Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Genetic markers

Genetic marker types
• Morphological marker
• Molecular marker
1
...
DNA marker

MOLECULAR MARKERS

DNA-based Molecular marker
• A DNA sequence that is readily detected and whose
inheritance can be easily monitored
...

• A marker is a gene of known function and location, that
allow the studying of the inheritance of the gene
...

• NB: polymorphism involves existence of different forms of
same gene in plants or population of plants
...
e
...

* Co-dominant inheritance: allow the discrimination between

homozygotes from heterozygotes
* Occurs frequently throughout the genome
* Highly reproducible: can be analyzed by different persons in

different labs
* Easy, fast and cheap to detect
* Selectivity neutral: all alleles have the same fitness
* High resolution with large number of samples

Desirable properties for a good molecular marker
*Not epistatic: one can determine the genotype of a phenotype
irrespective of the genotype of the other loci
...

* Evenly distributed throughout the genome
...


Co-dominant marker
Gel configuration
P1

P2

O1

O2

Dominant marker
P2

O1

Polymorphism
Parent 1 : one band

Gel configuration
P1

Polymorphism
-Parent 1 : one band
-Parent 2 : a smaller band
-Offspring 1 : heterozygote =
both bands
-Offspring 2 : homozygote
parent 1

O2

-Parent 2 : no band
-Offspring 1 : homozygote parent 1
-Offspring 2 : ????

Dominant versus Co-dominant molecular markers
Dominant:
No distinction between homo- and heterozygotes
possible
...

• The technique is mainly based on the special enzyme called
Restriction Endonucleases
...

• Specific binding patterns can be visualized by hybridization
with labelled probes
...


•
•
•
•
•

It is a PCR based technology
...

This procedure detects nucleotide sequence polymorphism in DNA
...

In RAPD the decamer (10 bases) primers will or will not amplify a
segment of DNA depending on the positions that are complimentary to
the primer sequence
...

• Amplified products are separated on agarose gel in presence of ethidium
bromide (ETBR )and viewed under UV
...
e
...
For example, no
fragment is produced if primers annealed too far apart or 3'
ends of the primers are not facing each other
...


How RAPD works

Template
DNA

 Primer binds to many locations on the template
DNA
 Only when primer binding sites are close and
oriented in opposite direction so the primers point
toward each other will amplification take place

RAPD-When can amplification fail?

Template
DNA

Primers point away
from each other, so
amplification
won’t happen

RAPD-When can amplification fail?

Template
DNA

> 2,000 bases

Primers too far
apart, so
amplification
won’t happen

RAPD(Random Amplified Polymorphic DNA)
DNA markers which developed by amplifying random sequence
of specific markers through the used of random primers

Random Amplified Polymorphic DNA (RAPD)

RAPD Polymorphisms among landraces of sorghum
Sequences of 10-mer
(10 nucleotides) RAPD
primers

RAPD gel configuration

Primer name

OP A08
OP A15
OP A 17
M
OP A19
OP D02

Primer Sequence

5’ –GTGACGTAGG- 3’
5’ –TTCCGAACCC- 3’
5’ –GACCGCTTGT- 3’
5’ –CAAACGTCGG- 3’
5’ –GGACCCAACC- 3’

Advantages of RAPD
 Amplifies anonymous stretches of DNA using arbitrary primers
...
Nearly all RAPD markers are dominant, i
...
it is not possible to distinguish
whether a DNA segment is amplified from a locus that is heterozygous (1
copy) or homozygous (2 copies)
...

2
...
Thus, the RAPD technique is
notoriously laboratory dependent and needs carefully developed laboratory
protocols to be reproducible
...
Mismatches between the primer and the template may result in the total
absence of PCR product as well as in a merely decreased amount of the
product
...


Microsatellites (SSR)

Microsatellites (SSR)
• The term microsatellites was coined by Litt & Lutty (1989)and it is
also known as Simple Sequence Repeats (SSRs)
...

• Microsatellite markers, developed from genomic libraries, can
belong to either the transcribed region (exons) or the non
transcribed region (introns) of the genome
...

• If nucleotide sequences in the flanking regions of the microsatellite
are known, specific primers can be designed to amplify the
microsatellite by PCR
...


Microsatellites (SSR)

Microsatellites (SSR)
5 repeats of ACT

DNA
ACT

ACT

ACT

ACT

ACT

4 repeats of ACT

DNA

ACT

ACT ACT

ACT

31

Microsatellites (SSR)
DNA markers which developed by amplifying microsatellite in
the genome
Sequence
ACTGTCGACACACACACACACGCTAGCT
TGACAGCTGTGTGTGTGTGTGCGATCGA

Primer
(AC)7

ACTGTCGACACACACACACACACGCTAGCT
TGACAGCTGTGTGTGTGTGTGTGCGATCGA

(AC)8

ACTGTCGACACACACACACACACACACGCTAGCT
TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGA

(AC)10

ACTGTCGACACACACACACACACACACACACGCTAGCT
TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA

(AC)12

Primers anneal to the flanking regions
of microsatellites
Forward primer

ACT

ACT

ACT

ACT

ACT

Reverse primer

1
...
PCR fragments separated by capillary
electrophoresis

Microsatellites (SSR)
P1

AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG

P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG
P1
Gel configuration

P2

Advantage of microsatellites
• The main advantages of microsatellites as genetic markers are
their locus-specificity and co-dominant nature
...

• Additional advantages of microsatellites are based on the fact
that they are very short DNA sequences (90 - 300 bp) easily
generated by PCR
...


Disadvantages of microsatellites
• The main disadvantage of microsatellites is related to their
high mutation rate and the fact that in species that are being
analyzed for the first time they need to be isolated de novo
(i
...
newly)
...

• Polymorphisms are detected from differences in the length of the
amplified fragments by polyacrylamide gel electrophoresis (PAGE)
• The technique involves four steps: (1) Restriction of DNA and
ligation of oligonucletide adapters, (2) Preselective
amplification , (3) Selective amplification (4) Gel analysis of
amplified fragments
...
These fragments are viewed on denaturing
polyacrylamide gels either through autoradiographic or
fluorescence methodologies
...
Once labeled, amplified products are
separated by electrophoresis
...

• To be visualized, DNA polymorphism, which is usually
made of small DNA fragments of few base pairs (up
to 500), must be amplified
...
DNA extraction: clean and high molecular weight DNA is a
prerequisite for AFLP
...
Restriction: Restriction fragments of the genomic DNA
are produced by using two different restriction enzymes:
a frequent cutter (the four-base restriction enzyme (MseI)
and a rare cutter (the six-base restriction enzyme EcoRI)
...

MseI

5’TTAA3’

EcoRI 5’GAATTC3’

Basic steps of AFLP fingerprinting
3
...

-One adaptor will complement to the Msel cut end, the
other will complement to the EcoRI cut end
...
Therefore, adapters are specific for
either the EcoRI site or the MseI site
...
Ligation of the adapter to the
restricted DNA alters the restriction site in order to prevent a
second restriction from taking place after ligation has occurred
...


Basic steps of AFLP fingerprinting
4
...
This first PCR, called Preamplification, allows a first selection of fragments by only
amplifying the DNA restriction fragments that have ligated
an adapter to both extremities
...
This
extra base enables another first selection by amplifying
¼ of the fragments that have ligated an adapter to
both extremities
...
6 %
agarose gel
...
Amplification: DNA fragments with MseI-EcoRI ends will be
selected as DNA template for amplification
...

-The PCR primers are labeled with radioactive or
fluorescence dye for detection of DNA bands on gels
...
For this second amplification, we added three more
nucleotides at the 3’ end of the primer sequence used for the preamplification (= adapters sequence + 3 nucleotides)
...

Moreover, one of the primers (usually the EcoRI primer) is labeled with
a fluorescent dye, and will allow the visualization of DNA during the
migration
...
Electrophoresis: polyacrylamide gel is used for separating
DNA bands
...


AFLP: Summary of basic steps

Procedures in AFLP:
- Digestion

- Adaptor Ligation
- Amplification

- Electrophoresis

Advantages of AFLP
•

High genomic abundance
...

• AFLPs can be analyzed on automatic sequencers
...

• Capability to amplify between 50 and 100
fragments at one time
...


Disadvantages of AFLP

•

Need for purified, high molecular weight DNA
...


Application of AFLP
• AFLPs can be applied in studies involving genetic identity,
parentage and identification of clones and cultivars
...

• AFLP markers have successfully been used for analyzing
genetic diversity in plant species
...

• AFLP markers are useful in genetic studies, such as
biodiversity evaluation, analysis of germplasm collections,
genotyping of individuals and genetic distance analyses

---