1. Genetic Positioning of Centromeres Using Half-Tetrad Analysis in a 4x-2x Cross Population of Potato Park et al. Genetics 176: 85-94 (May 2007)

2. CENTROMERES Important functional elements of eukaryotic chromosomes –Ensure proper cell division –Ensure stable transmission of genetic material Determining their composition and structure can provide insight into their functional roles Identification of genetic position important for: –Distinguishing chromosome arms –Identifying proximal and distal markers or genes –Providing fixed positions in genetic maps

3. RECENT CENTROMERE RESEARCH Centromeres sequenced and studied extensively in: –Arabidopsis –Maize –Rice –Wheat Little sequencing has been reported for potato.

4. MAIN GOALS OF RESEARCH Use half-tetrad analysis (HTA) to localize centromeres on 12 potato chromosomes –Compare results of HTA with UHD map proposing possible centromere positions based on marker density (van Os et al. 2006) Determine whether there is truly a single crossover per chromosome arm Previous publications on these issues were theoretical proposals or were limited in loci/chromosome number.

5. 4x – 2x Population Male parent –Diploid –Produced numerically- unreduced 2n pollen by first-division restitution (FDR) Female parent –Tetraploid Tetraploid mapping population

6. FDR vs. SDR FDR Abnormal orientation of spindles before anaphase II Non-sister chromatids end up in the same nucleus Efficiently transmits heterozygosity of original genotype SDR Premature cytokinesis before second meiotic division Sister chromatids end up in the same nucleus

7. FDR vs. SDR: Probability of Heterozygosity FDRSDR Centromere100%0% Telomere50%100%

8. FDR vs. SDR: Probability of Heterozygosity FDRSDR Centromere100%0% Telomere50%100%

9. van Os et al. 2006 Proposed centromere position –based on strong clustering of AFLP markers on UHD genetic map –AFLP markers tend to be clustered in centromeric regions in several other species Park et al. compared their HTA-based results with the proposed positions of van Os et al.

10. aaaa x ab Nulliplexaaaaheterozygosity NOT maintained Simplexaaabheterozygosity MAINTAINED Duplexaabbheterozygosity NOT maintained

11. aaaa x ab Nulliplexaaaaheterozygosity NOT maintained Simplexaaabheterozygosity MAINTAINED Duplexaabbheterozygosity NOT maintained D = [ f(aaaa) + f(aabb)] x 100 cM CROSSING OVER OCCURRED.

12. AFLP Marker Patterns A1: Six nulliplex offspring genotypes More crossing over, farther from centromere A2: One nulliplex offspring genotype Less crossing over, closer to centromere

13. Frequency of homozygosity Based on location on UHD map (van Os et al. 2006), grouped linkage groups Within linkage groups, arranged according to genetic position Calculated frequencies of alleles –233 genotypes –Map position: [f(aa) + f(bb)] x 100 [(# homozygous alleles)/233] x 100

14. Combining results: HTA and UHD Marker-to- centromere distance (% homozygosity) Gray box marks bin containing centromere Zero distance to centromere

15. Centromere Locations ChromosomeBin 113 21 435 546 617 768 822 931 1064 1249

16. Centromere Locations ChromosomeBin 113 21 435 546 617 768 822 931 1064 1249 Telocentric predominantly terminal location of the centromere

17. Centromere Locations ChromosomeBin 113 21 435 546 617 768 822 931 1064 1249 What about chromosomes 3 and 11?

18. Chromosomes 3 and 11 No markers with 100% heterozygosity Approaching zero

19. Other funny stuff: Chromosomes 1 and 5 100% heterozygosity found in the wrong bin (chromosomes 1, 5) 99.6% heterozygosity found in the “centromere” bin (chromosome 1)

20. Cross-over frequency Chromosome 2 (telocentric) –one arm analyzed Chromosome 4 (metacentric) –one arm analyzed Chromosome 6 (metacentric) –both arms analyzed Only one crossover per chromosome arm?

21. Cross-over frequency Boldface: all markers are heterozygous (centromeric) Block a: no crossovers Block b: one crossover Block c: two crossovers

22. Number crossovers No crossovers One crossover Two crossovers Chr21181105 Chr4167588 chr6135935 per 233 genotypes

23. Cross-over frequency Confirms that 2n pollen originated through FDR –Since some markers were entirely heterozygous, cannot have involved an SDR mechanism Fewer noncrossover events on telocentric chr2 –Telocentric chromosome shows more crossover? –No. X-squared not significant. Second crossover per chromosome arm very rare –5/233 for chr2, 8/233 for chr4, 5/233 for chr6 –Indicates strong interference

24. Summary of Results Used HTA to localize centromeres of most potato chromosomes Confirmed centromere positions with those in UHD map (van Os et al. 2006) Marker density approach w/ UHD can be used for positioning centromeres HTA in potato is also powerful for positioning centromeres