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Developing a Methodology to Determine the Relative Effectiveness of Male and Hermaphrodite Papayas in Cross-Pollination.

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Title:Developing a Methodology to Determine the Relative Effectiveness of Male and Hermaphrodite Papayas in Cross-Pollination.
Authors:Kawai, Yosuke
Contributors:Molecular Biosciences & Bioeng (department)
Keywords:qPCR
papaya
transgenic
DNA extraction
Date Issued:May 2017
Publisher:University of Hawaiʻi at Mānoa
Abstract:It has been almost two decades since the introduction of transgenic papaya saved the crop in Hawai’i from the devastating effects of the papaya ringspot virus. The virus-resistant papaya was rapidly adopted by farmers, and it now accounts for approximately 80% of papaya acreage in the State. Although transgenic fruits have passed USDA, EPA, and FDA testing to become an integral part of the papaya market, there are also concerns of uncontrolled transgene flow into organic production systems and crops bound for international markets. Pre-commercialization testing of new transgenic crops is becoming more stringent and researchers will need to have a better understanding of the crop’s reproductive behavior, so they can correctly assess the biological risk and design appropriate bio-containment procedures. Provided with these data, there will be less room for false speculations regarding gene flow between plants. For papaya, it is currently known that gene flow by seed is more significant than by pollen, but this situation could change if male plants from feral dioecious (male and female) populations become transgenic by out-crossing with transgenic varieties. Male plants generally produce more flowers than hermaphrodites, which suggests that the role of pollen in transgene flow would increase. Currently, no data exist regarding the relative effectiveness of male and hermaphrodite papaya as pollen contributors to female and hermaphrodite papaya that receive the pollen. The purpose of this study is to design a qPCR methodology for detecting fingerprints of male and hermaphrodite pollen in seed batches and quantifying the percent contribution from the two pollen sources.
The genetic contribution by male papaya is tracked by amplifying a product from a male-specific marker (PMSM-1), whereas the hermaphrodite contribution is tracked using the
transgenic coat protein gene. The primer and probe sequences of the coat protein gene has been previously published, so the first objective of the thesis is to design primer and probes that amplify a product from PMSM-1. Primers and probes that bind specifically to the male marker was successfully developed. Next, a CTAB-based DNA extraction procedure that can yield high amounts of high quality DNA for qPCR usage was developed. Through the combination of the traditional CTAB method and the DNeasy Plant Mini Kit, the method could yield approximately 70 ng/μL of amplifiable DNA with a purity of 1.64 to 1.97 (260/280 absorbance ratio).
A reliable and sensitive qPCR assay for detecting and quantifying the coat protein gene and the male marker in bulked seed and leaf tissue samples was developed in this thesis. There was a strong linear relationship in the standard curve between the measured fluorescence and the total DNA concentration when the template concentration was 100 to 1 ng per reaction. The data were normalized using the 2-ΔΔCt method, adjusting for variation in the amount of template DNA, provided that all primer efficiencies were operating at similar efficiencies. The sensitivity and reliability of the assay was validated by extracting DNA from known ratios of male DNA to hermaphrodite DNA, followed by qPCR detection and quantification.
The methodology outlined in this thesis is a reliable and sensitive assay allowing for the detection and accurate quantification of the pollen contribution by the male and hermaphrodite papayas. The results from this thesis can benefit future researchers by outlining a quick qPCR protocol in screening papaya seeds for different genetic markers. This will further add to the knowledge of the reproductive biology of papaya.
Description:M.S. Thesis. University of Hawaiʻi at Mānoa 2017.
URI/DOI:http://hdl.handle.net/10125/62550
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: M.S. - Molecular Biosciences and Bioengineering


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