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.
Inhaltsverzeichnis (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 The disease cycle of Phytophthora infestans
- 2.3 Origin and migration routes of Phytophthora infestans
- 2.3.1 Phytophthora infestans in Mauritius
- 2.3.2 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
- 2.1 Biology and Taxonomy of Phytophthora 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
- 3.1 Overview of Methodology
- 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
Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)
This project aims to optimize DNA concentration for use in RAPD fingerprinting of Phytophthora infestans isolates in Mauritius. The study utilizes RAPD-PCR as a molecular marker technique to characterize genetic diversity amongst the isolates.
- The biology and taxonomy of Phytophthora infestans.
- The disease cycle of Phytophthora infestans, focusing on the pathogen's spread and reproduction in Mauritius.
- The principles and applications of RAPD fingerprinting for genetic characterization of P. infestans isolates.
- The optimization of DNA concentration for reliable and robust RAPD-PCR results.
- The analysis of genetic diversity within the P. infestans isolates collected in Mauritius.
Zusammenfassung der Kapitel (Chapter Summaries)
Chapter 1 provides an introduction to the study, outlining the aim and objectives of the research. Chapter 2 presents a comprehensive review of relevant literature, covering the biology and taxonomy of Phytophthora infestans, the disease cycle, the use of molecular markers for genetic fingerprinting, and the optimization of RAPD-PCR methods. Chapter 3 describes the methodology employed in the research, including the isolation of P. infestans strains, DNA extraction techniques, screening of primers, and the implementation of RAPD fingerprinting. Chapter 4 presents the results of the study, encompassing the culture of Phytophthora infestans isolates, DNA concentration estimation, and the analysis of RAPD fingerprinting results across varying DNA template concentrations. Chapter 5 discusses the implications of the findings, analyzing the effectiveness of different DNA concentrations, the influence of other factors on RAPD assay, and the implications for understanding genetic diversity within the P. infestans isolates collected in Mauritius.
Schlüsselwörter (Keywords)
This research focuses on Phytophthora infestans, late blight, RAPD fingerprinting, DNA concentration optimization, genetic characterization, molecular markers, and the impact of Phytophthora infestans in Mauritius.
- 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
