Cacao BAC Library Constructionand

Physical Mapping

Tissue Harvesting

50 grams Young, expanding leaves were harvested after 12h of dark period

Nuclei Isolation

Chromosomal Band (Large in-tact HMW DNA)

Lambda HMW DNA ladder

50 kb

>500kb

Serial Digest with HindIIIPlugs 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

First Size SelectionThe 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

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.

Recombinant ColoniesBlue/White Screening: White colonies contain BAC vector + insert, Blue colonies vector with no insert

Automated Colony Arraying: Genetix Q-Bot

BAC Characterization Gel 1

Insert

Vector

96 Random BAC clones were digested with a rare cutter and resolved by PFGE for insert size determination

BAC Characterization Gel 2

Insert Size Distribution

Insets range from 60kb-450kbAverage insert size is 140kb0% Empty Vectors

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

Construction of Physical Map of Cacao

High-Information Content Fingerprinting (HICF) Technique:

• Restriction Digestion• SNaPshot Labeling• Capillary Electrophoresis• Fragment Analysis and Assembly

BAC library

DNA extraction

DNA digestion

DNA Labeling

Fragment analysis

FPC Capillary Electrophores

isLabeled DNA

samples run on 3730 DNA

analyzer

DNA extracted from BAC library using a 96 well alkaline-lysis

miniprep

5-enzyme restriction digest of BAC DNA

Digested DNA is labeled with 4 fluorescent dyes

(FingerPrinted Contigs)software

OVERVIEW

BAC Fingerprinting: Restriction Digest and SNaPshot Labeling

HindIII - Green

XbaI - Yellow

BamHI - Blue

XhoI - Red

HaeIII – blunt ends

AATTCGA

GATCCGG

TCGAGTC

TCAGATC

These enzymes may change once the protocol is optimized; but the overhangs would be the same.

Raw data from capillary electrophoresis processed by GeneMapper.

•High-throughput and automated•High level of accuracy

ABI3730 DNA Analyzer

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

Batch data analysis in FPMinerFragments are assigned a size and converted to a digitized fingerprint for contig assembly

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.

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 HindIII BAC library: Estimated completion – December 1, 2008

• Data analysis portion of the Physical Map available WEBFPC : Estimated completion – January 1, 2009