Identification of molecular marker for self-incompatibility in selected germplasm accessions of cocoa (Theobroma cacao L.)

Abstract

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 self-compatible 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.