Anchoring Linkage Groups to Chromosomes

Chromosome microdissectionMonosomic analysis

Chromosome Isolation and Microcloning

Steps:1) chromosome preparation2) microdissection/isolation3) purification and amplification

Microdissection Group:Rick Jellen – BYU, Provo, UtahEric Jackson, Becky Oliver – USDA, Aberdeen, IdahoRamesh Kantety, Ramesh Buyyarapu – Alabama A&M U.Nick Tinker – Agriculture & Agri-Food Canada, ECORCAndrzej 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 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

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

ExtracSure™ devices

ExtracSure™ devices attachto eppendorf tube and centrifugeto collect DNA sample

PicoPureTM extraction buffer

Incubate overnight

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 replicatedupstream

4.

4. The displaced strand then becomes a template for replication allowinghigh yields of high-molecular-weight DNA to be generated from the template

Potential Problems: Incomplete coverage Loses methylation

Results from REPLI-g of single chromosome dissections

DNA Clean-up DArT/SSR Analysis

Library 33

Library 82

Example Chromosome Picks

Library 62

Library 28

Example Chromosome Picks

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 IDTry other WGA protocols to improve coverageNew 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

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 chromosomesNew 454 sequencing facility at BYU in Fall

Additional EST sequencing for oat SNP developmentCrucial 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 OgleComplete for 6 of the monosomic lines; others in process

For DArT and SSR analyses

Currently, the 19 monosomics are under DArT/SSR analysisAnchor by single-dose marker quantification?SSR marker quantification in real-time

Jackson SSR-enriched librariesi.e., AB_AM_017 anchored to S-Mono-16

“Sorry we’re CLOSED” is

occasionally a good thing…

Hmmm…. Save the oat

cytogeneticists?