Identification of Certain Papaya Cultivars and Sex Identification in Papaya by DNA Amplification Fingerprinting (DAF)

Abstract

DNA amplification fingerprinting (DAF) was employed to study the relationships among fourteen cultivrs of the papaya (Carica papaya L.) collected from Srisaket and Chantaburi Horticultural Reserch Centre. Ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. Using the 11 primers, a total of 129 distinct fragments were mplified. Cultivars were scored for the presence or absence of DAF fragments and grouped by cluster analysis using sample matching coefficients of similarity and the unweighted pair-group method with rithmetic average (UPGMA). The results identified three groups of cultivars. The first group was coomprised of Cocoa Karn Dum, Apple, Tainung, Mae Hia, Florida Tolerant, Sunset and Mexico Amerilla. The third group was comprised of Taiwan, Kapoho Solo and Richter. The DAF Technology is a rapid, precise andsensitive techniques for genomic analysis, and is recomended as a accurate method to identify plantgenotypes within the same species. The best DAF reaction mixture contined 30 ng of genomic DNA, 5 mM magnesium ion, 2.5 mM primer per 20 ui reaction. A total of 52 primers were used inbulk segregate analysis (BSA) against of male, fermale andhermaphroditic plants. The OPA 06 (5' GGT CCC TGAC 3') primer could be used to identify the sex type of papaya plants. This primer produced two polymorphic bands: oneof ~ 365 base pirs (bP) from hermaphrodite bulk DNA and the other of ~ 360 bp from the male bulk DNA. Neither band was detected for females. Evaluation of the accuracy of OPA 06 analysis was done using 254 plants of different generations and their original parents, and the analysis correctly identify sex type for 88.18% of the plants. The final experiment, 47 tissue cultured hermaphrodite plants of the Khaeg Dum cultivar were examined using OPA 06 , and the sex type was identified correctly for 100% of teh plants.