Anna-Maria Botha

Analysis of genetic variation in Cucurbita moschata by random amplified polymorphic DNA (RAPD) markers.

Knowledge of genetic relationships among genotypes is essential for the effective utilisation of germplasm, especially for poorly characterised species. Random amplified polymorphic DNA (RAPD) analysis provides a quick and reliable method for resolving genetic relationships. Although Cucurbita moschata Duch, also known as tropical pumpkin, is one of the most important vegetable crops in Africa, being adapted to a wide range of climatic and soil conditions, it is a scientifically neglected species. The objectives of this study were to (1) analyse the amount of genetic diversity inC. moschata landraces grown in south-central Africa and (2) classify the landraces to assist in selection of parent genotypes for improvement of fruit characteristics. Cluster analysis, based on 39 polymorphic and 105 monomorphic DNA fragments amplified by 16 primers, was used to show relationships among 31 genotypes obtained from Zambia and Malawi. The analysis revealed four clusters, with genotypes from Malawi mainly clustering in three clusters while all genotypes from Zambia and three from Malawi clustered in one cluster. The pair-wise mean genetic distance was 0.32 ± 0.04 for samples from Malawi and 0.26 ± 0.04 for samples from Zambia. The possible application of the resulting classification in breeding of C. moschata is discussed.

A comparative study of molecular and morphological methods of describing genetic relationships in traditional Ethiopian highland maize

The comparison of different methods of estimating the genetic diversity could define their usefulness in plant breeding and conservation programs. In this study, a total of 15 morphological traits, eight AFLPprimer combinations and 20 simple sequence repeat (SSR) loci were used (i) to study the morphological and genetic diversity among 62 selected highland maize accessions, (ii) to assess the level of correlation between phenotypic and genetic distances, and (iii) to classify the accessions into groups based on molecular profiles and morphological traits. The analysis of variance of the morphological data revealed significant differences among accessions for all measured traits. The mean morphological dissimilarity (0.3 with a range of 0.1-0.68) was low in comparison to dissimilarity calculated using SSR markers (0.49 with a range 0.27-0.63) and AFLP markers (0.57 with a range 0.320.69). The correlation between the morphological dissimilarity matrix and the matrices of genetic dissimilarity based on SSR and AFLP markers was 0.43 and 0.39, respectively (p = 0.001). The correlation between SSRs and AFLPs dissimilarity matrices was 0.67 (p = 0.001). This congruence indicates that both marker systems are equally suited for genetic diversity study of maize accessions. Cluster analysis of morphological and marker distances revealed three groups of maize accessions with distinctive genetic profiles and morphological traits. This information will be useful for collections, conservation and various breeding programs in the highlands of Ethiopia.

Responses of phytoplankton upon exposure to a mixture of acid mine drainage and high levels of nutrient pollution in Lake Loskop, South Africa

The relationships between water quality and the phytoplankton community within Lake Loskop were studied during the late summer and autumn of 2008 to evaluate the impacts of acid mine drainage and high nutrient concentrations. The higher concentrations of metal ions and sulphate had adverse effects on certain phytoplankton species in the inflowing riverine zone of Lake Loskop, in comparison to the reference site in the lacustrine zone of the lake, which was dominated by the larger and slower growing late summer species of Coelastrum reticulum Nägeli, Straurastrum anatinum Meyen ex Ralfs and Ceratium hirundinella Müller. The high nutrient concentrations (nitrogen: 17 mg l(-1) and orthophosphate: 0.7 mg l(-1)) during the mid-summer peak of the rainy season were associated with the development of a bloom of the cyanobacterium Microcystis. Water quality data associated with the development of the Microcystis bloom suggest that the aquatic system of Lake Loskop has now entered an alternate, hypertrophic regime. This change overshadowed the adverse effects of high concentrations of heavy metal ions and low pH. Throughout this study, the reference site in the lacustrine zone of Lake Loskop had lower concentrations of metal ions and sulphate, and higher pH values. The response of phytoplankton bioassays on integrated water samples from the different sampling sites did provide potential answers to the reasons for the absence of the algal group Chlorophyceae in the phytoplankton community structure in the riverine zone of the lake.

Identification of toxigenic Microcystis strains after incidents of wild animal mortalities in the Kruger National Park, South Africa

The eutrophic process potentially caused by a high urine and faecal load resulting from an unusually high hippopotamus (Hippopotamus amphibious) density in the Nhlanganzwane Dam, Kruger National Park, South Africa, triggered a chain of events characterised by an increase in the growth of primary producers (Microcystis aeruginosa). This increase in M. aeruginosa biomass was followed by bio-intoxication incidents in wild animals. In this study, we determine if a M. aeruginosa bloom with a total microcystin level of 23,718microgl(-1) have been responsible for mortalities of megaherbivores in the Nhlanganzwane Dam. We further use microcystin molecular markers derived from the mcy gene cluster to identify potentially toxigenic environmental Microcystis strains in the dam during the occurrence of animal intoxications. The estimated total microcystin-LR daily intake by an adult male white rhinoceros (Ceratotherium simum) from cyanobacterial-contaminated water of the dam during the toxic event was an order of magnitude higher (754.29microgkg(-1)bw) in comparison with the lowest observed adverse effecting level (LOAEL) value measured for pigs in a previous study by other authors. In this study the presence of toxic cyanobacterial strains was confirmed with the use of molecular markers that detected the presence of the mcy gene cluster responsible for the production of toxin by M. aeruginosa.

Characterization and genetic distance analysis of cassava (Manihot esculenta Crantz) germplasm from Mozambique using RAPD fingerprinting

Twenty-eight cassava genotypes from Mozambique, along with seven genotypes from Angola, Madagascar, Nigeria, Togo, Columbia, and Thailand for comparison, were fingerprinted using random amplified polymorphic DNA (RAPD) analysis. The Mozambican material represented a wide range of landraces. A total of 311 scored RAPD loci were used to calculate genetic distances between the genotypes. This revealed an average genetic distance of 3.1% between all the germplasm. The average genetic distance between the Mozambiquen genotypes was 2.7%, whilst the seven accessions from the other countries showed an average distance of 3.4%. Neighbor-joining (NJ) method cluster analysis of the genetic distance yielded a tree that did not indicate a relationship between geographic distribution and genetic diversity.

Fractionated Extracts of Russian Wheat Aphid Eliciting Defense Responses in Wheat

Abstract It is hypothesized that the interaction between aphids and plants follows a gene-for-gene model. The recent appearance of several new Russian wheat aphid,Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), biotypes in the United States and the differential response of wheat,Triticum aestivum L., genotypes containing different resistance genes also suggest a gene-for-gene interaction. However, aphid elicitors remain unknown. This study was conducted to identify fractionated Russian wheat aphid extracts capable of eliciting differential responses between resistant and susceptible wheat genotypes. We extracted whole soluble compounds and separated proteins and metabolites from two Russian wheat aphid biotypes (1 and 2), injected these extracts into seedlings of susceptible wheat Gamtoos (dn7) and resistant 94M370 (Dn7), and determined phenotypic and biochemical plant responses. Injections of whole extract or protein extract from both biotypes induced the typical susceptible symptom, leaf rolling, in the susceptible cultivar, but not in the resistant cultivar. Furthermore, multiple injections with protein extract from biotype 2 induced the development of chlorosis, head trapping, and stunting in susceptible wheat. Injection with metabolite, buffer, or chitin, did not produce any susceptible symptoms in either genotype. The protein extract from the two biotypes also induced significantly higher activities of three defense-response enzymes (catalase, peroxidase, and β-glucanase) in 94M370 than in Gamtoos. These results indicate that a protein elicitor from the Russian wheat aphid is recognized by a plant receptor, and the recognition is mediated by theDn7-gene product. The increased activities of defense-response enzymes in resistant plants after injection with the protein fraction suggest that defense response genes are induced after recognition of aphid elicitors by the plant.

Transcript Profiling of Wheat Genes Expressed During Feeding by Two Different Biotypes of Diuraphis noxia

ABSTRACT Diuraphis noxia (Kurdjumov) (Russian wheat aphid) has severe economic impacts on wheat and barley production in the United States. The interaction between the Russian wheat aphid and its cereal hosts is poorly understood. However, the recent appearance of new biotypes in the United States showed that specific interactions exist between wheat resistance loci and Russian wheat aphid biotypes. At present, Dn7 is the only known gene in hexaploid wheat that confers resistance against all U.S. Russian wheat aphid biotypes. This study was conducted to investigate the molecular mechanism of Dn7-mediated resistance against two U.S. Russian wheat aphid biotypes (Russian wheat aphid 1 and Russian wheat aphid 2). Using GeneChip Wheat Genome Arrays, we compared transcript profiles of resistant and susceptible lines infested with either Russian wheat aphid 1 or Russian wheat aphid 2 using two time intervals (5 and 48 h after infestation). Russian wheat aphid feeding on hexaploid wheat led to the induction of groups of genes functioning in oxidative and general stress, photosynthesis, cell respiration and energy production, signal transduction, calcium-dependent signaling, pathogenesis related (PR) responses, and defense compound synthesis. The number of differentially expressed genes was higher in plants infested with Russian wheat aphid 1 compared with those infested with Russian wheat aphid 2. Although most genes involved in basic cellular functions were shared, unique genes were also obtained. This finding may indicate subtle differences in genes induced in response to different virulence proteins.

Cereal host interactions with Russian wheat aphid: A review

Abstract A lack of understanding the interaction between the Russian wheat aphid (RWA) and its host plant is a limitation in developing effective strategies for controlling the aphid. It is generally assumed that the interaction between aphid and plant is similar to that between plant and pathogen; that is, an elicitor from the insect is recognized by a protein from the host plant and a cascade of signal transduction events follows. However, evidence suggests that RWA feeding is eliciting both the SA- and JA/ethylene-dependent signaling pathways by mimicking aspects of both pathogen and herbivorous insect attacks. Results further suggest that phenotypic symptoms after RWA feeding are under regulation via two independent reactions, namely an immediate response (i.e., leaf rolling) and a downstream event (i.e., chlorosis). These defense responses enable a resistant host plant to defend itself and overcome the stress response, while their susceptible counterparts die. The processes involved in the onset of the defense response are discussed, and mechanisms enabling resistant plants to overcome the stress associated with the feeding process are presented as a working model for RWA-cereal host interaction. Knowledge of genes involved in wheats defense responses against the RWA and an understanding of their functions may provide additional strategies for developing broad-spectrum resistance in plants.

Genetic diversity in traditional Ethiopian highland maize accessions assessed by AFLP markers and morphological traits

In the highland regions of Ethiopia the heterogeneity of the land, the climate, and soil favors the presence of a large number of landraces. We analyzed a representative sample of 62 traditional Ethiopian highland maize accessions, using amplified fragment length polymorphism (AFLP®) markers and morphological traits with the aim to group the accessions based on the their genetic profiles and morphological traits, to study agroecological variation and to assess the level of correlation between phenotypic and genetic distances. Eight EcoRI/MseI primer combinations and 15 morphological traits were used. The accessions varied significantly for all of the measured morphological traits. Of a total of 650 AFLP markers that were scored, 89.5% were polymorphic. Pair-wise genetic distance estimates based on AFLP data revealed dissimilarity coefficients ranging from 0.32 to 0.69 (mean of 0.57). Cluster analysis of the AFLP data grouped most accessions collected from the Northern highlands into one major cluster. It, however, failed to separate the Western and Southern accessions into different clusters. Regardless of the large variation in environmental conditions between agroecologies, only 9% of the total genetic variation was found between agroecologies, whereas 91% was found within agroecologies in Ethiopia. This finding may be explained by long distance seed exchange, continuous seed introduction and gene flow between agroecologies. The relationship between morphological and AFLP-based distances was significant and positive. Based on this study, three groups of highland accessions, with distinctive genetic profiles and morphological traits were identified. This information will be useful for further collections and conservation of the unique diversity included in the highland maize landraces of Ethiopia.

Functional and comparative analysis of expressed sequences from Diuraphis noxia infested wheat obtained utilizing the conserved Nucleotide Binding Site

Russian wheat aphid (Diuraphis noxia, Morvilko; RWA) is a major pest on wheat, barley and other triticale in South Africa. Infestation by the RWA results in altered protein expression patterns, which is manifested as differential expression of gene sequences. In the present study, Russian wheat aphid resistant (Tugela DN, Tugela*5/SA2199, Tugela*5/SA463, PI 137739, PI 262660, and PI 294994) and susceptible triticale (Tugela) were infested and cDNA synthesized. A PCR based approach was utilized to amplify the nucleotide binding site conserved region to obtain expressed sequence tags (ESTs) with homology to resistance gene analogs (RGAs). The approach proved highly feasible when the isolation of RGAs is the main objective, since 18% of all obtained ESTs showed significant hits with known RGAs, when translated into their corresponding amino acid sequences and searched against the nonredundant GenBank protein database using the BLASTX algorithm.