The main goals of this study were to ascertain farmers' preferred traits in rice landraces and their perception of Rice yellow mottle virus, to collect rice landraces across Burkina Faso, investigate their genetic diversity, and to exploit this diversity in a search for varieties resistant and tolerant to RYMV, for their utilisation in rice breeding. Farmers' preferred traits, approaches to crop management, and disease perceptions were assessed using a Participatory Research Appraisal (PRA) approach. In the main rice growing regions of Burkina Faso, 330 rice landraces were collected. The agro-morphological diversity of the germplasms was evaluated in the field with 20 quantitative and 30 qualitative agro-morphological parameters. Thereafter, 22 Simple Sequence Repeat molecular markers were used to assess the genetic diversity and the population structure of the collection. Finally, the rice landraces were screened against four RYMV isolates to assess the susceptibility, tolerance and resistance of the landraces in the collection using visual assessment and Enzyme Linked Immunosorbent Assay. The PRA identified sweet taste, grain expansion when cooking, easy cooking and yield as paramount selection criteria in rural rice farming communities in Burkina Faso. Drought and disease resistance are characters that farmers wish to have in their varieties. The PRA also highlighted that farmers are conscious of RYMV disease in their fields. However, they are unaware about the epidemiology of the disease. An agro-morphological study of the phenotypic diversity of the collection confirmed the presence of the two cultivated rice species: O. glaberrima and O. sativa. There were more O. sativa accessions than O. glaberrima landraces. There were 48 O. glaberrima and 282 O. sativa accessions in the collection. Both species were divided into four clusters, reflecting the richness of the collection. The underlying genetic diversity of the collection was confirmed by the use of 22 Simple Sequence Repeat molecular markers. The neutral markers confirmed the existence of two substructures, namely O. glaberrima and O. sativa, and the presence of admixture varieties. However, a core collection of 52 individuals was developed. This included 13 O. glaberrima and 39 O. sativa accessions. It reflects the genetic diversity of the sub-clusters present in each species. This core collection contains 89% of the allelic richness of the collection. Its small size will facilitate the maintenance and active use of diversity of germplasm in the core collection. The entire collection was utilised to search for varieties resistant and tolerant to RYMV disease. The screening of the collection with different RYMV isolates exposed the susceptibility of most of the accessions in the collection. Most of the O. sativa indica accessions were highly susceptible. However, ten O. glaberrima accessions displayed a delay of symptom expression, and moderate resistance. However, their resistance was overcome later by a particularly virulent RYMV isolate BF1. Remarkably, a single moderately resistant cultivar, BM24, showed that partial resistance and tolerance to RYMV can be found in an O. sativa variety. Serological evaluation of this local variety in comparison with the partially resistant variety, Azucena, showed that BM24 and Azucena expressed similar resistance patterns. A genetic profile of both varieties showed that both had an identical allele status at RM101, which is a marker bracketed in the same zone as the QTL12. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Rice is a staple food in many West African countries, including Niger. However, both regional and national rice production have failed to meet demand due to several constraints, among which is the Rice yellow mottle virus (RYMV). Moreover, attempted intensification of rice cultivation and the introduction of modern cultivars are encouraging farmers towards abandoning local landraces for high yielding, but often susceptible varieties. The study was primarily oriented towards rice pre-breeding, and identifying priorities for rice breeding in Niger in relation to farmers' preferences and their environment. A secondary aim was the development and evaluation (for release at the regional level) of new breeding lines with resistance to RYMV. This study aimed to: 1) Establish farmers' perception of rice varieties as well as the main constraints on rice production in Niger and particularly those posed by RYMV; 2) Create a collection of rice species from Niger for ex- situ conservation, and to determine the phenotypic variability within this collection; 3) Determine the genetic diversity and population structure of the collection; 4) Screen the collection for resistance to RYMV, so that new sources of resistance could be detected; 5) Improve five elite varieties from West Africa for resistance to RYMV using marker-assisted selection (MAS). The germplasm collection and PRA of this study were conducted in 2008 and 2009 in Niger, while the field and the laboratory researches were conducted in 2008 and 2009 at the Africa Rice Center (AfricaRice) in Benin. For the PRA, data was obtained from a semi-structured group discussion carried out in 14 villages, individual questioning of 153 farmers and visits to farmers' field and storage facilities. The local farmers' union was the only formal seed dissemination system. Seed exchanges between farmers and the use of seeds from previous harvests were important. The RYMV and the bacterial leaf blight (BLB) were cited as the prevalent biotic stresses in the irrigated agrosystem, where the varieties IR1529-680-3 and Waihidjo were found to be the most popular. Flood, birds and hippopotamus were the most damaging agents in the lowland cropping system, and the landrace Degaulle/ D5237 was the preferred variety. Apart from the yield, farmers preferred varieties with good grain quality (milling quality and good taste), high market value, stress tolerance (drought, flood, disease, birds, rodents), and those recommended by the local farmers' association. These findings should be included in breeding goals, seed production and dissemination systems. During collection, a total of 270 rice accessions were assembled, comprising the two cultivated rice species Oryza sativa L. and O. glaberrima Steud. and its two wild relatives Oryza barthii A. Chev. and O. longistaminata Chev. et Roehr. The region of the Niger River and its tributary (the Dallol Maouri) provided the majority (80.7%) of the accessions. Apart from a few wild O. barthii accessions, the accessions found around Lake Chad and the Komadougou river (South-East) were also collected in the Niger River area. Farmers' naming and ecological classification of rice varieties was generally consistent. Three major phenotypic groups were found during the field trials, and the overall phenotypic variability of the collection (as measured by the Shannon-Weaver Diversity Index) was relatively high. There was no significant difference in diversity between the main eco-geographical zones of collection, as well as between the identified phenotypic groups, suggesting a high level of germplasm exchange between the regions in Niger. From the collection, 264 accessions were genotyped from the collection using 18 well distributed SSR markers and two main genetic compartments were detected, comprising O. sativa subsp. indica varieties and O. glaberrima and its wild relative O. barthii and O. longistaminata. The O. sativa group in Niger was divided into irrigated and floating rice, bound by lowland rice. The wild progenitor O. barthii was widespread but without any clear genetic differentiation from O. glaberrima, probably due to the presence of admixtures within the collected samples of O. barthii. Allelic diversity was relatively high, despite the geographical distance from the centre of domestication of African rice, and the points of entry of Asian rice to Africa. The findings reflect the underuse of Niger's rice landraces genetic potential for rice breeding, given that all the "improved" varieties released during the last 25 years in Niger were clustered together on the dendrogram. The response of a set of the rice collected from Niger and some accessions from Mali to inoculation by RYMV was evaluated using five different virus isolates from Niger (3), Benin (1) and Burkina Faso (1). All rice varieties were susceptible to the disease. However, depending on the virus strain, a few O. glaberrima accessions displayed partial resistance, similar to the highly resistant TOG5681. Allelic research based on primers derived from the RYMV1 gene revealed one accession with allele rymv1-3, and two accessions with allele rymv1-4, and one accession with a different resistance gene. The implications of the finding were discussed and a strategy proposed for breeding varieties with a comprehensive resistance to RYMV. After three generations of backcrossing, the major resistance gene of the variety Gigante was successfully introgressed into five elite rice varieties of West Africa by Marker-Assisted Backcross (MABC). The newly developed BC3F3 progenies were screened for resistance to RYMV in farmers' fields in Guinea and Mali and also under controlled conditions in a screenhouse in Benin. As shown by low virus content and level of disease incidence, low tiller number and plant height reduction, the transferred gene was fully functional in the new genetic background. Moreover, some lines also displayed a high level of resistance to rice blast (Pyricularia oryzae) and stem borer infestation in Guinea. Four of those lines are in the second year of multi-location trial in seven West African countries. Therefore, effective deployment of the newly developed varieties, coupled with good cultural practices, should reduce the damaging effects of RYMV in lowland and irrigated rice cropping systems and thereby increase the income of small scale farmers from rice cultivation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Pepper (Capsicum annuum L.) is an important vegetable crop grown and consumed worldwide. Potato virus Y (PVY) is a globally economically important pathogen which significantly reduces the yield and quality of cultivated pepper. The virus is considered as a major limiting factor to the economic production of pepper in the province of KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). Many applied practices to control the spread of PVY are ineffective to mitigate the losses incurred by many farming communities across the KZN province. Therefore, the objectives of this study was to determine the full genome sequence of a PVY isolate from KZN, to identify resistance alleles in commercially available pepper varieties in KZN and to develop a pepper hybrid variety with resistance to PVY using a molecular breeding strategy The first part of the study was conducted to determine the first full genome sequence of a PVY isolate (JVW-186) infecting pepper from KZN. The complete genome sequence of JVW-186 was assembled from overlapping RT-PCR clones using MEGA 5 software. Individual ORFs were identified using the nucleotide data base NCBI and aligned using CLUSTALW. RDP4 software was used to identify recombination junctions in the sequence alignment of JVW-186. CLC Main Workbench 6 software was used to determine the nucleotide sequence similarity of recombinant and non-recombinant fragments of JVW-186 in conjunction with ten PVY parental isolates. Based on sequence data, virus morphology and the coat protein size as determined by SDS-PAGE analysis, the identity of the isolate JVW-186 was confirmed as PVY. Phylogenetic trees were constructed from all recombinant and non-recombinant segments of the sequence by the maximum likelihood method using MEGA 5 software. The full length sequence of JVW-186 consisted of 9700bp. Two ORF’s were identified at position 186 and 2915 of the sequence alignment encoding the viral polyprotein and the frameshift translated protein P3N-PIPO, respectively. RDP4 software confirmed two recombination breakpoints at position 343 and 9308 of the sequence resulting in four segments of the genome. At each recombination event, a 1021-bp fragment at the 5’ end in the region of the P1/HC-Pro protein and a 392-bp fragment in the region of the coat protein shared a high sequence similarity of 91.8 % and 98.89 % to the potato borne PVYC parental isolate PRI-509 and the PVYO parental isolate SASA-110 respectively. The non-recombinant fragment 1 clustered within the C clade of PVY isolates; however the large 7942-bp fragment 3 did not cluster within any of the clades although it shared > 80% nucleotide sequence similarity to other PVY isolates used in this study. Our results suggest that isolate JVW-186 is a novel recombinant strain of PVY that could have evolved due to the dynamics of selection. The second part of the study aimed to evaluate different pepper lines for resistance to PVY. Two recessive alleles (pvr21 and pvr22) located on the pvr2-elF4E locus are known to confer resistance to the virus. To this end, six pepper lines were challenged with PVY infected Nicotiana tabacum cv. Xanthi leaf material using mechanical inoculation under greenhouse conditions. Each line was assessed for resistance to PVY by visual screening for disease severity and quantitative enzyme linked immunosorbent assay (ELISA) for virus load. Pepper lines were further characterized using tetra-primer ARMS-PCR (amplification refractory mutation system polymerase chain reaction) to identify and differentiate the presence of homozygous/heterozygous resistance alleles that confer PVY resistance. Evaluations revealed two resistant pepper lines (Double Up and Cecelia) and varying levels of susceptibility in the other four pepper lines challenged with PVY. The most susceptible pepper line was Benno, although high levels of susceptibility were observed in three other lines (IP, Mantenga and Excellence). The pvr2+ allele was positively identified in all the susceptible pepper lines using the T200A tetra-primer which confirms that the presence of this allele is dominant for PVY susceptibility. Double Up and Cecelia were genotyped homozygous pvr21/pvr21 and pvr22/pvr22 respectively, and remained asymptomatic throughout the trial which indicates that these alleles confer resistance to the isolate of PVY used in this study. The information generated in this study can be incorporated into breeding programs intended to control PVY on pepper in KZN. The final part of the study focused on the development of resistant varieties as the best alternative to manage PVY diseases on pepper. Homozygous F2 pepper lines were developed from local germplasm carrying PVY resistance genes (pvr21 and pvr22) using marker assisted selection (MAS). The F1 progeny was obtained by crossing a homozygous pvr21 (resistant) ‘Double Up’ cultivar with a heterozygous susceptible (pvr2+/pvr22) ‘Benno’ cultivar. F1 and F2 generations were assessed for the presence of PVY resistance/susceptibility alleles (pvr2+/pvr21/pvr22) at the pvr2-elF4e locus using the tetra primer amplification refractory mutation system – polymerase chain reaction (ARMS-PCR) procedure. Negative selection was carried out using the tetra-primer T200A marker to detect the pvr2+ (susceptible) allele. All F1 progeny displaying the pvr2+ allele were eliminated from further study. All 302 plants belonging to 29 F2 families expressing homozygous recessive traits were tested via mechanical inoculation for their response to PVY infection and resistance to PVY was confirmed in all selected families based on symptomatology in greenhouse house screens using double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). These results show that ARMS-PCR can be used to successfully screen pepper genotypes for alleles that confer PVY resistance thereby contributing to the improvement of pepper production using molecular breeding approaches. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV. / AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting (Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is, vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies. Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR) en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom (mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen). Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor- Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan insig verleen in die etiologie van siektes wat met GRSPaV verbind word.