Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
Fields of Applications
+ -
• 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
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
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
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
Molecular Markers
• The number and degree of the various types of mutations define the
genetic diversity within a species
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
• 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
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
• Must be polymorphic
• Co-dominant inheritance
• Randomly and frequently distributed throughout the genome
• Easy and cheap to detect
• Reproducible
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
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
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
RFLP
• + -
• Universal technique • Labor intensive
• Co-dominant • Requires relatively large
amounts of DNA
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
RFLP gel
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 • Dominant marker
• Cheap method • Unstable amplification
• Highly variable leads to poor repeatability
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
RAPD gel
AFLP
Amplified Fragment Length Polymorphism
AFLP
• + -
• Fast • Dominant marker
• Relatively inexpensive
• Highly variable
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
AFLP gel
AFLP gel
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
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
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
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
SSR
• + -
• Highly variable • Relatively expensive and
• Fast evolving time comsuming to develop
• Co-dominant • Desing of primers
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
SSR pattern
FACULTY OF TROPICAL CZECH UNIVERSITY
OF LIFE SCIENCES
Plant breeding and genetic resources conservation
AGRISCIENCES PRAGUE
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