small amount of

sequence knowledge

• Effort and expense

required to design

specific primers

for each locus

Inter-simple sequence repeats (ISSRs)

• They are regions found between microsatellite repeats• The technique is based on PCR

amplification ofintermicrosatellite sequences• Because of the known abundance of

repeat sequences spread all over the genome, it targets multiple loci

Identifying ISSR polymorphisms

• A typical PCR is performed in which primers have been designed, based on a microsatellite repeat sequence, and extended one to several bases into the flanking sequence as anchor points. • Different alternatives are possible:

• Only one primer is used• Two primers of similar characteristics are used• Combinations of a microsatellite-sequence

anchored primer with a random primer (i.e. those used for RAPD)

Designing primers for ISSR polymorphisms

• The diagram above presents three different items:

• The original DNA sequence in which two different repeated sequences (CA), inversely oriented, are identified. Both repeated sections are, in addition, closely spaced.

• If primers were designed from within the repeated region only, the interrepeat section would be amplified but locus-specificity might not be guaranteed. In the second row, a PCR product is shown as a result of amplification from a 3'-anchored primer (CA)n NN at each end of the interrepeat region. CA is the repeat sequence that was extended by NN, two nucleotides running into the interrepeat region.

• Alternatively, anchors may be chosen from the 5' region. The PCR product in the third row is a result of using primers based on the CA repeat but extended at the 5' end by NNN and NN, respectively

Advantages• Do not require prior sequence

information• Variation within unique regions of

the genome may be found at several loci simultaneously

• Tend to identify significant levels of variation

• Microsatellite sequence-specific• Very useful for DNA profiling,

especially for closely related species

Disadvantages• Dominant markers• Polyacrylamide gel

electrophoresis and detection with silver staining or radioisotopes may be needed

Latest strategies

DNA sequencing Expressed sequence tags (ESTs)

Microarray technology

Diversity array technology (DArT)

Single nucleotide polymorphisms

(SNPs)

References…

References

Plant Biotechnology and Molecular

Markers• P.S. Srivastava• Alka Narula

• Sheela Srivastava

Using molecular marker

technology in studies on plant genetic diversity• M. Carmen de

Vicente• Theresa Fulton

Molecular Markers

• Ashwani Kumar• Scientific Blogging 2.0