markers assisted breeding methods

1. Molecular Markers
DNA markers Techniques

2. DNA markers
Restriction fragment length polymorphism (RFLP)
Randomly amplified polymorphic DNA (RAPD)
Amplified fragment length polymorphism (AFLP)
SSR
SNP

3. RFLP
Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP)
To recognize:
Neutral variation at the DNA level
SNPs within a gene or between genes or
Variable number of tandem repeats present between genes
Out come:
Accelerated the construction of molecular linkage maps
Improved the accuracy of gene location and
Reduced the time required to establish a complete linkage map
Technique:
The digestion of purified DNA using restriction enzymes leads to the formation of
RFLPs - a molecular fingerprint - unique to a particular individual
Six base specific RE - cleave the DNA at every 4096 bases on average (46
)
A genome of 109
bases - produce around 250,000 restriction fragments of variable
length - a continuous smear image.

4. RFLP workflow from DNA extraction to radio-autograph
Molecular probes are DNA fragments isolated and individualized by
1. PstI Genomic Cloning – single copy fragments - 500 to 2000bp
2. cDNA cloning
3. PCR amplification

5. Limitations:
Requires large amounts of high quality DNA
Low genotyping throughput
Difficult to automate
Involves radioactive methods so its use is limited to specific laboratories
RFLP probes must be physically maintained and therefore difficult to share
between laboratories.
The level of RFLP is relatively low
selection for polymorphic parental lines is a limiting step, therefore a complete map
Advantages of RFLP
1. co-dominant
2. reproducible
3. simple methodology
4. requires no special instrumentation
5. Cleaved amplified polymorphic sequence (CAPS) marker (PCRRFLP)
- digesting a PCR-amplified RFLP fragment with one or several restriction
enzymes, and detecting the polymorphism by the presence/absence
restriction sites (Konieczny and Ausubel, 1993)

7. Molecular Basis of RFLP

8. RAPD
A single, random-sequence oligonucleotide primer in a low stringency PCR (35–45°C)
simultaneously amplifies several discrete DNA fragments
random amplified polymorphic DNA (RAPD) by Williams et al. (1990)
arbitrary primed PCR (AP-PCR) by Welsh and McClelland (1990)
DNA amplification fingerprinting (DAF) by Caetano-Anollés et al., (1991)
10-mer oligonucleotide
several discrete DNA products up to 3 kb are amplified (amplicons)
these are considered to originate from different genetic loci
visible in conventional agarose gel electrophoresis as the presence or absence of a
particular RAPD band
RAPDs predominantly provide dominant markers

10. Advantages:
(i)Neither DNA probes nor sequence information is required for the design of
primers
(ii)No blotting or hybridization steps – quick, simple and efficient technique
(iii)Small amounts of DNA (about 10 ng /rxn)
(iv)Can be automated
(v)Capable of detecting higher levels of polymorphism than RFLP
(vi)Can be applied to virtually any organism
(vii) The primers universal and so,can be used for any species
(viii)RAPD products of interest can be cloned, sequenced and converted into
PCR-based markers like
Sequence Tagged Sites (STS)
Sequenced Characterized Amplified Regions (SCAR)
(Paran and Michelmore, 1993)
Limitations: Reproducibility is questionable due to factors such as
PCR buffers
deoxynucleotide triphosphates (dNTPs)
Mg2+ concentration
cycling parameters
source of Taq polymerase
condition and concentration of template DNA
primer concentration

11. AFLP
(selective restriction fragment amplification - SRFA)
Amplified fragment length polymorphism (Zabeau and Voss, 1993; Vos et al., 1995)
The selective PCR amplification of restriction fragments from a gDNA double-digest
of under high stringency conditions
Combination of polymorphism
at RE sites and hybridization of arbitrary primers
50 to150 bp are amplified and polymorphism detected
small DNA samples (1–100 ng) only required
relatively reproducible across laboratories

14. LIMITATIONS TO THE AFLP ASSAY
(i)The maximum polymorphic information content for any bi-allelic marker is 0.5.
(ii)High quality DNA is needed
(iii)Proprietary technology is needed to score heterozygotes and ++ homozygotes
(iv) AFLP markers cluster densely in centromeric regions in species with large
genomes, e.g. barley (Qi et al., 1998) and sunflower (Gedil et al., 2001).
(v) Developing locus-specific markers from individual fragments can be difficult
(vi) AFLP primer screening is often necessary to identify optimal primer specificities
and combinations otherwise the assays can be carried out using off-the-shelf
technology.
(vii) There are relatively high technical demands in AFLP analysis including
radio-labelling and skilled manpower.
(viii) Marker development is complicated and not cost-effective
(ix) Reproducibility is relatively low compared to RFLP and SSR markers
genome-wide Bare-1 retrotransposon-like markers in barley (Waugh et al., 1997)
diploid Avena (Yu and Wise, 2000)

15. cDNA-AFLP technique (Bachem et al., 1996)
Application of standard AFLP protocol to a cDNA template
e.g.,
Transcripts with altered expression during race specific resistance reactions
For the isolation of differentially expressed genes from a specific
chromosome region using aneuploids
Construction of genome wide transcription maps

16. Simple Sequence Length Polymorphisms
(SSLP)
• SSLPs are arrays of repeat sequences that display length
variations, different alleles containing different numbers of
repeat units
• Unlike RFLPs that can have only two alleles, SSLPs can be
multi-allelic as each SSLP can have a number of different
length variants. There are two types of SSLP, both of which
were described in
• Minisatellites, also known as “variable number of tandem
repeats (VNTRs)”, in which the repeat unit is up to 25 bp in
length;
• Microsatellites or simple tandem repeats (STRs), whose
repeats are shorter, usually “dinucleotide” or “tetranucleotide”
units.

17. SSR
Variants
1. Microsatellites
2. short tandem repeats (STRs)
3. sequence-tagged microsatellite sites (STMS)
A. repeat units 1–6 bp long
B. Di-, tri- and tetranucleotide repeats – (CA)n, (AAT)n and (GATA)n
C. widely distributed in genomes (plants &animals (Tautz and Renz, 1984).
Advantages:
 high level of allelic variation
 Flanks of SSR motifs - templates for specific primers to amplify the SSR
alleles via PCR
 Referred to as simple sequence length polymorphisms (SSLPs)
 Mutation rates of SSR are about 4 × 104
–5 × 106
/allele/generation (Primmer
et al., 1996).
 Mutation mechanism -‘slipped strand miss pairing’ (Levinson and Gutman,
1987)

18. Molecular Basis of SSR

21. SSRs are characterized by:
Hypervariability
Reproducibility
Codominant nature
Locus specificity
random dispersion throughout most genomes
More variable than RFLPs or RAPDs.
The advantages:
Readily analysed by PCR and easily detected on PAGE
SSLPs with large size differences - detected on agarose gels
SSR markers can be multiplexed (by pooling independent PCR products or by
true multiplex-PCR)
genotyping throughput is high and can be automated
start-up costs are low for manual assay methods (once the markers have been
developed)
SSR assays require only very small DNA samples(ca.100 ng / individual)
The disadvantages
Labour intensive development process particularly when screening from G DNA
High start-up costs for automated methods.

22. SNP
Pronounced as snip - an individual nucleotide base difference
There are three types recognized
Transitions (C/T or G/A)
Transversions (C/G, A/T, C/A or T/G)
e.g., AAGCCTA
AAGCTTA The two alleles are C and T.
Indels
Human genome has at least 1.42 million SNPs
100 000 of which result in an RFLP
C/T transitions constitute 67% of the SNPs in humans
Similar is the case with plants (Edwards et al., 2007a)
2/3 of SNPs involve the replacement of C / T transitions
Single base variants in cDNA (mRNA) are also SNPs - insertions and
deletions (indels) in the genome.
Nucleotide base - the smallest unit of inheritance, SNPs
- Ultimate form of molecular marker.
1% of the population should have SNP
90% of all human genetic variation are SNPs and occur every 100–300 bases

23. Barley
Soybean
Sugarbeet
Maize
Cassava
Potato
Typical SNP frequencies are in the range of one SNP every 100–300 bp
SNPs may fall within
coding sequences of genes
- if same polypeptide then synonymous
- if different polypeptide then non-synonymous
non-coding regions of genes
gene splicing,
transcription factor binding
the sequence of non-coding RNA
the intergenic regions between genes at different frequencies in
different chromosome regions

24. Of the 3–17 million SNPs found in the human genome,
5% are expected to occur within genes.
Therefore, each gene may be expected to contain ca.6 SNPs.

25. Approaches adopted for discovery of novel SNPs:
I.in vitro discovery, where new sequence data is generated
II.in silico methods that rely on the analysis of available sequence data
III.Indirect discovery, where the base sequence of the polymorphism remains
unknown
SNP genotyping methods and chemistries:
Sobrino et al. (2005) classified SNP genotyping assays into 4 groups (based
on the molecular mechanisms)
Allele-specific hybridization
Primer extension
Oligonucleotide ligation
Invasive cleavage
Chagné et al. (2007) added three methods to this list
Sequencing
Allele-specific PCR amplification and
DNA conformation method
Enzymatic cleavage method -
Approaches for discovery of SNPs

26. 1. Allele-specific hybridization
Hybridization between two DNA targets differing at one nucleotide position
(Wallace et al., 1979)
Two allele-specific probes labelled with a probe-specific Fluor dye and a
generic Quencher that reduces fluorescence in the intact probe
5' exonuclease activity of Taq polymerase cleaves the copml probe
distancing the Quencher from flour
a) TaqMan assay
b) Molecular beacon
These can be used in high-density oligonucleotide chips
Approaches for discovery of SNPs

27. MOLECULAR BECONS

29. 2. Primer extension:
a) Mini-sequencing, single-base extension or the GOOD assay (Sauer et al., 2002)
b) Employs oligonucleotides which anneal immediately upstream of the query
c) SNP and are then extended by a single ddNTP (SBE) in cycle sequencing
reactions
d) Thermo stable proof-reading DNA polymerases ensure the complementary
ddNTP is incorporated.
e) ddNTP terminators that are labelled with different fluorescent dyes are used
SNaPshot (Applied Biosystem) uses differential fluorescent labelling of the four
ddNTPs in a SBE reaction
SNP-IT (Orchid Biosciences)
3. Oligonucleotide ligation (OLA)
ligase joins two oligonucleotides covalently when they hybridize next to one another
on a DNA template
Both primers must have perfect base pair complementarity at the ligation site which
makes it possible to discriminate two alleles at a SNP site

31. SNP
The advantages of SNPs are their abundant
numbers and the fact that they can be typed by
methods that do not involve gel
electrophoresis. This is important because gel
electrophoresis has proved difficult to
automate so any detection method that uses it
will be relatively slow and labor-intensive.
SNP detection is more rapid because it is
based on oligonucleotide hybridization
analysis.

32. Oligonucleotide hybridization analysis
An oligonucleotide is a short single-stranded
DNA molecule, usually less than 50 nucleotides
in length, that is synthesized in the test tube. If the
conditions are just right, then an oligonucleotide
will hybridize with another DNA molecule only if
the oligonucleotide forms a completely base-
paired structure with the second molecule. If there
is a single mismatch - a single position within the
oligonucleotide that does not form a base pair -
then hybridization does not occur.

33. Oligonucleotide hybridization
Oligonucleotide hybridization can therefore
discriminate between the two alleles of a SNP.
Various screening strategies have been devised
including “DNA chip” technology and
solution hybridization techniques