1. Molecular identification of living things

2. Molecular Markers Single locus marker Multi-locus marker RFLP Microsatellite DNA Fingerprinting AFLP RAPD

3. PCR reactions per se are only prelude to many forms of DNA methods: PCR-based methods RAPD: randomly amplified polymorphic DNA STR (= microsatellites): short tandem repeats AFLP: amplified fragment-length polymorphisms SNP: single nucleotide polymorphisms DNA sequencing

4. RAPD is a PCR-based method which employs single primers of arbitrary nucleotide sequence with 10 nucleotides to amplify anonymous PCR fragments from genomic template DNA What is RAPD?

5. RAPD technology A BC Genomic DNA + Taq polymerase + Arbitrary primers A + Nucleotides + Buffer PCR (under relaxed conditions)

6. Electrophoresis PCR 360 bp 260 bp 520 bp 260 bp 360 bp ABC ABC

7. A schematic picture of an agarose gel Plant AMarkerPlant B - + Plant C Monomorphic bands Polymorphic bands Presens of a band, ”1” Absence of a band, ”0”

8. RAPD bands are treated as independent loci: AA/ Aa aa bb BB/ Bb bb CC/ Cc cc CC/ Cc cc dd DD/ Dd dd DD/ Dd 1 2 3 4 5 6 7 8 91011121314 Locus A Locus B Locus C Locus D 1234567891011 121314

9. RAPD bands are scored for presens ”1” and absens ”0”. Only clear, consistent and polymorphic bands are usually used to create a binary matrix for future statistical analyses

10. Band 1 Band 2 Band 3 Band 4 Plant A 1 0 0 1 Plant B 0 1 0 1 Plant C 1 1 1 0 Plant D 1 1 0 1 Plant E 0 1 0 1 Plant F 1 0 0 1 Plant G 1 0 1 0 A binary matrix:

11. Amplified Fragment Length Polymorphism (AFLP) Restriction endonuclease digestion of DNA Ligation of adaptors Amplification of ligated fragments Separation of the amplified fragments via electrophoresis and visualization AFLPs have stable amplification and good repeatability

12. Technology with elemnts of PCR-based and RFLP-based methods Amplifies a subset of restriction fragments from a mixture of fragments produced by digestion of genomic DNA by two restriction endonucleases Fragments are linked to adapters sequences so that subsequent PCR amplifies only a subset of them (to manageable level) Polymorphisms due to length differences of fragments AFLP: amplified fragment length polymorphism

13. Restriction Fragment Length Polymorphism (RFLP) Genomic DNA digested with Restriction Enzymes DNA fragments separated via electrophoresis and transfer to nylon membrane Membranes exposed to probes labelled with P 32 via southern hybridization Film exposed to X-Ray

14. RFLP Restriction Fragment Length Polymorphism Cutting a DNA sequence using restriction enzymes into pieces  specific enzymes cut specific places Starting DNA sequence: 5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC 3’-ATTAAAGGCAATCAAGTTCGCAATAATGG Enzyme X 5’-TTC- 3”-AAG- Enzyme X 5’-TTC- 3”-AAG- 5’-TAATTT 3’-ATTAAA 5’-CCGTTAGTT 3’-GGCAATCAA 5’-CAAGCGTTAGGACC 3’-GTTCGCAATAATGG

17. Microsatellites

18. What are microsatellites? –Microsatellites (also known as SSR – Simple Sequence Repeats) Mononucleotide SSR (A)11 AAAAAAAAAAA Dinucleotide SSR (GT)6 GTGTGTGTGTGT Trinucleotide SSR (CTG)4 CTGCTGCTGCTG Tetranucleotide SSR (ACTC)4 ACTCACTCACTCACTC

19. Microsatellites What are microsatellites? –Homozygous …CGTAGCCTTGCATCCTTCTCTCTCTCTCTCTATCGGTACTACGTGG… 5’ flanking regionmicrosatellite locus3’ flanking region –Heterozygous …CGTAGCCTTGCATCCTTCTCTCTCTCTCTCT ATCGGTACTACGTGG… …CGTAGCCTTGCATCCTTCTCTCTCTCTCTCTCTCTATCGGTACTACGTGG…

20. Microsatellites Where are microsatellites found? Majority are in non-coding region

21. SSR Analysis

22. SSR: Simple Sequence Repeat or Microsatellite PCR based markers with 18-25 base pair primers SSR polymorphisms are based on no. of repeat units and are hypervariable SSRs have stable amplification and good repeatability SSR are easy to run and automate

25. Most accurate molecular technique, uses genetic blackprint (detects silent mutations and changes without length variation) Large selection of potential genes (mt DNA, nu DNA) but… expensive and time consuming and therefore only used for limited number of genes DNA sequencing

26. “PCR-mediated DNA sequencing has made some (but certainly not all) earlier methods for generating molecular markers, especially for phylogenetic purposes, nearly obsolete” (Avise, 2004) In recent years, DNA sequence information has increased explosively: by the beginning of 2003 nearly 25 million sequences representing 115 000 taxa in GanBank! DNA sequencing

27. Amplification of a gene fragment by polymerase-chain-reaction (PCR) with specific primer combinations DNA sequencing

28. Primers Common mtDNA genes: - 12S rRNA 16S rRNA (structural) - cytochrome oxidase I, cytochrome-b (coding) DNA sequencing

29. Purification and Sequencing DNA sequencing

30. Phylogenetic reconstruction distance methods parsimony methods likelihood methods DNA sequencing