How to measure genetic diversity at the molecular level?

1. How to measure genetic diversity at the molecular level? Thomas Geburek Department of Genetics Federal Research Centre for Forests, Natural Hazards, and Landscape (BFW) Austria Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

2. Objectives of this lecture • Introduce traditional molecular markers and novel genomic markers: their characteristics, advantages and disadvantages • Overview of relevant statistics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

3. Molecular genetic markers are major tools of conservation genetics • allozymes • restriction fragment length polymorphism (RFLP) • random amplified polymorphic DNA (RAPD) • microsatellites or simple sequence repeats (SSRs) • amplified fragment length polymorphism (AFLP) • cleaved amplified polymorphism (CAP), and others. Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

4. Allozymes Disadvantages: • limited number • biased • only nonsynonymous variation • hidden alleles • tissue demanding • automation is impossible Advantages: • genic • inexpensive • codominant • reproducible • easy to develop & assay • relatively polymorphic Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

5. Allozymes - applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

6. Restriction Fragment Length Polymorphism (RFLP) Disadvantages: • laborious • complex patterns • large amount DNA required • automation is difficult Advantages: • numerous • codominant • reproducible • representative • relatively polymorphic Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

7. RFLPs - applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

8. Microsatellites or Simple Sequence Repeats (SSRs) Disadvantages: • nongenic • expensive to develop • frequent return mutations • frequent null alleles • low transferability across genera Advantages: • numerous • codominant (mostly) • reproducible (within species) • relatively easy to assay • very polymorphic • automation is possible Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

9. SSRs -applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

10. Random Amplified Polymorphic DNA (RAPD) Advantages: • numerous • inexpensive • easy to develop & assay • very polymorphic Disadvantages: • low reproducibility • anonymous • dominant • low transferability across species Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

11. RAPDs-applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

12. Amplified Fragment Length Polymorphism (AFLP) Advantages: • numerous • moderately expensive • very polymorphic Disadvantages: • anonymous • dominant • difficult to score • technically demanding • requires high quality and quantity of DNA • questionable reproducibility • low transferability across species Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

13. AFLPs- applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

14. Cleaved Amplified Polymorphism (CAP) Advantages: • numerous • codominant • easy to assay • reproducible • polymorphic Disadvantages: • laborious and expensive to develop • requires upfront discovery of restriction site polymorphisms or sequencing • selective genotyping Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

15. EcoR V EcoR V Cleaved Amplified Polymorphism (CAP) Barley EST marker GBS0734 Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

16. CAP -applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

17. Single Nucleotide Polymorphism (SNP) Advantages: • numerous • codominant • easy to assay • reproducible • potentially suited for automated technology (DNA-chips) Disadvantages: • laborious detection • not universal Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

18. Single-stranded conformation (SSCP) Sequencing A G G T C C T G G Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

19. SNP -applications in conservation genetics Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

20. Measures of genetic variation within populations Actual genetic multiplicity - Number n of alleles at a single locus or several loci (allelic multiplicity) or number of different genotypes (genotypic multiplicity). Potential gametic multiplicity for m loci and n alleles per locus Potential genotypic multiplicity for m loci and n alleles per locus Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

21. Number of effective alleles at the locus k with allele frequency p Locus 1 Locus 2 0.9 p1 0.5 Mean number of effective alleles at m loci (genic diversity) p2 0.1 0.5 1.2 ν 2.0 ν = 2 x 1/ [ 1/2+ 1/1.2 ] = 1.5 Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

22. Observed (actual) heterozygosity (Ho) – relative number of heterozygotes Expected heterozygosity (He) (genetic diversity) Genetic distance (according Gregorius) Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

23. Measures of genetic variation between and among populations Genetic differentiation of a population (deme) to its compliment Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

24. Genetic differentiation among and within populations – the so-called F-statistics FST = (HT - HS)/ HT HT = expected heterozygosity of all populations. Allele frequences are weigthed with the respective population size. HS = mean expected heterozygosity. Individual population heterozygosity is wheighted by its size. Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

25. Example: N Frequency Frequnecy H allele 1 allele 2 Pop. 1 33 0.47 0.53 0.50 Pop. 2 42 0.78 0.22 0.34 0.36 0.64 Mean Solution: HT = 0.46; HS = 0.41; FST = 0.11 Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

26. Genetic diversity among plant populations Categories HS HT FST Annuals .322 .196 .355 Short-lived perennials herbaceous .301 .219 .253 woody .287 .243 .155 Long-lived perennials herbaceous .307 .228 .278 woody .283 .253 .084 Hamrick et al. (1992) Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

27. Genetic Evenness K=1 K=2 K=3 K=4 K=5 K=6 K=10 Example: Estimation of the eveness for 10 genes which are presented by 3 different allelic types with absolute freqencies of 6, 2 and 2. E = 1-0,2 = 0,8 Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

28. Don‘t worry ! There are several good PC programs available. and many others Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

29. By now you should know ........... Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

30. Chloroplast Genome • Size varies between 120 (gymnosperms) and 150 kb (angiosperms) • Approx. 100 to 120 genes (mainly for photosynthesis) • Genes are tightly packed, large non-coding regions are missing. • Order of genes is nearly identical among all plant species. • Mutation rate is two up to threefold lesser than in the nuclear DNA and up to four times larger than in mt-DNA Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

31. Chloroplast genome Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

32. Cp-DNA RFLP Fagus sylvatica Cp-DNA SSR Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

33. Plant history in oaks Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia

34. Measures of genetic variation between and among populations Genetic differentiation of a population (deme) to its compliment Mean genetic differentiation Training Workshop on Forest Biodiversity, Kuala Lumpur, Malaysia