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Molecular Genetics Molecular Breeding or Marker assisted selection July 8 th 2008

Molecular Genetics Molecular Breeding or Marker assisted selection July 8 th 2008. Why molecular markers?. Molecular markers mark (tag) a part (position) of a chromosome (locus) allowing the monitoring of that part in a specific individual. Markers can also be used to analyse diversity

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Molecular Genetics Molecular Breeding or Marker assisted selection July 8 th 2008

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  1. Molecular Genetics Molecular Breeding or Marker assisted selection July 8th 2008

  2. Why molecular markers? Molecular markers mark (tag) a part (position) of a chromosome (locus) allowing the monitoring of that part in a specific individual • Markers can also be used to analyse diversity • differences between individuals -genetics • identify individuals (forensics) Because Mol markers are DNA markers they can encode of traits or be genetically linked to such trait genes

  3. Marker types: Morphological (e.g. height, grain color, aroma of rice) Biochemical (e.g. isozymes) Cytological (e.g. banding pattern of chromosomes) Biological (e.g. host-pathogen interaction) DNA based and or molecular (e.g. AFLP, SSR)

  4. Genetic variation within species is abundant • Each human differs from other humans 1 in 1000 bp • Many organism show more variation 1/300 – 1/ 600 for Arabidopsis – potato 1 /50 • Other molecular differences between individuals are: • Chromosal rearrangements (relatively rare) • Insertions/deletions of shorter or large sequences • Number of repeated sequences in a stretch of DNA

  5. DNA variation can be used as markers • What do you need? • Technology to detect variation • Knowlegde about where is variation in the genome • Methods to use the marker data in genetics Technology (You will do this during your lab session!): • DNA isolation • NA amplification (PCR) • Detection of DNA (direct ethydium bromide, via labelling (radioactive or fluorescent dyes)

  6. What type of molecular marker systems? (see Peters et al. 2003) • RFLP- Restriction Fragment Length Polymorphisms • RAPD – Randomly Amplified polymorphic DNA • AFLP – Amplified Fragment Length Polymorphism • CAPS – Cleaved Amplified Polymorphic Sequences • SSR (= SSLP = microsatellite) - Single Sequence Repeat • SSLP – Single Sequence Length Polymorphism • SSCP – Single-Strand Conformation Polymorphism • INDEL – Insertion – Deletion Polymorphisms • SNP- Single Nucleotide Polymorphisms

  7. Polymerase Chain Reaction (PCR) -Principle

  8. PCR in practice You need: - Template DNA - Primers - Nucleotides 1: Making ssDNA 2: Primer annealing 3: Polymerase reaction

  9. Detection of PCR products PCR machine = thermocycler Separation on gels In general you have enough DNA to see it with ethydium bromide In complex reactions (e.g. AFLP) DNAs can be labelled and need specific detection systems

  10. SSR / microsatellites • Isolation of DNA fragments containing a simple sequence repeat (microsatellite), e.g. [GATA], [CGA], [GC] • Sequencing of regions flanking the SSR • Designing primers for border sequences • Testing in population for duplications and SSR polymorphism

  11. SSR - methodology Genotype A Genotype B [AT] [AT] 18 22 [AT] [AT] 18 22 PCR amplification with radiolabelled nucleotide Polyacrylamide Gel Electrophoresis of PCR products and autoradiography Genotype A Genotype B

  12. Amplified Fragment Length Polymorphism(AFLP) • Genomic DNA double digests with a 4-cutter (MseI) and a 6-cutter (EcoR1) • Ligate adapters to the EcoR1 and MseI RE sites • Primers complementary to Adapters with selective nucleotides at 3’ ends and perform PCR amplification • Separate DNA fragments on high-resolution gels • After detection, screen for band polymorphisms (e.g. earlier slide)

  13. Single Nucleotide Polymorphisms (SNPs) SNPs are abundant and important to analyse linked SNPs from which haplotypes can be derived Haplotype is a unique genotype for a specific part of the genome (a gene, a chromosome etc)

  14. Direct sequencing of PCR products gives SNPs and allows identification of haplotypes ( = chromosome part with the same DNA composition

  15. Haplotypes = chromosome parts with a specific DNA composition based on multiple polymorphisms (in this case SNPs) Haplotypes are more useful in genetics than single SNPs Haplotypes are real genetic units

  16. Thank you for your attention!! Please join me for the lab session on the 15th of July at 1 pm (we meet in the class room)! I hope to see you all!! Please bring any plant sample you wish to investigate and the read/bring the handouts!!

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