Anchoring Linkage Groups to Chromosomes

Anchoring Linkage Groups to Chromosomes • Chromosome microdissection • Monosomic analysis

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

Chromosome Preparation • Sterile germination of dehulled Ogle 1040 seeds on MS media • Metaphase arrest/condensation: ice-water, colchicine, N2O • 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 uLacetocarmine, 1-3 hrs • Tease apart fragile cell mass • Pipet ~10 uL onto surface of glass-membrane slide, spread on surface • Examine slide on ArcturusVeritas laser-capture microscope (LCM) • 100X, 200X, 600X brightfield objectives

Chromosome Isolation • Identify isolated chromosome (>20 um separation)

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

Chromosome Isolation • UV laser – physically separates chromosome from membrane

Chromosome Isolation • Remove cap containing chromosome sample

Chromosome Isolation • Verify presence of the isolated chromosome on the cap

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

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

Results from REPLI-g of single chromosome dissections DNA Clean-up DArT/SSR Analysis

Example Chromosome Picks Library 33 Library 82

Example Chromosome Picks Library 62 Library 28

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

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 • MethylationPstI-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

Monosomic Analysis • Fox et al. (2001) – minimal numbers of RFLP loci assigned to monosomic F1 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

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

Anchoring Linkage Groups to Chromosomes