1. Genetic and physical maps around the sex-determining M- locus of the dioecious plant asparagus Telgmann-Rauber et al. 2007

2. Background The M-locus controls sexual dimorphism –MM = supermale –Mm = male –mm = female In breeding male genotypes are desired –Higher yield –Longevity Markers at this locus would be useful to differentiate MM and Mm genotypes

3. Author’s goals Clone the region determining sex in asparagus from its position in the genome Develop markers in this region to use in asparagus breeding programs

4. Previous work The M-locus was fine-mapped to the sex chromosome L5 (Reamon-Büttner et al. 1998) 15 markers were used to construct a high density map around the M-locus (Jamsari et al. 2004)

5. Materials and Methods Bulked Segregant Analysis was used to develop new AFLPs –BSA uses two bulked pools of segregants differing for 1 trait, where the pools differ is likely to be the region controlling the trait 809 F 3 individuals were used for mapping –F 3 individuals were created by selfing 3 andromonoecious plants in the F 2 generation 1-2% plants are andromonoecious New markers were mapped using JoinMap

6. Materials and Methods Used a BAC library with 86,784 clones (5.5X coverage) BACs were screened with 5 AFLP primer combinations Positive clones were then aligned into contigs using FPC software 4 BACS were partially sequenced to identify consensus sequences –Sequences were then blasted to identify putative ORFs Also ran FISH for physical mapping

7. Results Found 12 novel AFLPs cosegregating with the M-locus 11 BACs identified using 5 AFLP combos 39 BACs identified by chromosome walking –Aligned BACS still miss the M-locus

8. Markers BAC s Figure 1 - Genetic and Physical Map

9. Figure 2 - sequencing results 53% of hits had homology to transposons/retrotransposons

10. FISH results Probes based off BACs were found to hybridize to centromeric and pericentromeric regions

11. Conclusions Created a detailed map with 26 markers spanning 8.01cM around the M-locus Could not close the gap around the M- locus –Marker density not high enough –M-locus not evenly represented in BACs

12. Conclusions Recombination frequency is reduced in the M-locus region Gene density in this region is low The sex locus is enriched for repetitive sequences The locus is likely to be near a cetromeric or pericentromeric region

13. Future direction Create more markers Use a different BAC library Expression profiling using subtractive hybridization/microarray technologies