1. PLANT GENETIC MARKERS Plant Biotechnology Dr.Ir. Sukendah, MSc

3. A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species.DNA sequencechromosomecells species A Variation (which may arise due to mutation or alteration in the genomic loci) that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites.single nucleotide polymorphism minisatellites What Genetic Marker -?

4. Marker - types Morphological marker Protein - based marker DNA - based marker MARKERS- PENANDA

5. Perfect marker * Close linkage with the trait of interest and marker - Reproducable - Easy to use and economical * Polymorphic * Multiallelic * Codominat * Non epistatic

6. 1. Morphological Marker colour, size, shape,…… Cheap and fast - but, influenced by environmental conditions Dalam coklat Dalam hijau

7. Protein-based marker Common protein marker: Isozymes Multiple forms of the same enzyme - allozyme: one enzyme and one locus - isozyme: one enzyme, more than one locus (gene duplication; gene families) To be useful as markers, isoforms must be electrophoretically resolvable, and detectable by in-gel assay methods

8. Limited to those enzymes that can detected in situ = thin coverage of the genome Dimeric and multimeric enzymes add complexity Pattern can be influenced by environment and tissue-type specific

9. DNA - based marker Advantages – not influenced by environment – expressed in all tissues RFLPs - restriction fragment length polymorphisms PCR-based markers RAPDs SSRs AFLPs

10. RFLPs - restriction fragment length polymorphisms Electrophoretic comparison of the size of defined restriction fragments derived from genomic DNA ** Cutting (restricting) DNA with one or more endonucleases Separation of restriction fragments according to molecular weight Denature the DNA Transfer by capillarity to a membrane Hybridize to a given probe Types of DNA and implications (mt, cp, nDNA )

11. RFLPs - restriction fragment length polymorphisms

12. PCR-based markers RAPDs: Random Amplified Polymorphic DNA markers Basis: Detection of differences in patterns of DNA amplification from short primers of arbitrary sequence Method: Compare PCR and RAPD Denaturation of DNA and annealing of primers Primer extension Repeat cycling for 20 x Electrophorese PCR products Stain and score. Variability is then scored as the presence or absence of a specific amplification product

13. RAPDs: Random Amplified Polymorphic DNA markers

14. Advantages More polymorphic than RFLPs Simple and quick Selective neutrality Disadvantages: RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. Reproducibility among labs may be a problem