Optimization of DNA concentration in RAPD fingerprinting of Phytophthora infestans

TABLE OF CONTENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

ACKNOWLEDGEMENTS

DISSERTATION DECLARATION FORM

ABSTRACT

LIST OF ABBREVIATIONS

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 R
4.3.4 Screening of OPE primers with Isolate R
4.3.5 Screening of OPL primers with Isolate R
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

References

Appendix

LIST OF TABLES

Table 3.1: The composition of the antibiotic mix added to 1 litre of Rye B Agar medium

Table 3.2 : The composition of Rye Agar B medium. Formula adjusted, standardized to suit performance parameters

Table 3.3 The composition of Extraction buffer

Table 3.4: The composition of TE buffer

Table 3.5: List of primers that were screened (Operon Technologies Inc., CA, USA)

Table 3.6: Composition of PCR Master mix

Table 3.7: PCR Cycling conditions

Table 4.1: Triplicate readings and mean value from spectrophotometric analysis of the DNA

Table 4.2: Purity of the DNA from the isolates

Table 4.3: DNA concentration for 3 isolates

Table 4.4: Rf values for the corresponding bands of the 50bp DNALadder

Table 4.5: The number of bands obtained with the OPB 5 primer, and their presence or absence at DNA concentrations 50,100 and 200ng/µl

Table 4.6: The number of bands obtained with the OPB 7 primer, and their presence or absence at DNA concentrations 50,100 and 200ng/µl

Table 4.7: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.8: The number of bands obtained with the OPE 3 primer, at a DNA concentration of 50ng/µl

Table 4. 9: The number of bands obtained with the OPE 4 primer, at a DNA concentration of 50ng/µl

Table 4.10: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.11: The number of bands obtained with the OPL 2 primer, and their presence or absence at DNA concentrations 20, 50 and 80ng/µl

Table 4.12: The number of bands obtained with the OPL 4 primer, and their presence or absence at DNA concentrations 20, 50 and 80ng/µl

Table 4.13: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.14: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPB 5 primer

Table 4.15: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.16: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPB 7 primer

Table 4.17: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.18: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPE 3 primer

Table 4.19: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.20: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPE 4 primer

Table 4.21: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.22: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPL 2 primer

Table 4.23: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.24: Band Scoring Table for isolates at concentrations 30, 50 and 70 ng/µl with OPL 4 primer

Table 4.25: Summary of the results obtained during the testing of DNA concentrations of 30, 50 and 70 ng/µl

Table 4.26: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.27: Band Scoring Table for isolate R1 at concentration of 40ng/µl with the six primers

Table 4.28: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4.29: Band Scoring Table for isolate PS1 at concentration of 40ng/µl with the six primers

Table 4.30: Rf values for the corresponding bands of the 50bp DNA Ladder

Table 4. 31: Band Scoring Table for isolate TS1 at concentration of 40ng/µl with the six primers

Table 4.32: Summary of the results obtained during the testing of DNA concentrations of 30, 40 and 50 ng/µl

LIST OF FIGURES

Figure 2.1: Increase in the number of described Phytophthora species over time (Kroon, 2012)

Figure 2.2: The relationship between the different supergroups and their kingdoms (Carris et al., 2012)

Figure 2.3: Life cycle of Phytophthora infestans (Agrios ., 2005, p.425)

Figure 2.4: Symptoms of late blight on the abaxial side of potato leaf (Picture taken and modified by Nesaratnam Alwar; Mare-Longue, 11th September 2014)

Figure 2.5: Lemon-shaped sporangia attached to sporangiophores, stained with lactophenol blue solution (isolate from Mare-Longue) (Picture taken by Nesaratnam Alwar with Leica DMIL LED Inverted Trinocular microscope with high definition camera; Biosciences Laboratory, University of Mauritius, 12th September 2014)

Figure 2.6: Summary of the steps involved in RAPD fingerprinting (White et al., 2007, p.67) (Modified by Nesaratnam Alwar)

Figure 3.1: Part of the infected leaf containing sporulating lesions. (Picture taken by Nesaratnam Alwar; Biosciences Laboratory, University of Mauritius, 12th September 2014)

Figure 3.2: Slice of potato placed on infected tissue. (Picture taken by Nesaratnam Alwar; Biosciences Laboratory, University of Mauritius, 12th September 2014)

Figure 3.3: Growth of P.infestans after 3 weeks (Picture taken by Nesaratnam Alwar; Biosciences Laboratory, University of Mauritius, 3rd October 2014)

Figure 3.4: Rye Agar B medium in petri dishes (Picture taken by Nesaratnam Alwar; Biosciences Laboratory, University of Mauritius, 2nd October 2014)

Figure 3.5: GeneRuler 1kb DNA ladder, ready-to-use (Source: Thermo Scientific, no date)

Figure 3.6: GeneRuler 50 bp DNA Ladder, ready-to-use (Source: Thermo Scientific, no date)

Figure 3.7: The ProFlex™ PCR System is a thermal cycler that has a triple block (3 x 32 wells). (Picture taken by Nesaratnam Alwar; Mare-Longue, 6th February 2015)

Figure 4.1: Isolates 'T11' and 'PS1' after 3 weeks of culture on Rye B Agar medium

Figure 4.2: Picture of gel showing an estimation of the amount of DNA yielded from DNA extraction

Figure 4.3: Thermo Scientific myImageAnalysis Software v2.0 is a program that measures the Rf values of the DNA bands

Figure 4.4: Screening of OPB primers at DNA concentrations 50,100 and 200ng/µl. The numbers 1-10 represent the different OPB primers (OPB 1- OPB 10)

Figure 4.5: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the bands obtained with the OPB primers

Figure 4.6: Screening of OPE primers at a DNA concentration of 50ng/µl. The numbers 1-10 represent the different OPE primers (OPE 1- OPE 10)

Figure 4.7: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the bands obtained with the OPE primers

Figure 4.8: Screening of OPL primers at DNA concentrations of 20, 50 and 80ng/µl. The numbers 1-10 represent the different OPL primers (OPL 1- OPL10)

Figure 4.9: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the bands obtained with the OPL primers

Figure 4.10: Pictorial explanation of how the work was set up. 3 different DNA concentrations of the 3 isolates were tested with a single primer at a time

Figure 4.11: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPB 5 primer

Figure 4.12: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPB 5 primer

Figure 4.13: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPB 7 primer

Figure 4.14: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPB 7 primer

Figure 4.15: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPE 3 primer

Figure 4.16: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPE 3 primer

Figure 4.17: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPE 4 primer

Figure 4.18: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPE 4 primer

Figure 4.19: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPL 2 primer

Figure 4.20: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPL 2 primer

Figure 4.21: RAPD fingerprints of isolates R1, PS1 and TS1 at concentrations 30, 50 and 70ng/µl with OPL 4 primer

Figure 4.22: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentrations 30, 50 and 70ng/µl with OPL 4 primer

Figure 4.23: Graph of the clarity of each isolate and their frequency of occurrence during testing of DNA concentrations 30, 50 and 70ng/µl. On the x-axis, there is the clarity (from 1-3) for each isolate. On the y-axis, there is the frequency of each occurring clarity for each of the isolate and on the z-axis, there is the different DNA concentrations (30, 50 and 70ng/µl)

Figure 4.24: Gel image of the isolate R1 with primers OPB 5, OPB 7, OPE3, OPE 4, OPL 2 and OPL 4 at DNA concentration of 40ng/µl

Figure 4.25: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentration of 40ng/µl for isolate R

Figure 4.26: Gel image of the isolate PS1 with primers OPB 5, OPB 7, OPE3, OPE 4, OPL 2 and OPL 4 at DNA concentration of 40ng/µl

Figure 4.27: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentration of 40ng/µl for isolate PS

Figure 4.28: Gel image of the isolate TS1 with primers OPB 5, OPB 7, OPE3, OPE 4, OPL 2 and OPL 4 at DNA concentration of 40ng/µl

Figure 4.29: Semi log graph of Molecular weight (bp) vs Rf value for the estimation of the molecular weight of the DNA bands obtained at DNA concentration of 40ng/µl for isolate TS

Figure 4.30: Graph of the clarity of each isolate and their frequency of occurrence during testing of DNA concentrations 30, 40 and 50ng/µl. On the x-axis, there is the clarity (from 1-3) for each isolate. On the y-axis, there is the frequency of each occurring clarity for each of the isolate and on the z-axis, there is the different DNA concentrations (30, 40 and 50ng/µl)

ACKNOWLEDGEMENTS

A completed project work bears only the name of the student; however the path leading to its completion is always achieved in combination with the dedicated action of other people. I hence wish to acknowledge my appreciation to certain people that have been of great help to me during these past several months

First, I would like to thank my parents and sister for their unconditional contributions and support.

I would also like to thank Dr N. Taleb-Hossenkhan, for her advices, continued guidance and assistance.

I am grateful to Keshav Gangadin from the Food and Agricultural Research and Extension Institute (FAREI) for his expert guidance on field.

Thanks also to Mrs. Anishta for her valuable help in the Molecular Laboratory.

DISSERTATION DECLARATION FORM

illustration not visible in this excerpt

ABSTRACT

Phytophthora infestans is a pathogenic oomycete which causes the late blight disease affecting both potato and tomato plantations The P.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 P.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.

The RAPD primers were screened at DNA template concentrations 20, 50, 80, 100 and 200ng/µl. 6 RAPD primers were selected and tested with DNA template concentrations of 30, 50 and 70ng/µl. DNA template concentrations of 30 and 50ng/µl gave consistent results with regard to clarity of amplified DNA and these were compared with a DNA template concentration of 40ng/µl. In this study, it was found that a DNA template concentration of 40ng/µl gave the best result in terms of clarity of the amplified DNA fragments and the reproducibility of RAPD ranged between 30 and 50ng/µl.

The RAPD protocol requires the optimization of all the parameters before valid claims can be made about the level of genetic diversity of P.infestans populations. Since RAPD is low cost and simple to undertake, it can be a useful tool to assess the diversity of strains of P.infestans causing late blight infections annually.

Keywords: Genetic characterization, late blight disease, optimization, Phytophthora infestans, Random Amplified Polymorphic DNA (RAPD)

LIST OF ABBREVIATIONS

illustration not visible in this excerpt

Chapter 1 Introduction

1.1 Introduction

Phytophthora infestans which originates from the mountainous regions of Central Mexico (Goss et al., 2014), causes the late blight disease. During the 1840s, late blight caused extensive damage in agricultural fields and food shortages throughout Europe. The severe impact of the disease at that time in Ireland had caused the “Irish Potato Famine”. Late blight disease can eradicate an entire field of potatoes in a few days under suitable weather conditions (Burges et al., 2005). Phytophthora infestans is considered to be an important pathogen of potato and tomato production systems worldwide (Grünwald & Flier, 2005) and a high chemical input is required in order to control the disease (Céspedes et al., 2012). It is mainly due to the fact that the pathogenic oomycete has been able to evolve and overcome a great majority of the control measures that have been introduced over the years. Late blight has consequently established itself as one of the major limiting factors affecting potato production in the world in recent years. Outbreaks of late blight are regularly reported each year in Mauritius in both potato and tomato growing areas.

In order to develop effective strategies for the management of late blight, it is vital to understand the epidemiology of the disease and to closely monitor the different strains present in the country. Identification of the oomycete under the microscope is possible through the presence of lemon shaped sporangia but it is impossible to distinguish between the different strains because they all look the same. Therefore, the differentiation of the strains relies uniquely on DNA-based sequence approaches (Martin et al., 2012). Genetically characterizing the local strains can therefore provide valuable information such as whether it is the same strains that are causing infection each year; the number of strains present on the island; how the different potato cultivars respond to these strains and to what extent those strains exhibit host specificity.

Rapid identification of the strains is important as new genotypes which are more resistant to systemic fungicides can appear. A variety of molecular markers are available to genetically characterize strains of Phytophthora infestans and one of the simplest methods now commonly used for genetic diversity studies is Random Amplification of Polymorphic DNA (RAPD). The speed, simplicity, low cost and quality of the RAPD technique have made it widely popular as a genetic characterization tool (Kumar & Gurusubramanian, 2011). RAPD technique enables the generation of a considerable amount of genetic markers by using small amounts of DNA and there is no need for any other form of molecular characterization, such as cloning or sequencing of the concerned genome (Bardakci, 2001). However, the use of RAPD-PCR as a genetic characterization tool requires an extensive optimization in terms of template DNA concentration, primer concentration and magnesium chloride concentration.

1.2 Aim

The aim of this project is to design and carry out a set of experiments in order to optimize the utilization of RAPD-PCR for the genetic characterization of local populations of P.infestans, with respect to the starting concentration of template DNA.

1.3 Objectives

The specific objectives of the study involve:

- Collection of potato leaves infected with P.infestans strains from the field.
- Culture of the different P.infestans strains obtained from infected leaves and available isolates on Rye B medium.
- Performing DNA extraction from the cultured strains.
- Screening of 30 RAPD primers to identify which ones provide the best interpretable results and these will be selected for further testing on a range of DNA concentrations.
- Finding the optimum DNA concentration for RAPD-PCR through testing of various DNA template concentrations.

Chapter 2 Literature Review

2.1 Biology and Taxonomy of Phytophthora infestans

2.1.1 Biology of Phytophthora infestans

Phytophthora infestans was named by Anton de Bary, who had also elucidated the life cycle of this fungus in 1876. Formerly, the fungus was named Botrytis infestans in the mid-19th century by Jean Montagne, however, de Bary discovered that the late blight fungus did not share the same characteristics as those species in the Botrytis genus and he thus created the Phytophthora genus. The name Phytophthora is generated from the combination of Greek words: ‘ Phyto ’ meaning plant and ‘ phthora’ meaning destroyer (Schumann & D’Arcy, 2000).

illustration not visible in this excerpt

By the year 2010, as shown in the graph above, 101 Phytophthora species had been successfully described and recognized (Kroon, 2012). Of all these species, Phytophthora infestans is the most famous one, due to its damaging effects on potato and tomato cultivations worldwide. Also, unlike other species of Phytophthora which cause rotting mainly at the level of the roots, late blight infections affect the leaves, stems, potato tubers and tomato fruits.

Phytophthora infestans is a eukaryote as it contains nuclei and other membrane bound organelles. It produces microscopic, hyaline and lemon shaped asexual spores which are known as sporangia (Shihab & Ahmad, 2014). These sporangia are thin-walled and have a comparatively short lifespan outside living host tissue. P.infestans is assigned to the kingdom Stramenopila because it produces zoospores that have two hollow hair-like flagella and which is a particular feature of that kingdom. A whiplash flagellum is located on the posterior end of the zoospore to allow forward movement and a tinsel flagellum which is fibrous and ciliated, is found on the anterior end to pull the spore through water (Volk, 2001). Phytophthora infestans is grouped together with some plant pathogens that are commonly known as “water molds”, as they have a high affinity for water (Schumann & D’arcy, 2000).

For a long period of time in the past, Phytophthora infestans had traditionally been considered as a member of the kingdom Fungi because of its biological, ecological and epidemiological characteristics. For example, they produce hyphae, they acquire their nutrients by absorption and they produce filamentous threads known as mycelium which is a characteristic of true fungi. Nonetheless, present-day molecular and biochemical investigations advocate that oomycetes possess a very limited taxonomic relationship with the true fungi but they instead fit in the kingdom Stramenopila which is one of several eukaryotic kingdoms (Kamoun, 2003). Furthermore, the size of P.infestans genome is estimated to be about 240Mb whereas the genome size of the true fungi is about 40Mb (Haas et al., 2009; Kupfer et al., 1997).

2.1.2 Taxonomy of Phytophthora infestans

illustration not visible in this excerpt

In the above phylogenetic classification, it can be seen that the true fungi share a closer evolutionary relationship with the kingdom Animalia in the supergroup Opisthokonta. The oomycetes on the other hand, are classified in the supergroup Chromalveolata and in the kingdom Stramenopila. rRNA based studies confirm the fact that the oomycetes share a closer phylogenetic relationship with the diatoms and brown algae (Sogin & Silberman, 1998).

In the fourth edition of ‘Introductory Mycology’, Alexopoulos et al. (1996, cited in Soni & Soni, 2010, p.103) assigned the genus Phytophthora to the:

- Kingdom Stramenopila,
- Phylum Oomycota,
- Class Oomycetes,
- Order Perenosporales,
- Family Pythiaceae

The family Pythiaceae is further subdivided into two genera, Phytophthora and Pythium. The genus Phytophthora contains many species including P.infestans that cause damage to commercially important crops like potato, tomato, strawberries and papaya.

2.2 Origin and migration routes of Phytophthora infestans

The identification of the centre of origin of Phytophthora infestans was subjected to vigorous debates in the 1990s when two different established theories on the origin of the first inoculum collided: the Andean theory and the Mexican theory. Analyses of microsatellite markers and sequences of four nuclear genes provided information that suggests that P.infestans is of Mexican origin (Goss et al., 2014). The center of genetic diversity of P.infestans in the Toluca Valley, Mexico, along with the certitude that it undergoes sexual reproduction and that both mating types occur in equal frequencies in that region also favour the Mexican theory (Grünwald and Flier, 2005). The findings support the idea that the Andean population is a descendant of the populations in the Central Valley of Mexico (Goss et al., 2014).

The A1 and A2 mating types of P.infestans were both identified in Mexico, however, populations that had been sampled outside Mexico were made up of the A1 type and were thus asexual (Shaw & Wattier, 2003, p. 23). Investigations at the level of nuclear DNA markers and mitochondrial haplotype of the oomycete by researchers at Cornell University in the 1990s revealed a common lineage of P.infestans named US-1 which occurred in 13 different countries across four continents (Goodwin et al., 1994). The A1 mating type which is widespread in the United States of America, Canada and the European region is associated with the US-1 genotype. Many plant pathologists had hypothesized that the migration of P.infestans occurred from the Mexican land to the United States of America and then to Europe in the 1840s and had caused severe crop damage on a global scale (Goodwin et al., 1994). However, the results obtained from analyses of strains that had been sampled during the period of the epidemic in the 1840s exposed a different genotype from the US-1 lineage (Ristaino, 2002).

The A2 mating type was observed in the late 1970s in parts of Europe (Fry et al., 1993). The dissemination of Phytophthora species occurred through the maritime transportation of potatoes from Mexico towards Europe. The migration of both the A1 and A2 strains from Mexico and their asexual or sexual mating induced greater variations in the populations of P.infestans. The confirmation of new strains having increased aggressiveness, the ability to resist to fungicides and different responses to environmental parameters has accumulated (Fry and Smart, 1999).

In the early 1980s, after the discovery of the A2 mating type, identification of ‘new’ populations of A1 mating type were identified using genetic markers. The ‘old’ US-1 population was displaced by the ‘new’ mating type populations by an incredible speed indicating that the ‘new’ population had a fitness advantage as compared to the ‘old’ one (Guo et al., 2009)

2.2.1 Phytophthora infestans in Mauritius

Several strains of the pathogen are known to exist and the ones present in Mauritius have not yet been fully characterized. Molecular characterization of Phytophthora infestans at Cornell University, USA, showed that isolates from Solanum tuberosum and S. lycopersicum received from Mauritius belonged to the US-1 genotype, the most widely distributed strain (Mauritius Sugar Industry Research Institute, Annual Report, 2000, p.48).

2.3 The disease cycle of Phytophthora infestans

illustration not visible in this excerpt

P. infestans can undergo both asexual and sexual reproduction as shown in Figure 2.3. The asexual cycle enables a rapid growth of the population. The damages caused by the late blight disease are due to the ability of the oomycete to produce asexual spores that are airborne or waterborne and that propagate from one host to another in the environment. The propagation of late blight epidemics relies on humidity and temperature of the environment, whereby a high humidity and temperature range between 15-25°C will favour the growth of the oomycete whereas at higher temperatures the growth is halted (Agrios, 2005, p.421).

2.3.1 Asexual reproduction of Phytophthora infestans

The fungus reproduces asexually when it has absorbed sufficient nutrients and if the atmospheric conditions are suitable. Late blight infections are initiated from the sporangia which release zoospores or produce germ tubes. Dispersal of the sporangia occurs over long distances by wind (Aylor et al., 2001) or on short distances when the sporangia get splashed by rain and land on the surface of potato leaves. These biflagellated zoospores swim for a while until they locate the leaf stomata. They eventually attach themselves; encyst, whereby the zoospores lose their flagellum and form germ tubes on the leaf surface. The penetration into the leaf occurs when the apex of the germ tube forms an appressorium which enables the invasion of the underlying host cells (Birch & Whisson, 2001). This type of asexual reproduction is known as indirect germination (MetaPathogen, no date). When the temperature is above 15°C the sporangia undergoes direct germination to form the germ tubes (MetaPathogen, no date). Within the leaf, growth of the hyphae produces haustoria; structures that are specialised in retrieving nutrients from the host plant (MetaPathogen, no date). The fungus will establish a biotrophic interaction up to 48 hours without causing any visible lesions and is followed by necrotrophic interaction. Lesions are thus formed on the leaves. The mycelia will grow through the stomata after 3 to 5 days and produce new sporangia (Kamoun et al., 1998). About 100,000 sporangia can be produced by a single lesion within a day and these sporangia may reach the tubers or neighbouring leaves of other plants and trigger infection during wet and cool conditions.

2.3.2 Sexual reproduction of Phytophthora infestans

Phytophthora infestans has the ability to reproduce both sexually and asexually. The pathogen was thought to be asexual until the 1950s (Shaw and Wattier, 2003, p.23). This oomycete is heterothallic and it consists of the A1 and A2 mating types. When specific hormones are produced from the mating types, sexual differentiation occurs and the oogonia and the antheridia are formed within a mating zone in which asexual reproduction is inhibited (Ilarionova, 2006). Gametogenesis occurs within each gametangium to produce haploid nuclei with n number of chromosome. Two haploid nuclei from each gametangia fuse via a process known as karyogamy and a diploid thick-walled oospore with a nucleus is formed. The offspring of one of the mating types then starts developing from the germinated oospore to form a sporangia and the asexual cycle starts all over again (MetaPathogen, no date) The oospores have a dual role, that is, they act as tough structures that resist adverse environmental conditions and they also produce genetic variation by sexual recombination.

The presence of sexual reproduction leads to a more diverse population of P. infestans at the genetic level , as a result of genetic mitotic recombination (Cooke & Lees, 2004). Thus, the emergence of A2 mating type coupled with the appearance of ‘new’ aggressive A1 population has subsequently resulted in an increased genetic diversity which is closely associated with an increased level of resistance to metalaxyl fungicide (Mazakova et al., 2006).

2.4 Symptoms of Late Blight

Phytophthora infestans damages the foliage, stems and tubers of potato plants (Solanum tuberosum). The initial symptoms of late blight appear as small, water soaked spots , often with a chlorotic halo, on the stems and leaves (The Pennsylvania State University, 1998). As the disease progresses, necrotic lesions enlarge into a purple-black colour, which later spreads across the whole leaves to the petioles and the stem.

Zones of white masses of sporangia form on the abaxial side of leaves which is visible to the naked eyes (Birch & Whisson, 2001). As the infected leaves die, the aerial parts rot away giving a characteristic odour. In dry conditions, the lesions stop enlarging, darken and wither. The sporangia may detach and fall onto the tubers. The infection of the tuber in its early stages presents slightly brown or purple patches on the skin and there is a rapid decaying of the tuber before harvest (Birch & Whisson, 2001). When the infection of the tuber occurs, there is also the initiation of secondary fungal or bacterial invasion which is known as ‘wet rot’ (Birch & Whisson, 2001).

illustration not visible in this excerpt

2.5 Molecular Markers and Genetic Fingerprinting

Molecular markers can measure genetic relatedness more accurately than other types of markers, namely, morphological and biochemical. Molecular markers are simply indicators of sequence polymorphism among individuals which can be due to multiple bases being inserted or deleted, or it can be due to single nucleotide polymorphisms (Edwards & Mogg, 2001, p. 1; Brookes, 1999). Molecular markers do not possess any significant biological effect but they act as landmarks in the genome and are linked to or are part of a gene (Semagn et al., 2006). They are inherited from one generation to the next. Knowledge of the position of markers on a chromosome and their proximity to genes that code for desirable traits is of economic and agronomic importance. There are various molecular characterization tools that differ in their principles and a thorough consideration is therefore required when choosing a molecular marker for genetic studies (Roychowdhury et al., 2013). The choice of marker may ultimately depend on the type of application, the availability of laboratory facilities, expertise, time and a suitable budget.

2.5.1 RAPD Fingerprinting

Progresses in molecular biology techniques have made a considerable number of highly useful DNA markers available for the detection of genetic polymorphism (Bardakci, 2001). During the previous decade, the Random Amplification of Polymorphic DNA (RAPD), which is a variant of the Polymerase Chain Reaction (PCR), has been extensively used to develop DNA markers (Kumar & Gurusubramanian, 2011). The RAPD markers are anonymous DNA sequences which have been amplified at random in a thermocycler and which make use of single, short oligonucleotide primers and therefore, it is not required to have a prior knowledge of the DNA sequence being studied (Bardakci, 2001). The amplified DNA sequence with primers is primarily formed by the interaction between DNA polymerase, RAPD primer and template annealing sites (Semagn et al., 2006). In comparison, Restriction Fragment Length Polymorphism (RFLP) assay which requires restriction enzyme digestion and connected with DNA hybridization is a slow process (Bardakci, 2001). The widespread use of RAPD is due to the benefit of generating a considerable amount of genetic markers from only small amounts of DNA, therefore, any other forms of molecular characterization such as cloning or sequencing of the genome of the species being examined is not required (Gupta et al., 2010). Nonetheless, due to the fact that DNA amplification by RAPD is of a random nature and generated from primers at random, it is fundamental to optimize and preserve constant reaction conditions in order to develop a standard protocol.

2.5.2 Process of RAPD fingerprinting

STEP 1: DNA is isolated from the sample of cultured P.infestans.

STEP 2: It involves the PCR amplification of the DNA with the primers and dNTPs. The denaturing of the DNA takes place to produce a single strand of DNA. It is followed by the annealing of the RAPD primers and their extensions.

The idea of RAPD technique is that, a short primer that binds to different loci on the genomic DNA, is used for amplification of random sequences. At low annealing temperatures, the primers which are short sequences anneal to their complementary sequences on both DNA strands. Even though arbitrary primers are used, two important rules should be considered: the use of primers with a minimum of 40% GC content and the absence of palindromic sequence (Williams et al., 1990).

STEP 3: When the amplified DNA bands (0.5–5 kb size range) obtained after gel electrophoresis using ethidium bromide staining, are observed with the help of UV light, specific banding patterns known as genomic fingerprints are produced as stated by Jones et al. (1997, cited in Kumar et al., 2009). Also, among the amplified DNA bands obtained, are the ones that may be amplified from some genomic DNA of a particular organism only but not from others which therefore indicates that the presence or absence of the amplified fragment is polymorphic in the population of the organism tested.

illustration not visible in this excerpt

2.5.3 Optimization of RAPD-PCR Method

A few key factors need to be taken into consideration concerning the optimization of the RAPD process in order to achieve a standardised protocol. Quality of the result for the RAPD-PCR method depends on an array of variables. RAPD-PCR is an enzymatic reaction and is laboratory-dependent (Kumar & Gurusubramanian, 2011). The following key variables must be carefully controlled: the quality and concentration of the template DNA; the quality of PCR components used; primer size and the percentage GC content; PCR cycling parameters (more specifically the annealing temperature); the concentration of Taq DNA polymerase enzyme; the concentration of Magnesium chloride; the PCR cycling conditions and the type of thermocycler used (Kumar & Gurusubramanian, 2011). The careful standardization of the technique and reagents is hence required.

2.5.4 DNA template concentration in RAPD fingerprinting

An efficient protocol for RAPD fingerprinting should be resistant to variations in concentration of the DNA template (Skoric et al., 2012). RAPD amplification of DNA fragments does not take place under a certain amount of DNA template concentration .It is also vital to take into consideration that amplification of DNA fragments in this process can be inhibited by high genomic DNA concentrations or due to the presence of PCR inhibitors remaining after DNA extraction (Dias-Neto et al., 1993). In order to improve the RAPD fingerprinting method, it is important to obtain good quality, high molecular weight DNA. The DNA should be quantitated and tested for reproducibility of profiles with at least a two-fold dilution and two-fold concentration of the optimum.

2.5.5 Applications of RAPD-PCR for genetic characterization of P.infestans

RAPD fingerprinting has been used worldwide since the last two decades for the genetic characterization of P.infestans populations. A study by Atheya et al. (2005) using RAPD in India and Himalayan hill was focused on the level of genetic diversity of populations of P.infestans occurring either on hills or plains. Similarly, RAPD analysis of P.infestans isolates in China evaluated the relationship among isolates occurring in different provinces (Xiao Qiong et al., 2006). Outbreaks of late blight disease in Turkey during the year 1997 led to a survey carried out between the year 1999 and 2000 where 25 isolates from different locations had been genetically characterized using 21 RAPD primers (Yildirim et al., 2007).

Chapter 3 Methodology

3.1 Overview of Methodology

3.1.1 Protocol used

The protocol used for DNA extraction was adapted from the online ‘Laboratory Manual for P. infestans work at CIP’ by Fry et al. (2007) which is accessible from the following website:

https://research.cip.cgiar.org/typo3/web/fileadmin/icmtoolbox/ICM_Toolbox/Files/Manual_draft1.pdf

3.1.2 Outline of the Methodology for this project

The methodology of this work is divided into five main steps:

1) Collection of infected leaves on the site of Mare-Longue.
2) Culture of the different P.infestans isolates obtained from infected leaves and from already available cultures on Rye B medium.
3) DNA extraction from the cultured isolates.
4) Screening of 30 primers
5) RAPD fingerprinting using different concentrations of DNA template.

3.1.3 Sources of the isolates

The first isolate was obtained from a potato field at Mare-Longue and was named ‘MLo’. Existing cultures of the fungus were obtained from the Biosciences Laboratory and were named ‘T11’, ‘TS1’, ‘PS1’, ‘R1’ and ‘StP’. T11’ and ‘TS1’ were originally isolated from tomato. ‘PS1’, ‘R1’ and ‘StP’ were previously isolated from potato.

3.2 Isolation of P.infestans strains from the field

Infected potato leaves were collected at Mare-Longue on the 11th September 2014. The infected potato plants were from the variety Delaware and Spunta. The infected leaves were excised and placed in Ziploc bags along with moist pieces of cotton wool. The moist condition was required to preserve the oomycete due to the hot and dry climate on that day. The infected leaves were incubated at 15°C overnight until fresh sporulation appeared.

Isolation:

1. The next day, potatoes of the varieties Delaware and Spunta were thoroughly washed and cut into slices of about 1cm thick.
2. In a completely sterilised laminar flow hood, the sporulating borders of lesions were cut using a pair of sterile scissors (dipped in 90% alcohol and flamed).
3. The pieces of infected leaves were then placed in Petri dishes containing filter papers which have been dampened with sterile distilled water.
4. A piece of potato disc was then placed on the leaves. It is recommended to put the infected tissues underneath the slice of potato as it favours sporulation.
5. 16 petri dishes containing the Delaware potato variety and 16 petri dishes containing the Spunta potato variety were labelled accordingly and sealed with PARAFILM M film. The petri dishes were then incubated at 17°C. The incubation lasted for 3 weeks until there were enough sporulation on the upper side of the potato slices.

Figure 3.1: Part of the infected leaf containing sporulating lesions. (Picture taken by Nesaratnam Alwar; Biosciences Laboratory, University of Mauritius, 12th September 2014).

illustration not visible in this excerpt

3.3 Preparation of Cornell medium

The following antibiotic stock mix was prepared with 10ml Dimethyl sulfoxide (DMSO) and aliquoted in 1 ml Eppendorf tubes. The Eppendorf tubes were then stored in the freezer at -20°C. 1ml of the antibiotic mix was to be used per litre of agar being prepared for sub culture. The antibiotic mix has a brick red colour.

Table 3.1: The composition of the antibiotic mix added to 1 litre of Rye B Agar medium.

illustration not visible in this excerpt

(Source: Laboratory Manual for P. infestans work, International Potato Center, 2007)

3.4 Preparation of Rye B Agar medium

3.4.1 Composition of Rye B Agar medium

Rye B Agar is used for sporulation of Phytophthora infestans.

Table 3. 2: The composition of Rye Agar B medium. Formula adjusted, standardized to suit performance parameters.

illustration not visible in this excerpt

(Source: HiMedia Laboratories Technical Data, 2011)

Rye is a cereal grain which supplies the elements manganese, phosphorous, magnesium and the amino acid tryptophan to the pathogen. Sucrose is the carbohydrate source and Beta sitosterol helps in sporulation of the oomycete.

3.4.2 Preparation Rye Agar B medium

1. 23.76g of Rye Agar B powder was put into 4 conical flasks of 1000ml capacity each. This made a total of 95.05g of Rye Agar B powder.
2. 250ml of sterile distilled water was added to each conical flask using a measuring cylinder.
3. The 2 previous steps were carried out instead of putting 95.05g of Rye Agar B powder in only one conical flask then adding 1 litre of sterile distilled water. This was to ensure that foaming and spillage of the agar did not take place in the autoclave machine.
4. The medium in the 4 conical flasks was stirred to dissolve the light brown hygroscopic soft lumps.
5. Each conical flask was plugged with cotton wool and covered with aluminium foil.
6. They were put in autoclave at 121°C for 20 minutes.
7. In the meantime, the laminar flow hood where the agar would be put in petri dishes, was cleaned with 70% alcohol and 5% javel. A gas cartridge camping stove was lit to provide a sterile environment.
8. After removal from the autoclave, the flasks were left to cool down to about 60°C in the laminar flow hood.
9. The contents of the 4 conical flasks were poured in a sterile conical flask of 1000ml capacity and 1ml of the antibiotic mix was added. The mixture was then stirred until the brick red colour of the antibiotic mix was evenly distributed throughout the agar medium.
10. 75% of alcohol was sprayed on both hands before pouring the agar onto the plates.
11. The molten agar was then poured in 20 plastic petri dishes and left to set for 45 minutes. This also gave time for the moisture on the petri lids to evaporate.
12. The petri dishes were then sealed with PARAFILM M film.
13. The petri dishes were left overnight in the laminar flow hood.

Note: The neck of the conical flask was flamed from time to time for a few seconds. This was not done to kill microorganisms but to produce an upward flow of air from the flask such that any microorganism in the area will not fall in the flask.

illustration not visible in this excerpt

3.5 Subculture technique

3.5.1 1st Subculture

While waiting for the isolate ‘MLo’ to fully grow, the five other isolates were subcultured on the agar medium. The process was carried out in the laminar flow hood.

1. A sterile scalpel (dipped in 90% alcohol and flamed) was used to collect five small puffs of mycelia from the plates and putting them on agar. Care was taken not to scrape off the potato tissues or the agar medium of the cultured isolate.
2. The petri dishes were then sealed with PARAFILM M film and incubated and 17°C.

The scalpel is constantly dipped in alcohol and flamed especially when culturing various strains so as to avoid cross contamination.

3.5.2 2nd Subculture

After 2 weeks of maintenance of the subcultures and removal of contaminants, a 2nd subculture was made which involved all the six isolates: ‘MLo’, ‘T11’, ‘TS’, ‘PS’, ‘R1’ and ‘StP’.

3.6 DNA Extraction

1. The Petri with the purest culture was chosen and mycelia were harvested by gently scraping the aerial portion surface of the agar plate.
2. Approximately 100mg of the mycelia was weighed using an electronic balance and it was put in a mortar. 1ml of extraction buffer was added and the mycelia were grounded until fine particulates were obtained. This was done to break the cells and release the nucleus.
3. Using a micropipette, the mixture was collected from the mortar and put in 1ml Eppendorf tubes. They were incubated for 1 hour at 65 °C. Once or twice during that time, the contents were gently mix by inverting the tubes.
4. 333µl of Potassium acetate was added and the tubes shaken vigorously and put on ice for 20 minutes.
5. The tubes were spun at 14,000rpm in the Hettich MIKRO 200R Centrifuge for 10 minutes. This was the last step in the preparation of cell extract and it involved the formation of a pellet consisting of cell debris and partially digested organelles and leaving the cell extract as a clear supernatant.
6. The Eppendorf tubes then contained an aqueous clear layer containing DNA and grayish pellet which consisted of cell debris. The aqueous supernatant was removed carefully using a micropipette and put into a sterile 2ml Eppendorf tubes. Care was taken not to include the grayish pellet.
7. Purification of DNA from cell extract: 800µl of cold isopropanol was added, the tubes were inverted to mix the content and the tubes were put on ice for 30 minutes. This step favours nucleic acid precipitation.
8. The tubes were then centrifuged at 14,000rpm for 5 minutes. The DNA was precipitated as pellets and the supernatant was discarded. The pellets were dried by placing the tubes on a heat block.
9. For a 2nd precipitation, the pellets were resuspended in 700µl of TE buffer.
10. 75µl of 3M Sodium acetate and 500µl of isopropanol were then added. The contents were mixed by inversion and spun down for 30 seconds. The tubes are then stored at -20°C for overnight precipitation.
11. After overnight precipitation, the tubes were centrifuged at 13,000rpm for 30 minutes.
12. The supernatants were dumped and the pellets were washed with 75% ethanol and centrifuged again at 13,000rpm for 10 minutes.
13. The supernatants were discarded and the pellets were dried by placing the tubes on a heat block.
14. The pellets were resuspended in 50µl of TE buffer and stored at -20°C.

3.6.1 Solutions used in DNA extraction of Phytophthora infestans

It is important to note that when preparing the Extraction and TE buffer, all of their components were 10 times more concentrated and kept as stock solutions.

1. Extraction buffer

Table 3.3 The composition of Extraction buffer.

illustration not visible in this excerpt

(Source: Laboratory Manual for P. infestans work, International

Potato Center, 2007)

The Extraction buffer helps in maintaining the structure of DNA during breakage and purification steps. It also causes the inactivation of DNA degrading enzymes present in the cells.

Components of the Extraction buffer

- Ethylenediaminetetraacetic acid (EDTA)
EDTA is a chelating agent that binds with Mg2+ which is an essential cofactor of DNases, thereby inhibiting the activities of the enzymes (Allers & Lichten, 2000).
- Sodium dodecyl sulfate (SDS)
SDS breaks down the cell membrane by emulsifying lipids and denaturing proteins. This hinders the polar interactions occurring in the membrane (Simon Fraser University (sfu), no date).
- Sodium chloride (NaCl)
NaCl increases the stability of the solution as Na+ forms a cloud of positive charges around the DNA more specifically, it shields the negative phosphate ends of the DNA strand. This causes the strands to regroup and the nucleic acid to precipitate out of organic solutions.
- Beta-Mercaptoethanol (ß-ME)
Beta-mercaptoethanol (ß-ME) is a reducing agent that breaks disulfide bonds and causes the denaturation of RNases, hence inhibiting the activities of the enzyme (QIAGEN - Sample & Assay Technologies, no date).
- Tris pH 8.0

Tris protects the DNA from changes in pH

2. TE buffer

Table 3.4: The composition of TE buffer.

illustration not visible in this excerpt

(Source: Laboratory Manual for P. infestans work, International

Potato Center, 2007)

3. Isopropanol

Isopropanol is used to precipitate DNA.

4. Ethanol

Ethanol is particularly a better choice when carrying out DNA precipitation. Often, ethanol is used if small volumes of DNA are being precipitated. In this way, larger volumes of DNA can be recovered without having to worry about salt contamination as when using isopropanol because the salt remains soluble even at low temperatures in ethanol (Molecularcloning, 2012).

3.6.2 DNA Quality & Concentration investigated by Gel Electrophoresis

1. Preparation of TBE buffer (x10)

Reagents needed:

10.8g Tris base

5.5g Boric acid

4ml 0.5 EDTA (pH 8)

100ml sterile distilled water

10.8g Tris base, 5.5g Boric acid and 4ml 0.5 EDTA (pH 8) were added to 100ml sterile distilled water to prepare a stock solution of TBE buffer (x10).

Dilution procedure:

The TBE buffer was then diluted to x1 by adding 20ml of the stock solution (x10) to 180ml of sterile distilled water.

2. Preparation of small size 1.5% agarose gel

0.75g agar powder was dissolved in in 50ml of 1 x TBE buffer in a conical flask. The mixture was heated for about 45-50 seconds in a microwave. It was swirled and heated again until a clear solution was obtained. A few drops of Ethidium bromide was then pipetted in the conical flask.

The agar was left to cool on the bench and then it was poured in a gel tray. Immediately, the comb was inserted and the gel was left to set for 15 minutes.

3. Preparation of DNA samples of the isolates

5µl of DNA sample from an isolate was put in 0.2ml Eppendorf tube. 2µl of 6 x DNA Loading Dye (Fermentas) was added to the DNA sample. The process was repeated for other DNA samples.

4. Loading of the agarose gel

- The agarose gel was put in the tank and 1 x TBE buffer was poured over the gel up to the graduated level in the tank.

- 3µl of 1kb DNA Ladder (GeneRulerä, Fermentas) was loaded in the well first and the remaining wells were loaded with 7µl of DNA samples each. The 1kb DNA Ladder (GeneRulerä, Fermentas) is ideal for both DNA sizing and approximate quantification. Care was taken not to damage the well with the tip of the micropipette.

illustration not visible in this excerpt

Figure 3.5: GeneRuler 1kb DNA ladder, ready-to-use (Source: Thermo Scientific, no date).

- A few drops of Ethidium bromide was added to the running buffer.
- The tank was closed and the gel was run at 100V for 1hour and 30 minutes.
- After the running time, the gel was visualized under UV transillumination.

à Precautions should be taken when dealing with Ethidium bromide as it is a suspected carcinogen. Additionally, after the electrophoresis, the gel and the buffer were discarded in special waste bucket.

3.6.3 Determination of DNA concentration by spectrophotometric estimation

An easy way of determining the concentration of DNA is by spectrophotometric analysis. The bases present in the DNA strand absorb UV light, therefore the concentration of the DNA solution is positively correlated to the amount of UV light being absorbed.

- Concentration of pure double-stranded DNA with an A260 of 1.0 = 50 µg/ml

The following formula can be used to determine the DNA concentration of a solution (Promega, no date).

Unknown ng/µl (equivalent to µg/ml) = 50 ng/µlx Measured A260 x dilution factor

1. The spectrophotometer was blanked with 1 ml sterile TE buffer first.

2. 5 µl of the DNA preparation from an isolate was diluted with 995 µl sterile TE buffer (dilution factor x 200) and readings were taken at 260 nm and 280 nm respectively.

3. Triplicate readings for each isolate were taken at each wavelength and a mean value of DNA concentration was worked out.

4. The purity of the DNA was evaluated using the A260/A280 ratio.

5. The concentration of DNA was calculated using the above formula (in bold character).

3.7 Screening of Primers

Before testing different concentrations of DNA, the primers (Operon Technologies Inc., CA, USA) needed to be screened and those giving the best results were selected. For this project work, 30 primers were screened. RAPD primers act as both forward and reverse decamers.

Table 3.5: List of primers that were screened (Operon Technologies Inc., CA, USA)

illustration not visible in this excerpt

3.8 Storage and manipulation of the DNA templates

The DNA of the isolates, each of a capacity of 30µl (remaining after spectrophotometric analysis and gel electrophoresis) were stored at -20°C. Storing DNA at high concentration prevents its degradation. When required, a certain amount of the DNA from each isolate was taken and diluted in TE buffer. Each time, a range of concentration was tested and the clarity of the bands were evaluated.

3.9 RAPD fingerprinting

The following stock solutions were first set up:

- 50 µl of 2.5mM dNTPs (Fermentas) (a mix containing 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP)
- 50 µl of 10 µM primer (Operon Technologies Inc., CA, USA)
- Sterile distilled water

Each RAPD-PCR reaction should ALWAYS have the following 6 components:

1. Sterile distilled water
2. 5 x PCR buffer (containing MgCl2) (Thermo Scientific)
3. dNTPs (Fermentas)
4. RAPD primer (acting as both forward and reverse primer)
5. Taq polymerase (Thermo Scientific)
6. DNA template

Here we varied the concentration of the DNA template only

The master mix was prepared in stock with one extra unit amount (to compensate for pipetting errors) and then it was distributed to the respective 0.2ml Eppendorf tubes. The appropriate RAPD primers and DNA template were added to the respective tubes and the contents were mixed.

Table 3.6: Composition of PCR Master mix

illustration not visible in this excerpt

Table 3.7: PCR Cycling conditions

illustration not visible in this excerpt

The PCR products were then run on a 2% agarose gel at 80V for 1 hour and 30 minutes. A 50bp DNA Ladder was used.

illustration not visible in this excerpt

Chapter 4 Results

4.1 Culture of Phytophthora infestans isolates

The isolates ‘T11’ and ‘PS1’ grew very well with minimal to no contamination at all. The isolates ‘StP’ did not grow at all or were heavily contaminated. The isolates ‘R1’ and ‘TS1’ grew abundantly on some plates but were spoiled on the majority of the other plates. The isolate ‘MLo’ which was collected on field at Mare-Longue grew much better on potato tissue than on Rye B Agar medium.

illustration not visible in this excerpt

4.2 Estimation of presence of DNA by fluorescence of Ethidium bromide

Agarose gel electrophoresis is a way to quickly estimate the presence of DNA after DNA extraction. The intensity of fluorescence of Ethidium bromide (ethidium bromide intercalates itself within the DNA) under UV light after electrophoresis was used to estimate the amount of DNA obtained and the DNA quantity was estimated by comparing its level of fluorescence with the intensity of fluorescence of the DNA ladder. This method may not be accurate but it gives a good indication about the quality of the product obtained.

The products of DNA extraction fluoresced much more than the DNA ladder. This gave an indication that a high proportion of DNA was present. (But since the exact values of concentration were calculated from the spectrophotometric values, we did not rely on the fluorescence method much. It only served as an indicator.)

illustration not visible in this excerpt

4.2.1 Result of Spectrophotometric analysis

DNA extraction was carried out on isolate PS1, R1 and TS1. Triplicate readings for each isolate were taken at wavelength A260 and A280 and the mean value (obtained from the two closest values) was used to calculate the purity and concentration of the DNA yielded.

Table 4.1: Triplicate readings and mean value from spectrophotometric analysis of the DNA.

illustration not visible in this excerpt

(Note: In order to calculate the mean values, only the 2 closest values (in bold) from the triplicate readings were taken.)

4.2.2 Purity of DNA

The purity of the DNA was assessed by the ratio A260/A280. A value between 1.8 and 1.9 is generally regarded as “pure” for double stranded DNA as stated by Sambrook et al. (1989, cited in Ejaz et al., 2014). The DNA obtained from the isolates was hence considered as pure since the ratios were neither to low nor high.

Table 4.2: Purity of the DNA from the isolates.

illustration not visible in this excerpt

4.2.3 Concentration of DNA yielded

The concentration of DNA for each isolate was calculated using the formula:

Unknown (ng/µl) = 50 ng/µl x Measured A260 x dilution factor (Promega, no date)

where the dilution factor is 200 since 5µl of DNA was added to 995µl of TE buffer.

Table 4.3: DNA concentration for 3 isolates.

illustration not visible in this excerpt

These concentrations of DNA from each isolate act as stock solution. In order to prepare several concentrations of DNA, the stock is then diluted with TE buffer.

4.3 Screening of Primers

All the 30 primers were screened with the isolate R1. All the gel pictures obtained during the laboratory work were analysed and the Rf values of the bands were calculated by using the computer program Thermo Scientific myImageAnalysis Software v2.0 and DNA standard curves were made using Microsoft® Excel 2010.

The screening of primers was also a step where a certain DNA template concentration range was tested. In this way, the best primers but also the range of DNA concentrations to be analysed were obtained.

4.3.1 Measurement of Rf values

illustration not visible in this excerpt

Figure 4.3: Thermo Scientific myImageAnalysis Software v2.0 is a program that measures the Rf values of the DNA bands.

Thermo Scientific myImageAnalysis Software v2.0 has a user-friendly graphical interface and enables a rapid detection of the bands and the calculation of the Rf values for each of the bands present. It also has preset calibrations for every DNA Ladders such that there is no need for the user to insert each amount of base pairs manually. This software also allows the exportation of the data obtained to Microsoft® Excel 2010 to allow a graphical representation of the information. This software was used in this work to increase the accuracy of data.

4.3.2 Generation of Molecular Weight vs Rf value Semi Log Graph

After obtaining the Rf values for the DNA ladder in each picture, these data were entered in Microsoft® Excel 2010 to generate a Molecular Weight vs Rf value semi log graph. The graph is in the Semi-Log form with an exponential trendline passing through the data points. It should be noted that when using the exponential trendline, the R2 value should be close to 1 (values close to 1 indicate that all points lie approximately on a straight line with minimal scatter. Therefore, knowing the value of x enables the prediction of the value of y). The molecular weights of the bands are then obtained from the equation of the trendline.

4.3.3 Screening of OPB primers with Isolate R1

For the first screening of primers, DNA concentrations of 50,100 and 200 ng/µl were used.

illustration not visible in this excerpt

Table 4.4: Rf values for the corresponding bands of the 50bp DNA Ladder.

illustration not visible in this excerpt

From the screening of the OPB set of primers (OPB1 - OPB10) using the R1 DNA template at concentrations 50,100 and 200ng/µl, 2 primers, OPB 5 and OPB 7 gave positive results at concentration 50ng/µl as shown in figure 4.4, table 4.5 and 4.6. The presence of bands was not detected at DNA template concentrations of 100 and 200ng/µl, Instead, smears were produced at those concentrations.

Table 4.5: The number of bands obtained with the OPB 5 primer, and their presence or absence at DNA concentrations 50,100 and 200ng/µl .

illustration not visible in this excerpt

Table 4.6: The number of bands obtained with the OPB 7 primer, and their presence or absence at DNA concentrations 50,100 and 200ng/µl.

illustration not visible in this excerpt

4.3.4 Screening of OPE primers with Isolate R1

For the second screening of primers, a DNA concentration of 50ng/µl was used.

illustration not visible in this excerpt

Table 4.7: Rf values for the corresponding bands of the 50bp DNA Ladder.

illustration not visible in this excerpt

From the screening of the OPE set of primers (1-10) using the R1 DNA template at a concentration of 50ng/µl, 2 primers, OPE 3 and OPE 4 gave positive results as shown in figure 4.6, table 4.8 and 4.9.

Table 4.8: The number of bands obtained with the OPE 3 primer, at a DNA concentration of 50ng/µl.

illustration not visible in this excerpt

Table 4. 9: The number of bands obtained with the OPE 4 primer, at a DNA concentration of 50ng/µl.

illustration not visible in this excerpt

[...]