Maize (Zea mays L.) streak virus disease (MSD) is the most important virus disease in Africa but farmers are unaware of its status. A project was initiated to assess the current status of MSD and to breed for its resistance. Four populations comprised of two BC1F1 and two F2 progenies developed by backcrossing and selfing the F1 progenies of two crosses between a donor line (CMl 202) and two susceptible lines (CMl 321 and CMl 384) were developed. Conventional and molecular marker assisted selection (MAS) methods were used to screen for resistance to MSD in each of the four populations. To facilitate unbiased comparison, separate screening nurseries were established for MAS and conventional screening. The objectives of the study were five-fold; 1) to assess the status of MSD in Uganda and understand farmers' preferences and varietal selection criteria for maize using a participatory rural appraisal (PRA), 2) to screen for MSD resistance in early generations of segregating maize populations using conventional method, 3) to screen for resistance to MSD using SSR marker assisted selection , 4) to compare the effectiveness of marker assisted selection and conventional methods for selection for resistance to MSD, and 5) to compare costs associated with MAS and conventional selection methods. Results of PRA showed that unreliable rainfall and insect pests were the dominant constraints to maize productivity in Uganda. Diseases were ranked fifth among the production constraints . Maize streak virus disease was considered the most important disease constraint. Farmers showed common preference for high yielding and early maturing cultivars. However, farmers had other special preferences which were diverse and included large, white and high test density kernels for marketing, and sweet taste, particularly for home consumption. Farmers' research priorities included tolerance to drought, resistance to insect pests and diseases, sweetness, prolificacy, resistance to lodging, and drooping leaves because theyt cover the soil fast and prevent weed growth. Conventional screening for resistance to MSD showed that backcross and selfing populations segregated in 1:1 and 3:1 Mendelian ratios confirming the presence of one major gene with simple inheritance . Severity and incidence of disease were positively correlated suggesting a non-reference by the insects. In the selfing populations, the presence of complete esistance against MSD was suggested because frequency distribution patterns were highly skewed in favour of resistance. There was a decrease in disease severities with selection from BC1F1 to BC2F1 and from F2 to F3 generations indicating that high response to selection was achieved. On the other hand, one marker, umc1917, consistently polymorphic and eo-dominant was selected and used in MAS protocol. Results showed that the observed outcomes fitted the expected ratio of 1:2:1 for a F2 population and 1:1 for a BC1F1 population (X2 not significant). Evaluation of F3 and BC2F1 progeny selected using markers showed low disease severity suggesting that marker assisted selection was effective. However, the study showed that the presence of the O'Tl, was not consistent with symptom expression in the field. Evaluation of lines in three-way crosses identified ten potential lines that were high yielding, highly resistant to MSD and stable across three locations. Both MAS and conventional selection were equally effective in identifying high yielding lines although resistance was higher under MAS. Costs of MAS and conventional method varied depending on the units for comparison. The total costs of conventional method were higher than that of MAS in both first and second selection cycles. Comparing costs per row for conventional and costs per plant or data point for MAS showed that conventional selection was 2.4 times more expensive than costs per sample for MAS. However, costs per plant for MAS were 6.6 times higher than for conventional selection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006

Despite the increasing number of farmers growing cassava in Zambia, yield per hectare has remained low at 5.8 t ha-1. The major constraints contributing to low yields are pests and diseases of which cassava mosaic disease (CMD) caused by East Africa cassava mosaic virus (EACMV), Africa cassava mosaic virus (ACMV) and South Africa mosaic virus (SACMV) is the most important. Breeding of cassava is restricted by limited information on viruses and associated satellites, and farmer preferences. Most of the farmers cannot manage to institute control strategies that require buying of chemicals. The most feasible option remains improving existing cultivars through resistance breeding. The study therefore was conducted to: i) establish farmers’ perception and knowledge of CMD; ii) to identify viruses of cassava occurring in Luapula province; iii) evaluate the performance of local and improved cultivars for agronomic traits; iv) evaluate the performance of F1 progenies for CMD resistance; and v) determine general combining ability and specific combining ability for CMD resistance. The studies were carried out between 2008 and 2011 at different locations in Zambia. The information generated was important in formulating a local breeding strategy for CMD resistance. A participatory rural appraisal and a structured survey was conducted in Mansa, Samfya and Mwense districts in Luapula province involving farmers to ascertain farmers’ perceptions of CMD. The results of the study showed that the majority of the respondents (97.6%) were not aware of CMD. Most of the farmers grew landraces on small pieces of land. Although, the cultivars (local and improved) were widely grown, they were susceptible to CMD. The farmers preferred cultivars with high yielding and early bulking characteristics among others. A CMD survey conducted between April and May 2009 in Samfya, Mansa, Mwense, Kawambwa and Nchelenge districts in Luapula province established East Africa cassava mosaic virus (EACMV), and Africa cassava mosaic virus (ACMV) as the most prominent viruses in the area. Symptoms of satellites were also observed in the farmers’ fields in most of the areas visited. Satellite II and III were detected in leaf samples. The CMD incidence (59.1%) and severity (2.4) was moderate across the districts surveyed. The CMD symptoms on the cassava plants were variable with plants showing mild and severe symptoms characterised with narrowing and reduced leaf blades. The transmission of CMD infections was mainly through cuttings rather than via whitefly infection which means that most of the planting materials used by the farmers were infected. Evaluation of cassava cultivars for CMD resistance was conducted in 2009/2010 and 2010/11 seasons at Mansa Research Station in Luapula province using a 4 x 4 α lattice design. Both introduced and locally grown cultivars had significant (P<0.001) differences in their reaction to CMD. Bangweulu, Namuyongo, Kalaba, Chikula, Mwakamoya, Chila7 and Chila11 were the most susceptible genotypes. Mweru, Tanganyika, and Nalumino were moderately tolerant to CMD. Eight hundred F1 genotypes developed using a North Carolina II mating design were evaluated in a 4 x 5 α lattice design in 2011 at Mansa Research Station, Luapula province to determine combining ability for reaction to CMD, yield and yield components. The plants were harvested 7 months after planting (MAP). Significant (P<0.001) general combining ability and specific general combining ability were recorded for CMD. The SCA effects were more important for CMD than GCA effects suggesting that non-additive gene action was more prominent than the additive gene action in determining CMD reaction. Parent lines with desired significant, negative GCA effects for reaction to CMD were Bangweulu, Kampolombo, Nalumino and TME2. In general, the survey and participatory rural appraisal established CMD as one of the constraints to cassava production and created a basis for the research study. The findings indicate opportunities that exist in creating genotypes with tolerance to CMD. The study identified cassava lines with resistance to CMD. The lines that expressed the above trait should be selected and tested further for release to the farmers in Zambia. Since the clonal evaluation trial was harvested at 7 MAP, there is need to investigate further for earliness trait in best performing lines in different locations. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Cassava (Manihot esculenta Crantz), is the world’s most widely grown starch storage root crop. It is a principal food staple in sub-Saharan Africa where it accounts for approximately one-third of the total production of staple food crops. It plays a key role as a food security and an income-generating crop for millions of smallholder farmers. In Uganda, cassava ranks second to bananas (Musa spp.) in terms of area occupied, total production and per capita consumption; however, nearly 5% of the total population experiences hunger with the prevalence of food energy deficiency at the country level standing at 48%. Cassava is a crop with high potential to alleviate food shortages and energy deficiencies, owing to its unique advantages of producing acceptable yields and starch on infertile soils amidst erratic rainfall, when most other crops would fail. Hoewever, its yield potential has not been fully realised since most of the cassava cultivars grown are susceptible to pests and diseases, low yielding and late bulking. The main objective of the research was to develop high yielding, early bulking cassava genotypes that combine resistance to cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) with farmer preferred traits for cultivation in Uganda. The specific objectives were to: (i) evaluate farmers’ attitudes to and/or perceptions of cassava early bulking, production constraints and cultivar preferences; (ii) determine the extent of genetic variability in storage root bulking and other important traits of selected cassava genotypes; (iii) assess the effects of genotype x environment interaction on early bulking and related traits of selected cassava genotypes; (iv) develop and evaluate cassava F1 families for early bulking in terms of the attainment of early, high fresh storage root yield (FSRY) and resistance to CBSD and CMD; and (v) determine the combining ability and gene action controlling early bulking and yield-related traits, as well as resistance to CBSD and CMD. Through the farmer participatory survey, a number of cassava production constraints were identified, key of which were: diseases, especially CBSD and CMD; lack of early bulking cultivars; rodents and insect pests. Farmers rated early bulking as the second most important preferred trait after FSRY, but suggested that early bulking should be complemented with high dry mass content (DMC), sweetness, high FSRY and resistance to pests and diseases. The analysis of variance of 12 cassava genotypes selected for evaluation in three diverse locations and at five different harvest times indicated significant variation among genotypes, harvest times, locations and their interactions for FSRY and most of the other traits evaluated. Fresh storage root yield and the other traits evaluated were predominantly under the control of genetic variation, indicating that genetic advance would be achieved through hybridisation of the test genotypes. Additive main effects and multiplicative interaction (AMMI) analysis of the data collected at nine months after planting (MAP) indicated a non-significant GEI for early FSRY, but significant GEI for other traits assessed. Eight of the 12 genotypes analysed had relatively low interaction with locations for early FSRY, signifying that these genotypes were relatively stable for early FSRY. Thirty-six F1 families were generated from a 9 x 9 diallel and exhibited a high degree of variation between and within families for all the traits assessed at the seedling evaluation stage. Diallel analysis at the seedling evaluation stage at 10 MAP indicated that additive gene effects were predominant in the expression of early FSRY and most of the other traits analysed. At the clonal evaluation stage, the 36 families were assessed for early FSRY at 8 MAP and this trait together with most of the other traits assessed were found to be predominantly under the control of non-additive gene effects. High mid- and better-parent heterosis for early FSRY was recorded in most families at the clonal evaluation stage with NASE3 x Nyara, Nyara x B11 and NASE3 x B11 recording the highest. Selection from the 36 families at the clonal evaluation stage based on farmers’ top two preferred traits, viz. early bulking for FSRY and DMC, plus resistance to CBSD and CMD identified 50 genotypes that had early FSRY of ≥25 t ha-1 at 8 MAP compared to the best parent, CT1 that had 15.9 t ha-1 at 8 MAP. The selected genotypes also had high DMC and dual resistance to CMD and CBSD. Advancement of the selected genotypes should go a long way towards increasing cassava yield per unit time, reducing food shortages and increasing the income of smallholder farmers in Uganda. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Maize (Zea mays L.) supports millions of livelihoods in sub-Saharan Africa (SSA) in terms of food and feed. Production of the crop is however limited by several factors, among these, maize streak virus (MSV) disease. Although extensively studied, MSV remains a serious problem in SSA due to several challenges in breeding MSV resistant maize varieties. These include integration of MSV resistant germplasm from different backgrounds, reliance on a few resistant sources, and genotype x environment interactions. This study was designed to assess the breeding potential of several MSV resistant lines in hybrid combinations. Understanding architecture of genetic divergence and background of these genotypes would greatly aid in breeding high yielding and stable MSV resistant hybrids. Experiments were conducted during 2010 to 2012 seasons in Kenya. Diallel crosses and SSR markers were used to characterize MSV resistant maize inbred lines from three programs of CIMMYT, KARI and IITA. In general, this study revealed that MSV is still an important problem in Kenya with high incidence and severity levels in the farmers’ fields. The levels of MSV resistance in locally grown hybrids needs to be improved. Farmers challenged breeders to develop new hybrids that combine early maturing, high yield potential and MSV resistance. The study was successful in identifying the best eight inbred lines for use in breeding new maize hybrids with MSV resistance. The nature of gene effects was established for the first time, in particular the role of epistasis and G x E in conditioning MSV resistance in hybrids. Results indicate serious implications for previous models that ignored epistasis in studying MSV resistance in maize. The inbreds Z419, S558, CML509 and Osu23i, displayed high levels of epistasis for MSV resistance. Unless strong sources of MSV resistance, such as MUL114 and CML509, are used, breeding resistant hybrids will require parents that carry dominant resistance genes. The additive-dominance model was adequate to explain northern leaf blight (NLB) resistance in hybrids, indicating fewer complications in breeding NLB resistant hybrids. The study also reveals that SSR genetic distance data can be used to predict hybrid performance, especially when the correct set of markers is used. Many previous studies have not found any significant relationship between genetic distance and heterosis, due to large G x E and use of a wrong set of markers. The diallel analysis and SSR data established the important heterotic groups, which will be exploited for efficient development of MSV resistant maize hybrids. These strategies will be recommended to programs that emphasize MSV resistance in maize hybrids. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination. / AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose. / AFRIKAANSE OPSOMMING: Volgende-generasie tegnologie om basis volgordes van nukleiensure te bepaal, word al meer gebruik in metagenomiese studies. Dit is veral weens die hoe data-omset kapasiteit en onbevooroordeelde aanslag in die bepaling van die genetiese samestelling van onbekende omgewingsmonsters. Hierdie studie het die aanwending van die Illumina volgende-generasie volgorde-bepalingsplatform in 'n metagenomiese studie van wingerdvirusse, ondersoek. Dit het ten doel gehad om die eerste volledige virus profiel, of viroom, van 'n geinfekteerde wingerd saam te stel. Blaarmateriaal is verkry vanaf 44 lukraak-gekose wingerdstokke in 'n rolblad-geinfekteerde wingerd in Suid-Afrika. Monster materiaal is saamgevoeg en dubbelstring-RNS geekstraheer. Die dubbelstring-RNS is onderwerp aan gepaarde-ent volgorde-bepaling deur gebruik te maak van die Illumina volgorde-bepaling-deur-sintese tegniek. Meer as 19 miljoen volgorde reekse, ekwivalent aan ongeveer 837 megabasisse volgorde data, is verkry. Van hierdie data kon ongeveer 400 megabasisse saamgevoeg word in 449 konstrukte ("scaffolds"), deur gebruik te maak van die de novo samesteller Velvet. Hierdie konstrukte is onderwerp aan BLAST soektogte teen die NCBI databasisse en die hoogste trefslag-telling is gebruik vir virus identifikasie. Op grond van die "BLAST" resultate is geskikte volgordes geselekteer vanaf die NCBI databasis en gebruik as verwysingvolgordes in MAQ kartering-analises. Met die bioinfomatika analises kon die virus spesies teenwoordig, asook die mees prominente variante en relatiewe voorkoms van elk, bepaal word. Vier bekende virus wingerdpatogene is geidentifiseer. Grapevine leafroll-associated virus 3, verteenwoordig deur 59% van die geanaliseerde kort-reeks volgorde data, is identifiseer as die mees prominente virus spesie. Drie variante van die virus is in die wingerdmonster opgespoor: GP18 kom die mees algemeen voor, gevolg deur 'n CL-766/NY1 variant en 'n potensiele nuwe wingerd rolblad-geassosieerde ampelovirus. 'n Enkele Grapevine rupestris stem pitting-associated virus variant, soortgelyk aan SG1, en 'n Grapevine virus A variant, 'n lid van molekulere groep III, is geidentifiseer. Hierdie studie is ook die eerste om die teenwoordigheid van Grapevine virus E (GVE) in Suid-Afrikaanse wingerde te rapporteer. Grapevine virus E is verder geneties gekarakteriseer en die genoomvolgorde van GVE isolaat SA94 is bepaal. Die GVE SA94 genoomvolgorde, 7568 nukleotiede lank, is die eerste volledige genoomvolgorde vir hierdie virus spesie. Die genoomorganisasie is tipies van vitivirusse, maar in kontras met ander RNA virusse is die AlkB domein binne-in die helikase domein van oopleesraam 1 (ORF 1) geleë. Grapevine virus E SA94 deel byna 100% nukleotied identiteit met die Japannese TvP15 isolaat en GVE 3404, 'n de novo konstruk gegenereer vanaf die metagenomiese volgorde data. Bioinformatika analises van die metagenomiese volgorde data het verder die teenwoordigheid van drie swam-infekterende virus families, die Chrysoviridae, Totiviridae en ongeklassifiseerde dubbelstring-RNS virus, Fusarium graminearum dsRNA mycovirus 4, aangetoon. 'n Virus van die Chrysoviridae familie, soortgelyk aan Penicillium chrysogenum virus, het die tweede meeste voorgekom in die wingerd monster. Hierdie studie demonstreer die suksesvolle toepassing van 'n kort reeks volgorde-bepalingstegnologie soos die Illumina platform, vir die opstel van 'n virusprofiel van 'n geinfekteerde wingerd. Sover ons kennis strek is hierdie die eerste aanwending van die Illumina tegnologie vir hierdie doel.

Made available in DSpace on 2014-06-11T19:34:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-18Bitstream added on 2014-06-13T20:27:47Z : No. of bitstreams: 1 moura_mf_dr_botfca.pdf: 469933 bytes, checksum: eccaa27134acf7a66cc0606cbff1deaa (MD5) / Os potyvírus constituem cerca de 90% das espécies conhecidas da família Potyviridae. No Brasil ocasionam sérios entraves em alface (Lactuca sativa L.) e em pimentão (Capsicum annuum L.), onde se pode citar o Lettuce mosaic virus – LMV e o Pepper yellow mosaic virus (PepYMV), respectivamente. Com o intuito de melhor compreender o reservatório natural de potyvírus em plantas invasoras, amostras foram coletadas em áreas produtoras de pimentão e analisadas utilizando-se antissoro anti-potyvirus (Agdia). Entre estas plantas positivas, destacou-se Solanum americanum Mill, onde foi verificada infecção mista do Cucumber mosaic virus e do Potato virus Y, e Commelina benghalensis L. em que foi encontrado um possível novo potyvírus com a maior identidade de nucleotídeos da proteína capsidial (62%) com a espécie Hardenbergia mosaic virus. Este potyvírus não foi transmitido por extrato vegetal, bem como por afídeos para plantas de pimentão e Nicotiana tabaccum TNN. Na região codificadora para a proteína capsidial do potyvirus não foi encontrado o domínio DAG, relacionado a transmissão por afídeos. Visando encontrar possíveis fontes de resistência ao Lettuce mosaic virus - LMV, genótipos foram inoculados com o isolado LMV-AF-199 (LMV-Most) e o fator de iniciação de tradução eucariótico eIF4E destes genótipos analisado. Em Calona e Salinas-88, conhecidas previamente como portadoras dos genes recessivos mol1 e mol2 foram observados sintomas em todas as plantas inoculadas e verificado o padrão típico do eIF4E1 e eIF4E2, respectivamente... / The Potyvirus genus corresponds to 90% of known species of the Potyviridae family. In Brazil potyviruses causes serious problems in lettuce (Lactuca sativa L) and in pepper crops (Capsicum annuum L.), which we can highlight Lettuce mosaic virus – LMV and Pepper yellow mosaic virus (PepYMV), respectively. To increase knowledge about the natural reservoir of potyviruses in weeds, samples were collected from a pepper producer area and analyzed for potyvirus using antiserum anti-potyvirus (Agdia). Solanum americanum Mill was identified as a host for Cucumber mosaic virus and Potato virus Y. In Commelina benghalensis L. a possible new species of potyvirus was found with higher nucleotide identity of the coat protein (62%) with Hardenbergia mosaic virus. This potyvirus could not be transmitted by aphids to sweetpepper and... (Complete abstract click electronic access below)

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA). A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control. A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN. The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN. Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.