BREEDING PROGRAM
ADVANCEMENTS IN
CACAO
INTRODUCTION
• Cocoa is an important crop belonging to the genus Theobroma in
the family Malvaceae.
• The crop was domesticated in Central America in pre-Columbian
times, and the native Indians considered it to be of divine origin.
Consequently, in 1753, Linnaeus designated its scientific name,
Theobroma, meaning food of the gods.
• Cacao is a species that has two sets of chromosomes, making it diploid
with a chromosome count of 20. It has a genome size of approximately
411 to 494 million base pairs, which is relatively small compared to
other species
• In spite of economic importance of cacao, genetic improvement of
cacao has not advanced as rapidly as that of many annual and
biennial crops and other tropical perennial crops such as rubber
(Hevea brasiliensis).
• Progress in cacao breeding has been hindered by a long-generation
cycle, and limitations in land availability and other necessary
resources. However, accelerated progress in cocoa breeding is still
required to meet cocoa farmers’ needs for high-yielding, diseaseresistant, drought and other abiotic stress tolerant genotypes with
good favor potential.
FUNCTIONAL GENOMICS ASSISTED DEVELOPMENT OF
GENE MARKERS
• In 2015,
Cacao’s genes and physical Characteristics are being
looked into a study by applying the latest technology in functional
genomics.
• The project aims to produce cacao variety with durable and
sustainable resistance to pest and diseases and high bean quality
for chocolate production.
GENOMIC VARIATION OF FIVE INDONESIAN CACAO
(THEOBROMA CACAO L.) VARIETIES BASED ON
ANALYSIS USING NEXT GENERATION SEQUENCING
• The objective of the study was to characterize genomic
variation of five superior Indonesian cacao varieties using
next generation sequencing.
• Genetic materials used were five Indonesian cacao varieties, i.e.
ICCRI2, ICCRI3, ICCRI4, SUL2 and ICS13.
METHODS
• Genomic DNA was isolated from healthy young leaf taken from
the first fully open leaf from the shoot tip of each cacao variety by
using acetyl trimethyl ammonium bromide.
• Cacao genomic library construction was conducted using the
Illumina TruSeq DNA low throughput (LT) protocol.
• Whole Genome DNA Sequencing of Cacao Genomic Libraries
was done using NGS HiSeq2000.
• Resequencing data of the five cacao varieties were bioinformatically
analyzed by aligning the sequences with that of cacao genome
reference derived from Criollo variety (Argout et al. 2011) using
Bowtie2 software (Langmead and Salzberg 2012).
• Alignment of the resequence data derived from five superior
Indonesian cacao varieties with the cacao reference genome
sequence of Criollo variety resulted a total of 2.6 million DNA
variations consisted of 2.3 million SNPs and 0.3 million Indels.
• The DNA variation obtained from the study is very useful for high
throughput DNA marker development to expedite national cacao
breeding programs.
VARIETY DIFFERENTIATION
DEVELOPMENT OF A CRISPR DETECTR METHOD FOR THE DETECTION
OF CACAO AND ALMOND VARIATIONS (SNPS)
• In this study, it presents CRISPR-Cpf1 DETECTR-based assay for
the differentiation of plant ingredients in sweet confectionary like
fine and bulk-cocoa, or bitter and sweet almonds.
METHODS
• DNA Isolation
• Template Generation by PCR
• Agarose Gel Electrophoresis (AGE)
• Target Selection
• CRISPR-DETECTR System
• Lateral Flow Assay
DNA ISOLATION
• Cocoa: DNA was isolated using the peqGOLD Plant DNA Mini
Kit according to the manufacturer’s protocol for fresh and frozen
samples.
Template Generation by PCR
• Generates templates using specific primers
AGAROSE GEL ELECTROPHORESIS (AGE)
• For agarose gel electrophoresis (AGE) prestained agarose gels with
a concentration of 1.5% agarose solved in TAE buffer (40 mM Tris
acetate, 2 mM EDTA, pH 8.0) were used.
• All PCR templates were purified using the Monarch PCR and
DNA cleanup kit from New England BioLabs Inc.
TARGET SELECTION
• Cocoa: The chloroplast genome sequences (cp Genome) of Arriba
and CCN-51 were used to find appropriate SNPs.
• All sequences used as templates in this study can be found in NCBI
under the accession numbers OM436774-OM436777 (Locus nt
75587; 5’-TT[T/G]G-3’) and OM416212-OM416215 (Locus nt
129451; 5’-[T/G]TTG-3’).
CRISPR-DETECTR SYSTEM
• DNA
Endonuclease
Targeted
CRISPR
Trans
Reporter (DETECTR) are diagnostic tools that can be used to
detect specific RNA/DNA at low attomolar concentrations.
THANK YOU !!