Identification of certain papaya cultivars and sex identification in papaya by DNA amplification fingerprinting (DAF)

摘要

DNA amplification fingerprinting (DAF) was employed to study the relationships among fourteen cultivars of the papaya (Carica papaya L.) collected from Srisaket Horticultural Research Center and Chantaburi Horticultural Research Center. 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 amplified. 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 arithmetic average (UPGMA). The results identified three groups of cultivars. The first group was comprised of 'Pak Chong', 'Khaeg Dum', 'Mexico Mammy' and 'Khaeg Nuan'. The second group was comprised of 'Cocoa KarnDum', '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 and sensitive technique for genomic analysis, and is recommended as an accurate method to identify plant genotypes within the same species. The best DAF reaction mixture contained 30 ng of genomic DNA, 5 mM magnesium ion, 2.5 mM primer per 20 micro l reaction. A total of 52 primers were used in bulk segregate analysis (BSA) against of male, female and hermaphroditic 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: one of ~365 base pairs (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 identified sex type for 88.18% of the plants. In the final experiment, 47 tissue cultured hermaphrodite plants of 'Khaeg Dum' were examined using OPA 06, and the sex type was identified correctly for 100% of the plants..