Off-types Identification in Fingerprinting Indonesia Cocoa Clones

Rany Agustina Susanti*, Juan Carlos Motamayor, Donald Livingstone III**, Smilja Lambert***
*Mars Indonesia, Makassar Indonesia
**Mars-USDA, Miami United States
*** Mars Australia, Ballarat Australia

4. Result
1. The Problem
Indonesia has problem with low cocoa yield and pests / diseases
Breeding and selection process for superior cocoa clones needs long time and big
efforts
Cocoa germplasm collections are badly maintained with many mis-identification
(off-types)
Need to identify best and right individual trees (true-to-type) to be used as
parental clones

MRCC Parental Trees Summary

Total sample : 13 clones, 113 trees
Off-types trees : 5 trees (4.42% from total trees)
Off-types trees has been found in PBC123, M05, and BB01 clones

5. Follow up research
All the true-to-type parental trees will be used for pollination and
flowering trial research

Data is shared publications

Mars Makassar, Indonesia

DNA Lab

Mars Miami DNA Lab

2. Objectives
To have faster and more accurate breeding and selection process for
producing new, high yielding and resistant cocoa clones
To identify the right individual trees to be used as parental clones (trueto-type)
To complement phenotypic data with genetic information for higher
accuracy and to speed up the selection process
Data is shared in public domain to help cocoa breeder and farmers

This work is being supported by the Cocoa Genome Project where Mars Inc. have
sequenced and annotated the whole cocoa genome and release data in the public
domain to be accessible to all cocoa scientists on the World to use it for developing
better cocoa varieties for cocoa farmers around the world. In 2010, the results were
placed on the Web by the intellectual property organization Public Intellectual
Property Resource for Agriculture (PIPRA) which helps developing countries gain
access to agricultural science they can use. Any scientist can access the genome
after registering on the website. By making the results publicly available, scientists
will have access to key learnings to advance plant science, while plant breeders and
farmers around the world will be able to develop cacao trees that are higher yielding
and resistant to Pests/diseases, this way assuring sustainable cocoa production into
the future.

3. Method

DNA Extraction
1. Samples collecting in the field
2. Cutting and homogenizing leaf sample
3. Centrifuge to take supernatant
4. Binding DNA with binding matrix
5. Eluting DNA with pure water

http://www.cacaogenomedb.org/

SNP method
(Large amount sample, shorter time)
Using Dapeng 24 Markers (Mat_150, Mat_Tc1060,

Mat_Tc75, Mat_Tc151,
Mat_Tc139,
Mat_Tc1126, Mat_Tc364, Mat_Tc90, Mat_Tc230, Mat_Tc891, Mat_Tc1439, Mat_Tc591, Mat_Tc174,
Mat_Tc534, Mat_Tc1484, Mat_Tc372, Mat_Tc998, Mat_Tc329, Mat_Tc702, Mat_Tc144, Mat_Tc602,
Mat_Tc899, Mat_Tc226, Mat_Tc878)

Fluidigm Technology

http://cocoasustainability.com/

Laboratory Activities

Summary
We were able to extract DNA and genotype more than 4033 cocoa trees from
Indonesia (from Mars Regional Cocoa Center research station, Luwu South
Sulawesi) in less than 6 months
We succeeded in identifying and defining a strategy that allowed us to quantify
and
normalize large amount of DNA samples in short time
We succeeded in identifying the leading laboratories which used the latest
technologies and in defining a strategy that allowed us to genotype
Collaboration between research institutes are now integrated in studying and
correcting individual trees of cocoa germplasm collection in the field