Cacao BAC Library Construction and Physical Mapping

1. Cacao BAC Library ConstructionandPhysical Mapping

2. Tissue Harvesting 50 grams Young, expanding leaves were harvested after 12h of dark period

3. Nuclei Isolation Lambda HMW DNA ladder Chromosomal Band (Large in-tact HMW DNA) >500kb 50 kb

4. Serial Digest with HindIII Plugs were subject to a serial partial digestion using HindIII for 20 minutes varying the amount of enzyme. The ideal amount produces a smear from 100kb to 350kb. 1.5U HindIII for 20’ was chosen for this library. 0 2 4 6 10 100 350kb 100kb

5. First Size Selection The gel section removed was not subject to EtBr staining and UV exposure. The DNA in the size range from 150kb to 350kb was cut into 3 sections and loaded into the second size selection gel. 350kb 100kb

6. Second Size Selection The center part of the gel consists of Low Melting Point Agarose. The DNA migrates into the LMP removing small, trapped fragments and will be eluted for ligation and cloning.

7. Recombinant Colonies Blue/White Screening: White colonies contain BAC vector + insert, Blue colonies vector with no insert

8. Automated Colony Arraying: Genetix Q-Bot

9. BAC Characterization Gel 1 Insert Vector 96 Random BAC clones were digested with a rare cutter and resolved by PFGE for insert size determination

10. BAC Characterization Gel 2

11. Insert Size Distribution Insets range from 60kb-450kb Average insert size is 140kb 0% Empty Vectors

12. Next Steps • Decondense 384-well plates to 96-well format • Test different restriction enzymes to determine which combination gives the most bands per clone • Optimize protocol for each BAC library • Begin High-Throughput Fingerprinting

13. Construction of Physical Map of Cacao High-Information Content Fingerprinting (HICF) Technique: • Restriction Digestion • SNaPshot Labeling • Capillary Electrophoresis • Fragment Analysis and Assembly

14. BAC library DNA digestion DNA extraction DNA Labeling FPC Capillary Electrophoresis Fragment analysis OVERVIEW 5-enzyme restriction digest of BAC DNA DNA extracted from BAC library using a 96 well alkaline-lysis miniprep Digested DNA is labeled with 4 fluorescent dyes (FingerPrinted Contigs) software Labeled DNA samples run on 3730 DNA analyzer

15. BAC Fingerprinting: Restriction Digest and SNaPshot Labeling TC AGATC These enzymes may change once the protocol is optimized; but the overhangs would be the same. GATCC AA GG TTCGA BamHI - Blue HindIII - Green HaeIII – blunt ends TCGAG TC XbaI - Yellow XhoI - Red

16. ABI3730 DNA Analyzer • High-throughput and automated • High level of accuracy Raw data from capillary electrophoresis processed by GeneMapper.

17. Samples plot data is exported to be further analyzed in FPMiner.

18. Batch data analysis in FPMiner Fragments are assigned a size and converted to a digitized fingerprint for contig assembly

19. Example of digitized fingerprint data in FPC. This data is the contig (clones assembled because of similar banding patterns) of one of the two control clones we use in each plate.

20. Milestones and Timeline • BAC Library Construction • 1st BAC library (HindIII)— Completed • 2nd BAC library (EcoRI) — Completed • 3rd BAC library — Completed • Preliminary data collection – In progress • Begin fingerprinting of HindIII BAC – September 8, 2008 • Total number of clones to fp: 36,864 • CUGI will process 4,608 clones per week • Wetlab portion of the Physical Map of the HindIIIBAC library: Estimated completion – December 1, 2008 • Data analysis portion of the Physical Map available WEBFPC : Estimated completion – January 1, 2009