1. 1 Chapter 8: Fingerprints, diversity analysis, specific markers Cultivar identification (fingerprint) Specific markers Distance analysis (genetic relatedness) Diversity analysis (genetic variation)

2. 2 RAPD: Species specific markers in Lolium perenne / L. multiflorum. Here used to check cross-fertilisation. M MP1P2 1650 bp 950 bp ** * RAPD fingerprints of P1 : Lolium multiflorum parent P2 : Lolium perenne parent Offspring harvested on P1 M: length marker The upper band (1650bp) is L. perenne specific Most plants are a result of a cross between L. multiflorum and L. perenne * indicates plants which are obtained by selfing the L. multiflorum plant. Fingerprint

3. 3 Jacaratia C. papaya V. monoica V. palandensis V. parviflora V. weberbaueri ‘carv’ E coRI GA / MseI ACAA clear distinction between Vasconcellea species species-specific markers (not always possible) Fingerprint AFLP = real fingerprint! (Many polymorphic bands)

4. 4 sp240 V. omnilingua Fingerprint Clustering can reveal closest relatives of unknown specimen

5. 5 Specific marker DNA markers for specific genes are often mapped (=the genetic map location is known, see chapter 9), but this is not necessary Example : ASO marker for cystic fibrosis WT & mutant allele WT mut

6. 6 Estimation of genetic distance Quantitative measure of genetic difference that is calculated between individuals, populations or species Usually based on data from multiple loci (= multiple markers =multiple variables) Useful in breeding to select 2 parents that are genetically distant DISTANCE  SIMILARITY Distance 0 = completely identical Similarity 1 = completely identical Similarity = 1- distance (and reverse) Distance

7. 7 population B population A population C Between genotypesBetween populations  Distance

8. 8 How many loci must be analysed? No formal rules exist –Smith et al. (1991) used 200 RFLP markers dispersed across the maize genome to fingerprint 11 inbred lines. They concluded that accuracy (in this experiment) was sufficient with 100 or more markers In general, the estimation of genetic similarity between two individuals is affected by the number of markers sampled, but increasing the number of markers produces disminishing return It is better if the markers are very polymorphic and dispersed across the genome, but this is not possible in many cases AFLP and multilocus STR preferred methods  Distance

9. 9 Distance

10. 10 1 0 0 1 1 0 1 0 1 0 0 0 0 1 0 1 1 1 0 0 0 1 1 0 1 0 1 0 1 0 Scoring an AFLP-gel: Coding 1/0 (1= AFLP-fragment present; 0= AFLP-fragment absent) Distance

11. 11 Distance Scoring an AFLP-gel: Dominant 1 = fragment present in two copies (homozygote dominant) or in one copy (heterozygote) 0 = fragment absent (homozygote recessive) Multilocus For each individual several markers (loci) are visualised in one gel. Scoring is put into a matrix

12. 12 samples markers

13. 13 Distance Calculation of Genetic Distance/Similarity Nei and Li: 1979 Jaccard: Simple Matching: = With a = present in x and y d = absent in x and y b = present in x, absent in y c = present in y, absent in x 2a+b+c 2a a+b+c a a+b+c+d a + d = Dice coefficient

14. 14 Distance Software programmes are then used to make a distance or a similarity matrix and these data are then used to perform a clustering analysis, with a tree as result. Alternatively a PCO (or PCA) analysis can be performed and the different groups plotted in the different dimensions. Examples of software programmes:  NT-SYS http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html  AFLP-surv (Vekemans et al. 2002)

15. 15 Genetic similarity: usually very high within species (diagonal) Vasconcellea species and related genera Nei and Li Distance

16. 16 Distance

17. 17 Example: plotting of genetic distances from wild and domesticated apple samples M. sylvestris M. x domestica PCO in NT-SYS PCA and PCO are used to derive a 2- or 3-dimensional scatter plot of individuals, such that the geometrical distances among individuals in the plot represent the genetic distances among them with minimal distortion  Distance

18. 18 spin dig  pap ■ cun  chrys ◊ bab  nm?? stip  can  mon  carv  pal  carp ∆ parv  web ⇒ 3 genera clearly distinct Carica Jacaratia Vasconcellea ⇒ relations Vasconcellea spp.: 5 groups ⇒ V.  heilbornii plants in 2 groups with the putative parents Example: PCO on Vasconcellea and related species

19. 19 Richness: –Total number of alleles in the population (or sample) Mean number of alleles/locus % polymorphic loci (PLP) => These values are sensitive to presence/absence of rare alleles (<5%); high sampling errors are possible Evenness –Frequency of different alleles in a population (or sample) Observed heterozygosity (H o ): the higher the heterozygosity the higher the ‘diversity’ Expected heterozygosity (H E ): can be calculated from allelic frequencies, and assuming Hardy Weinberg equilibrium Effective no. of alleles =>These values are not sensitive to rare alleles  Diversity How to measure genetic variation?