Assignment Work

On
Molecular Markers


GP-509 (Biotechnology for Crop
Improvement )











Course Teacher :
Dr.K.K.Tiwari
Assistant professor ,
C.P.College of Agriculture,
S.D.A.U. , Sardar Krushinagar




Submitted by :
Satyendra singh
04-agrma-01157-2013
M.Sc. (Agri.) GPB
C.P. College of Agriculture,
S.D.A.U. , Sardar Krushinagar


Marker
Marker can be any sign or character which are considered to be associated with a particular trait
morphologically and/or genetically.
Criteria for desirable markers
1. High level of genetic polymorphism.
2. Co- dominance (so that heterozygotes can be distinguished from homozygotes).
3. Clear distinct allele features (so that different alleles can be identified easily).
4. Even distribution on the entire genome.
5. Neutral selection (without pleiotropic effect).
6. Easy detection (so that the whole process can be automated).
7. Low cost of marker development and genotyping.
8. High duplicability (so that the data can be accumulated and shared between laboratories).

Types of Markers
Genetic markers fall into one of the three broad classes:
1. Morphological Markers
2. Biochemical Marker
3. Molecular Markers

1. Morphological Markers
These are based on visually assessable traits (morphological and agronomic traits).
Morphological markers represent genetic polymorphisms which are visible as
differences in appearance.
Pink colour of mango related to sweetness.
Spotted guava is considered good in quality.
Morphological markers are usually mapped by classical two- or three-point linkage tests.

Advantages Limitations
-Readily available
-Less expensive
-Skilled labour and laboratory not required.
-Limited in number
-Narrow in diversity
-We cannot combine 3-4 markers
-Unknown genomic information


2. Biochemical Marker (Isozyme): Tanksley and Orton 1983
These are based on gene product.
Two or more enzymes that catalyse the same reaction but are encoded by different
genes are called isozymes.
Isozymes originate through amino acid alterations, which cause changes in net charge,
conformation of the enzyme molecules and their electrophoretic mobility. This change in
electric charge and conformation can be detected by gel electrophoresis.
Applications:
Allozymes can be applied in many population genetics studies, including measurements
of out crossing rates, population structure and population divergence.
Allozymes are particularly useful at the level of conspecific populations and closely
related species, and are therefore useful to study diversity in crops and their relatives.
Allozymes have been used, in concert with other markers, for fingerprinting purposes,
and diversity studies, to study interspecific relationships, the mode of genetic inheritance, and
allelic frequencies in germplasm collections over serial increase cycles in germplasm banks,
and to identify parents in hybrids.
Advantages Limitations
-Useful for evolutionary studies
-Isolation lot easier than that of DNA
-Can be used across species
-No radioactive labelling
-No need for sequence information
-Tedious in handling
-Limited in polymorphism
-Expensive (each system is unique)
-Have to know the location of the tissue
-Influenced by environment
-stage specific

3. Molecular Markers
These are relying on a DNA assay.
Molecular markers are constant landmarks in the genome. They are identifiable DNA
sequences, found at specific locations of the genome, and transmitted by the standard laws of
inheritance from one generation to the next.

DNA markers are the best candidates for efficient evaluation and selection of plant
material. Since DNA markers segregate as single genes and they are not affected by the
environment.
The uses of molecular markers are based on the naturally occurring DNA
polymorphism.
Genetic polymorphism is defined as the simultaneous occurrence of a trait in the same
population of two discontinuous variants or genotypes.
Basis of Genetic polymorphism
1. Unequal recombination
2. Replication slippage
3. Single nucleotide polymorphism
4. INDEL

Use of genetic markers in plant breeding
1. Gaining a better understanding of breeding materials and breeding system.
Molecular markers can be used to characterize germplasm, develop linkage maps, and
identify heterotic patterns.
2. Rapid introgression of simply inherited traits.
Introgression of genes into another genetic background involves several rounds of
tedious backcrosses.
When the source of desirable genes is a wild species, linkage drag becomes a problem.
Using markers and QTL analysis we can reduce linkage drag.
3. Early generation testing.
Breeding for compositional traits, such as high lysine and high tryptophan genes in
maize, can be advanced with early detection and selection of desirable segregants.
4. Unconventional problem solving.
The recessive linkage drag was removed by using DNA markers flanking the
introgression to pre-select for individuals that were recombinant in the vicinity of the gene.
Breeding Lettuce resistant to aphid Nasonovia ribisnigi from a wild lettuce Lactuca
virosa by repeated backcross assisted by molecular markers.
The life span of new cultivars can be extended through the technique of gene
pyramiding (i.e., transferring multiple disease resistance genes into one genotype) for
breeding disease resistant cultivars.
Marker assisted backcross can be used to achieve this rapidly, especially for genes
with indistinguishable phenotypes.
5. Plant cultivar identification.
Molecular markers are effective in cultivar identification for protecting proprietary
rights as well as authenticating plant cultivars


Classification of molecular markers
a) Method of analysis
(Hybridization-based or PCR based markers).
b) Mode of gene action
(Dominant or co-dominant markers).
c) Mode of transmission
(biparental nuclear inheritance, maternal nuclear inheritance, maternal organelle
inheritance, or paternal organelle inheritance).

DNA markers and related major molecular techniques
Southern blot-based markers / hybridisation based markers
Restriction fragment length polymorphism (RFLP)
Single strand conformation polymorphic RFLP (SSCP-RFLP)
Denaturing gradient gel electrophoresis RFLP (DGGE-RFLP)
PCR-based markers
Randomly amplified polymorphic DNA (RAPD)
Sequence tagged site (STS)
Sequence characterized amplified region (SCAR)
Random primer-PCR (RP-PCR)
Arbitrary primer-PCR (AP-PCR)
Oligo primer-PCR (OP-PCR)
Single strand conformation polymorphism-PCR (SSCP-PCR)
Small oligo DNA analysis (SODA)
DNA amplification fingerprinting (DAF)
Amplified fragment length polymorphism (AFLP)
Sequence-related amplified polymorphism (SRAP)
Target region amplified polymorphism (TRAP)
Insertion/deletion polymorphism (Indel)
Repeat sequence-based markers
Satellite DNA (repeat unit containing several hundred to thousand base pairs (bp) )
Microsatellite DNA (repeat unit containing 25 bp)
Minisatellite DNA (repeat unit containing more than 5 bp)
Simple sequence repeat (SSR) or simple sequence length polymorphism (SSLP)
Short repeat sequence (SRS)
Tandem repeat sequence (TRS)
mRNA-based markers
Differential display (DD)
Reverse transcription PCR (RT-PCR)
Differential display reverse transcription PCR (DDRT-PCR)
Representational difference analysis (RDA)
Expression sequence tags (EST)
Sequence target sites (STS)
Serial analysis of gene expression (SAGE)
Single nucleotide polymorphism-based markers
Single nucleotide polymorphism (SNP


Classification of markers
S.No. Name of the
Technique
Discoverer
A. Biochemical markers Allozymes
Tanksley and Orton 1983;
Kephart 1990; May 1992
B. Molecular markers

i) Non-PCR
2
based
techniques
Restriction Fragment Length
Polymorphisms (RFLP)
Botstein et al. 1980; Neale and
Williams 1991

Minisatellites or Variable Number of
Tandem Repeats (VNTR)
Jeffreys et al.. 1985
ii) PCR-based techniques
DNA sequencing
Multi-copy DNA, Internal Transcribed
Spacer regions of nuclear ribosomal
genes (ITS)
Takaiwa et al. 1985; Dillon et al.
2001

Single-copy DNA, including both
introns and exons
Sanger et al. 1977; Clegg 1993a

Sequence-Tagged Sites
(STS)
Microsatellites, Simple Sequence
Repeat (SSR), Short Tandem Repeat
(STR), Sequence Tagged
Microsatellite (STMS) or Simple
Sequence Length Polymorphism
(SSLP)
Litt and Lutty (1989),Hearne et
al. 1992; Morgante and Olivieri
1993; Jarne and Lagoda 1996

Amplified Sequence Length
Polymorphism (ASLP)
Maughan et al. 1995

Sequence Characterized Amplified
Region (SCAR)
Michelmore et al. (1991); Martin
et al. (1991); Paran and
Michelmore 1993

Cleaved Amplified Polymorphic
Sequence (CAPS)
Akopyanz et al. 1992;
Konieczny and Ausubel 1993

Single-Strand Conformation
Polymorphism (SSCP)
Hayashi 1992

Denaturing Gradient Gel
Electrophoresis (DGGE)
Riedel et al. 1990

Thermal Gradient Gel Electrophoresis
(TGGE)
Riesner et al. 1989
Heteroduplex Analysis (HDA) Perez et al. 1999; Schneider et al.
1999
Denaturing High Performance Liquid
Chromatography (DHPLC)
Hauser et al. 1998; Steinmetz et
al. 2000; Kota et al. 2001
Multiple Arbitrary Amplicon Profiling (MAAP) Caetano-Anolles 1994;
Caetano-Anolles et al. 1992

Random Amplified Polymorphic DNA
(RAPD)
Williams et al. 1990; Hadrys et
al. 1992
DNA Amplification Fingerprinting
(DAF)
Caetano-Anolles et al. 1991

Arbitrarily Primed Polymerase Chain
Reaction (AP-PCR)
Welsh and McClelland 1990;
Williams et al. 1990
Inter-Simple Sequence Repeat (ISSR)
Zietkiewicz et al. 1994; Godwin
et al. 1997
Single Primer Amplification Reaction
(SPAR)
Staub et al. 1996

Directed Amplification of
Minisatellites DNA (DAMD)
Heath et al. 1993; Somers and
Demmon 2002

Amplified Fragment Length
Polymorphism (AFLP)
Vos et al. 1995

Selectively Amplified Microsatellite
Polymorphic Loci (SAMPL)
Witsenboer et al. 1997



Molecular basis of major DNA markers. Parts AE show different ways in which
DNA markers (listed below each diagram) can be generated. The cross in part A indicates
that mutation has eliminated the priming site. Abbreviations: as defined in Table ; VNTR,
variable number of tandem repeat; CAPS, a DNA marker generated by specific primer PCR
combined with RFLP; ISSR, inter simple sequence repeat.
Hybridization-based molecular markers
DNA-based marker systems can be described as hybridization-based makers when the
DNA profiles are visualized by hybridizing fragments to labelled probes.
It include:
I. Restriction fragment length polymorphisms (RFLPs)

Restriction fragment length polymorphisms (RFLPs):
Botstein et al. 1980
The restriction fragment length polymorphisms (RFLPs) marker technology is the first
generation of DNA markers and one of the best for plant genome mapping.
Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms
are differentiated by analysis of patterns derived from cleavage of their DNA. When two
different organisms are digested with similar Restriction Endonucleases, it cut within genome
at a particular sequence in both the organisms. The similarity of the patterns generated can be
used to differentiate species from one another.
RFLP is the most widely used hybridization-based molecular marker. RFLP markers
were first used in 1975 to identify DNA sequence polymorphisms for genetic mapping of a
temperature-sensitive mutation of adeno-virus serotypes (Grodzicker et al., 1975). It was then
used for human genome mapping (Botstein et al., 1980), and later adopted for plant genomes
(Helentjaris et al., 1986; Weber and Helentjaris, 1989).
Procedure for RFLP
(i) DNA isolation a significant amount of DNA must be isolated from the sample and
purified to a fairly stringent degree as contaminants can often interfere with the restriction
enzyme and inhibit its ability to digest the DNA.
(ii) Restriction Digest - Restriction enzyme is added to purified genomic DNA under
buffered conditions. The enzyme cuts at recognition sites throughout the genome and leaves
behind hundreds of thousands of fragments.
(iii) Gel electrophoresis The digest is run on a gel and when visualized appears a smear
because of the large number of fragments.
(iv) Southern blotting-transfer to nitrocellulose or nylon membrane filter
(v) Probe visualization Because of the large number of fragments, probes must be
constructed to visualize more specific bands in the digest. These probes consist of radio
labelled oligonucleotide sequences which will anneal to the fragment sequences so that that
they may be visualized on photographic paper using a technique called autoradiography.
(vi) Analysis- Number of RFLP loci can be analysed after autoradiography.





RFLP workflow from DNA extraction to radio-autograph.Modified from Xu and Zhu (1994).


Advantages Limitations
Easy to score
Sequence used as probe need not be known
Transferable across populations
High genomic abundance
Highly reproducible
Good genome coverage
Can be used across species
Needed for map based cloning
requires the presence of high quantity and quality of
DNA
depends on the development of specific probe libraries
Technique is not amenable for automation.
Level of polymorphism is low, and few loci are
detected per assay.
Time consuming, laborious, and expensive
Usually requires radioactively labelled probes.

PCR-based molecular markers
PCR-based molecular markers operate through a PCR process i.e. directly amplifying a
specific short segment of DNA without the use of a cloning method. An attractive feature of a
PCR-based marker system is that only a minute amount of DNA is needed for a task and
yields higher throughput than RFLP
PCR-based techniques are of two types depending on the primers used for amplification:
1) Arbitrary primed PCR techniques that developed without prior sequence information.
e.g., AP-PCR, DAF, RAPD, AFLP, ISSR

2) Site-targeted PCR techniques that developed from known DNA sequences.
e.g., EST, CAPS, SSR, SCAR, STS

Arbitrarily Amplified DNA Markers
RAPD (random amplified polymorphic DNA), AP-PCR (arbitrarily primed PCR) and
DAF (DNA amplification fingerprinting) are collectively termed as multiple arbitrary
amplicon profiling (MAAP; Caetano-Annolles, 1994).
These three techniques were the first to amplify DNA fragments from any species
without prior sequences information.
The key innovation of RAPD, AP-PCR and DAF is the use of a single arbitrary
oligonucleotide primer (10mer primer) to amplify template DNA without prior knowledge of
the target sequence.

The difference among MAAP techniques include:
Modifications in amplification profiles by changing primer sequence and length,
annealing temperature
The number of PCR cycles used in a reaction
The thermo-stable DNA polymerase used
Enzymatic digestion of template DNA or amplification products and
Alternative methods of fragment separation and staining.

Random amplified polymorphic DNA (RAPD):
Williams et al. 1990
RAPD is a PCR-based marker system in which the total genomic DNA is amplified
using a single short (10mer) random primer. It differs from traditional PCR analysis in that it
does not require specific knowledge of the DNA sequence of the target organism.

Since it is a dominant marker, polymorphisms can be detected by simply presence or
absence band on gel.

The RAPD protocol usually uses a 10 bp arbitrary primer at constant low annealing
temperature (generally 34 37
o
C).

Criteria for RADP primers giver by William et. al.(1990) :
I. A minimum of 40% GC content (50 - 80% GC content is generally used), and
II. The absence of palindromic sequence (A base sequence that reads exactly the same
from right to left as from left to right).

Because G-C bond consists of three hydrogen bridges and the A-T bond of only two, a
primer-DNA hybrid with less than 50% GC will probably not withstand the 72
o
C temperature
at which DNA elongation takes place by DNA polymerase.
The resulting PCR products are generally resolved on 1.5- 2.0% agarose gels and
stained with ethidium bromide (EtBr).

Advantages Limitations
High genomic abundance
Good genome coverage
No sequence information
Ideal for automation
Less amount of DNA (poor DNA acceptable)
No radioactive labelling
Relatively faster
No probe or primer information
Dominant markers
Not reproducible
Cannot be used across species
Not very well-tested
Difficult to analyse
Low transferability
AFLP (amplified fragment length polymorphism):
Vos et al. 1995
AFLP technique combines the power of RFLP with the flexibility of PCR-based
technology by ligating primer recognition sequences (adaptors) to the restricted DNA.
The key feature of AFLP is its capacity for genome representation: The simultaneous
screening of representative DNA regions distributed randomly throughout the genome. AFLP
markers can be generated for DNA of any organism without initial investment in
primer/probe development and sequence analysis.
Both good quality and partially degraded DNA can be used for digestion but the DNA
should be free of restriction enzyme and PCR inhibitors

Procedure for AFLP


Polyacrylamide gel electrophoresis (PAGE) provides maximum resolution of AFLP
banding patterns to the level of single-nucleotide length differences, whereas fragment length
differences of less than ten nucleotides are difficult to score on agarose gels(2%-2.5% gel).
(i) DNA is cut with two specific restriction
enzymes, one frequent cutter (3 bp
recognition site) and one rare cutter (6 bp
recognition site).
(ii) Oligonucleotide adapters are ligated to
the ends of each fragment. One end with a
complimentary sequence for the rare cutter
and the other with the complimentary
sequence for the frequent cutter. This way
only fragments which have been cut by the
frequent cutter and rare cutter will be
amplified
(iii) Primers are designed from the known
sequence of the adapter, plus 1-3 selective
nucleotides which extend into the fragment
sequence. Sequences not matching these
selective nucleotides in the primer will not
be amplified.
(iv) PCR performed

(v) Visualized on agarose gels with ethidium
bromide OR on denaturing polyacrylamide
gels with autoradiography AgNO3 staining
OR automatic DNA sequencers.


Advantages Limitations
Highly reliable and reproducible.
DNA sequence information not required.
Information-rich due to its ability to analyse a
large number of polymorphic loci
simultaneously (effective multiplex ratio) with
a single primer combination on a single gel

Co-migrating AFLP amplification products
are mostly homologous and locus specific


more number of steps are required.
Requires template DNA free of inhibitor
compounds that interferes with the restriction
enzyme.
This technique requires the use of
polyacrylamide gel in combination with
AgNO3 staining, radioactivity, or fluorescent
methods of detection, which will be more
expensive and laborious than agarose gels.
It involves additional cost to purchase both
restriction and ligation enzymes as well as
adapters.
AFLP loci are dominant, hence does not
differentiate dominant homozygotes from
heterozygotes. This reduces the accuracy of
AFLP markers in population genetic analysis,
genetic mapping, and marker assisted selection.



ISSR (inter-simple sequence repeat): Zietkiewicz et al. 1994

ISSR involves amplification of DNA segments present at an amplifiable distance in
between two identical microsatellite repeat regions oriented in opposite direction.
The technique uses microsatellites as primers in a single primer PCR reaction targeting
multiple genomic loci to amplify mainly inter simple sequence repeats of different sizes.
The microsatellite repeats used as primers for ISSRs can be di-nucleotide, tri-
nucleotide, tetra-nucleotide or penta-nucleotide. The primers used can be either unanchored
or more usually anchored at 3` or 5` end with 1 to 4 degenerate bases extended into the
flanking sequences.
ISSR uses longer primers (1530 mer) as compared to RAPD primers (10 mer), which
permit the subsequent use of high annealing temperature leading to higher stringency.
The annealing temperature depends on the GC content of the primer used and ranges from 45
to 65
o
C. The amplified products are usually 2002000 bp long and amenable to detection by
both agarose and polyacrylamide gel electrophoresis.
The technique is simple, quick, and the use of radioactivity is not essential. ISSR
markers usually show high polymorphism.

Detection of ISSR

Polyacrylamide gel electrophoresis (PAGE) in combination with radioactivity was
shown to be most sensitive, followed by PAGE with AgNO3 staining and then agarose gel
(2%-2.5% gel) with EtBr system of detection.


Advantages Limitations
Show high polymorphism
Simple
Quick
Use of radioactivity is not essential

Reproducibility,
Dominant inheritance
Homology of co- migrating amplification
products



Site-targeted PCR techniques

Microsatellites: Litt and Lutty (1989
Microsatellites, also known as, Simple Sequence Repeat SSRs, are tandemly repeated
units of short nucleotide motifs that are 16 bp long. Di-, tri- and tetra-nucleotide repeats
such as (CA)n, (AAT)n and (GATA)n are widely distributed throughout the genomes of
plants and animals.
Presence of high level of allelic variation, make them to utilise as genetic markers.
Microsatellite sequences are especially suited to distinguish closely related genotypes;
because of their high degree of variability, these are, favoured in population studies and for
the identification of closely related cultivars.

PCR fragments are usually separated on polyacrylamide gels in combination with
AgNO3 staining, autoradiography or fluorescent detection systems. Agarose gels (usually
3%) with EtBr can also used when differences in allele size among samples is larger than 10
bp.

SSR markers are characterized by their hypervariability, reproducibility, codominant
nature, locus specificity and random dispersion throughout most genomes. SSRs are reported
to be more variable than RFLPs or RAPDs.

Origin of SSR
The predominant mutation mechanism in microsatellite tracts is slipped-strand
mispairing.
When slipped-strand mispairing occurs within a microsatellite array during DNA
synthesis, it can result in the gain or loss of one, or more, repeat units depending on whether
the newly synthesized DNA chain loops out or the template chain loops out, respectively. The
relative propensity for either chain to loop out seems to depend in part on the sequences
making up the array, and in part on whether the event occurs on the leading (continuous DNA
synthesis) or lagging (discontinuous DNA synthesis) strand . SSR allelic differences are the
results of variable numbers of repeat units within the microsatellite structure. The repeated
sequence may be, consisted of two, three or four nucleotides (di-, tri-, and tetra-, nucleotide
repeats, respectively).
Method of SSR genotyping
Four methods:
SSRs on agarose gel: These assays can usually
distinguish alleles which differ by 24 bp or
more.

PAGE based: to separate radio-labelled or
silver-stained PCR products by denaturing or
non- denaturing PAGE using ethidium
bromide or SYBR staining.


Semi-automated SSR : genotyping can be carried
out by assaying fluorescently labelled PCR
products for length variants on an automated DNA
sequencer.



Drawback of fluorescent SSR genotyping is the
cost of end-labelling primers with the necessary
fluorophores. e.g. 6-carboxy-fluorescine (FAM),
hexachloro- 6-carboxy-flurescine (HEX) or tetra
chloro- 6-carboxy-fluorescine (TET).

SSRs assayed on polyacrylamide gels typically
show characteristic stuttering. Stutter bands are
artefacts produced by DNA polymerase slippage.
Stutters are multiple near-identical ladders of
PCR products which are one or two nucleotides
shorter or longer than the full length product
Stuttering reduces the resolution between alleles
such that 2- or possibly 4-bp differences between
alleles cannot be sharply or unequivocally
distinguished on polyacrylamide gels.



Advantages Limitations
High genomic abundance
Highly reproducible
Fairly good genome coverage
High polymorphism
No radioactive labelling
Easy to automate
Multiple alleles
Cannot be used across species
Need sequence information
Not well-tested
High mutation rate
Primer preparation is time consuming



CAPS (cleaved amplified polymorphic sequence):
Akopyanz et al. 1992
CAPS is a combination of the PCR and RFLP, and it was originally named PCR-RFLP
(Maeda et al., 1990). The technique involves amplification of a target DNA through PCR,
followed by digesting with restriction enzyme.
CAPS markers rely on differences in restriction enzyme digestion patterns of PCR
fragments caused by nucleotide polymorphism between samples.
Critical steps in the CAPS marker approach include DNA extraction, PCR conditions,
and the number or distribution of polymorphic sites.
The ability of CAPS to detect DNA polymorphism is not as high as SSRs and AFLPs
because
Nucleotide changes affecting restriction sites are essential for the detection of DNA
polymorphism by CAPS.
The development of CAPS markers is only possible where mutations disrupt or create
a restriction enzyme recognition site.

A technique known as derived-CAPS (dCAPS) is developed that can eliminate the
problems related with CAPS markers by generating mismatches in a PCR primer, which are
subsequently used to create a polymorphism based on the target mutation.






SCAR (sequence characterized amplified region):
Michelmore et al. (1991)
A SCAR marker is a genomic DNA fragment that is identified by PCR amplification
using a pair of specific oligonucleotide primers of 15-30 pb size.
By using longer PCR primers, SCARs do not face the problem of low reproducibility
generally encountered with RAPDs.
SCARs are derived by cloning and sequencing the two ends of RAPD markers that
appeared to be diagnostic for specific purposes (e.g., a RAPD band present in disease
resistant lines but absent in susceptible lines).


Advantages Limitations
Quick
Easy to use
High reproducibility
Locus-specific.
Due to the use of PCR, only low quantities of
template DNA are required (10100 ng per
reaction).

need for sequence data to design the PCR
primers


STS (sequence tagged site): Olsen et al. (1989)

STS was first developed by Olsen et al. (1989) as DNA landmarks in the physical
mapping of the human genome, and later adopted in plants.
STS is a short, unique sequence whose exact sequence is found nowhere else in the
genome. Two or more clones containing the same STS must overlap and the overlap must
include STS.

STS markers are co-dominant, highly reproducible, suitable for high throughput and
automation, and technically simple for use.



Advantages Limitations
Useful in preparing contig maps
No radioactive labelling
Fairly good genome coverage
Highly reproducible
Can use filters many times
Laborious
Cannot detect mutations out of the target sites
Need sequence information
Cloning and characterization of probe are
Required




SNP (single nucleotide polymorphism): Tautz and Renz (1984)
A single nucleotide polymorphism SNP is an individual nucleotide base difference
between two DNA sequences.
SNPs can be categorized according to nucleotide substitution as:
Transitions (C/T or G/A) or
Transversions (C/G, A/T, C/A or T/G).
A single base variation in cDNA (mRNA) is considered as SNPs and therefore SNPs
provide the ultimate form of molecular marker.

For a variation to be considered a SNP, it must occur in at least 1% of the population.
Two of every three SNPs involve the replacement of cytosine (C) with thymine (T).

SNPs may fall within coding sequences of genes, non-coding regions of genes or in the
inter-genic regions between genes at different frequencies in different chromosome regions.


SNPs within a coding sequence will not necessarily change the amino acid sequence of
the protein that is produced due to redundancy in the genetic code.
A SNP in which both forms lead to the same polypeptide sequence is termed
synonymous, while if a different polypeptide sequence is produced they are non-
synonymous.
SNPs that are not in protein coding regions may still have consequences for gene
splicing, transcription factor binding or the sequence of non-coding RNA.

Discovery of SNP
Three general categories are possible for discovery of SNP:
1. In-vitro discovery, where new sequence data is generated;
2. In-silico methods that rely on the analysis of available sequence data; and
3. Indirect discovery, where the base sequence of the polymorphism remains unknown.
A convenient method for detecting SNPs is RFLP (SNP-RFLP) or by using the CAPS
marker technique. i.e. cut with a restriction enzyme and analyse the sequence data stored in
the major databases and identify SNPs.
Software approach for SNP discovery
SNP discovery by alignment of sequence traces obtained from direct sequencing of
genomic PCR products. It is not always possible to distinguish between sequence artefacts
and true polymorphism, when two peaks are present at one position.
Box1: top sequence homozygote AA, middle sequence heterozygote AG, bottom
sequence homozygote GG.
Box 2: The polymorphism detection software has considered the top and bottom
sequences as heterozygote CT and the middle one as homozygote CC. Clonal sequence
removes many of such ambiguities, since any double peak is a sequence artefact.


Method of SNP genotyping
Sobrino et al. (2005) classify majority of SNP genotyping assays to one of four groups based
on the molecular mechanisms:
A. Allele-specific hybridization,
B. Primer extension,
C. Oligonucleotide ligation
D. Invasive cleavage

(A) Hybridization with allelic specific oligonucleotides (ASO):
Two ASO probes are hybridized with the target DNA that contains the SNP. Under
optimized conditions, only the perfectly matched probe-target hybrids are stable.

(B) Primer extension reactions:
Mini sequencing (B1) and allelic-specific extension (B2). (B1) mini sequencing: a
primer anneals to its target DNA immediately upstream to the SNP and is extended with a
single nucleotide complementary to the polymorphic base. (B2) allelic-specific extension: the
3 end of the primers is complementary to each allele of the SNP. When there is a perfect
match the primer is extended.




(C) Oligo-nucleotide ligation assay (OLA):
Two allelic-specific probes and one common ligation probe are required per SNP. The
common ligation probe hybridized adjacent to the allelic specific probe. When there is a
perfect match of the allelic-specific probe, the ligase joins both allelic-specific and common
probes.

(D) Invasive cleavage:
The oligonucleotides required called invader probe and allelic-specific probes, anneal
to the target DNA with an overlap of one nucleotide. When the allelic-specific probe is
complementary to the polymorphic base, overlaps the 3 end of the invader oligonucleotide,
forming the structure that is recognized and cleaved by the Flap endonuclease, releasing the
5 arm of the allelic-specific probe.

Chemistry, demultiplexing, detection options in SNP genotyping



Advantages Limitations
Low mutation rate
Easy to automate
Cross-study comparison easy SNP

Time consuming
Expensive
Low information content of a single
Ascertainment bias








DArT (diversity arrays technology)

Diversity array technology (DArT) is a novel type of DNA marker which employs a
microarray hybridization-based technique developed by CAMBIA (http://www.diversity
arrays.com) that enables the simultaneous genotyping of several hundred polymorphic loci
spread over the genome.
DArT can be used to construct medium-density genetic linkage maps in species of various
genome sizes.























.


Schematic representation of DArT.

(A) Generation of diversity panels:
Genomic DNAs of specimens to be studied are pooled together. The DNA is cut with a chosen restriction
enzyme and ligated to adapters. The genome complexity is reduced in this case by PCR using primers with
selective overhangs. The fragments from representations are cloned. Cloned inserts are amplified using
vector-specific primers purified and arrayed onto a solid support.

(B) Contrasting two samples using DArT:
Two genomic samples are converted to representations using the same methods as in (A). Each representation
is labeled with a green or red fluorescent dye, mixed and hybridized to the diversity panel. The ratio of
green: red signal intensity is measured at each array feature. Significant differences in the signal ratio
indicate array elements (and the relevant fragment of the genome) for which the two samples differ.
(Jaccoud et al., 2001).

DArT markers are biallelic and behave in a dominant (present versus absent) or
codominant (two doses versus one dose versus absent) manner. DArT detects single-base
changes as well as indels.
Advantages Limitations
does not need prior sequence information
high throughput, quick, and highly
reproducible method.
Cost effective, tenfold lower than SSR
markers
Genetic scope of analysis is defined by the
user and easily expandable
Not covered by exclusive patent rights, but on
the contrary open-source
DArT is a microarray-based technique that
involves several steps, so it demands an
extensive investment both in laboratory facility
and skilled manpower.
Marker dominance
Being a novel technique, it is not in use on
large scale.
SUMMERY
Links between the signal generation and detection: Vignal et al., 2002



Methods used to detect products generated by the allele-specific reactions are:
1: PCR-RFLP
2: Oligonucleotide ligation assay (OLA)
3: Good Assay
4: Minisequencing techniques
5: single stranded conformation polymorphism (SSCP)
6: denaturing high performance liquid chromatography (DHPLC)
7: Pyrosequencing
8: SNP it
9: exonuclease detection (Taqman)
10: Invader Assay,
11: Microarray or DNA chips





Comparison of the five most widely used DNA markers in plants

RFLP SSR RAPD AFLP ISSR
Genomic abundance high medium very high very high medium
Part of genome
surveyed
low copy coding
regions
whole genome whole genome whole genome whole genome
Amount of DNA
required
high low low medium low
Type of polymorphism single base changes, changes in length of
repeats
single base
changes,
single base changes, single base
changes,
insertion, deletion insertion,
deletion
insertion, deletion insertion, deletion
Level of
polymorphism
medium high high very high high
Effective multiplex
ratio
low medium medium high medium
Marker index low medium medium high medium
Inheritance Co-dominant Co-dominant dominant dominant dominant
Detection of alleles yes yes no no no
Ease of use labour intensive easy easy difficult initially easy
Automation low high medium medium medium
Reproducibility
(reliability)
high high intermediate high medium to high
Type of
probes/primers
Low copy genomic
DNA or cDNA clone
specific repeat DNA
sequence
Usually 16 bp
random
nucleotide
specific sequence specific repeat
DNA
sequence
Cloning and/or
sequencing
yes yes no no no
Radioactive detection usually yes no no yes/no no
Development/start-up
costs
high high low medium medium
Utility for genetic
mapping
species specific species specific cross specific cross specific cross specific
Proprietary rights
status
No No (some are
licensed)
licensed licensed no