basic methods of molecular biology

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PRAGUEPlant breeding and genetic resources conservation

Basic methods of molecular biology

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

• Markers are codominant

-

• Only reveals small proportion of

DNA variation

• 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 substitutionBase substitutionGATCCGAGTGATCCGAGTAATCGCAATTAGCATCGCAATTAGCAGATCCGAGTGATCCGAGTGGTCGCAATTAGCATCGCAATTAGCA

DeletionDeletionGATCCGAGTAGATCCGAGTATCGCATCGCAATTAGCAATTAGCAGATCCGAGTAATTAGCAGATCCGAGTAATTAGCA

InsertionInsertionGATCCGAGTATCGCAATTAGCAGATCCGAGTATCGCAATTAGCAGATCCGAGTATCGCAGATCCGAGTATCGCAGCGCATTAGCAATTAGCA

DuplicationDuplicationGATCCGAGTATCGCAATTAGCAGATCCGAGTATCGCAATTAGCAGATCCGAGTATCGATCCGAGTATCTCTCGCAATTAGCAGCAATTAGCA

InversionInversionGATGATCCGCCGAGTATCGCAATTAGCAAGTATCGCAATTAGCAGATGATGCCGCCAGTATCGCAATTAGCAAGTATCGCAATTAGCA


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• The number and degree of the various types of mutations define the

genetic diversity within a species

Molecular Markers


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• 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

Codominant vs. Dominant Markers


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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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RFLPRestriction 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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RAPDRandom 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


Plant breeding and genetic resources conservation


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AFLPAmplified 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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SSRSimple 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

• 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:https://netstorage.czu.cz/NetStorage/http://www.its.czu.cz/cs/?r=841


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Tato prezentace byla vytvořena za finanční podpory grantu č. FRVŠ 1940/2012 ´Vytvoření laboratorních úloh z molekulárníbiologie pro praktickou výuku v předmětu „Seed productionand plant breeding’’

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 ’’


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