Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Molecular characterisation and carotenoid quantification of pro-vitamin A biofortified genetically modified bananas in Uganda

In Uganda, a significant proportion of the population depends on the micronutrient poor East African highland banana as a food staple. Consequently, micronutrient deficiencies such as vitamin A deficiency are an important health concern in the country. To reach most vulnerable rural poor populations, staple crops can be biofortified with essential micronutrients though conventional breeding or genetic engineering. This thesis provided proof of concept that genetically modified East African highland bananas with enhanced provitamin A levels can be generated and fully characterised in Uganda. In addition, provitamin A levels present in popular banana varieties was documented.

Shaping development through mechanical strain: The transcriptional basis of diet-induced phenotypic plasticity in a cichlid fish

Adaptive phenotypic plasticity, the ability of an organism to change its phenotype to match local environments, is increasingly recognized for its contribution to evolution. However, few empirical studies have explored the molecular basis of plastic traits. The East African cichlid fish Astatoreochromis alluaudi displays adaptive phenotypic plasticity in its pharyngeal jaw apparatus, a structure that is widely seen as an evolutionary key innovation that has contributed to the remarkable diversity of cichlid fishes. It has previously been shown that in response to different diets, the pharyngeal jaws change their size, shape and dentition: hard diets induce an adaptive robust molariform tooth phenotype with short jaws and strong internal bone structures, while soft diets induce a gracile papilliform tooth phenotype with elongated jaws and slender internal bone structures. To gain insight into the molecular underpinnings of these adaptations and enable future investigations of the role that phenotypic plasticity plays during the formation of adaptive radiations, the transcriptomes of the two divergent jaw phenotypes were examined. Our study identified a total of 187 genes whose expression differs in response to hard and soft diets, including immediate early genes, extracellular matrix genes and inflammatory factors. Transcriptome results are interpreted in light of expression of candidate genesmarkers for tooth size and shape, bone cells and mechanically sensitive pathways. This study opens up new avenues of research at new levels of biological organization into the roles of phenotypic plasticity during speciation and radiation of cichlid fishes.

Sweet potato chlorotic stunt virus: Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Bottom trawl codend mesh selectivity for Bagrus docmac (Forskahl) from Lake Victoria, with some remarks on the proposed trawl fishery

The methods of estimating codend selectivity for alternate hauls are briefly described and applied to Bagrus docmac (forskahl) data from Lake Victoria. Certain inconsistencies are noted and it is postulated that they may be due to differential escapement of larger Bagrus docmac through the trawl meshes when the different codends were fitted. The calculated 50% selection lengths yielded a fair regression on measured weigh give a fork length selection factor of 2.33, consistent with data from the smallest codend mesh size used. The implications of the findings are briefly discussed in relation to the proposed Haplochromis trawl fishery 1971.

Catalogue of EAFFRO scientific archives

Besides the formal publications held in the library (books, annual reports. Journals and reprints) a considerable quantity of unpublished Information on subjects relevant to freshwater fisheries in East Africa is held on various departmental files, etc.in Jinja. This material is extremely valuable: firstly because it is usually of immediate reference to current research work at EAFFRO, and secondly because it usually exists only as original material: no other copies are available elsewhere.

The establishment of a commercial fishery for Haplochromis in the Uganda waters of Lake Victoria

Lake Victoria, straddling the Equator, is the second largest lake in the world, with a surface area of approximately 26,000 square miles (41,500 square kilometers) and a maximum depth of about 300 feet. Uganda possesses most of the north end of the lake which consists of a long, indented coastline and a chain of offshore islands on the edge of a 'continental shelf' separating relatively shallow sheltered inshore waters from the deeper open waters of the lake. At the present time the lake is harvested mainly by a native gill net fishery confined almost entirely to the shallow sheltered inshore waters. The annual production of all species from Uganda waters is in the region of 24,000 tons per annum, and Tilapia(Cichlidae)is commercially the most important genus. Haplochromis, a close relative of Tilapia, but generally much smaller, contributes only a small amount to this annual production; see Table 1, although they are probably the most abundant group of fish present in the lake. Through international aid programmes Uganda has been offered a canning plant and it is thought that Haplochromis is the most suitable type of fish to be utilized by such a plant. The Uganda Fisheries Department are conducting research into processing techniques and marketing and the East African Freshwater Fisheries Research Organization was asked to conduct a survey of the Haplochromis stocks of the lake with a view to estimating the ability or otherwise of these stocks to support a commercial canning industry.

A brief report on a survey of the fish and fisheries of the Tana river, with special reference to the probable effects of the proposed barrages

The construction of several barrages in order to develop the hydroelectric and irrigation potential of the Tana river has been proposed and the probable effects of these developments upon the fish and fisheries of the area' has been investigated. Briefly in the highest reaches the sport fishery will be unaffected, in the middle reaches the sparse subsistence fisheries will be only slightly inconvenienced but in the terminal reaches of the river the subsistence and commercial fishing enterprises are expected to be seriously reduced by the progressive re-regulation of river-flow. However each new dam will support a new and productive reservoir fishery and with proper development the annual yield of fish from the Tana basin is expected to increase considerably.

An analysis of records of gillnet fishing in Pilkington Bay, Lake Victoria

Among the commercially valuable species occurring in Lake Victoria the two endemic species of Tilapia, T. esculenta (Graham) and T. variabilis (Boulenger) still provide the basis for the fishery in many areas, though in some places the non-cichlid species are more important, for example in the Kagera river area where large numbers of Labeo victorianus and Schilbe mystus migrate seasonally into the river. Although certain species show these habitat preferences. Most of the coastline of Lake Victoria supports a wide variety of species, all of which contribute to the commercial fishery. Over the past ten years the East African Fisheries Research Organisation has fished experimentally a number of stations within a radius of fifty miles from Jinja. These stations have been selected so as to cover a variety of habitats which range from sheltered bays to exposed coastlines. The records discussed in this paper are the result of fishing operations carried out in Pilkington Bay over the period December 1954 to November 1956. This series of fishing operations was carried out in an attempt to assess the composition of the fish populations contained in this area. To this end a variety of nets of differing mesh sizes were used. Pilkington Bay is about ten square miles in extent and is an irregular and deep indentation on the north coast of Buvuma Island. It lies within the sleeping siclmess area where fishing by Africans is prohibited. When selecting an experimental fishing ground, it is important to have some idea of the amount of commercial fishing being carried out in the area. Throughout the period of this experiment a few African fishermen were seen operating illegally in the area, but it is unlikely that their efforts greatly affected the results obtained. Pilkington Bay was fished by E.A.F.R.O. in 1949 and 1950 (Lowe McConnell 1956) but since then till the present experiment, fishing in the area has been infrequent.

On the Catch Assessement Survey (CAS) of Lake Victoria

If recent estimates are accurate the world’s human population can be expected to double in the next thirty years. The rate of growth will likely be even greater in many African nations, yet food supplies in these countries especially of essential animal proteins, are even not; inadequate Clearly increased production of food for domestic consumption must become a high- priority development goal. The inland fisheries of Africa will play an increasingly important role in augmenting protein supplies. In 1970, production of the inland fisheries was already-about 1.4 million metric tons, and had increased some 71 per cent in the previous six years. With further development and more affective fishery management a two-fold increase 1n output over the present level can reasonably be expected. Effective management of the fisheries at optimum exploitation levels end development of under utilized fish resources will neccessite major improvement in the stastistical systems employed to produce information on the fish stocks and fisheries. More reliable and detailed information on the catch, effort and other important aspect of the fishing enterprises will be required.

Technical guidelines for management of fisheries resources, biodiversity and environment of Victoria basin lakes

Lake victoria is the second largest lake in the world.the lake is shatred between three East African countries (Kenya,Uganda and Tanzania) the lake basin is estimatedto have about 30 million people who depend on it as a source of fish for food,employment,income and recreation.the lake is transport locally and regionally is used for recreation and is recongnised internationally for its high fish species diversity of ecological and scientific value. This document in the first in a series to be produced on different fish production systems in Uganda and should stimulate discussions and comments to guide application of scientific findings into the policy environment.

A report of the Fisheries Catch Assessment Survey in the Ugandan waters of Lake Victoria for the August 2007 survey

The monitoring of fish stocks as well as the magnitude, distribution and trends of fishing effort and fish catches is required for sound fisheries resources management. Conducting regular Catch Assessment Surveys (CASs) in Lake Victoria is one of the ways through which the partner states sharing the lake are generating information to contribute to the above process. The EU funded Implementation of a Fisheries Management Plan (IFMP) project for Lake Victoria through the Lake Victoria Fisheries Organisation (LVFO) is supporting the implementation of regionally harmonised CASs in Lake Victoria . The CASs under IFMP are following a statistical design laid down in Standard Operating Procedures (SOPs) agreed by the three partner states of the East African Community sharing the lake. In the Ugandan part of the lake, the CASs are carried out at 54 fish landing sites selected in the eleven riparian districts sharing the lake. The National Fisheries Resources Research Institute (NAFIRRI), Jinja; the Department of Fisheries Resources (DFR), Entebbe; and the Districts of Busia, Bugiri, Mayuge, Jinja, Mukono, Wakiso, Kampala, Mpigi, Masaka, Kalangala and Rakai jointly conduct the surveys. The CAS enumerators are recruited from the fishing communities and work under direct supervision of subcounty Fisheries Officers. NaFIRRI seeks to continue strengthening the engagement of the Beach Management Units (BMUs) and other fisheries Co-management Institutions in fisheries data collection .

DIFFERENT RATES OF MITOCHONDRIAL-DNA SEQUENCE EVOLUTION IN KIRK DIK-DIK (MADOQUA-KIRKII) POPULATIONS

We have investigated evolutionary rates of the mitochondrial genome among individuals of Madoqua kirkii using the relative rate test. Our results demonstrate that individuals of two chromosome races, East African cytotype A and Southwest African cytotype D, evolve about 2.3 times faster than East African cytotype B. Cytogenetic changes, DNA repair efficiency, mutagens, and more likely, hitherto unrecognized factors will account for the rate difference we have observed. Our results suggest additional caution when using molecular clocks in the estimation of divergence time, even within lineages of closely related taxa. Rate heterogeneity in microevolutionary timescales represents a potentially important aspect of basic evolutionary processes and may provide additional insights into factors which affect genome evolution. (C) 1995 Academic Press, Inc.

Uganda Freshwater Fisheries Research Organization Annual Report 1977

Following the collapse of the East African Community on 30th June, 1977 Ugandan fisheries research employees who were stationed in Kenya and Tanzania were recalled by the Uganda Government. The return of these officers strengthened the staff position of the Department. Formally the Headquarters of the East African Freshwater Fisheries Research Organization (EAFFRO), the institution was re-named the Uganda Freshwater Fisheries Research Organization (UFFRO) and the Uganda Government took up the full responsibility for running and maintaining the Department. The Annual report presents activities and achievements during the reporting period 1977.