PCR-SRAP/ITAP FOR MOLECULAR CHARACTERIZATION OF THE Theobroma GENUS
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Abstract
Genetic improvement of cocoa (Theobroma cacao L.) could be assisted by low-cost and highly informative molecular techniques. In this study, primers for the detection of sequence-related amplified polymorphism (SRAP) and intron targeted amplified polymorphism (ITAP) were used for the molecular characterization of the genus Theobroma. Genotypes Carmelo, Lagarto, PMCT 58, CATIE R1 and CATIE R6 were evaluated through genomic DNA using 43 combinations of SRAP-type primer pairs and 55 combinations of ITAP-type primer pairs by PCR. Once the highly polymorphic SRAP and ITAP primers were selected, their usefulness was verified to distinguish between genotypes of Criollo, Trinitarian and Forastero cocoa, and species related to the genus Theobroma. To analyze the polymorphism, the SRAP and ITAP bands of equal size and mobility generated by the same combination for the evaluated genotypes were considered as identical for that locus. A binary matrix was generated with the record of absence (0) or presence (1) of bands for each locus. With the matrix, the Jaccard’s genetic similarity was calculated. With the similarity values, a dendrogram was generated using the unweighted pair group method with arithmetic means (UPGMA). Likewise, the polymorphic information content (PIC) and the resolution power (RP) were calculated per primer combination. With the SRAP technique, 312 bands were generated, and an average polymorphism of 44 %, a PIC of 0.17, and an RP of 1.92 were found. With the ITAP technique 931 bands were generated, as well as an average polymorphism of 69 %, a PIC of 0.27, and an RP of 7.5. The individual analysis generated for each primer combination allowed to select 10 pairs for SRAP and 11 for ITAP. The data generated by combinations Me1/Em7, Me6/Em3, and Me6/Em7 of SRAP in interaction with combinations Itpr3/Em10 and Itpr4/Em3 of ITAP showed greater ability to distinguish between genetic types of cocoa and species related to the genus Theobroma.