MSc

Cocoa (Theobroma cacao L.) is a diploid species (2n = 2X = 20) with a
small genome size of 380Mbp. It belongs to the family of Malvaceae
(alternatively Sterculianceae). Theobroma cacao L. is a highly cross pollinated
crop due to its peculiar flower structure and existence of self-incompatibility.
Self-incompatibility is exploited in hybridization process of breeding programmes
and also in selecting clones for polyclonal garden. Traditional method to access
self- incompatibility is by hand pollinating 100 flowers per tree. This is very
tedious and time consuming method. This necessitated the identification of a
marker associated with self-incompatibility.
The cocoa germplasm accessions are maintained at Cocoa Research
Centre, KAU, Vellanikkara. The centre has identified and maintained Selfincompatible
and self-compatible cocoa accessions. Self-incompatibility was
manually assessed by selfing 100 flowers per accessions. The cocoa accession
setting fruits on selfing classified as self-compatible and which do not set fruit as
self-incompatible. Five self-incompatible [IMC 20, PNG 299, IMC 105, ICS 5
and R 10 (MEX)] and five self-compatible cocoa accessions (NA 149, EET 397,
SIC 5, POUND 18, GV 13.5) were selected for the present study.
Isolation of good genomic DNA from all self-incompatible and selfcompatible
accessions of cocoa was carried out from the young leaves; using
Doyle and Doyle (1987) method was used for analysis of RAPD, ISSR and SSR
markers.
Random Amplified Polymorphic DNA (RAPD) fragments were generated
in the bulked DNA samples in order to identify markers that were polymorphic
between self-incompatible and self-compatible plants. Among the 76 RAPD
primers screened, 15 primers were selected based on amplification. A 550 basepairs
(bp) DNA fragments were generated with RN-6 primers in three selfcompatible
specific genotypes. The polymorphic band was cloned and sequenced
at SciGenom Pvt. Ltd., Cochin. The sequence analysis gave no information to
develop SCAR marker.
In Inter Simple Sequence Repeats (ISSR) assay, among the 42 primers
screened initially, 20 primers were selected for the study. There was no
polymorphism between self-compatible and self-incompatible genotypes.
Additionally Simple Sequence Repeats (SSR) assay was also performed
with genome specific primers. Total genomic DNA of the self-incompatible and
self-compatible accessions was amplified with 11 SSR primers and out of them
seven primers were selected. The primer mTcCIR 33 yielded distinctly
polymorphic band of 350-400bp size in self-incompatible genotypes. mTcCIR 33
SSR primer also produced polymorphic band obtained on PAGE. The PCR
product was directly sequenced at SciGenom Pvt. Ltd., Cochin. The sequence
analysis showed 86 per cent identity to Theobromo cacao.
More number of genus specific primers has to be screened to develop
better markers to distinguish self-comptability and self-incompatability in cocoa
or an attempt to biochemical markers can also be resorted.