1. Anchoring Linkage Groups to Chromosomes Chromosome microdissection Monosomic analysis

2. Chromosome Isolation and Microcloning Steps: 1) chromosome preparation 2) microdissection/isolation 3) purification and amplification Microdissection Group: Rick Jellen – BYU, Provo, Utah Eric Jackson, Becky Oliver – USDA, Aberdeen, Idaho Ramesh Kantety, Ramesh Buyyarapu – Alabama A&M U. Nick Tinker – Agriculture & Agri-Food Canada, ECORC Andrzej Kilian – CAMBIA, Australia

3. Chromosome Preparation Sterile germination of dehulled Ogle 1040 seeds on MS media Metaphase arrest/condensation: ice-water, colchicine, N 2 O Digest with 0.1% pectolyase Y-23, 0.2% cellulase R-10 (Onozuka), 1 hr Fix in 75% acetic acid-25% methanol, 1 hr Stain in 100 uL acetocarmine, 1-3 hrs Tease apart fragile cell mass Pipet ~10 uL onto surface of glass-membrane slide, spread on surface Examine slide on Arcturus Veritas laser-capture microscope (LCM) – 100X, 200X, 600X brightfield objectives

4. Chromosome Isolation Identify isolated chromosome (>20 um separation)

5. Chromosome Isolation IR laser – melts polymer on cap to sample surface

6. Chromosome Isolation UV laser – physically separates chromosome from membrane

7. Chromosome Isolation Remove cap containing chromosome sample

8. Chromosome Isolation Verify presence of the isolated chromosome on the cap

9. DNA Extraction Using PicoPure TM DNA Extraction Kit (Veritas TM ) CapSure LCM Caps ExtracSure™ devices ExtracSure™ devices attach to eppendorf tube and centrifuge to collect DNA sample PicoPure TM extraction buffer Incubate overnight

10. Multiple Displacement Amplification (MDA) technology Non-PCR-based Whole Genome Amplification using REPLI-g (QIAGEN) 1. 1. exonuclease-resistant random hexamer primers bind to the template strand 2. 2. Phi 29 DNA polymerase moves along replicating DNA 3. 3. As Phi 29 continues it displaces complementary strands being replicated upstream 4. 4. The displaced strand then becomes a template for replication allowing high yields of high-molecular-weight DNA to be generated from the template Potential Problems: Incomplete coverage Loses methylation

11. Results from REPLI-g of single chromosome dissections DNA Clean-upDArT/SSR Analysis

12. Library 33 Library 82 Example Chromosome Picks

13. Library 62 Library 28 Example Chromosome Picks

14. Conclusions and Future Work with Chromosome Libraries Non-random distribution of DArT marker “hits”: Libraries biased toward Ogle 1040 markers As Nick discussed Protocol alterations: 8-hydroxyquinoline to improve chromosome ID Try other WGA protocols to improve coverage New libraries of Ogle 1040 plus TAM 0-301, Ogle, Kanota Increase distribution of DArT marker hits Andrzej Kilian, CAMBIA

15. Conclusions and Future Work with Chromosome Libraries Methylation decreases marker number? Dramatic increase in number of DArT markers amplifying in Ogle 1040 between genomic vs. chromosome libraries Methylation PstI-digest “screen” in making DArT genomic libraries “Screen” mitigated by WGA protocol in chromosomal libraries CONSEQUENCE: noise in interpreting DArT array data! Methylation-mediated inactivation of homeologous genes? Sequencing of library-specific chromosomes New 454 sequencing facility at BYU in Fall Additional EST sequencing for oat SNP development Crucial for coalescence of linkage groups

16. Monosomic Analysis Fox et al. (2001) – minimal numbers of RFLP loci assigned to monosomic F 1 plants (Kanota or Sun II x Ogle) – Rines/Phillips labs New monosomic hybrids with Ogle 1040, TAM 0-301, Kanota, and Ogle Complete for 6 of the monosomic lines; others in process For DArT and SSR analyses Currently, the 19 monosomics are under DArT/SSR analysis Anchor by single-dose marker quantification? SSR marker quantification in real-time Jackson SSR-enriched libraries i.e., AB_AM_017 anchored to S-Mono-16

17. “Sorry we’re CLOSED” is occasionally a good thing… Hmmm…. Save the oat cytogeneticists?