Lawrence Chibandamabwe thesis

Molecular characterisation of the P1, P3 and Coat protein genes
of a Sweet Potato Feathery Mottle Virus (SPFMV) isolate found
in Zimbabwe
By
Chibandamabwe Lawrence
A thesis submitted in partial fulfillment of the requirements of the Bachelor of Science
Honors degree in Agriculture (Crop Science)
Department of Crop Science
Faculty of Agriculture
University of Zimbabwe
May 2015

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UNIVERSITY OF ZIMBABWE
FACULTY OF AGRICULTURE
The undersigned certify that they have read and recommended to the Department of Crop
Science for acceptance, the thesis entitled:
MOLECULAR CHARACTERISATION OF P1, P3 AND CP GENES OF SWEET
POTATO FEATHERY MOTTLE VIRUS ISOLATE FOUND IN ZIMBABWE
(SPFMV)
Approved
----------------------------------------------------------
Ms. Masekesa (Supervisor)
Date----------------------------------
Dr. U. Mazarura (Chairman)
Date----------------------------------

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ABSTRACT
A study was conducted at the Crop Science Department at the University Of Zimbabwe to
characterise the only virus currently affecting sweet potato in Zimbabwe. To obtain the virus,
total plant RNA extraction was done using the ZR Plant RNA MiniPrep kit and the result ran
on a 1% agarose gel. A distinct 10Kb band was produced indicating the presence of SPFMV.
The remaining RNA was then subjected to reverse transcription using SPFMV specific
primers. The cDNA generated was then used in PCR to amplify the P1 and P3 gene of
SPFMV. The result was ran on a 1% agarose gel. Since multiple bands resulted from a single
primer pair. PCR products of the right fragment length were cut from the gel and purified
using a kit. The purified PCR products were digested using Xba1 to create sticky ends. The
same was done for pUC 19. The two digested products were then ligated together and used to
transform E.coli TG1 cells. The resultant transformation was used to generate a blue/white
colony screen for the presence or absence of P1 and P3 transformed cells. To characterise the
Coat protein in pCambia 1305, double digestion of the plasmid carrying the coat protein
insert was done using Xba1 in combination with EcoR1, Ascl, Xhol and BamH1. For both
RNA extraction and PCR the results of running products on the 1% gel showed distinct bands
confirming success of both procedures. The P1 was found to be approximately 951 bp and P3
was approximately 1041 bp and both of them were in range with other potyvirus. The
pCambra plasmid with CP of the SPFMV isolate found in Zimbabwe was characterized using
Restriction Fragment Length Polymorphism (RFLP). The enzymes Xba1/EcoR1 produced 3
fragments, Xba1/ Ascl produced 1 fragment, Xba1/Xhol produced 1 fragment and
Xba1/BamH1 produced 2 fragments. The fragments generated were compared with strains O,
S, RC, EA and the C strain. Results generated from this study using RFLP shows that the
SPFMV isolate found in Zimbabwe is slightly related to the Common strain of SPFMV and
control strategies for strain C can also be used for SPFMV-Z.

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ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my supervisor Miss Masekesa for her
guidance, inspiration, finances and intellectual support during the running of this research
project. My greatest thanks to Dr. Gasura for his guidance, intellectual support, and his time
and to Dr. Mabasa for his encouragement and motivation. Special regards goes to Tsitsi,
Nyasha, Tendai and Wisdom for their coordinated commitment in providing technical
assistance in the Physiology lab during their internship at the University of Zimbabwe and to
Dr. Ngadze for her useful lectures that helped me gain a deeper insight into the project.
Finally I would like to acknowledge members of staff in the departments of Biochemistry and
Crop Science for the help and kind use of equipment and reagents used in the project.

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DEDICATION
I dedicate this project to all my family members, friends, relatives and my fellow classmates
for their love, support and great mentorship.

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TABLE OF CONTENTS…………………………………………………..........v
ABSTRACT………………………………………………………………........ii
ACKNOWLEDGEMENTS…………………………………………………….. iii
DEDICATION…………………………………………………………………………......iv
LIST OF TABLES.......................................................................................................….....viii
LIST OF FIGURES……………………………………………………………………….viii
LIST OF APPENDICES………………………………………………………………… viii
CHAPTER ONE
Introduction................................................................................................................................1
1.1 Background..........................................................................................................................1
1.2 Problem statement................................................................................................................3
1.3 Justification..........................................................................................................................4
1.4.1 Main objective ..................................................................................................................4
1.4.2 Specific objectives ............................................................................................................4
1.5 Hypothesis............................................................................................................................4
CHAPTER TWO
2.0 Literature review..................................................................................................................5
2.1 Importance of sweet potato..................................................................................................5
2.2 Sweet potato production constraints....................................................................................5
2.2.1 Socio-economic constraints ..............................................................................................5
2.2.2 Abiotic constraints ............................................................................................................6
2.2.3 Biotic constraints ..............................................................................................................6
2.3 Sweet potato feathery mottle virus classification ................................................................7
2.4 Geographical distribution and ecology................................................................................7
2.5 Strains of SPFMV................................................................................................................7
2.5.2 The symptoms on sweet potatoes .....................................................................................9
2.6 SPFMV infection cycle........................................................................................................9
2.7 SPFMV RNA genome .......................................................................................................10
2.7.1 The known functions of the potyvirus genes ..................................................................11

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2.7.2 Importance of the P1 gene in SPFMV perpetuation .......................................................12
2.7.3 Importance of the P3 gene in SPFMV perpetuation .......................................................12
2.7.4 Properties of the SPFMV virions....................................................................................12
2.8 Detection and diagnosis of SPFMV...................................................................................13
2.8.1 Molecular based method.................................................................................................13
2.8.1.1 Nucleic acid spot hybridisation....................................................................................13
2.8.1.2 Polymerase Chain Reaction (PCR) Method ................................................................13
2.8.1.3 Restriction Fragment Length Polymorphism...............................................................14
2.8.2. Serological tests .............................................................................................................14
2.8.2.1 Thin section immunohistochemistry............................................................................14
2.8.2.2 Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA)..15
2.8.2.3 Membrane Immuno-Binding Assay (MIBA)...............................................................15
2.8.2.4 Electron Microscopy....................................................................................................16
2.9 Current work on inducing transgenic resistance to SPFMV in sweet potatoes .................16
CHAPTER THREE
MATERIALS AND METHODS ..........................................17
3.1 Study site............................................................................................................................17
3.2 Total plant RNA Extraction (ZR Plant RNA MiniPrep™ Protocol).................................17
3.3 cDNA Synthesis.................................................................................................................18
3.4 PCR Amplification.............................................................................................................18
3.5 Agarose gel electrophoresis ...............................................................................................21
3.6 Competent Cell Preparation (Calcium Chloride Treatment) materials..............................21
3.6.1 Luria Bertoni broth preparation ......................................................................................22
3.6.2 Luria Bertoni Agar medium preparation.........................................................................22
3.6.2 Competent cell preparation procedure............................................................................22
3.6.3 Plasmid and PCR product digestion and clean up ..........................................................22
3.6.6 Transformation................................................................................................................23
3.6.7 Stock solution..................................................................................................................23
3.7 Purification of plasmid DNA from 1.5 ml cultures of E.coli- miniprep procedure...........24
3.7.1 Cell lysis..........................................................................................................................24
3.7.2 DNA purification ............................................................................................................24

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CHAPTER FOUR
4.1 Total plant RNA extraction................................................................................................25
4.2 Amplification of P1 and P3 using different primer combinations.....................................26
4.2 Cloning (blue white colony screening)..............................................................................27
4.3 Characterisation of Coat Protein using RFLP....................................................................27
CHAPTER FIVE
5.1 Total plant RNA extraction................................................................................................30
5.2 Amplification of P1 and P3 using different primer combinations.....................................30
5.3 Cloning (blue white colony screening)..............................................................................31
5.4 Characterisation of Coat Protein using RFLPs ..................................................................32
CHAPTER SIX
6.1 Conclusion .........................................................................................................................33
6.2 Recommendations..............................................................................................................33
LIST OF TABLES
Table 2.1 The known functions of the potyvirus genes...........................................................11
Table 3.3 Primer combinations used for P1 gene amplification..............................................19
Table 3.3 Primer combinations used for P3 gene amplification..............................................20
Table 4.1 Comparison of CP genes by RFLP of SPFMV in Zimbabwe with other strains ....29
LIST OF FIGURES
Fig. 1 Diagram to show the organisation of SPFMV genome ................................................10
Fig 4.1 Image showing the bands of total RNA extraction………………………………… 38
Fig 4.2 Image showing the bands P1 and P3 genes .................................................................26
Fig 4.3 Image showing blue white colony screening...............................................................27
Fig 4.4 Image showing characterisation of CP using RFLPs ..................................................28

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LIST OF APEPENDICES
Appendix A1: Restriction analysis of CP of strain C ..............................................................41
Appendix A2: Restriction analysis of CP of strain EA............................................................41
Appendix A3: Restriction analysis of CP of strain O..............................................................42
Appendix A4: Restriction analysis of CP of strain RC............................................................42
Appendix A5: Restriction analysis of CP of strain S...............................................................43

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CHAPTER ONE
INTRODUCTION
1.1 Background
Sweet potato, Ipomea batatas (L) is a dicotyledonous perennial plant grown as an annual that
is found in the Morning Glory or Convulvuceae family (Karyeija et al., 2000). The crop ranks
fourth in position of importance after rice, wheat and maize in developing countries and ranks
seventh in position worldwide (Karyeija et al.,1998). Sweet potato has an average world
production of 122 million metric tonnes per annual and the biggest producer of the crop is
China. China produces 102 metric tonnes/year, which translates to 80% of total world
produce. The African continent as a whole produces 7.5 million metric tonnes per year and
this contribution is 6% of total world production (Bailey et al., 2009). According to Factfish
(2014), Zimbabwe is ranked number 86 on world sweet potato production and contributes
0.01% to the world production.
Sweet potato is of great importance to human health as the tubers and leaves contain large
quantities of beta-carotene and protein of between 5 to 18.5% of dry matter (Degras, 2003).
The purple fleshed sweet potato varieties are high in peonidins and cyanidins anthocyanins
between (350-410mg/100g of dry matter). These anthocyanins serve as antioxidants and anti-
inflammatory factors in human bodies. Orange fleshed sweet potatoes have high amounts of
vitamin A, a figure capable of meeting 30 to 38% of vitamin A requirements in people. Other
vitamins and minerals present in sweet potato are, vitamin C at 20-25 mg/100g fresh weight,
vitamin B1, B2, B5, B6 between 0.05-060mg/100g fresh mass, niacin, dietary fibre and
phosphorous (Ukpabi et al., 2012).
Sweet potato is also used in the fuel industries for production of biofuels as a complement to
non-renewable fossil fuels to prevent their depletion as sweet potato tubers can produce
11 300 L/ha of ethanol. The sweet potato tuber is also used as a raw material in the beverage
industry, for instance China produces 45 000-55 000 t/yr of alcohol (Degras, 2003) from the
crop alone. Some countries also use sweet potato vines and tubers for livestock feed thus
helping to reduce the small holder farmers’ expenditure.

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Despite the great potential for use in industry and at household level, sweet potato production
in the country is still very low as it is prone to both biotic and abiotic strains which negatively
reduce tuber yield. One of the major biotic constrains in sweet potato production is viral
infections which accounts for 50% crop loss in Zimbabwe (Chavi et al., 1997). Chavi et al.
(1997) while conducting an epidemiology study on viruses infecting sweet potato in
Zimbabwe, identified only one virus as being the only virus or at least the major one
currently infecting Zimbabwean sweet potato. Chavi positively identified the Sweet potato
feathery mottle virus (SPFMV) as being the main virus contributing to yield loss in
Zimbabwe. SPFMV is a virus in the Potyviridae family in the genus Potyvirus and this is the
largest plant viral genus with over 180 members (Revers et al., 1999). The potyvirus has long
virions that measure between 820 to 865 nm in length. The viral genome is a single stranded,
positive sense RNA molecule which also serves as the mRNA for translation into viral
protein. The nuclear structure of SPFMV was first determined by Dootlittle and Hartler in
USA in 1945, while in Africa the first report of the virus was made in Tanzania, then Kenya
and Uganda in 1957 (Karyeija et al., 1998).
SPFMV is ubiquitous and is found were ever sweet potato is grown. Infection by a single
virus causes marked yield losses as compared to when the virus forms synergistic infection
with other sweet potato viruses which can lead to total yield loss. The economic losses in
sweet potato production in Africa by SPFMV is because it causes sweet potato virus diseases
(SPVD) (Karyeija et al., 1998) a result of dual infection between sweet potato feathery
mottle virus (SPFMV) and sweet potato chlorotic stunting virus (SPCSV), a virus in the
Closteroviridae family and genus Crinivirus (Karyeija et al., 2000b). The symptoms of the
infected sweet potatoes are mosaics, leaf purpling, plant stunts, vein clearing, mottling and
feathery and also leaf distortion. The presence of symptoms such as leaf distortion and
mosaics reveals that the SPFMV dominates in SPVD (Karyeija et al., 1998). The SPVD
attacks the economic part of sweet potatoes, the roots, causing internal corkiness and external
cracking (Ndunguru et al., 2009). SPVD can cause losses ranging from 45-90% and can even
go up to 98% (Gasura and Mukasa, 2011). In Africa, SPFMV is considered as a major
problem in sweet potato cultivation (Ndunguru et al., 2009).
Chavi et al. (1997) conducted pioneering work in sweet potato viral disease epidemiology.
His work lead to the partial characterisation of SPFMV strain infecting sweet potato in

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Zimbabwe. Chavi et al. (1997) was able to elucidate the nucleotide sequence of the coat
protein gene of SPFMV but unfortunately could not sequence the remaining nine genes of the
virus. The aim of this study is to clone, characterise and hopefully conduct sequencing of the
P1 and the P3 genes of SPFMV. Analysis will also be done on recently isolated SPFMV that
was developed into coat protein clones to try and see if the virus identified by Chavi et al.
(1997) is still the same, has mutated or is completely different from the potyvirus that they
worked with. Sequencing, which is determining the order of nucleotides sequence on the
genome (Acquaah, 2012) will also be attempted and this will be done to increase the
understanding of SPFMV variability (Mukasa et al., 2003).
1.2 Problem statement
Despite the potential for use of sweet potato in the fuel industry, as the crop is high yielding
(up to 18 tonnes/ha) as well as having a high nutritional content for the achievement of food
and nutritional security, the UN MGD 1 goal, SPFMV which causes SPVD in sweet potato
has not been studied extensively in Zimbabwe in recent years. There is little scientific
research studies on the genome of sweet potato feathery mottle virus strain found in
Zimbabwe that will provide knowledge for circumventing the problems that are caused by
SPFMV. Since the isolate was characterised in 1997 there was no other characterisation up to
now (Chavi et al., 1997). There is high probability of having new strains or mutations on the
isolate already characterised by Chavi et al. (1997). The Zimbabwean SPFMV isolate was
partially sequenced and revealed an addition of 22 amino acids at the N-terminal of coat
protein (CP) which shows great divergence from other SPFMV strains (Chavi et al., 1997).
Basing on the CP sequence of the SPFMV-Zim in the Genebank, differences with other
SPFMV strains were also confirmed (Mukasa et al., 2003, Souto et al., 2003) but further
characterisation of CP with RFLP and molecular characterisation of P1 and P3 genes is
required to enhance the understanding of the genetic variability of SPFMV-Zim from other
SPFMV strains. There is need to confirm the differences using other protein coding regions
on the RNA genome of the SPFMV isolate found in Zimbabwe.

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1.3 Justification
According to Chavi et al. (1997), further cloning and molecular characterisation of the
SPFMV-Z genome will reveal its position within the Potyvirus family. The molecular
characterisation of P1, P3 and CP genes will allow the development of fast, sensitive and
reliable PCR tests to monitor or manage the development of viral infections in fields with
new virus-free sweet potato clones or tissue cultured sweet potatoes. Cloning and
characterisation of the P1, P3 and CP genes of SPFMV and assessment of the genetic and
evolutionary relationship with other sweet potato viruses will add to the body of knowledge
of potyviruses genomics and increase information about SPFMV variability. This will also
help to control the SPFMV by conventional breeding and genetic engineering of sweet
potatoes both of which hold promise for inducing tolerance to the virus. This ability depends
mostly on characterisation. It has to be noted however that genetic engineering is not largely
supported in developing countries and lacks public support although Zimbabwe is hopeful of
harvesting the benefits of biotechnology (Masekesa and Gasura, 2013). This will increase the
quality and quantity of sweet potato.
1.4.1 Main objective
 To characterize the Coat protein gene of SPFMV using RFLPs.
1.4.2 Specific objectives
1) To detect and amplify the P1 and P3 genes of a SPFMV found in Zimbabwe using PCR
based methods.
2) To clone the P1 and P3 viral genes of SPFMV into pUC 19.
1.5 Hypothesis
1) It is possible to detect and amplify the P1 and P3 genes of SPFMV.
2) The P1 and P3 viral genes of SPFMV can be cloned into pUC 19.
3) It is possible to use RFLPs to characterize the coat protein gene of SPFMV found in
Zimbabwe and possibly classify the virus as either a common, Severe, Ordinary, Russet
Crack or East African strain.

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CHAPTER TWO
LITERATURE REVIEW
2.1 Importance of sweet potato
Sweet potato (Ipomea batatas) originated in south America or central America 5000 years
ago (Kivuva, 2013) and is one of the most important food crops in Africa. For Sub Saharan
countries it plays a significant role in food security and holds promise for being
commercialised (Kivuva et al., 2014). In developing countries it is ranked fourth in position
after rice, wheat and maize and it is also ranked third most important root crop after potato
and cassava (Karyeija et al., 1998). Sweet potato is grown in tropical and subtropical regions
of the world and this is usually under marginal conditions with low inputs but yields are
often high (40-50 t/ha) (Valverde et al., 2007). Sweet potato is also becoming important in
Zimbabwe especially in both rural and urban societies. In the rural areas of Zimbabwe
consumption is at 2-5 kg per capita and in the urban areas, the consumption rate is 0.5-6.5 kg/
capita and about 10.5% of rural sweet potato production is consumed in urban areas
(Mutandwa, 2008). Sweet potato as a starchy root crop has a composition of about 1.2-8.5%
protein, 3.2-6% crude fibre, 1.3-6.0 ash and 0.2-1.8% lipid and orange fleshed has 38.60-
66.30 μg/g β-carotene which is important for human health (Ukpabi, 2012). Sweet potato can
be boiled and eaten as breakfast food, milled to flour, chipped and also used to prepare baby
weaning foods. Sweet potatoes are used to generate income by small holder farmers by
selling the roots and vines as planting material. Some companies tin the processed tuber for
export and the light industries use sweet potato tubers as raw material for fermented products
such as ethanol, wine, and butanol. Sweet potato is used to produce natural colorants, starch
and can even be used as livestock feeds in some countries (Kivuva et al., 2014).
2.2 Sweet potato production constraints
2.2.1 Socio-economic constraints
Sweet potato production is reduced by socio-economic constraints which includes poor
agronomic practices poor extension, poor post-harvesting knowledge and lack of clean virus
free vegetative propagation vines (Kivuva et al., 2014). Most farmers dig holes for tuber
storage and this is a poor tuber storage facilities (Mutandwa, 2008) which leads to huge post

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harvest losses. Potential production is never achieved due to unavailability of high yielding
varieties and disease resistant varieties (Ndunguru et al., 2009). According to Kivuva et al
.(2014), most the of sweet potato production farmers are women and children .
2.2.2 Abiotic constraints
Abiotic constraints also greatly reduces sweet potato production. For example drought and
inherent low fertility soils in the tropics and sub-tropical regions of Africa greatly affect
yield. Although sweet potatoes are drought tolerant (Rukundo et al., 2013), extreme drought
caused yield loss as a result of disruption of the source sink relationship and the vines are
more prone during crop establishment (Kivuva, 2013). The continuous cropping without
addition of manure and fertilisers for nutrients replenishment also decreases productivity as
cited by (Ngailo et al., 2013).
2.2.3 Biotic constraints
Despite its benefits and potential uses, sweet potato production remains very low with yield
potential of 9t/ha instead of 50t/h in the tropics (Kivuva et al., 2014). In Zimbabwe, the main
growing agro-ecological regions are 1, 2 and 3 and the main production zones are
Mashonaland Central, East and West, Manicaland, Masvingo and Midlands and yield is
around 0.5 tonnes per hectare (Mutandwa, 2008). The sweet potato weevils, Cylas
formicarius are devastating as they feed on the root and vines thereby reducing quality and
quantity of the root tuber and may cause up to 100% yield losses as cited by Ngailo et al.
(2013). Alternaria leaf spot, weeds, stem blights, nematodes, bacterial rot and Fusarium rot
are also biotic constrains affecting sweet potato production (Ngailo et al., 2013; Kivuva et al.,
2014). The low yields are due to production constraints such as insects, weed and viral
diseases (Ndunguru et al., 2009). Sweet potato virus disease caused by the co-infection of
Sweet potato chlorotic stunt and Sweet potato feathery mottle virus causes yield loss of up to
98% (Karyeija et al., 1998).

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2.3 Sweet potato feathery mottle virus classification
Sweet potato feathery mottle virus (SPFMV) belongs to the family Potyviridae and the genus
Potyvirus (Untiveros et al., 2010; Tugume et al., 2010; Karyeija et al., 1998; Kreuze 2002).
The family Potyviridae is the largest taxon among viruses affecting plants and has six genera
which are Potyvirus, Mancluravirus, Rymovirus, Bymovirus, Tritimovirus and the
Ipomovirus. Most of the members are monopartite excluding the Bymovirus which is bipartite
(Mukasa, 2004). The Potyvirus genus has virions which are flexuous filamentous rods of
800-850 nm and have positive-sense single stranded RNA genomes (Sakai et al., 1997).
2.4 Geographical distribution and ecology
SPFMV is distributed worldwide and is found where sweet potatoes are produced (Kreuze,
2002a) although the disease is most pronounced where continuous cropping is practiced in
the tropical and subtropical regions (Kerlan, 2006). The climate in the temperate regions
allow Potyvirus to survive on perennial and vegetative propagated crops but the restricted
host range and few alternative host species makes the disease to be of minor importance
(Kerlan, 2006). SPFMV is transmitted by cotton aphid (Aphis gossipi) and green peach aphid
(Myzus persicae) in a non-persistent manner (El-din, 2008; Kreuze, 2002; Mukasa, 2004;
Revers et al., 1999; Shukla and Ward 1988; Souto et al. 2003; Ngailo et al. 2013; Gasura and
Mukasa, 2011; Karyeija et al., 2000b). The rate of SPFMV spread and severity is greatly
increased by proximity and occurrence of the virus source for example volunteer crops and
the number and activity of the aphids as vector species (Kerlan, 2006).
2.5 Strains of SPFMV
The phylogenetic analysis of the coat protein reveals that the isolates of the SPFMV are
divided into four strains which are C (common), RC (russet crack), O (ordinary) and EA
(East African) (Tugume et al., 2010). The symptoms, host range of virus and serological tests
have been extensively used to group the SPFMV into different strains. The strains RC, O and
the EA are phylogenetically identical and different from strain C (Untiveros et al., 2010). The
Russet Crack causes internal corkiness to the affected sweet potatoes for example the Jersey
variety. This strain has been found in Japan, Korea and China (Kreuze, 2002a). The RC

8
causes local lesions, necrosis of roots and cracking of root tubers whilst the C strain does not
cause necrosis and root cracking but offers cross protection against the RC strain (Karyeija et
al., 2000a). The O strains are usually found in Argentina, Nigeria, USA and the C strains are
found in Argentina, China and USA (Okada et al., 2008) and shows up to 78.3% sequence
identity with other strains. The isolates from East Africa have been placed into the fourth
group, the EA and no other SPFMV strain besides EA has been found in East Africa (Okada
et al., 2008).
The complete nucleotide sequence of SPFMV-S was obtained from complementary DNA
clones, (Sakai et al., 1997) and also by sequencing the viral RNA. The RNA genome of
SPFMV-S was found to be 10 820 bases excluding the poly (A) tail. The SPFMV strain C
has been completely sequenced together with the 5’ region of other strains (Untiveros et al.,
2010). They observed a hypervariable domain on the large protein coding region of P1 gene.
The SPFMV LSU-2 and SPFMV-Zim have 22 amino acids at the N-terminal of the coat
protein gene which is absent in other SPFMV strains (Souto et al., 2003) and the SPFMV-
Zim is grossly diverged from all SPFMV strains (Mukasa et al., 2003).
2.5.1 Phylogenetic relationships and genetic variability of SPFMV
Phylogenetic relationships using the representative CP-encoding region sequence shows that
SPFMV-Z with the NCBI accession number AF016366, is distinctly different in the N-
proximal from other SPFMV isolates. The comparison of the Coat protein amino acid
sequences reveals high identity of the C-proximal sequences in the SPFMV-Z and SPVG
isolate in Egypt. The N –proximal part of the coat protein is different between the SPFMV-Z
and the SPVG. The SPVG isolate from Ethiopia has 355 amino acids on the CP and 221 nt on
the 3’UTR and phylogenetic relationship analysis reveals Ethiopian and SPVG isolates had a
closer similarity with sweet potato virus 11 (SPV 11) and the Zimbabwean SPFMV
(SPFMV-Z) (Anon 2004). Chavi’s isolate SPFMV-Z isolate reveals an amino acid sequence -
SSETSEIKDAGATPTKFPKTSGKGTGTQTTTPLEKPPGAVDPTIPPP on its N-terminal
part of the CP gene and this sequence data propose that the isolate is a distinct potyvirus
(Souto et al., 2003). However the detection of the SPFMV-Z with antisera using the
immunogold labelling electron microscopy revealed that the virus shared common epitopes
and has serological relationship with SPFMV-RC (Chavi et al., 1997).

9
2.5.2 The symptoms on of SPFMV on sweet potato
The symptoms caused by single infection with SPFMV are mild and the symptoms are vein
clearing and feathery and also spots on the older leaves (Karyeija et al. 2000b). The
economic yield loss by SPFMV is external cracking and internal corkiness of the root tuber
(Karyeija et al., 2000b). When SPFMV occurs in dualist infections, the SPVD causes
chlorotic, malformed leaves, stunted growth and yield reduction although infection by a
single strain causes little yield loss (Ngailo et al., 2013). The yield losses of SPVD can be up
to 98% (Ngailo et al., 2013) and it can led to the extinction of some elite varieties (Gasura et
al., 2010). The decrease in root diameter in SPVD shows dominance of SPFMV. According
to Ndunguru et al. (2009), there is lower incidence and severity of SPVD in cooler agro-
ecological regions as compared to warmer and prolonged hot and dry spells breaks the natural
transfer of virus between crops (Ngailo et al., 2013).
2.6 SPFMV infection cycle
The SPFMV is acquired by the aphid through its stylet during feeding and the acquisition
take place in a matter of seconds and its transmissibility is lost after some minutes (Kreuze,
2002a). The virus acquisition by the aphid depends on the DAG box, amino acid motif,
Aspartate-Alanine-Glycine on the N-terminal of the coat protein, the KITC box with the
amino acid motives, Lysine-Isoleucine-Threonine- Cysteine on the N-terminal of the HC-Pro
(Sakai et al., 1997) and also the PTK box with the Proline-Threonine-Lysine amino acid
motives in the N-terminal of the helper component protein (HC-Pro). The HC-Pro make
changes on the conformation of the coat protein allowing binding to the stylet (Kreuze,
2002a). The aphid inoculates new host by regurgitating its saliva during feeding. After
infection, the virus starts to disassemble and by a process called co-translational disassembly
it is then translated and replicating into new viral proteins with a single open reading frame
which is then autocatalically processed into functional proteins. After replication it starts to
move from cell to cell into the vascular tissue with the aid of the movement proteins which
supress the plant defence mechanisms by preventing plant cell-to-cell communication
(Kreuze, 2002a).

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2.7 SPFMV RNA genome
Fig. 1 Diagram to show the organisation of SPFMV genome
Adapted from (Revers et al., 1999)
SPFMV has the viral protein (VPg) which is covalently linked to the 5’ end (Revers et al.,
1999). The RNA genome has a single open reading frame (ORF) and the proteins products
are indicated in boxes parted by lines indicating putative cleavage positions of the
polyprotein. The SPFMV RNA genome encodes a large polyprotein processed to useful
proteins which are P1, HC-Pro (Helper component), P3, 6K1, Cl (Cytoplasmic Inclusion),
6K2, Nla (Nuclear Inclusion a), Nlb (Nuclear inclusion b) and the CP (Coat Protein) (Sakai et
al., 1997).
SPFMV possess elongated rods between 800-860nm with a single-stranded RNA up to 15%
larger than other typical potyvirus and the RNA genome is also approximately 10.6Kb (Wang
et al., 2007). The functions of the genes of SPFMV genome among other Potyviruses have
been elucidated although the functions of the P1 ,P3 and the two 6K2 are not well
understood in detail (Sakai et al., 1997). The SPFMV isolates from the Central Taiwan, CY1
and CY2 had been sequenced and the CP genes contained 313 and 315 amino acid residues
respectively and the important CP domain was DAG box positioned between amino acids 9-
11 from N-terminal of the CP used in aphid transmission (Wang et al., 2007). The complete
nucleotide sequence of SPFMV-S which was said to be in EMBL and the Genebank data of
nucleotide sequence accession number D86371 shows that SPFMV-S includes the P1 (74K),
P3 (46K) and the coat protein 35K. The CP has 664 amino acids and is the biggest and least
resemblance to other potyviruses.

11
2.7.1 The known functions of the potyvirus genes
Table 2.1 The known functions of the potyvirus genes
Gene Known function Reference
P1 Potyviral genome amplification
Proteinase
ssRNA binding activity and movement protein
in Tobacco vein mottling virus (TVMV)
(Revers et al., 1999)
(Sakai et al., 1997)
HC-Pro Posttranscriptional gene silencing (PTGS)
Genome amplification and symptom
development
Vascular movement protein
Aphid transmission
(Savenkov and Valkonen
,2001;Karyeija et al., 2000b)
(Sakai et al., 1997;Savenkov
and Valkonen 2001)
(Dolja et al., 1993)
P3 Potyvirus genome amplification (Revers et al., 1999)
6K1 Involvement in viral replication (Mukasa ,2004;Kreuze, 2002b)
Cl Virus replication and assists viral movement (Sakai et al., 1997;Revers et al.
1999)
6K2 Participate in viral replication
RNA helicase
Long distance movement
(Merits et al., 1999)
Rajamaki and Valkonem 1999
cited by( Mukasa, 2004)
Nla/VPg
Nla/Pro
Cell to cell movement and long distance
movement
Proteinase
Nlb Replicase
RNA polymerase activity used for viral
replication
CP Aphid transmission
RNA replication

12
2.7.2 Importance of the P1 gene in SPFMV perpetuation
The P1 gene is suggested to be involved in host selection and also functions as an RNA
silencing suppressor in the SPFMV ancestry (Li et al., 2012). The conservation of the P1 N-
domain in SPFMV also suggests it to be important for its fitness in sweet potatoes infection.
The P1-Pro protein is a serine protease which is involved in polyprotein processing by
cleaving at the P1-Pro/ HC-Pro junction (Kerlan ,2006). The P1 gene targets RNA silencing
which helps to defend the plant against the virus. In tobacco vein mottling virus, P1 has a
proteinase activity at its C-terminal autoprocessing (Kreuze, 2002b) and is also involved in
genome amplification (Sakai et al., 1997).
2.7.3 Importance of the P3 gene in SPFMV perpetuation
The P3 gene is associated with the cylindrical inclusion protein therefore involved in
replication. The P3 function is not well known (Sakai et al., 1997) but it is believed to be
involved in inducing wilting. The P3 is also involved replication or virus propagation and
movement (Kreuze, 2002b).
2.7.4 Properties of the SPFMV virions
SPFMV has numerous biological and cytological similarities with other Potyviruses and also
has intrinsic viron with a length approximately 810-860nm. The coat protein encoding region
has a Mr of 3.8 x 10⁴ (Moyer and Cali 1985). Most particles of the Potyviruses have a length
between 600-700nm but some can be as long as 800nm after relaxation with divalent positive
ions and few have particles with a length above 800nm even without the addition of divalent
cations and these extra length of the viral particles encapsidate up to 15% more RNA genome
which might encode an additional polypeptide.
.

13
2.8 Detection and diagnosis of SPFMV
2.8.1 Molecular based method
2.8.1.1 Nucleic acid spot hybridisation
Nucleic acid hybridisation is a method that utilises labelled viral DNA or RNA in
combination with radioactive or non-radioactive labels (Valverde et al., 2007). The RNA is
extracted from the samples and base-denatured in NaOH and the samples blotted to a nylon
membrane. The membrane is fixed by ultraviolet (UV) or heat and hybridised with a
characterised probe consisting of cDNA of the required SPFMV. If the required SPFMV is
present it is hybridised with the DNA extracts in the sample. The use of non-radioactive
techniques such as dioxigenin-anti-digoxigenin (DIG) and horseradish peroxidase (HRS) are
also used to label the nucleic acid used as probes to detect pathogens. Nucleic acid spot
hybridisation is able to detect single stranded and double stranded virus nucleic acid but it is
more laborious than ELISA (Wasswa 2012). The use of riboprobes that are complementary to
the RNA has been identified as being more sensitive (Dje and Diallo, 2005) than serological
assays, labelled cDNA or the immunobinding assay due to its ability to remove non-
hybridised probe and also specificity. For SPFMV detection, in-vitro transcribed RNA
probes were developed from cDNA with 3’ terminal region of the capsid protein cistron
(Chavi et al. 1997). The riboprobes also overcomes the intervention of the host factors that
reduces reliability of the immunodiagnostic assay (Dje and Diallo ,2005).
2.8.1.2 Polymerase Chain Reaction (PCR) Method
The nucleic acid spot hybridisation (NASH) and PCR based protocols have been developed
to cater for the diagnosis of sweet potato viruses and they are present in advanced
laboratories (Mwanga, 2001). PCR protocol is able to detect distinct potyviruses in the sweet
potato infected plants (Chavi et al. 1997). Multiplex complex PCR techniques have been
developed for the detection of multiple complex sweet potato viruses simultaneously. The
multiplex PCR technique for detection of SPFMV and Sweet potato chlorotic stunt virus
(SPCSV) has been developed (Opiyo et al., 2010). The PCR method starts by extraction of
DNA or RNA from the infected plant tissue using the available kit following the
manufactures procedure (Wasswa, 2012). The PCR technique encompasses RT-PCR to make

14
cDNA and involves synthesising many copies of DNA by in-vitro reactions in the presence of
Taq DNA polymerase enzyme. The multiplex PCR is able to detect many viruses in a single
sample simultaneously (Wasswa, 2012). RT-PCR utilises specific primers or universal
primers that are designed on the basis of the conserved regions that are present in all viral
strains and it detects SPFMV infected material irrespective of the viral isolate (El-din, 2008).
The forward oligonucleotide primers bind to opposite ends 3’-5’ and reverse primers bind to
5’-3’ on the region of interest of the virus nucleic acid. The restriction fragments are
separated by gel electrophoresis in 1.5% agarose gel (Rännäli et al., 2008) and the bands
stained using ethidium bromide (Colinet, 1994) and visualised using UV light (Wasswa,
2012).
2.8.1.3 Restriction Fragment Length Polymorphism
The use of PCR/RFLP is a broad spectrum method that is used for detection of virus
complexes for instance SPFMV and Sweet potato leaf curl virus (SPLCV). RFLP is used as a
primary approach for rapid confirmation and differentiation of potyvirus and sweepovirus
infections (Clark et al., 2012). RT-PCR is also used to identify different strains in sero-
positive plants by revealing the amplification products by RFLP (Tairo, 2006).
2.8.2. Serological tests
Serological techniques are most preferred to date as compared to other methods that are being
used in the detection of viruses (Shukla and Ward, 1988) and (El-din, 2008). Serological
techniques are more popular because they are inexpensive, simple, and efficient and
reproducibility is high. To control the spread of SPFMV infection, highly sensitive and
reliable techniques for virus diagnosis should be used (El-din, 2008).
2.8.2.1 Thin section immunohistochemistry
Thin leaf pieces of the suspected SPFMV infected sweet potato plant are cut with a scalpel
and instantly immersed into (4% formaldehyde), a fixative agent over a night. The fixed
samples are treated in Histo-Clear and implanted in paraffin (Savenkov and Valkonen 2001).
The embedded thin leaf piece of about 7µm is cut with microtome and shifted to micro slides.
The samples are enclosed with poly-L-lysine and incubated at 37ºC over the night (Savenkov
and Valkonen, 2001). Paraffin is removed by washing with xylene twice then the sample is

15
rehydrated and then washed with Phosphate buffered saline (PBS). The samples are pre-
incubated in PBS with 4% Bovine Serum Albumin (BSA) for half an hour and then incubated
with monoclonal antibody diluted in PBS with 4% BSA for about 1 hour with temperature
maintained at room temperature. After the samples washed with PBS, the thin sections are
incubated with the rabbit anti-mouse monoclonal antibodies combined with an alkaline
phosphatase at room temperature for about half an hour. The samples are then washed with
PBS and stained with Fresh Fuchsin solution(Savenkov and Valkonen, 2001) and tissue
counter stained with Mayers reagent (Karyeija et al., 2000b). This method is preferred as it is
able to detect low titres of virus in the affected plant even at latent infection (Savenkov and
Valkonen, 2001).
2.8.2.2 Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA)
The Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) is
done using rabbit polyclonal antibody which is raised against SPFMV and also a polyclonal
alkaline phosphatase-labelled goat anti-rabbit is used as secondary antibodies. The results can
be visualised using the Bio-Rad microplate reader 3550 (El-din, 2008). The sensitivity and
reliability of DAS-ELISA in SPFMV diagnosis is decreased if there is low virus
concentration in the infected plant tissue (El-din, 2008). The inhibitory factors which includes
the phenols and latex which interferes with the specific binding of virus and specific
antiserum results in difficulties of distinguishing healthy and infected plants (Aritua et al
.,2005) cited by (El-din, 2008).
2.8.2.3 Membrane Immuno-Binding Assay (MIBA)
Membrane Immuno-binding Assay is a method of detection of SPFMV that is efficient and
specific. The basis of the immune-fingerprinting method on the nitrocellulose membrane is
the recognition specificity between the viral antigens and the polyclonal antibody against
them (Dje and Diallo, 2005). The viral concentration in the sampled plant tissue allows is
directly proportional to the spots or dark bands to be observed after reactions on the
membrane. The MIBA technique is highly sensitive which allows detection of many viruses
and also specific which allows detection of the known virus. It is very useful SPFMV
diagnosis method as it is able to detect between the healthy and the infected plants (Dje and
Diallo, 2005).

16
2.8.2.4 Electron Microscopy
The SPFMV can be detected by visualising its particles on the electron microscopy. By
visualising potyviruses on the electron microscopy, the cylindrical inclusion (Cl) is observed
to form aggregates called pinwheels in the cytoplasm of the diseased cells (Revers et al.,
1999). The infected plant tissue sample is negatively stained with 2% sodium phosphate-
tungstate (PTA) which is a dense electron stain or uranyl acetate and visualised on an
electron microscopy. Microscopy detects a wide range of virus and do not require specific
virus reagent. According to Schoepp et al. (2004) cited by (Wasswa, 2012), electron
microscopy detected virus in infected samples preserved for decades in unknown solutions.
2.9 Current work on inducing transgenic resistance to SPFMV in sweet potatoes
Although sweet potatoes have their own natural resistance to SPFMV, it is of limited use
hence transgenic resistance holds promise. The Japan International Research Centre for
Agriculture and the International Centre for Potato (CIP) work on augmentation of natural
resistance by incorporating transgenic resistance of sweet potatoes to SPFMV. They are using
the virus coat protein and the cysteine proteinase inhibitor (Kreuze, 2002a). The
protein/pathogen mediated resistance (PDR) is induced by incorporating the virus coat
protein in the transgenic plant. The expression of the viral coat protein in the plant interfere
with the uncoating of the virus upon infection and restricts its movement (Kreuze, 2002a).
The use of foreign cysteine inhibitors is another approach that is currently used whereby the
rice cysteine inhibitor is used to inhibit proteolysis of polyprotein in SPFMV which interfere
with replication (Cipriani et al., 2000). To cater for the co-infection of sweet potato feathery
mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV), their NIb gene (RNA
dependant RNA polymerase) sequences are fused and the inverted repeat of this fused
segment separated by intron (CSFMhr) is then transformed to sweet potatoes. The
transcription of this fragment leads to the formation of dsRNA which is specific to the two
virus hence preventing SPVD (Kreuze, 2002a).

17
CHAPTER THREE
MATERIALS AND METHODS
3.1 Study site
Experiments were conducted in the Physiology Laboratory and Plant Breeding at the
Department of Crop Science and in the department of Biochemistry Laboratory at the
University of Zimbabwe.
3.2 Total plant RNA Extraction (ZR Plant RNA MiniPrep™ Protocol)
The fresh 2 grams of visible virus symptom sweet potato new leaves were pounded and the
plant sample was filled into a 2 ml ZR BashingBread™ Lysis Tube and 800 µl Lysis buffer
was added to the sample using a micropipette. The sample in a ZR bashing bead lysis tube
was centrifuged at 12 000 x g for a minute. To a Zymo-spin lllC Column in a collection tube,
400µl of a supernatant was transferred and centrifuged at 8000 x g for 30 seconds. To the
through-flow in a collection tube 320µl of 95% ethanol was added to 400µl sample. The
mixture was transferred to a Zymo-Spin™ llC⁴ in a collection tube and it was centrifuged at
12 000 x g for 30 seconds and the flow-through was discarded. RNA Prep Buffer of 400µl
was added into the column and the mixture was centrifuged at 12 000 x g for 1 minute. The
flow-through was discarded and the Zymo-Spin™ llC Column was replaced back into the
collection tube.
To the column, 800µl of RNA wash buffer was added and centrifuged at 12 000 x g for 30
seconds. The flow-through was discarded and the Zymo-Spin™ llC Column was replaced
back into the collection tube. The process of washing was repeated with 400µl RNA wash
buffer. The Zymo-Spin™ llC was centrifuged at 12 000 x g for 2 minutes in the emptied
collection tube to ensure complete removal of the wash buffer. The Zymo-Spin™ was
removed from the collection tube and placed into an enzyme free tube. To the column matrix,
25µl of DNase/RNase-free water was added left stand for 1 minute. The mixture was
centrifuged at 10 000 x g for 30 seconds to elute the RNA from the column. The eluted RNA
sample was transferred to the Zymo-Spin™ lV-HCR Spin Filter in a DNase/RNase-Free tube
and centrifuged at 8 000 x g for 1 minute. The extracted RNA was then stored at -70°C for
cDNA synthesis, PRC amplification and gel-electrophoresis.

18
3.3 cDNA Synthesis
The synthesis of a cDNA was carried out using the Thermo Scientific RevertAid First Strand
cDNA Synthesis Kit according to the manufactures instructions. An amount of 1µl total
RNA, 1µl gene specific primer and 1µl of nuclease-free water were into the sterile nuclease
free tube on ice. To the tube, 4µl of 5x reaction buffer, 1µl of RiboLock RNase Inhibitor, 2 µl
of 10 mM dNTP mix and 1µl of RevertAid M-MuLV RT (200 U/µl) was added to make a
total of 20µl PCR master mix. The contents were mixed gently and centrifuged at 8000 for 10
seconds. The contents were incubated at 42°C for 60 minutes and the reaction was terminated
by heating at 70 °C for 5 minutes.
3.4 PCR Amplification
During PCR amplification a master mix was made and the reagents were added into an
eppendorf tube, the template and the primers were not added to the master mix but were
added later using different primer combinations to amplify the P1 and P3 gene portions.
Every reagent was multiplied by 13 since they were 11 reactions and the extra 2 was to carter
for pipetting errors. And each PCR tube contained 22.7 5µl of contents before adding primers
and RNA template. An amount of 21.5µl of master mix was added to each PCR tube using a
micropipette. The tubes were labeled 1 to 11, cDNA template of 2µl was added to each tube
except the negative control and instead of template 2µl of buffer was added. Primers used
were specific to the P1 and P3 genes.
After the addition of master mix, primers and template, the PCR tubes were loaded into the
PCR machine for amplification. The conditions were set in which the initial denaturation
temperature was 94o
C for 5 minutes. The denaturation temperature was set at 94o
C for 30
seconds and the annealing temperature was 53o
C for 30 seconds. The extension temperature
was 72o
C for 2 minutes and the final extension was 72o
C for 8 minutes. The PCR was left at
hold for 4o
C.
The following reagents were added to make up 25µl of reaction for PCR, 2.5µl of x10 buffer,
2µl of 20mM MgCl₂, 0.5µl of 10mM dNTP, 0.75µl of Forward primer, 0.75µl of Reverse
primer, 0.5µl of Taq Polymerase, 2µl of Template and 17.25µl of water.

19
Table 3.3 Primer combinations used for P1 gene amplification
Tube Prime name and sequence Primer name and sequence
1 ProP1genefwd
CGCTCTAGAAAGGATCCATGGCAW
CYGYNATCBGYATYTGYGAA
Old HC-Pro fwd.
2 proPotyanchor/P1fwd
CGCTCTAGAGTACTGAACCTGCGT
GACAGTCGTC
Old HC-Pro fwd
3 P1 fwd.
Pro HC-pro fwd
CGCTCTAGATGYGAYAAYCARY
TNGAYNNNAAYG
GACAGTCGTC
Pro HC-pro fwd
CGCTCTAGATGYGAYAAYCARY
TNGAYNNNAAYG
5 ProP1genefwd
proHCpro-rev
CGCTCTAGAGANCCRWANGART
CNANNACRTG
6 proPoty anchor/P1fwd
GACAGTCGTC
proHCpro-rev
CGCTCTAGAGANCCRWANGART
CNANNACRTG
7 ProP1genefwd
oldHC-prorev
CGCTGATCTAGAGCGTACGGGT
CCTCAGATA

20
GACAGTCGTC
oldHC-prorev
CGCTGATCTAGAGCGTACGGGT
CCTCAGATA
Table 3.3 Primer combinations used for P3 gene amplification
Tube oldHC-prorev
CGCTGATCTAGAGCGTACGG
GTCCTCAGATA
proCyIind lnc fwd
CGCTCTAGAGGNVVNGTNGGNWSNGG
NAARTCIAC
9
10 oldHC-prorev
CGCTGATCTAGAGCGTACGG
GTCCTCAGATA
proCyIind lnc fwd
NAARTCIAC
11 proHCpro-rev
CGCTCTAGAGANCCRWANG
ARTCNANNACRTG
proCyIind lnc fwd
NAARTCIAC

21
3.5 Agarose gel electrophoresis
Buffer solution, 50X TAE of 10ml (Tris-acetate-EDTA) was diluted with 490ml of distilled
water to make a 1X TAE concentration and 1g of agarose powder was measured and filled
into 100mL of 1xTAE to make 1% agarose in a conical flask. The 1% agarose-TAE solution
was filled into a flask and was boiled in a microwave for 3min until the agarose dissolved.
The 1% agarose solution was left to cool down to 55o
C for 5 minutes. Using a micropipette,
5µl of ethidium bromide (EtBr) dye was then added to the molten 1% agarose solution. The
well comb was first put on the negative side of the gel tray and the agarose (agarose with
EtBr) was poured slowly to avoid bubbles which can disrupt the gel into the tray. The poured
gel was left at room temperature for 1 hour to solidify. When gel had solidified, the comb
was carefully removed not to disrupt the wells and the buffer 1X TAE was then added to
cover the gel. A fast ruler DNA ladder of 5 µl was loaded onto the first lane of the gel using
micropipette. The other wells were added with 20µl sample after adding 5 µl of loading dye
to each PCR tube and a negative control was included for all the gels. The gel was run at 100
volts, 500 Amps for a period of 35 minutes. The power was switched off after 35 minutes and
the electrodes were disconnected, and the gel was carefully removed from the gel box. The
DNA fragments were then visualized using UV light under a dark cabin wearing goggles.
3.6 Competent Cell Preparation (Calcium Chloride Treatment) materials
 LB broth
 Water bath
 Inoculation loop
 Microfuge tubes
 Polypropylene tubes
 Micro pipettes and tips
 Culture plates
 Ice cold CaCl2.2H2O (1 M)
 Ice cold MgCl2 CaCl2 solution
 Shaking incubator ( Department of Biochemistry Laboratory)
 Vortex mixer
 Centrifuge

22
3.6.1 Luria Bertoni broth preparation
An amount of 2.5g tryptone, yeast extract of 1.25g and 2.5g of NaCl were added into 250ml
of water and dissolved by mixing using a stirrer and autoclaved for 5 minutes. Ampicillin of
10µl was also added per each 10ml LB.
3.6.2 Luria Bertoni Agar medium preparation
Bacto tryptone of 2.5g, bacto yeast of 1.25g and 10g of NaCl were first added into 250ml of
distilled water and dissolved by mixing using stirer. After dissolving the chemicals, agar was
added and the mixture was placed in a microwave to dissolve the agar for 5 minutes.
3.6.3 Competent cell preparation procedure
To the TG1 colonies that have been streaked in petri dishes, a single colony was inoculated
into 50 ml 2x YT. The colony was incubated overnight in a shaker at 37°C at 200rpm in
rotary shaker. The entire overnight culture was then inoculated into 1 litre of the pre-warmed
2x YT. The contents were incubated in a shaker at 37°C at 200rpm for 3 hours. The cells
were spinned for 5 minutes at 5 Krpm and resuspended in 10 ml and then 290 ml to make it
300 ml ice cold 100mM MgCl2 and then incubated for 10 minutes on ice. The cells were
spinned for 5 minutes at 5 Krpm and resuspended by vortexing in 300 ml ice cold 100 mM
CaCl2 and incubated for 30 minutes on ice. The cell suspension was spinned down and
resuspended in 100 ml ice cold 100 mM and 20% Glycerol, (suspended in 10 ml then in the
remaining 90 ml). The cells, 0.5 ml per eppy tube were dispersed into aliquot on ice and the
aliquots were transferred to -70 °C freezer.
3.6.4 Plasmid and PCR product digestion and clean up
Reagents for pUC 19 digestion were 2 µl of DNA, 2 µl of buffer, 15µl of water and 1µl of
enzyme to make a total of 20µl. The reagents for PCR product digestion were 15µl of DNA,
2 µl of buffer, 2µl of water and 1µl of enzyme to make a total of 20 µl. After digestion for 2
hours 30 minutes, 2 µl at each of the digests were mixed with 10 µl TE and 2 µl 6X LD and
run on a 1% agarose gel, alongside uncut pUC 19. The remaining 18 µl of pUC 19 and 18 µl
of PCR product to make a total of 36 µl was added up to 200 µl by adding 164 µl of water to
top up the volume. To this volume, 200 µl of chloroform, vortexed and centrifuged 10 g. The
solution was then transferred into a clean test tube and 20 µl of 3M Sodium Acetate and 400

23
µl of Ethanol were added and stored at -20o
C for 2 hours. The tubes were spun at 10 K for 5
minutes and decanted to air dry the pellet.
3.6.5 PCR insert plus pUC 19 ligation
The pellet was resuspended to a final volume of 20 µl of 15 µl water, 4 µl 5x T4 DNA buffer
and 1µl of T4 DNA ligase enzyme. The contents were gently mixed by flicking with a finger
and incubated at 45o
C for 24 hours.
3.6.6 TG1 competent cell transformation
To the aliquots of 200µl competent E.coli cells, 20µl of the ligation mixture was added to the
tube. The contents were incubated on ice for 30 minutes without swirling. To a 42°C water
bath, the transformed E.coli were placed for 90 seconds without shaking and immediately
placed on ice for 2 minutes until chilled. The contents were transferred to a 15 ml sterile tube
and 800µl of YT with no Ampicillin added. The falcon tube was placed in a 37°C water bath
for 45 minutes to allow recovery of the transformed cells. The tubes were taped at an angle to
ensure shaking. After the 45 minutes, the contents were spread out onto 100µl LB plates with
IPTG, X-Gal and Ampicillin. The plates were inverted and incubated overnight at 37°C in an
incubator.
3.6.7 Stock solution
Amp →→ 1ml/ml
X-gal →→ 1ml/ml
IPTG →→ 50ml/ml
3.6.8 Blue/ white colony screening
Only the white colonies were selected and placed on an LB ampicillin plate grid and streaked.
A single colony was isolated and inoculated into LB liquid broth containing ampicillin.

24
3.7 Purification of plasmid DNA from 1.5 ml cultures of E.coli- miniprep procedure.
3.7.1 Cell lysis
The purification of the plasmid DNA from cultures of E.coli was done using the FlexiPrep
Kit miniprep procedure following manufactures instructions. The overnight cultured of the
white E.coli cells picked from the blue white colony screening, was transferred to the
microcentrifuge tube and centrifuged at 14 0000 x g for 30 seconds for pelleting the cells.
The supernatant was removed by decanting and left for drying in the air. The pelleted cells
were resuspended in 200µl of solution 1 by vortexing using the vortex machine. The solution
ll of 200µl was added and the tubes were inverted vigorously to mix the contents. At this
stage, the bacterial suspension becomes clear because cells were lysing. To the tubes with the
cells, 200µl of solution lll was added, mixed by inverting and centrifuged at 14 000 x g for 5
minutes and the supernatant transferred to fresh centrifuge tubes. To the supernatant, 450µl of
isopropanol was added, mixed by vortexing and incubated at room temperature for 10
minutes. To pellet the plasmid DNA, the tubes were centrifuged at 14 000 x g for 10 minutes,
supernatant removed by aspiration and tubes left to drain by inverting them on a clean filter
paper.
3.7.2 DNA purification
The Sephaglas FP slurry was suspended by shaking the glass and 150µl of the suspension
was transferred to DNA pellet, vortexed softly for 1 minute for dissolving the pellet. The
contents were centrifuged for 15 seconds at 14 000 x g and without disturbing the Sephaglas,
the supernatant was removed. The Sephaglas pellet was washed by adding 200µl wash buffer,
vortexed, gently, centrifuged at full speed and the supernatant removed. To the Sephaglas,
300µl of 70% ethanol was added, vortexed to resuspend, centrifuged at 14 000 x g for 15
seconds and supernatant removed without disturbing the pellet. The tubes were vortexed to
disperse the Sephaglas pellet and allowed to dry at room temperature for 10 minutes. The
bounded DNA was eluted by adding 50µl of TE buffer, pellet resuspended by vortexing,
incubated for 5 minutes at room temperature and vortexed infrequently for keeping the
Sephaglas suspended. The contents were centrifuged at 14 000 x g for 1 minute and the
supernatant transferred to clean microcentrifuge tubes and lastly 20µl of supernatant
transferred to PCR tubes for amplification and run the gel.

25
CHAPTER FOUR
RESULTS
4.1 Total plant RNA extraction
The total plant RNA extraction was carried out using the Zymo plant RNA MiniPrep kit to
extract the SPFMV RNA and it was successful as highlighted by the distinct band in 2.
M 1 2 3 C
Fig 4.1 Image showing the bands of total RNA extraction: Photograph of 1% Agarose gel
stained with 5µl of Ethidium Bromide dye showing the SPFMV RNA genome isolated from
virus infected samples. The isolates were from the infected sweet potato vines and uninfected
sweet potato. The lanes are: M- Molecular marker, 1, 2, 3 were the infected plant material
and C being the uninfected tomato. The gel was run at 100 V, 500 Amps and for 35 minutes.
SPMFV
23 kb Ladder

26
4.2 Amplification of P1 and P3 using different primer combinations
The results showing the amplification of the P1 and P3 genes using different primer
combinations for increasing the efficiency of detection of SPFMV.
Fig 4.2 Image showing the amplified bands of P1 and P3 genes: Photograph of 1%
Agarose gel showing PCR amplification from cDNA generated from RNA extracted from
infected sweet potato. M- 10kb ladder and the wells 1 to 8 were used for amplification of P1,
well 15 was used as a negative control ,wells 16 and 18 were used for P3 and other wells
were used for detection of CI genes although they were not of my interest. The gel was
stained with Ethidium Bromide and also 5µl of blue loading dye was used to load the
samples. The gel was run at 100 V, 500 Amps and for 35 minutes.
10 kb
P1
1 kb
250
bp
P3
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

27
4.2 Cloning (blue white colony screening)
The presence of white colonies shows the success of the cloning process due to high
proportions of white to blue colonies. The white colonies are the E.coli TG1 transformed
cells with a plasmid with a gene of interest and the blue colonies are the ones with a plasmid
without a gene of interest.
Blue colonies
White colonies
Fig 4.3 Image showing blue white colony screening: Photograph showing the blue white
colony screening after an overnight digest of E.coli TG1 cells in a Luria Bertoni media with
Ampicillin →→ 1ml/ml ,X-gal →→ 1ml/ml and IPTG →→ 50ml/ml.
4.3 Characterisation of Coat Protein using RFLP
The Fig 4.4 shows the results of characterisation of CP of SPFMV isolate found in Zimbabwe
using Restriction fragment Length Polymorphism. Different combination of enzymes were
used for double digestion of the pCambia 1305 plasmid with a coat protein insert and it was

28
partial digestion for 30 minutes. The digested fragments were compared with other strains of
SPFMV.
Fig 4.4 Image showing characterisation of CP using RFLPs: The photograph shows the
double digestion of pCambra plasmid with the CP gene. The plasmid was double digested 1-
Xba1/EcoR1 2- Xba1/Ascl 3- Xba1/Sal, 4-Xba1/Xho1, 5- Xba1/BamH1. The gel was 1%
Agarose stained with Ethidium Bromide.
Enzyme
combinations used
1 Xbal and EcoR1
2 Xbal and Ascl
3 Xbal and Sal
4 Xbal and Xhol
5 Xbal and BamH1
Cut fragments of
CP
23 kb
Plasmid

29
Table 4.1 Comparison of CP genes by RFLP of SPFMV in Zimbabwe with other strains
Virus strain (CP) Ascl EcoR1 BamH1 Sal Xhol
ZIM-CP - + + + +
Severe Strain (S) - + - - -
Russet Crack (RC) - + - - -
East Africa (EA) - - - - -
Ordinary (O) - - + - -
Common (C) - + + - -
Key: + digest
- No digest
The above table shows the comparison of Restriction enzymes which digested the coat
protein of the SPFMV strains (RC, C, O, S and EA) above in comparison with the Coat
protein of the isolate found in Zimbabwe. Basing from the strains compared to, the strain C is
slightly closer to the Coat protein of SPFMV in Zimbabwe. The EA was different from all the
strains whilst strain S and RC were closer.

30
CHAPTER FIVE
DISCUSSION OF RESULTS
5.1 Total plant RNA extraction
SPFMV was successfully extracted from the infected sweet potato vines as revealed by the
band around 10 kb on the gel profile. According to Revers et al. (1999) the SPFMV is 10kb
long and it was in line with the results obtained in the study. The success was a result of use
of the sweet potato vines with chlorotic and mottle virus symptoms. The use of newly
infected leaves also contributed to the success. There was no band in the uninfected tomato
used as a control. It can therefore be used as a partial means of detecting and diagnosing
SPFMV infection in conjunction with other methods.
5.2 Amplification of P1 and P3 using different primer combinations
The SPFMV RNA was reverse transcribed from total RNA using the SPFMV gene specific
primer. This increased the proportion of SPFMV to any other virus on the plant. A
combination of different primers were used to amplify the P1 and P3 for increasing chances
of detection of the virus at PCR level. For P1 coding region the primers used were P1 forward
+ old HC-pro forward, Anchor + old HC-pro forward, P1 forward + pro HC-pro forward,
Anchor + pro HC-pro forward, P1 forward + pro HC-Pro reverse, Anchor + pro HC-Pro
reverse, P1 forward + old HC-Pro reverse and lastly Anchor + old HC-Pro reverse were used.
For these primer combinations the P1 forward + pro HC –pro forward, P1forward + pro HC –
pro reverse, and P1 forward and old HC-Pro reverse produced best amplification bands which
were expected. From the electrophoresis profile of P1 gene products, the bands revealed
around 951 bp with 317 amino acids for P1 coding region. The number of amino acids
calculated by dividing the number of bases by 3 because an amino acid is coded for by 3
bases. According to Coombos et al. (2007), the length of P1 gene was 951 bp for Telosma
mosaic virus (TelMV) using the anchor25dT fwd and virus 5’RACE reverse PCR primers
and this was the same with the amplified P1 of the SPFMV isolate in Zimbabwe.
For P3, old HC pro rev + proCyIind Inc fwd, Old HC-pro rev+ proCyIind Inc fwd and pro
HC pro rev+ proCyIind Inc fwd were used and produce distinct bands in positive
amplification of P3 gene. From the amplification of P3 it revealed 1 041 bp with 347 amino
acids for P3 coding region. The major advantage of these primers was the ability to amplify

31
and produce distinct bands for P3 in the multiple bands produced in the SPFMV. From the
results of this study, it showed that the P1 coding region of the SPFMV-Z was in the range
with the P1 genes of other potyviruses. According to Sakai et al. (1997), the range of P1
genes for potyviruses is between 237 to 664 amino acids and the P1 gene for SPFMV-Z with
317 amino acids was in range. The P3 gene has 347 and it showed 5 amino acids less from
SPFMV-S which has 352 amino acids (Sakai et al., 1997).
From the results obtained in this study the P1 of SPFMV-Z is proposed to be an accessory
factor in the amplification of the virus genome. This is because it is in the range of P1 of the
potyvirus and for tobacco etch potyvirus besides being a serine proteinase , it also function in
genome amplification (Verchot and Carrington, (1995). The P1 of Turnip mosaic virus is
involved in ss RNA and ds RNA binding and the P1 for SPFMV-Zim is also speculated to be
involved in ss RNA binding activity. The P3 gene is believed to be involved in virus
replication or propagation as described by Kreuze (2002). It has been shown in Potato A
Potyvirus that there is biochemical and genetic evidence for the interaction of P1 and P3
proteins with other proteins that are shown to be part of the replication complexes in
potyvirus genome (Merits et al., 1999). All in all the P1 and P3 genes for SPFMV isolate in
Zimbabwe were in range with other potyvirus.
5.3 Cloning (blue white colony screening)
The E.coli strain TG1 was used because the bacteria cost little to maintain. The bacteria is
also easy and quick to grow. The E.coli was successfully transformed as they took the
exogenous pUC 19 DNA plasmid showed by many white colonies in the LB media. Many
colonies expressed ampicillin resistance because they had origin of replication and antibiotic
resistance ( ᵝ lactamase) (Berg et al., 1988). The selective pressure of ampicillin, IPTG and
Xgal makes white colonies of the transformed E.coli with an exogenous pUC DNA and the
blue colour shows simple visual symptoms of unsuccessful bacteria. The clones can be used
to develop transgenic resistance strategies for example the P1 gene clone of rice was used to
produce a cysteine-inhibitor which inhibits the proteolysis of polyprotein in potyvirus and
this interferes with the virus’s replication thereby transforming a SPFMV susceptible
Jonathan sweet potato into being a resistant variety (Cipriani et al., 2000). The P1 and P3
clones would provide a ready source of P1 and P3 for uses in virology studies.

32
5.4 Characterisation of Coat Protein using RFLPs
The use of SPFMV specific primers in RT-PCR is able to discriminate in favour of any
SPFMV strain that might be present in extracted RNA. However, this is not enough to detect
multiple infections of closely related SPFMV strains especially if one is in higher
concentrations compared to those in low titres (Tairo, 2006). Therefore mixed strains can be
revealed by inclusion of RFLP (Tairo, 2006) and this might have contributed to the success of
this project. From the research, the SPFMV-Z Coat protein insert in pCambia 1305 plasmid,
was cut into 3 fragments by EcoR1, no fragment was generated by Ascl, 1 fragment by Xhol
and 2 fragments by BamH1. A distinct banding pattern was produced. This was then
compared to the results generated from Serial cloner version 2.5, which was used to digest
SPFMV strains S, O, C, RC and EA to generate a restriction digest profile using the same
restriction enzymes used on the Zimbabwean isolate. RFLP digestion of strains Russet crack,
and Severe produced the same fragments pattern to each other whilst EA was different to all
strains used. The EcoR1 and BamH1 cut the Zimbabwean isolate and the Common strain in
the same manner. From the representatives of the strains used, it is speculated that the
SPFMV isolate in Zimbabwe is likely to be a descendant or related to strain C although from
phylogenetic analysis of the Coat protein, SPFMV-Z was found to be grossly diverged from
all strains (Mukasa et al., 2003). This however shows that the isolate in Zimbabwe could be
another strain or the isolate that had been already characterised by Chavi et al. (1997) had
suffer mutation.

33
CHAPTER SIX
CONCLUSION AND RECOMMENDATIONS
6.1 Conclusion
The use of restriction fragment length polymorphism (RFLP) is more efficient and accurate
in diagnosis of closely related SPFMV. SPFMV-Z can also be diagnosed and detected in
infected vines by using PCR based methods using a combination of primers P1 forward + pro
HC –pro forward, P1forward + pro HC –pro reverse, and P1 forward and old HC-Pro
reverse for amplification of the P1 region and all primers combinations for used for P3
amplification can also be used. As proven by the blue/white colonies, the P1 and P3 genes
have been cloned into pUC 19 and these can be used as infectious clones for testing potyvirus
resistance transgenic plants. The isolate found in Zimbabwe was suggested to be slightly
related to the strain C of SPFMV and this showed that the isolate characterised before by
Chavi et al. (1997) could have suffered mutation or another isolate had emerged in the
country.
6.2 Recommendations
 All SPFMV isolates and the cloned P1 and P3 plasmids should be sent for sequencing
to enhance knowledge about SPFMV variability.
 There is also need for characterisation of SPFMV in all sweet potato growing regions
and its wild hosts in Zimbabwe and characterise them using both RT-PCR
amplification and RFLP methods.

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41
APPENDECES
Appendix A1: Restriction analysis of CP of strain C
Appendix A2: Restriction analysis of CP of strain EA

42
Appendix A3: Restriction analysis of CP of strain O
Appendix A4: Restriction analysis of CP of strain RC

43
Appendix A5: Restriction analysis of CP of strain S

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