Phytophthora infestans is a pathogenic oomycete which causes the late blight disease affecting both potato and tomato plantations The Phytophthora infestans populations in Mauritius have not yet been genetically characterized to assess the possible strains present on the island. Random Amplified Polymorphic DNA (RAPD) is a low cost and simple genetic characterization tool that can be used to genetically characterize the different strains of Phytophthora infestans and lead towards a better management of the late blight disease. However, the RAPD fingerprinting is one which requires an extensive optimization in terms of the conditions and the adherence to a stringent protocol.
The aim of this study was to design and apply a series of experiments to optimize the RAPD protocol through the use of a set of DNA template concentrations. In this study, genomic DNA was extracted from 2 P.infestans isolates originating from potato and 1 P.infestans isolate emanating from tomato. The genomic DNA obtained from each isolates was diluted to obtain a set of DNA concentrations which were used for the screening of 30 RAPD primers and for further testing to identify the best DNA template concentration. The clarity of the amplified DNA fragments obtained during electrophoresis was used to determine the optimal DNA template concentration in this study.
Table of Contents
Chapter 1 Introduction
1.1 Introduction
1.2 Aim
1.3 Objectives
Chapter 2 Literature Review
2.1 Biology and Taxonomy of Phytophthora infestans
2.1.1 Biology of Phytophthora infestans
2.1.2 Taxonomy of Phytophthora infestans
2.2 Origin and migration routes of Phytophthora infestans
2.2.1 Phytophthora infestans in Mauritius
2.3 The disease cycle of Phytophthora infestans
2.3.1 Asexual reproduction of Phytophthora infestans
2.3.2 Sexual reproduction of Phytophthora infestans
2.4 Symptoms of Late Blight
2.5 Molecular Markers and Genetic Fingerprinting
2.5.1 RAPD Fingerprinting
2.5.2 Process of RAPD fingerprinting
2.5.3 Optimization of RAPD-PCR Method
2.5.4 DNA template concentration in RAPD fingerprinting
2.5.5 Applications of RAPD-PCR for genetic characterization of P.infestans
Chapter 3 Methodology
3.1 Overview of Methodology
3.1.1 Protocol used
3.1.2 Outline of the Methodology for this project
3.1.3 Sources of the isolates
3.2 Isolation of P.infestans strains from the field
3.3 Preparation of Cornell medium
3.4 Preparation of Rye B Agar medium
3.4.1 Composition of Rye B Agar medium
3.4.2 Preparation Rye Agar B medium
3.5 Subculture technique
3.5.1 1st Subculture
3.5.2 2nd Subculture
3.6 DNA Extraction
3.6.1 Solutions used in DNA extraction of Phytophthora infestans
3.6.2 DNA Quality & Concentration investigated by Gel Electrophoresis
3.6.3 Determination of DNA concentration by spectrophotometric estimation
3.7 Screening of Primers
3.8 Storage and manipulation of the DNA templates
3.9 RAPD fingerprinting
Chapter 4 Results
4.1 Culture of Phytophthora infestans isolates
4.2 Estimation of presence of DNA by fluorescence of Ethidium bromide
4.2.1 Result of Spectrophotometric analysis
4.2.2 Purity of DNA
4.2.3 Concentration of DNA yielded
4.3 Screening of Primers
4.3.1 Measurement of Rf values
4.3.2 Generation of Molecular Weight vs Rf value Semi Log Graph
4.3.3 Screening of OPB primers with Isolate R1
4.3.4 Screening of OPE primers with Isolate R1
4.3.5 Screening of OPL primers with Isolate R1
4.4 Testing of DNA Template Concentrations (Part 1)
4.4.1 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPB 5 primer
4.4.2 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPB 7 primer
4.4.3 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPE 3 primer
4.4.4 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPE 4 primer
4.4.5 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPL 2 primer
4.4.6 Isolates R1, PS1, TS1 at concentrations 30, 50 and 70ng/µl with OPL 4 primer
4.4.7 Synthesis of result obtained at DNA concentrations 30, 50 and 70ng/µl
4.5 Testing of DNA Template Concentrations (Part 2)
4.5.1 Isolate R1 at DNA concentration 40ng/µl
4.5.2 Isolate PS1 at DNA concentration 40ng/µl
4.5.3 Isolate TS1 at DNA concentration 40ng/µl
4.5.4 Synthesis of result obtained at DNA concentrations 30, 40 and 50ng/µl
Chapter 5 Discussion
5.1 Analysis of Results from DNA extraction
5.2 Primers used
5.3 Analysis of the Results from testing of DNA concentrations
5.3.1 DNA template concentration of 70ng/µl
5.3.2 DNA template concentrations of 30, 40 and 50ng/µl
5.4 Other potential key factors affecting the RAPD assay
5.4.1 The annealing temperature
5.4.2 TE buffer for storage of DNA and its effect on Mg2+ availability
5.5 Isolates used in this study and Genetic diversity
Chapter 6 Conclusion & Recommendations
6.1 Conclusion
6.2 Recommendations
Research Objectives and Focus
This study aims to optimize the Random Amplified Polymorphic DNA (RAPD) protocol for the genetic characterization of local Phytophthora infestans populations in Mauritius, specifically by determining the most effective starting DNA template concentration to improve reproducibility and signal clarity.
- Isolation and culture of P. infestans strains from infected potato and tomato sources.
- Comprehensive screening of 30 different RAPD primers to identify those yielding the most robust and interpretable results.
- Experimental optimization of DNA template concentration (tested across a range of 20ng/µl to 200ng/µl).
- Evaluation of band clarity and reproducibility to establish an optimized PCR protocol.
- Assessment of genetic diversity across different local isolates using the optimized RAPD assay.
Excerpt from the Book
4.4 Testing of DNA Template Concentrations (Part 1)
Once the screening step was completed, an experimental set-up was designed to test which DNA template concentration yielded best results in RAPD-PCR. A concise procedure was followed. Bearing in mind that the RAPD procedure has a problem of reproducibility with the results varying from laboratory to laboratory and day to day, 3 different DNA template concentrations (30, 50 and 70ng/ µl) were tested on the same gel with one primer at a time.
Scoring of the bands for each testing was made and the clarity of bands (i.e. band intensity relative to the background) in each lane was assessed on a scale of 1 to 3 with 1 being the best visual appearance and 3 the least.
Summary of Chapters
Chapter 1 Introduction: Provides a background on Phytophthora infestans and the importance of molecular characterization for managing late blight.
Chapter 2 Literature Review: Details the biology, taxonomy, disease cycle, and history of P. infestans, as well as the principles of RAPD fingerprinting.
Chapter 3 Methodology: Describes the materials and procedures used for isolation, DNA extraction, and the setup of the RAPD-PCR experiments.
Chapter 4 Results: Presents the raw data, gel images, and comparative analysis of different DNA template concentrations and primer screenings.
Chapter 5 Discussion: Interprets the findings regarding DNA extraction quality and the impact of template concentration and annealing temperature on PCR results.
Chapter 6 Conclusion & Recommendations: Summarizes the optimal parameters found and suggests future research directions for genetic characterization.
Keywords
Genetic characterization, late blight disease, optimization, Phytophthora infestans, Random Amplified Polymorphic DNA, RAPD-PCR, DNA template concentration, gel electrophoresis, molecular markers, primer screening, genetic diversity, spore isolation, PCR reproducibility, botanical pathology, Mauritius.
Frequently Asked Questions
What is the core purpose of this research?
The core purpose is to optimize the RAPD-PCR protocol for identifying and characterizing Phytophthora infestans strains in Mauritius, specifically by finding the ideal DNA template concentration that ensures reproducible results.
Which organisms are the primary focus of this study?
The study focuses on Phytophthora infestans, the pathogenic oomycete responsible for the devastating late blight disease in both tomato and potato crops.
What is the primary research question?
The research seeks to determine which starting concentration of genomic DNA provides the most consistent and clear genetic fingerprints when using the RAPD-PCR method.
What methodology is employed to analyze the genetic material?
The project utilizes the Random Amplified Polymorphic DNA (RAPD) technique, involving DNA extraction from fungal cultures, PCR amplification with arbitrary decamer primers, and visualization through agarose gel electrophoresis.
What main content is covered in the experimental section?
The experimental section covers the systematic isolation of strains, the screening of 30 unique primers, and the testing of varying DNA template concentrations to assess band clarity and reproducibility.
Which keywords best describe this study?
Key terms include Genetic characterization, late blight disease, optimization, Phytophthora infestans, RAPD, DNA concentration, and molecular markers.
Why is a DNA concentration of 40ng/µl identified as optimal in this study?
Based on the scoring of band intensity and reproducibility, 40ng/µl consistently yielded the highest frequency of "clarity 1" results (the best visual appearance) across the tested isolates.
How does the extraction protocol affect RAPD outcomes?
The study emphasizes that the extraction method and the absence of residual PCR inhibitors are crucial; different protocols can result in varying amounts of high-quality genomic DNA, which directly influences the success of the subsequent PCR reactions.
Are there genetic differences between the tested isolates?
The study notes differences in RAPD fingerprints between isolates originating from potato and tomato, though it cautions that a larger set of primers would be needed to definitively quantify genetic diversity.
- Quote paper
- Nesaratnam Alwar (Author), 2015, Optimization of DNA concentration in RAPD fingerprinting of Phytophthora infestans, Munich, GRIN Verlag, https://www.grin.com/document/344961
