DNA fingerprinting of selected cocoa (Theobroma cacao L.) varities of Kerala agricultural university

Abstract

Cocoa (Theobroma cacao L.), is an important tropical tree-crop belonging to the family Malvaceae. It has originated in South America (Wood and Lass, 1985) and was introduced to India, in 1798 at Courtallam in Tirunelveli district of the old Madras state (Ratnam, 1961). Now, cocoa is extensively cultivated in South Indian states and its cultivation is slowly extending to North Eastern states. Central sub-committee on crop standards has made the DNA fingerprinting data mandatory for the national release and notification of varieties. DNA markers, act as active tool to categorize and distinguish accessions and cultivars which have similar morphological characteristics. DNA fingerprinting is effectively used for cultivar identification, assessing seed purity of hybrids and to compare genetic similarities between the cultivars. DNA fingerprints act as a powerful tool to protect Plant Breeder’s Rights (PBR). In the present study, seven cocoa varieties, CCRP 3 (selection), CCRP 10, CCRP 11, CCRP 12, CCRP 13, CCRP 14 and CCRP 15 (hybrids) were fingerprinted. Molecular characterization was performed with genomic DNA isolated using modified Delloporta method (Ileana, 2005). Thirty five ISSR (inter simple sequence repeats) primers and 30 SSR (simple sequence repeats) primer combinations were screened for marker polymorphism, of which 23 ISSR and 17 SSR primer combinations were selected for further study. ISSR and SSR amplification patterns differed among the varieties and thus the DNA fingerprints from each primer combination were developed. ISSR primer UBC810 was found to distinguish CCRP 3 from other genotypes. Whereas, UBC835 and UBC857 produced unique amplicons in variety CCRP 10. Primer UBC810, UBC826, UBC841 and UBC854 generated unique amplicons and formed specific DNA fingerprints of the hybrid CCRP 11. Primer ISSR3, UBC815, UBC827, UBC854 and Oligo 05 gave highest (6) unique bands in CCRP12, whereas UBC 854 produced specific band in CCRP 13. Hybrid CCRP 14 generated unique amplicons with primers UBC855 and Oligo 07 which formed specific fingerprint of the hybrid. Similarly, CCRP15 generated three unique fingerprints with the primer UBC844 and Oligo05. In SSR marker analysis, all the seven genotypes have at least one unique band. mTcCIR40 generated unique amplicons at 250bp length in CCRP 3 and SHRSTc53 at 230bp in CCRP 10. mTcCIR10, mTcCIR8, mTcCIR11 and mTcCIR121 generated distinct bands in CCRP 11. In CCRP 12 genotype, primer mTcCIR18 (345bp), mTcCIR40 (200bp) and mTcCIR42 (210bp) produced specific band. Unique bands were generated by primers mTcCIR10(340bp), mTcCIR11 (310bp), mTcCIR12(270bp), mTcCIR22(200bp), mTcCIR24 (150bp), mTcCIR33 (300bp) and SHRSTc53 for CCRP12. DNA fingerprint generated for CCRP 14 revealed that unique bands were developed at 250bp (mTcCIR37) and 200bp (SHRSTc64). Similarly, CCRP15 developed specific fingerprint at 355 bp (mTcCIR18). ISSR amplicons shared by maximum of three varieties and SSR shared in four varieties were considered for developing final DNA fingerprint profile. The present study had facilitated to characterize the selected cocoa varieties of KAU and the data generated will be useful for varietal notification and in case of any third party litigations.