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Plant Genomics 2019
June 13-14, 2019
Berlin, Germany
Asian Journal of Plant Science & Research
ISSN: 2249-7412
Page 20
Plant Genomics
5
th
Edition of International Conference on
The development of a novel SNP genotyping assay to
differentiate cacao clones
Jocelyn De Wever
Ghent University, Belgium
P
lant genetic diversity studies are of high importance
for efficient plant conversation and resource
strategies (eg. tackling mislabeling, conserving valuable
geneticmaterial, parentageanalysis, andgeneticdiversity
studies) as they contribute to an increased knowledge on
the genetic background and diversity of specific plants.
These studies are most commonly analyzed through
simple and effective genotyping methods making use
of genetic markers, such as SSRs, however SNPs are
gaining more interest. Recently, a cost-effective qPCR-
based method has been proposed for SNP genotyping
purposes, coined double-mismatch allele-specific
(DMAS) qPCR as cheap alternative to other methods.
It’s an accurate and fast multi-sample and multi-locus
method,basedonstraightforwardreadoutofDNA-binding
dye based qPCR technology. Its design, optimization,
validation and application on
Theobroma cacao
L., an
important cash crop involved in the chocolate production,
has shown successful. It offered valuable knowledge
on the background of cocoa which is often plagued by
mislabeling and inefficient and limited management
resources. The method, optimized here, showed 98.05%
efficient in calling the right cacao genotype and identified
15.38% off-types and two duplicates in an internationally
recognized cacao population (n=65), using a limited
amount of markers (n=42). Furthermore, only 13 markers
were needed to differentiate all analyzed accessions.
Notably, the described method can easily be optimized
and implemented in any molecular biology lab for a
wide range of objectives and organisms e.g. mutation
detection and to facilitate gene mapping and marker-
assisted selection for breeding purposes.
Methodology and Theoretical Orientation:
In this study,
first theneed formoregenotyping inplant specificstudies
withfocusoncocoaiselucidated,focusedontheavailable
markers and methods, together with their advantages
and disadvantages. DMAS-qPCR SNP genotyping seems
a cheap and reliable alternative for such analysis and
has been analysed. In this study, the design (PrimerXl),
optimization and validation (sequencing and database
dependent) of the DMAS qPCR SNP genotyping method
is pointed out specifically for cocoa. In addition, several
genotyping models have been optimised, of which two
can be automated, to translate the retrieved Cq values
from DMAS qPCR assay to allele calls and finally a
genotype. Thereafter, its applicability on cocoa genetic
diversity and mislabelling studies, using GenAIEx v6.5,
has been analysed and confirmed on a Vietnamese
cocoa population.
Findings:
Cocoa DMAS-qPCR based SNP genotyping
method has been optimized and consist of 42 SNP
markers, which showed 98.05% as efficient in calling
correct genotypes. In addition 15.38% off-types and
two duplicates have been identified in an internationally
recognized cacao population (n=65). Furthermore, three
genotyping models have been proposed, of which two
could be used in an automated set-up starting from
the qPCR data retrieved. Thereafter, key descriptive
analysis of the markers, representing the applicability
of this method in cocoa genetic diversity studies, using
GenAIEx v6.5 has been described in more detail. From
this analysis it has been concluded only 13 SNP markers
from the DMAS- qPCR assay were needed to differentiate
all accessions individually.
Jocelyn De Wever, AJPSKY 2019, Volume 09