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Basic methods of molecular biology
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What are molecular markers?
• Marker is a piece of DNA molecule or protein that is
associated with a certain trait of an organism
• Specific fragments of DNA that can be identified within
the whole genome
• DNA markers = direct reflection of genotype
• Heritable DNA sequence differences (polymorphisms)
• Identified by many techniques
• Phenotypically neutral, developmentally and
environmentally stable
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Fields of Applications
• Genetic tool for plant genotyping and gene mapping
• Cultivar identification (fingerprinting)
– Useful to control the identity of reproductive material (seeds, grafts,
bulbs…)
– Useful to control the non-authorized use of cultivars from other
breeders
• Marker assisted breeding
– DNA-markers allow the breeder to introduce into their cultivated
plant only the gene(s) of interest from a related species
• Understanding relationships
• Analysis of diversity
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DNA markers: desired properties
• Highly polymorphic: able to detect many different alleles
• Highly informative; if one individual carries two different
alleles we can visualize both (co-dominant)
• Occurrence throughout the studied genome, at high
densities but not clustered
• Easy, fast and inexpensive to screen
• Reproducible within and between laboratories
No single technique fulfills all these criteria
Choice of DNA analysis technique depends upon the
infrastructure, technical expertise and operational funds
available as well as requirements of the experiment
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Types of Molecular Markers
• Due to rapid developments in the field of molecular genetics, a
variety of molecular markers has emerged during the last few
decades
• Biochemical Markers
– Protein Variation
• Molecular Markers
– DNA sequence Variation
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Allozyme (biochemical marker)
• The alternative forms of a particular protein visualized on a gel as
bands of different mobility
• Polymorphism due to mutation an amino acid has been replaced,
the net electric charge of the protein may have been altered
Technique: Electrophoresis and enzyme staining
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+
-
• Unexpensive
• Only reveals small proportion of
DNA variation
• Markers are codominant
• Many DNA variants do not result in
changes in amino acid sequence
• Some changes in amino acid
sequence do not result in changes
in mobility on the gel
1st locus
1 alelle
2nd locus
5 alelles
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Molecular Markers
• Molecular markers are based on naturally occurring polymorphisms
in DNA sequences (i.e. base pair deletions, substitutions, additions
or patterns)
Base substitution
GATCCGAGTATCGCAATTAGCA
GATCCGAGTGTCGCAATTAGCA
Deletion
GATCCGAGTATCGCAATTAGCA
GATCCGAGTAATTAGCA
Insertion
GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCGCAGCATTAGCA
Duplication
GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCTCGCAATTAGCA
Inversion
GATCCGAGTATCGCAATTAGCA
GATGCCAGTATCGCAATTAGCA
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Molecular Markers
• The number and degree of the various types of mutations define the
genetic diversity within a species
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Codominant vs. Dominant Markers
• Codominant
– A marker in which both alleles are expressed, thus heterozygous
individuals can be distinguished from either homozygous
– Allozymes, microsatellites, RFLP
• Dominant
– A marker shows dominant inheritance with homozygous
dominant individuals indistinguishable from heterozygous
individuals
– AFLP, RAPD, ISSR
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There are 5 conditions that characterize a suitable
molecular marker
•
•
•
•
•
Must be polymorphic
Co-dominant inheritance
Randomly and frequently distributed throughout the genome
Easy and cheap to detect
Reproducible
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Types of DNA Molecular Markers
•
•
Non-PCR based marker
–
RFLP (Restriction Fragment Length Polymorphism)
PCR-based markers – analysis of DNA fragments
–
Analysis of whole genome
• RAPD (Randomly Amplified Polymorphic DNA)
• AFLP (Amplification Fragment Length Polymorphism)
• ISSR (Inter Simple Sequence Repeats)
–
Analysis of certain parts of genome
• PCR-RFLP (Polymerase Chain Reaction‐RFLP)
• SSR (Simple Sequence Repeats (Microsatellites)
• SSCP (Single Strain Conformation Polymorphism)
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RFLP
Restriction Fragment Length Polymorphism
• The technique centres around the
digestion of genomic DNA with restriction
enzymes
• These enzymes consistently cut DNA at
specific base pair sequences (recognition
sites)
• DNA fragments separated via
electrophoresis (yield a characterictic
pattern) and transfer to nylon membrane
• Membranes exposed to probes
(radioactively labelled) via Southern
hybridization
• Film exposed to X-Ray
•
Video available on URL: http://www.youtube.com/watch?v=1Q-_qgCtk3c
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RFLP
• +
• Universal technique
• Co-dominant
•
•
Labor intensive
Requires relatively large
amounts of DNA
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RFLP gel
Virtual gel pattern
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RAPD
Random Amplified Polymorphic DNA
• The first developed PCR-based molecular marker technique
• The far simplest (just PCR and electrophoresis)
• Uses primers of random sequence (10-12 base pairs) to amplify DNA
fragments by PCR
• Amplified fragments run in agarose gel detected by EtBr
+
• Fast
• Cheap method
• Highly variable
• Dominant marker
• Unstable amplification
leads to poor repeatability
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RAPD gel
•
Video available on URL: http://www.youtube.com/watch?v=amSMffMJL60&feature=related
RAPD DNA Finger printing of Rice wmv.wmv
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AFLP
Amplified Fragment Length Polymorphism
•
•
•
•
Combination of RFLP followed by PCR of selected fragments
Four steps
– Restriction endonuclease digestion of DNA (by enzymes)
– Ligation of adaptors
– Preamplification of ligated fragments
– Amplification of ligated fragments
Separation of the amplified fragments via electrophoresis and
visualization
AFLPs have stable amplification and good repeatability
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• Genomic DNA is digested by
restriction enzymes and
adapters are ligated to the
restriction fragments.
• A subset of the ligated
fragments are amplified by
PCR, using primers with
selective nucleotides at the
3´-end.
• Polymorphism is revealed by
running the amplified
products of various samples
on a denaturing
polyacrylamide gel
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Available on URL: http://www.youtube.com/watch?v=-0xa_nACSMM&playnext=1&list=PL74F325416CBE43D1&feature=results_main
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AFLP
•
•
•
•
+
Fast
Relatively inexpensive
Highly variable
• Dominant marker
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AFLP gel
DNA fragments labeled with P33
DNA fragements fluorescently labeled
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AFLP gel
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SSR
Simple Sequence Repeat or Microsatellite
• Tandem repeated sequences with a 1-6 repeat motif
Dinucleotide
Trinucleotide
Tetranucleotide
•
•
•
•
•
•
•
(CT)6
(CTG)4
(ACTC)4
- CTCTCTCTCTCT
- CTGCTGCTGCTG
- ACTCACTCACTCACTC
SSRs are presented in the genome of all eukaryotes
Tandem repeats are very polymorphic, scattered through out genomes
Genomes typically contain hundreds of SSRs
PCR based markers with 18-25 base pair primers
SSR polymorphisms are based on number of repeat units and are
hypervariable
SSRs have stable amplification and good repeatability
SSR are easy to run and automate
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Where are microsatellites found?
Majority are found in non-coding regions
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SSR
•
SSR-PCR. Figure showing detection of polymorphism using microsatellite analysis. The arrows
represent forward and reverse primers for the (CA)n repeats for the same locus. The gel pattern of
the amplification products with different combination of alleles is shown in the box
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SSR
•
•
•
•
+
Highly variable
Fast evolving
Co-dominant
•
•
Relatively expensive and
time comsuming to develop
Desing of primers
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SSR pattern
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Video available on URL:http://www.youtube.com/watch?v=dl0lTCBxNgE&feature=relmfu
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Comparison of some molecular marker systems using for plant genome
analysis
(modified by Farooq and Azam, 2002; Harris, 2003; Semagn et al., 2006; Park et al., 2009)
RFLP
RAPD
AFLP
SSR
Abundance
Medium
Very high
Very high
High
DNA quality
High
Medium
High
Medium
DNA sequence information
Not required
Not required
Not required
Required
Level of polymorphism
Medium
High
High
High
Inheritance
Co-dominance
Dominance
Dominance
Co-dominance
Reproducibility
High
Low
Medium
High
Technical complexity
High
Low
Medium
Low
Developmental costs
High
Low (none)
Low
High in start
Costs ($ per assay)
High (2.00)
Low (1.00)
Medium (1.50)
Low (1.00)
Automation possible
No
Yes/No
Yes/No
Yes/No
Applications
Genetic
diversity,
polyploidy,
hybridization,
phylogeny,
mating systém
Fingerprinting,
genetic
diversity,
polyploidy,
hybridization,
phylogeny
Fingerprinting,
Genetic diversity
Genetic
diversity,
mating
system
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References
• Farooq, S.; Azam, F. (2002). Molecular Markers in Plant Breeding-II. Some Pre-requisites for Use. Pakistan
Journal of Biological Sciences 5 (10): 1141-1147
• Harris, K.; Subudhi, P. K.; Borrell, A. K.; Jordan, D.; Rosenow, D.; Nguyen, H.; Klein, P.; Klein, R.; Mullet, J.
(2007). Sorghum stay-green QTL individually reduce post-flowering drought-induced leaf senescence. Journal of
Experimental Botany 58: 327–338
• Liu X. J.; Ren, J. Y.; Zong, X. X.; Guan J. P.; Zhang X. Y. (2007). Establishment and optimization of AFLP for
faba bean. Journal of Plant Genetic Resources 8(2): 153-158
• Park, Y. J.; Lee, J. K.; Kim, N. S. (2009). Simple Sequence Repeat Polymorphisms (SSRPs) for Evaluation of
Molecular Diversity and Germplasm Classification of Minor Crops. Molecules 14: 4546-4569
• Reddy, M.P.; Sarla, N.; Siddiq, E.A. (2002). Inter simple sequence repeats (ISSR) polymorphism and its
application in plant breeding. Euphytica 120: 9–16
• Semagn, K.; Bjørnstad; Ndjiondjop, M. N.(2006). An overview of molecular marker methods for plants. African
Journal Biotechnology 5: 2540–2568
• Staub, J. E.; Serquen, F. C.; Gupta, M. (1996). Genetic markers, map construction, and their application in
plant breeding. HortScience 31(5): 729–739
• Wolfe, A. D.; Liston, A. (1998). Contributions of PCR-based methods to plant systematics and evolutionary
biology. In: Soltis, D. E.; Soltis P. S.; Doyle, J. J. (Eds.) Plant Molecular Systematics II. Kluwer Academic
Publishers, Dordrecht, The Netherlands: 43–86
•URL: biology.about.com
This presentation is available on:
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Elaboration of this presentation was financially supported by the
grant of Fund of development of universities FRVŠ 1940/2012
´Vytvoření laboratorních úloh z molekulární biologie pro praktickou
výuku v předmětu „Seed productionand plant breeding ’’
Tato prezentace byla vytvořena za finanční podpory grantu č.
FRVŠ 1940/2012 ´Vytvoření laboratorních úloh z molekulární
biologie pro praktickou výuku v předmětu „Seed productionand
plant breeding’’
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Thank you for your attention!