Metodologia de Biologia Molecular

FACULTY OF TROPICAL CZECH UNIVERSITY
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Plant breeding and genetic resources conservation
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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 Deletion
GATCCGAGTATCGCAATTAGCA GATCCGAGTATCGCAATTAGCA
GATCCGAGTGTCGCAATTAGCA GATCCGAGTAATTAGCA
Insertion
GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCGCAGCATTAGCA
Duplication Inversion
GATCCGAGTATCGCAATTAGCA GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCTCGCAATTAGCA 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 • Labor intensive
• Co-dominant • 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
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• Fast • Dominant marker
• Cheap method • Unstable amplification
• Highly variable 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 • Dominant marker
• Relatively inexpensive
• Highly variable
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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 (CT)6 - CTCTCTCTCTCT
Trinucleotide (CTG)4 - CTGCTGCTGCTG
Tetranucleotide (ACTC)4 - 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 • Relatively expensive and
• Fast evolving time comsuming to develop
• Co-dominant • 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 Fingerprinting, Fingerprinting, Genetic
diversity, genetic Genetic diversity diversity,
polyploidy, diversity, mating
hybridization, polyploidy, system
phylogeny, hybridization,
mating systém phylogeny
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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:

https://netstorage.czu.cz/NetStorage/
http://www.its.czu.cz/cs/?r=841
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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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FACULTY OF TROPICAL CZECH UNIVERSITY

Basic methods of molecular biology

What are molecular markers?

• Marker is a piece of DNA molecule or protein that is

• Genetic tool for plant genotyping and gene mapping

DNA markers: desired properties

Types of Molecular Markers

Allozyme (biochemical marker)

Technique: Electrophoresis and enzyme staining

Codominant vs. Dominant Markers

There are 5 conditions that characterize a suitable

Types of DNA Molecular Markers

• Non-PCR based marker

Virtual gel pattern

• Video available on URL: http://www.youtube.com/watch?v=amSMffMJL60&feature=related

RAPD DNA Finger printing of Rice wmv.wmv

• Combination of RFLP followed by PCR of selected fragments

• Genomic DNA is digested by

Available on URL: http://www.youtube.com/watch?v=-0xa_nACSMM&playnext=1&list=PL74F325416CBE43D1&feature=results_main

DNA fragments labeled with P33 DNA fragements fluorescently labeled

• SSRs are presented in the genome of all eukaryotes

Where are microsatellites found?

Majority are found in non-coding regions

Video available on URL:http://www.youtube.com/watch?v=dl0lTCBxNgE&feature=relmfu

Comparison of some molecular marker systems using for plant genome

This presentation is available on:

Elaboration of this presentation was financially supported by the

Tato prezentace byla vytvořena za finanční podpory grantu č.

Thank you for your attention!

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