Program

08:00 Registration

08:30 Welcome and introduction: Prof Don Cowan, Director

08:45 PLENARY LECTURE: Prof Michael Pepper, Department of Immunology and Institute for Cellular and Molecular Medicine:

The Southern African Human Genome Programme

09:30 L1: Melvin Ambele, Department of Immunology and Institute for Cellular and Molecular Medicine:

Potential biomarkers characterizing adipocyte differentiation revealed from genome-wide gene expression studies of human adipogenesis

09:45 L2: Desre Pinard, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI):

The regulation of carbon compartmentation and metabolism in Eucalyptus wood formation

10:00 L3: Evelien Adriaenssens, Centre for Microbial Ecology and Genomics, Department of Genetics:

Extreme viruses: A wide range of ssDNA viruses and a new lineage of haloarchaeal viruses in Namib Desert salt pans

10:15 L4: Jennifer Wayland, Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI):

The Optimization and Application of a High-Throughput Sequencing – Based Diagnostic system for the Detection of Viruses of Grapevines

10:30 Morning tea

11:00 L5: Velushka Swart, Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI):

Functional characterisation of the Cercospora zeina CTB oxidoreductase gene deletion by Agrobacterium tumefaciens-mediated transformation

11:15 L6: Keneilwe Peloakgosi, Department of Life and Consumer Sciences:

De novo assembly and annotation of Babesia rossi transcriptome in dogs diagnosed with canine babesiosis

11:30 L7: Ronishree Mangwanda, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI):

Transcriptional profiling of Eucalyptus grandis and Chrysoporthe austroafricana elucidates host defence mechanisms and putative pathogenicity strategies

11:45 L8: Philippa Franzini, Centre for Microbial Ecology and Genomics, Department of Genetics:

The microbiomes of two closely-related desert beetle species feeding on different diets

12:00 L9: Jonathan Botha, Centre for Microbial Ecology and Genomics, Department of Genetics:

Hyperthermophiles: A source of CAZymes for industrial lignocellulosic Degradation

12:15 L10: Cheryl Stewart, Institute for Cellular and Molecular Medicine and the Department of Immunology:

A genomics-driven public health solution to the cystic fibrosis problem in Africa

12:30 L11: Professor Dave Berger, Department of Genetics, Department of Plant Science, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI):

Systems genetics of the maize-grey leaf spot pathosystem

12:45 Lunch

13:30 L12: Martin P. Wierzbicki, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI):

Systems genetics of xylan modification in Eucalyptus

13:45 L13: Marco Alessandrini, Department of Immunology and the Institute for Cellular and Molecular Medicine:

Initiation of an HIV gene therapy clinical trial in South Africa

14:00 L14: Jasmin Mertens, Centre for Microbial Ecology and Genomics:

From Antarctica to Enhancing the Resistance of Crops to Drought Stress - The long journey of the WHy domain

14:15 L15: Drew Behrens, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI):

Discovery and transcriptional dynamics of the small noncoding RNA transcriptome in source, transport and woody sink tissues in Eucalyptus grandis

14:30 L16: Nanette Christie, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI):

Bioinformatics resources for analysis and visualization of genetic mapping data.

14:45 L17: Sandra Phoma, Centre for Microbial Ecology and Genomics:

Disentangling the drivers of taxonomic and functional community structure in the Southern Ocean

15:00 L18: Mokgoadi Trevor Molemane, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science:

The evaluation of the effect of cattle and buffalo host cells on gene expression in buffalo-derived and cattle-derived Theileria parva isolates

15:15 Afternoon tea

15:45 L19: Riëtte van Biljon, Department of Biochemistry, Centre for Sustainable Malaria Control:

Insights into the unique molecular mechanisms controlling asexual proliferation and sexual differentiation of malaria parasites from deep-transcriptome analyses.

16:00 L20: Jeanne van Rensburg, Department of Immunology and Institute for Cellular and Molecular Medicine:

Cystic fibrosis and rarefaction: Unification through diversity.

16:15 L21: Stanford Kwenda, Forestry and Agricultural Biotechnology Institute (FABI):

Identification and characterization of potato long noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection

16:30 L22: Megan Harris, Department of Microbiology and Plant Pathology:

Detection of Grapevine Leafroll Associated Virus 3, Viti- and Foveaviruses in Vitis Rootstocks.

16:45 L23: Olivier Zablocki, Centre for Microbial Ecology and Genomics:

Virus diversity and biogeography in Namib Desert soils

17:00 L24: Liberata Mwita, Centre for Bioinformatics and Computational Biology:

Specificity of gene regulation in Bacillus atrophaeus UCMB5137 during plant colonization compared to Bacillus amyloliquefaciens FZB42, the paradigm of plant growth promoting Bacillus

17:15 Evening Cocktail Function at the Plant Science Auditorium – Roof Top

LECTURES

PLENARY LECTURE

The Southern African Human Genome Programme

1 Michael S. Pepper and 2Fourie Joubert

1Dept. Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences; and 2Bioinformatics and Computational Biology Unit, Dept. Biochemistry, Faculty of

Natural and Agricultural Sciences, University of Pretoria

The Southern African Human Genome Programme (SAHGP) was initiated in January 2011 with funding from the Department of Science and Technology (DST). The objectives of the SAHGP are:

• To develop capacity for genomic research in southern Africa• To translate knowledge into improvements in human health• To contribute to our understanding of the origin of humankind• To promote public education regarding genomics research

Phase I, which included the initiating stakeholder meeting January 2011 and the preparation of a research project application, set the stage for phase II which was to sequence 24 whole genomes and to build bioinformatics capacity, again with funding from the DST. This work has now been completed and we have sequenced and analysed 24 genomes at 50x coverage. In order to do this a number of processes had to be followed including obtaining ethics approval, sample collection, sequencing and data analysis.

This presentation will highlight the important areas that we have needed to address in this journey including those mentioned above. We will also present some of the findings that have emerged from the analysis of the data recognizing that complete disclosure is not possible at this stage. We will also discuss issues related to data storage and access as part of the ethical, legal and social issues (ELSI) that need to be addresses in a project of this nature.

Phase III of the SAHGP includes a move towards precision medicine and the establishment of a database of southern African exomes. There has been a strong call from the DST for translation and innovation which requires the identification of areas of importance for the country and region. This is a lengthy process and is it is recognized that there are very few “low hanging fruits” but that without an investment into a project such as the SAHGP, the range of benefits to be derived by the country will be slow in forthcoming.

In summary, we have established and maintained a productive and highly interactive group based on a spirit of collegiality, and the project stands to benefit all of the people of our country and region as we reap the benefits of the genomics revolution.

L1: Potential biomarkers characterizing adipocyte differentiation revealed from genome-wide gene expression studies of human adipogenesis

Melvin Ambele1, Carla Dessels1, and Michael Pepper1

1Department of Immunology and Institute for Cellular and Molecular Medicine, University of Pretoria; SAMRC Extramural Unit for Stem Cell Research and Therapy

Increased adipogenesis in human white adipose tissue leads to obesity, which is a major risk factor for cardiovascular diseases, type 2 diabetes and cancer. Genome-wide studies of this process in human adipose-derived mesenchymal stromal cells (ASCs) may reveal novel biomarkers which characterize this process and could serve as good candidate molecules for the development of therapies that combat obesity.

RNA isolated from both adipogenic induced ASCs and their respective controls on days 1, 7, 14 & 21 were hybridized to Affymetrix HuGene 2.0 ST arrays. Bioinformatics tools were used to analyze gene expression.

It was observed that 61, 124, 138 and 149 genes were significantly up-regulated (fold change ≥4, p˂0.05 and FDR <0.5) on day 1, 7, 14 & 21 respectively. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors (TFs) were up-regulated throughout adipogenesis. In silico analysis revealed these TFs interact with SIRT1 AKT1, HDAC1 & NCOR2 which altogether are essential for driving adipogenesis. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB TFs were up-regulated only from day 14 – 21, which coincides with adipocyte maturation. These TFs could serve as biomarkers in characterizing this stage in adipocyte differentiation and could possibly be good candidates for controlling fat accumulation in and size of the mature adipocyte. In a similar manner, we identified genes significantly up-regulated only from day 1 -7 and day 7-21 which could serve as potential biomarkers for early-stage and general adipocyte differentiation respectively. Genes up-regulated during adipogenesis were also associated with neural and blood vessel development, migration of leukocytes, growth, tumor invasion and metastasis while down-regulated genes were associated with osteogenesis and immune response. Furthermore we observed genes involved in adipocyte differentiation to share common pathways with certain pathophysiological conditions, some of which have previously been described as being obesity related such as cancer, cardiovascular and metabolic diseases. Hence, this study reveals potential biomarkers for the different stages in ASC adipogenic differentiation which could serve as good candidates to modify adipogenesis in order to combat obesity. Furthermore, our study links obesity to certain pathophysiological conditions.

L2: The regulation of carbon compartmentation and metabolism in Eucalyptus wood formation

Desre Pinard 1 , Ana Carolina Fierro Gutiérrez3, Yves Van de Peer1, 5, Kathleen Marchal3,4, Alexander Myburg1,2, Eshchar Mizrachi1,2

1 Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private bag X20 Hatfield, Pretoria 0028, South Africa

2 Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Hatfield, 0028, South Africa

3 Dept. of Plant Biotechnology and Bioinformatics, U.Ghent, Dept. of Information Technology (INTEC, iMINDS), U.Ghent, Technologiepark 927, B-9052 Ghent, Belgium

4 Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

5 VIB, Department of Plant Systems Biology, Ghent University, Bioinformatics & Systems Biology, Technologiepark 927 , B-9052 Ghent, Belgium

The process of wood formation in trees requires extensive transcriptional and metabolic regulation to ensure the coordinated processing of precursors, polymerization and deposition of secondary cell wall biopolymers. An aspect of xylogenesis that is currently unknown is the regulation of the cellular compartmentation of carbon metabolism and the roles of plant organelles in carbon partitioning towards SCW biopolymers. We identified the predicted cellular locations for Eucalyptus genes that are expressed in 156 developing xylem transcriptomes of a E. urophylla x grandis hybrid population, and found that ~30% of proteins involved in carbon metabolism are predicted to be localized to the plastid and mitochondria. In this study, we constructed high-resolution query-based co-expression networks of plastid and mitochondrial carbon metabolic genes in order to analyse the transcriptional coordination and integration of carbon compartmentation in developing xylem cells. Network construction using a combination of correlation and reciprocal rank, followed by community detection clustering produced structured co-expression networks for plastids (2,550 genes) and mitochondria (2,117 genes) with seven and five clusters, respectively, and some overlap in coregulation. GO biological process term enrichment shows that the clusters have distinct biological functions, including SCW biopolymer synthesis. Analysis of the clusters has revealed that the metabolic shift from primary to secondary cell wall metabolism is encoded by distinct co-expression clusters, along with the circadian regulation of starch metabolism during wood formation. Identification of transcription factors, signalling molecules, and transport proteins in these gene clusters are expanding our understanding of carbon metabolism during wood formation.

L3: Extreme viruses: A wide range of ssDNA viruses and a new lineage of haloarchaeal viruses in Namib Desert salt pans

Evelien Adriaenssens 1 , Lonnie van Zyl2, Marla Tuffin2 and Don Cowan1

1Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria2Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape

Namib Desert salt pans or playas, are largely unexplored environments, with a salinity range that creates a potential home to halotolerant and halophilic microorganisms. This makes them ideal study sites for unique viral populations and sites of interest for bioprospecting of novel enzymes. In this study, we have sequenced the metaviromes of two different salt pans, the Hosabes playa (near the Gobabeb Research Station) and the Eisfeld playa (near the town of Swakopmund). In general, playas found in the Namib Desert are moist, salt-covered, sediment-filled depressions which form in drainage channels whose surface and groundwater flow is obstructed by linear bedrock outcrops. They have a salinity of 3 to 15% depending on the distance from the source, the depth of the pool and the time of day (evaporation), featuring halite and gypsum crusts.

Viral communities of these two salt pans were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harboured largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.

L4: The Optimization and Application of a High-Throughput Sequencing – Based Diagnostic system for the Detection of Viruses of Grapevines

Jennifer Wayland1, Anna E. C. Jooste2 and Gerhard Pietersen1,2

1Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa 2ARC-Plant Protection

Research Institute, Pretoria 0121, South Africa

Grapevine (Vitis) is an important agricultural commodity in many countries, including South Africa. More than 70 viruses have been reported infecting grapevine, of which many are associated with diseases. Certification schemes worldwide share the aim of producing healthy vines for the vegetative propagation of grapevine, mainly through virus elimination by heat therapy treatment and in vitro meristem culture. In South Africa, plants derived from these methods are tested for the presence of specific viruses by biological indexing, enzyme-linked immunosorbent assays (ELISA) and polymerase chain reactions (PCR), and only when found free of them, are further propagated. In this study we present a diagnostic system based on poly-specific PCRs in combination with high-throughput sequencing (HTS) for the detection and identification of 40 viruses in 10 genera. For the reliable interpretation of HTS data within this diagnostic system, a standard data analysis pipeline was determined using CLC genomics workbench.We recommend a threshold for percentage read count of 0.4 % during reference mapping to discern between presence and absence of viruses associated with reads. Various criteria for the evaluation of the BLAST results were identified based on virus hits, E-value, percentage overlap and percentage identity. The virus population of grapevine samples were determined to the genus level through the application of the poly-specific PCRs used within this study without the HTS component. This system will contribute to the detection and identification of novel and previously undetected viruses in grapevine and other crops.

L5 Functional characterisation of the Cercospora zeina CTB oxidoreductase gene deletion by Agrobacterium tumefaciens-mediated transformation

Velushka Swart 1 , Bridget Crampton1 and Dave Berger1

Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria

The maize foliar pathogen, Cercospora zeina causes the agriculturally devastating disease, grey leaf spot (GLS). Members of the genus Cercospora cause disease in a variety of economically important crops worldwide and their pathogenicity is linked to the production of the phytotoxin, cercosporin. Cercospora zeina fails to produce cercosporin in vitro. Identification and annotation of the C. zeina cercosporin toxin biosynthetic (CTB) gene cluster, demonstrated the presence of an in-frame deletion in one of the CTB genes. The aim of this study was to determine whether complementing this deletion with the full length gene from the sibling species, Cercospora zeae-maydis, could induce in vitro cercosporin production. Following Agrobacterium-mediated transformation, putative transformants were screened using PCR and studied with regards to in vitro cercosporin production. One of the over-expression mutants showed accumulation of a red pigment when grown on 0.2xPDA, which was subsequently confirmed to be cercosporin based on the KOH assay and thin-layer chromatography. These findings support the hypothesis that the CTB oxidoreductase gene is the bottleneck for in vitro cercosporin production in C. zeina. The results indicate that C. zeina has all the other machinery for synthesizing cercosporin-like molecules, and thus C. zeina may produce a structural variant of cercosporin in vitro.

L6: De novo assembly and annotation of Babesia rossi transcriptome in dogs diagnosed with canine babesiosis

Keneilwe Peloakgosi 1 , Kgomotso Sibeko², Andrew Leisewitz3 and Tshepo Matjila 2

¹Department of Life and Consumer Sciences, University of South Africa²Department of Veterinary and Tropical Diseases, University of Pretoria

3Department of Companion animal studies, University of Pretoria

Babesia rossi is a pathogenic apicomplexan parasite responsible for causing the most complicated form of canine babesiosis in domestic dogs (Jacobson, 2006). In addition, canine babesiosis induced by B. rossi still remains the cause of mortality and morbidity in South African domestic dogs (Schoeman, 2009). Yet, the transcriptome of this important species has not been investigated. Thus, this study reports the sequencing, de novo assembly and annotation of the whole transcriptome of the genotypes (19, 29 and 31). Total RNA was prepared from blood samples infected with Babesia rossi genotypes 19, 29 and 31, obtained from sick domestic dogs diagnosed with canine babesiosis. The RNA was sequenced using the Illumina. Subsequently, de novo assembly was performed using the CLC Genomic Workbench Version 7.5.1. The contig sequences generated from transcriptome assemblies were functionally annotated using Blast2GO version 2.8.0 software. A total of 2, 790; 2,520 and 4,090 contig sequences were obtained from genotypes 19, 29 and 31 respectively, after the removal of host sequences and de novo assembly. Functional annotation of the three genotypes revealed the same annotation pattern defined by the association of the majority of contig sequences with biological processes, followed by cellular component, finally the molecular function. No significant variation was observed in Babesia rossi genotype 19, 29 and 31 assembled transcriptomes based on the GO terms. However, this study presents the first transcriptomic resource for Babesia rossi, which will highly contribute to our genetic understanding of Babesia rossi and provide a platform for future gene expression studies in Babesia rossi genotype 19, 29 and 31.

ReferencesJacobson, L.S. (2006). The South African form of severe and complicated canine babesiosis: Clinical advances 1994-2004. Vet. Parasitol, 138: pp.126-139.Schoeman, J.P. (2009). Canine babesiosis. Onderstepoort J. Vet. Res, 76: pp. 59–66.

L7: Transcriptional profiling of Eucalyptus grandis and Chrysoporthe austroafricana elucidates host defence mechanisms and putative pathogenicity strategies

Ronishree Mangwanda1, 2, Albe van der Merwe 1, 2, Alexander Myburg 1, 2 Sanushka Naidoo 1, 2

1Department of Genetics, University of Pretoria , South Africa.2Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

Eucalypts are extensively propagated for their desirable wood properties, but this species is also being investigated as a potential source for biofuel production. Throughout their lifetime, these woody species are exposed to various pathogens that can cause severe losses to the industry. Among these pathogens is Chrysoporthe austroafricana, which causes the development of stem cankers on eucalypts. The established pathosystem of E. grandis and C. austroafricana can be used as a model system to elucidate the defence strategies of the host as well as to decipher pathogenicity mechanisms of the pathogen. To investigate the defence responses of E. grandis, a susceptible clone (EgrS) and a moderately resistant clone (EgrR) were inoculated with C. austroafricana and stem material, harvested 3 days post inoculation (dpi), was sent for transcriptome profiling. In vitro growth of fungi on differential media can mimic a stress response and reveal genes involved in pathogenicity. Thus, transcriptome profiling was performed on C. austroafricana grown in vitro on nutrient limited media and nutrient rich media. A Cuffdiff analysis was performed between the two media compositions as well as between the nutrient rich media and the in planta samples to identify potential pathogenicity mechanisms towards Eucalyptus.

Data analysis of E. grandis challenged with C. austroafricana revealed 1539 and 1495 differentially expressed genes in EgrR and EgrS respectively when compared to the controls. Further investigation of these genes suggested a role for gibberellin signaling in facilitating susceptibility. Pathogenicity mechanisms elucidated by transcriptome profiling of C. austroafricana included cell wall degrading enzymes, fungal effectors and genes such as ent-kaurene oxidase and salicylate hydroxylase that may manipulate gibberellin and salicylic acid signaling respectively. The ability of a host to fine-tune its defence responses is crucial in determining the outcome of a pathogen incursion and the responses identified in this study provide a glimpse into the complexity of these responses activated in Eucalyptus.

L8: The microbiomes of two closely-related desert beetle species feeding on different diets

Franzini, P.Z.N., Ramond, J-B., Ronca, S. and Cowan, D.A.

Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria

Microbial communities inhabit many environmental niches including the nutrient-rich gut system of animals. It is generally believed that the diet of the host animal plays an important role in the structure of the gut microbiome. In this study we investigated the role that host diet might have in microbial community structure in two closely related species feeding on different substrates. Species of the insect genus Pachysoma MacLeay (Coleoptera: Scarabaeidae) feed on dry dung pellets, plant detritus or both. Two Pachysoma spp, P. endroedyi (mixed feeder) and P. striatum (dung feeder) were collected from Namaqualand, South Africa. Whole guts from five insects of each species were dissected and metagenomic DNA extracted. Microbial community structure was determined by 454 sequencing of the bacterial 16S gene and fungal ITS gene region.

Bacterial community structure varied significantly between the two species. 14 and 6 phyla were detected within the gut of the mixed feeder and dung feeder, respectively. Proteobacteria, Firmicutes, Bacteriodetes and Actinobacteria were highly represented within the dung feeder, with low abundances of Planctomycetes and Deferribacteres. The same four dominant phyla were abundant in the mixed feeder gut along with high abundances of Planctomycetes, Synergistes and Elusimicrobia. Other phyla present within the plant feeder gut were Cyanobacteria, Deferribacteres, Fusobacteria, Lentisphaerae, Tenericutes, Candidate phylum BD1 5 and Candidate phylum TM7. Planctomycetes is commonly detected in termite and other lignocellulosic insect guts but in low abundances. Several other phyla in the mixed feeder gut were common termite gut phyla. Such differences in diversity at the phylum level are reported in insects of the same genera but, unlike Pachysoma, is typically restricted to minor phyla. Only 12 OTUs were shared between the two Pachysoma spp., the majority of which were most similar to strains isolated from feaces and skin. A high gut bacterial diversity correlates with a complex diet (plants, multiple substrates) whereas a simple diet (dung fluids, sap, pollen) generally reflects a low gut bacterial diversity. Fungal communities could not be amplified from the mixed feeder, suggesting a low fungal abundance within the gut of this species. Fungi within the dung feeder gut were classified into four classes within Ascomycota and one within Basidiomycota, with a large percentage of fungal reads unclassified at the phylum level. Low fungal diversity is common within insect groups, with Ascomycota most commonly reported. The two most abundant fungal OTUs, Preussia australis and Trichosporon ovoides, are most likely associated with the food source.

L9: Hyperthermophiles: A source of CAZymes for industrial lignocellulosic degradation

Jonathan Botha 1, 2, Eshchar Mizrachi 2, 3, Alexander A. Myburg 2, 3, Don Cowan1, 2*

*Corresponding author: Don.Cowan@up.ac.za1Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria,

Private Bag X20, Pretoria, 0028, South Africa2Department of Genetics, University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa

3Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa

4Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa

Currently, harsh conditions and expensive enzymes and chemicals make the extraction of biopolymers from lignocellulosic biomass economically unfeasible [1]. Hyperthermophiles (organisms which grow between 80⁰C to 120⁰C) are an important source of thermostable enzymes for use in industrial processes. In this study, we characterise and compare the CAZyme abundance and diversity within and between hyperthermophile genomes, and evaluate their ability to degrade lignocellulosic biomass. To do this, hyperthermophilic organisms with completely sequenced and published genomes were identified using GOLD (gold.jgi-psf.org) and HMMER scans were performed on their complete proteomes to identify CAZyme domains, which were quantified using custom bioinformatic scripts. Functional data for each domain was obtained from the CAZy web database (http://www.cazy.org/) and literature. In total, 66 hyperthermophilic proteomes were used in the study, ranging across the domains of Bacteria and Archaea. 4,191 CAZyme domains were identified, comprising all CAZyme classes. 41 CAZyme families were found uniquely in one of the proteomes investigated. Of these families, two xylan binding CBMs were found only in Ignisphaera aggregans (Archaeon) and Caldicellulosiruptor owensis (Bacterium), respectively, and four cellulose-binding CBMs were found in Caldivirga maquilingensis and Caldisphaera lagunensis (Archaea), while two were found only in Spirochaeta thermophila (Bacterium). Some cellulose targeting GHs were present in Bacteria but not Archaea. Conversely, one cellulose targeting CBM was present in Archaea but not Bacteria. Neither GH10 nor GH11 (xylan targeting) were observed in Archaea, though GHs with both xylanolytic and cellulolytic activity were present. Together, this shows that Archaea and Bacteria may have unique capacities to degrade lignocellulosic biopolymers. The high number of unique domains also suggests that as more hyperthermophilic organisms are identified and sequenced, new CAZyme domains may be discovered. This study provides a set of protein domains which may be used to design and synthesise enzymes for biotechnological applications under extreme conditions [3, 4].

L10: A genomics-driven public health solution to the cystic fibrosis problem in Africa

Cheryl Stewart 1 and Michael Pepper1

1Institute for Cellular and Molecular Medicine and the Department of Immunology, University of Pretoria

Cystic fibrosis (CF) is the most common autosomal recessive disease globally. Although CF has been known to affect Africans since as far back as 1959, the majority of investigations have been Euro-centric. As a result, African CF patients tend to be diagnosed late leading to premature death and high treatment costs. To date, only 12 African countries have investigated which CF-causing mutations are present in their populations. This is necessary since using a population-specific genetic test for CF would yield an unambiguous diagnosis and allow clinicians to implement an appropriate treatment regimen. Additionally, with the advent of class-specific drugs, identifying the mutations a patient carries can determine what drug therapies should be administered.

Since a key part of diagnosing and treating CF patients is being able to identify the mutations present, we investigated the molecular epidemiology of CF in Africa. Most of the molecular work has been conducted in North Africa (where 1,334 chromosomes were tested) and in Southern Africa (where 760 chromosomes were tested). A total of 2,344 chromosomes have been screened which has led to the identification of 79 variants. Of these 39 are known empirically to cause CF while 21 are unique to Africa. Only 12 variants were found in more than one country, highlighting the population-specific nature of these mutations. It should be noted that 51% of the chromosomes were tested using methods that could not identify novel or country-specific mutations. The majority of the investigations did not rely solely on sequencing meaning that some mutations may not have been detected.

African CF patients live about half as long as their peers from other parts of the world. A public health policy would be useful to improve the life expectancy and well-being of these patients. However, given the lack of molecular data on the continent, the diagnostic and therapeutic arms of this policy would need to be genomics-driven.

L11: Systems genetics of the maize-grey leaf spot pathosystem

Nanette Christie1, Zander Myburg1, and Dave Berger2

1Department of Genetics, 2Department of Plant Science, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria

Systems genetics is the integration of trait phenotype, genotype, and global cellular data (e.g. transcript, protein or metabolite) to dissect complex traits. Understanding quantitative resistance and susceptibility of crops to pathogens is a worthy goal of systems genetics. Our research is focused on grey leaf spot (GLS) disease of maize caused by Cercospora spp. fungi. GLS is a threat to maize production in Africa and most maize producing regions globally. We took a systems genetics approach to the problem by combining phenotyping (GLS disease scoring), genotyping (genetic mapping) and transcriptomics (Agilent whole genome microarrays) of a maize population segregating for GLS susceptibility. We carried out (i) expression QTL analyses and (ii) weighted gene co-expression network analysis. Data integration allowed us to explore the genetic basis for coordinated expression responses to GLS disease in susceptible maize plants. A customized bioinformatics pipeline for eQTL analysis was developed in the Galaxy platform. The pipeline is applicable to systems genetics analysis of complex traits using transcriptome data (microarrays or RNAseq) from segregating populations of other crop species.

L12: Systems genetics of xylan modification in Eucalyptus

Martin P. Wierzbicki 1 , Shawn D. Mansfield2 , Nanette Christie1, Eshchar Mizrachi1 and Alexander A. Myburg1

1 Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Pretoria, South Africa

2 Department of Wood Science, University of British Columbia, Vancouver, BC, Canada

Xylan is a short, branched polysaccharide component of the secondary cell wall (SCW) of woody dicots such as Eucalyptus and Populus. This molecule aids in stabilising and hardening the SCW and making the SCW resistant to pathogen attack. Xylan coating and cross-linking of cellulose microfibrils however, impedes biopolymer extraction for use in bio-based products such as textiles and bioplastics. It is proposed that the acetyl and (methyl)glucuronic acid modifications of the xylan backbone and side branches affect extractability, however the genetic regulation of these modifications remains unknown. We are using population genomic and transcriptomic data obtained from E. grandis and E. urophylla interspecific backcross populations to elucidate the regulatory interactions of xylan modification genes and identify novel genes involved in the process. Phylogenetic analysis and evolutionary reconstruction was performed on all four protein families with members known to be involved in xylan modification in model plants using the protein coding sequences found in Eucalyptus and the two model organisms Populus and Arabidopsis. Candidates were chosen on phylogeny but selection was aided by expression profiling, domain architecture and subcellular localisation within the cell. All candidate genes from these families found in Eucalyptus were subjected to co-expression analysis and expression QTL (eQTL) mapping in the E. grandis x E. urophylla backcross population. We used eQTLs shared between candidates to identify potential regulatory polymorphism(s) leading to co-regulation of these genes. Our results are allowing us to dissect the genetic architecture of xylan modification and identify biotechnology targets for genetic engineering of woody biomass traits in Eucalyptus.

L13: Initiation of an HIV gene therapy clinical trial in South Africa

Marco Alessandrini 1 and Michael S. Pepper1

1Department of Immunology and the Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria, South Africa; South African Medical Research Council

Extramural Unit for Stem Cell Research and Therapy.

Over 35 million people worldwide are affected with HIV/AIDS. There is no cure for the disease, and although significant progress has been made in its management, several disadvantages still exist. These include life-long drug adherence and the associated side effects, and the emergence of resistant strains in patients with poor compliance. Innovative approaches are therefore required to relieve the burden of this disease. As part of a collaborative effort, we have developed an HIV gene therapy aimed at curing HIV. The therapy is microRNA based and targets CCR5 expression, hence preventing HIV from entering the cell. The approach entails harvesting haematopoietic stem cells (HSCs) from an HIV infected patient, transfecting these cells with lentiviral vectors carrying CCR5-targeted microRNA, and re-introducing these gene–engineered cells back into the patient via intravenous infusion. Pre-clinical data derived from a humanised mouse model have been promising, and the next steps are thus to test safety and efficacy in humans. An in-depth evaluation of the requirements for implementing a clinical trial of this nature in South Africa is underway, which includes addressing several ethical and regulatory aspects, facilities for preparation of the gene therapy, and fund raising. Initial discussions with specialist clinicians in South Africa suggest that patients with HIV-related lymphoma may be an appropriate study population. However, a protocol for treating HIV patients who are stable and on antiretroviral treatment is also under consideration. Production of the lentiviral vectors and transfection of HSCs will require cleanroom facilities operated according to current Good Manufacturing Practices (cGMP). The latter part of this preparation is planned to take place in a newly established cleanroom facility based at the University of Pretoria. Once the protocol is finalised, approval from both the Research Ethics Committee of the Faculty of Health Sciences at the University of Pretoria and the South African Medicine’s Control Council (MCC) will be required before trial initiation. In conclusion, the initial steps towards implementing an HIV gene therapy clinical trial in South Africa have been taken. Raising the necessary funds is also underway and it is our intention to initiate a Phase I/II human clinical trial in 2016/2017.

L14: From Antarctica to Enhancing the Resistance of Crops to Drought Stress - The long

journey of the WHy domain

Jasmin Mertens1, Eloy Ferreras1, Dominique Anderson2, Don A Cowan1,2

1 Centre for Microbial Ecology and Genomics (CMEG), Genomics Research Institute, University

of Pretoria, South Africa

2 Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape,

Cape Town, South Africa

A serious consequence of global climate change is the increased desertification of those

regions which are already impacted by water deficit. Equatorial areas of the African continent

are most likely affected with increased negative impacts on crop range and productivity.

Therefore, an urgent need exists to identify innovative approaches for enhancing cropping

ranges in regions with water deficits. One such approach is the development of new crop

cultivars which have enhanced drought resistance, through the identification and in planta

expression of novel stress-response elements. Functional metagenomics library screening is

one approach that has already become an established method for the identification of novel

genes and gene products.

Screening of Antarctic soil metagenomic libraries identified a novel bacterial gene, homologous

to known Water and Hypersensitivity (WHy) domains. The WHy domain is a typical component

of Late Embryogenesis Abundant (LEA) proteins which occur widely in prokaryotes as well as in

eukaryotes (e.g. bacteria, archaea and plants) and are expressed under different stress

conditions. Our studies have shown that this bacterial protein elicits significant protection

against freeze and cold stress in recombinant E. coli (1). We are now investigating the question

of whether this novel WHy protein can be functionally expressed in Arabidopsis and whether it

will confer cold- and drought-resistance in planta.

(1) Anderson D, Ferreras E, Trindade M, Cowan D (2015) A novel bacterial Water Hypersensitivity-like protein shows in vivo protection against cold and freeze damage. FEMS Microbiology Letters, 362, 2015, fnv110

L15: Discovery and transcriptional dynamics of the small noncoding RNA transcriptome in source, transport and woody sink tissues in Eucalyptus grandis

D. Behrens 1 , A.A. Myburg1, E. Mizrachi1

1Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI) and Genomics Research Institute, University of Pretoria, South Africa

Rapid progress is being made in understanding the transcriptional networks and interactions governing xylogenesis and the regulation of lignocellulosic biopolymer synthesis in plants . Despite this, much is still unknown regarding the small noncoding RNAs (sRNAs) that post-transcriptionally regulate gene expression through complementary sequence targetting and RNA degradation. miRNAs are a well known category of this RNA species, and several highly conserved miRNAs are known to play essential roles in vascular tissue differentiation (Trumbo et al. 2015). The siRNAs are more complex, diverse, yet highly pervasive RNA type in plant genomes (Axtell 2013), and may indeed contribute significantly to species-specific biology. Here, we produce and quantify a comprehensive catalog of E. grandis regulatory sRNAs – including miRNAs and siRNAs – in leaves, secondary phloem and immature xylem, representing major points in carbon sequestration, transport and utilization for lignocellulosic biomass, respectively. Expression analysis of these genes has revealed tissue specific clusters occuring in vascular and leaf tissue (including high-confidence examples of known miRNA:target interactions). Target prediction and gene set enrichment analysis revealed new regulatory interactions in the xylogenesis transcriptome. In parallel, long noncoding RNA (lncRNA) discovery and expression analysis is being performed to gain insight into potential competition and sequestration of these sRNA species. These putative competitive endogenous (ce)RNAs (Salmena et al. 2011), their diversity and species-specificity in regulating the xylogenesis transcriptome, remain largely unexplored. This work will allow for a revision of the multidimensional transcriptional modules underlying xylogenesis, and may provide insights necessary for the genetic improvement of wood production in this species.

References

Axtell M. J., 2013 Classification and comparison of small RNAs from plants. Annu. Rev. Plant Biol. 64: 137–59.

Salmena L., Poliseno L., Tay Y., Kats L., Pandolfi P. P., 2011 A ceRNA hypothesis: The rosetta stone of a hidden RNA language? Cell 146: 353–358.

Trumbo J. L., Zhang B., Stewart C. N., 2015 Manipulating microRNAs for improved biomass and biofuels from plant feedstocks. Plant Biotechnol. J.: n/a–n/a.

L16: Bioinformatics resources for analysis and visualization of genetic mapping data

Nanette Christie 1 , Karen van der Merwe1, Dave Berger2, Zander Myburg1

1Department of Genetics, 2Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria

Expression quantitative trait loci (eQTL) mapping concerns finding genomic regions that contain DNA polymorphisms that underlie variation of transcript abundance of one or more genes (1). Microarray and RNA-seq technology allow genome-wide quantification of transcript levels facilitating eQTL analysis of tens of thousands of genes, an analysis which requires high-throughput computational and bioinformatics capacity. We have developed bioinformatics resources for the identification, analysis and visualisation of eQTLs as well as for the co-localisation of eQTLs with trait or metabolite QTLs. The eQTL detection and analysis pipelines were implemented in Python and R, and developed as workflows in the online data analysis platform Galaxy. eQTL results can subsequently be queried from a MySQL database via the Eucalyptus Genome Integrative Explorer (EucGenIE) interface (2) and visualised using networks in Cytoscape or as genomic projections in Circos plots. This platform, together with gene co-expression and correlation analysis, provides an excellent basis for the study of molecular networks underlying phenotypic traits of interest. These tools are currently used in systems genetics studies to analyse and interpret eQTL data in maize and Eucalyptus populations.

References1. Jansen, R. C. and Nap, J. P. (2001) Genetical genomics: the added value from segregation.

Trends in Genetics 17(7):388–91.2. Hefer C, Mizrachi E, Joubert F, Myburg A (2011) The Eucalyptus genome integrative

explorer (EucGenIE): a resource for Eucalyptus genomics and transcriptomics. BMC Proceedings 5(Suppl 7):O49.

L17: Disentangling the drivers of taxonomic and functional community structure in the Southern Ocean

Sandra Phoma, Surendra Vikram , Don Cowan and Thulani MakhalanyaneCentre for Microbial Ecology and Genomics (CMEG), Department of Genetics, Natural Sciences

2, University of Pretoria, Hatfield, Pretoria, 0028

Global climate change is expected to dispropotionatly affect marine ecosystems, due to increases in atmospheric CO2 which will lead to changes such as lower ocean pH. The factors which influence the structure of microbial communities remain unclear, and crucially, how they will respond to these environmental changes. The Southern Ocean (SO) surrounds the Antarctic continent and is a pivotal ecosystem in terms of its role in regulating the Earth’s climate [1, 2]. However, due to a number of reasons, we know very little regarding the correlation between microbial diversity and functional processes in this ecosystem and, more specifically, how depth may influence this relationship. To reduce this knowledge deficit we applied Illumina based amplicon pyrosequencing coupled with Shotgun metagenomic analysis to assess microbial diversity and functional capacity in the SO. Ocean water samples (27 in total) from the Crossroads (CR) monitoring line were collected during the SANAP Marion Island Relief cruise (15th April – 9th May 2015) on the SA Agulhas II polar research vessel equipped with a CTD/bottle and rosette sampler. Samples were collected at pre-determined depths: (a) 9 deep ( 10 m above seafloor), (b) 9 middle (oxygen minimum) and (c) 9 surface (fluorescence maximum). We found high taxonomic richness in surface and deep samples, with generally low numbers for middle samples, corresponding to oxygen minimum zones. ANOSIM analysis revealed marked differences between the three sample types (i.e. surface, middle, deep) dominated by marine bacterial (Proteobacteria, Firmicutes and Bacteroidetes) with smaller proportion of eukaryotes (Opisthokonta and Viridiplantae) and archaeal (Euryarchaeota) lineages. The results of functional annotation mirrored the taxonomic data with surface and deep samples showing the highest proportions of functional genes. Our data showed the first evidence of extensive biogeochemical capacity (C, N, S) in SO systems, with a large proportion showing homology to those of Proteobacteria (Rhizobiales), and Cyanobacteria (genus Synechoccus). Taken together, our results reveal important functional cues for biogeochemical cycling in the SO and provide a solid baseline for understanding future purtabations and consequent impacts on biogeochemical cycling.

References:

1. Mayewski, P.A., et al., State of the Antarctic and Southern Ocean climate system. Reviews of Geophysics, 2009. 47(1).

2. Chown, S.L., et al., The changing form of Antarctic biodiversity. Nature, 2015. 522(7557): p. 431-438.

L18: The evaluation of the effect of cattle and buffalo host cells on gene expression in buffalo-derived and cattle-derived Theileria parva isolates

Mokgoadi Trevor Molemane 1 , Frans Jongejan1,2, Nicola Collins1, Kgomotso Sibeko-Matjila1

1Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa

2Utrecht Centre for Tick- borne Diseases, Faculty of Veterinary Medicine, Utrecht University, The Netherlands

Theileria parva is a haemoprotozoan parasite that affects cattle in eastern, central and southern countries of Africa causing serious mortality. The African buffalo is the natural reservoir host of T. parva which is not affected by the parasite. The cattle-derived T. parva is responsible for East Coast fever (ECF) while the buffalo-derived parasites cause Corridor disease (CD). Transcriptome profiles of two T. parva isolates representing ECF- and CD-causing parasites were investigated. The transcriptome analysis revealed differentially expressed genes (DEGs) (n=1048); however, it is suspected that the observed variations in gene expression could have been influenced by the hosts of origin, namely; cattle and buffalo. Hence, the aim of this study was to evaluate the effect of cattle and buffalo host cells on gene expression in T. parva isolates. A microarray comprising of 4061 T. parva genes was developed to analyse gene expression profiles for comparison of cattle-derived and buffalo-derived T. parva isolates. Five isolates were maintained in cattle cell cultures and one (1) isolate was maintained in buffalo cell cultures. Thus the analysis was based on three groups of parasites maintained in different host cells including cattle-derived T. parva isolates maintained in cattle cell cultures, buffalo-derived isolates maintained in cattle cell cultures and buffalo-derived isolates maintained in buffalo cell cultures. The analysis of cattle-derived isolates maintained in cattle cells against buffalo-derived isolates maintained in cattle cells revealed 1864 differentially expressed genes. Most these DEGs were up-regulated in the buffalo-derived isolates. The analysis of buffalo-derived maintained in cattle cells against buffalo-derived maintained in buffalo cells identified fewer DEGs (n=646); the expression of these genes could be driven by factors associated with the host, either for recruiting proteins that can be used by the parasite for survival in the host or for protecting the host against infection. The preliminary data analysis indicates that gene expression in the parasite varies based on the host of origin and isolate type. The impact of the differential expression profiles is yet to be determined through functional annotation of the affected genes.

L19: Insights into the unique molecular mechanisms controlling asexual proliferation and sexual differentiation of malaria parasites from deep-transcriptome analyses.

Riëtte van Biljon 1 , Lindsey Altenhofen2, Jandeli Niemand1, Manuel Llinas2, Lyn-Marié Birkholtz1

1Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria, Private bag x20, Hatfield, Pretoria, South Africa, 0028;

2Pennsylvania State University, University Park, PA 16802, USA

Malaria is the most important parasitic disease affecting the global population. Traditionally, malaria chemotherapy was targeted against the persistent asexual blood stage forms, but current malaria elimination plans involve also targeting the sexual, gametocyte form of the parasite, as gametocytes are transmitted from the human host to the mosquito vector, leading to the spread of the disease. Therefore, parasite control is currently focused not only on developing new chemotherapies of the asexual red blood cell stages of the parasite (to treat people with the disease), but also on blocking transmission stages of the parasites (to prevent spread of the disease in a community). The causative agent of the most deadly form of this disease, the Plasmodium falciparum parasite, is still poorly characterised in several aspects of its biology; this hampers new drug discovery programs. The parasite has a complicated lifecycle and employs extraordinary mechanisms to enable progression through its asexual cell cycle and sexual maturation. Particularly, the parasite as a uniquely regulated transcriptome during asexual development, allowing just-in-time production of transcripts throughout its development. Through transcriptomic analysis across the asexual and sexual stages of the parasite, we have aimed to increase understanding of the molecular events enabling the parasite to fulfil its biological functions. An in-depth study of the cyclic asexual cell cycle was undertaken as well as a complete study of the extended maturation period of sexual development in the parasite. We identified, for the first time, uniquely expressed gene clusters associated with either asexual or sexual development. Within these, several molecular descriptors and regulators of the asexual cell cycle were identified as signalling events enabling the parasite to respond to quiescence-proliferation decision making. Interestingly, the parasite has unique mechanisms that enables it to undergo terminal differentiation during sexual development and these are disassociated from the normal proliferation regulatory mechanisms. Application of this knowledge could lead to identification of unique drug targets in the parasite for treatment of the disease or preventing transmission of the disease.

L20: Cystic fibrosis and rarefaction: Unification through diversity

Jeanne van Rensburg1; Marco Alessandrini1; Mark Robertson2; Michael Pepper1.

(1) Department of Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria

(2) Department of Zoology and Entomology, Centre for Environmental Studies, Faculty of Natural and Agricultural Sciences, University of Pretoria

Cystic fibrosis (CF), the most common autosomal recessive monogenic disorder, is associated with nearly 2,000 known variants in the CFTR gene. Although the frequencies of various CF variants in different countries is known, the diversity of CF variants across different world regions is not known. The aim of this project was therefore to determine the diversity of CF variants in different countries through the application of the rarefaction diversity method. Through the use of web-based searches, CF variant data from several countries was collected and databased. Data stratification was performed according to CF variants observed in South Africa and resulting data matrices were subjected to individual-based rarefaction analysis using EstimateS (v.9.1.0). CF variant data, represented by 206 different CF variants, was collected from 52 countries. Comparing this information to 17 CF variants observed in South African CF populations, three separate data matrices were constructed. Rarefaction analysis on each of the matrices revealed that significant differences exist in the diversity of CF variants in different countries. Rarefaction analysis is not influenced by variation in sample sizes, and provides a platform from which CF variant diversity can be determined and compared across different world regions. The method is robust and can be applied to multiple population groups and also to other disorders. This method has the potential to aid in the development and refinement of population specific CF screening panels.

References:

Goldman, A, Graf, C, Ramsay, M (2003) Molecular diagnosis of cystic fibrosis in South African populations. S. Afr. Med. J. 93:518-519.

Bobadilla, JL, Macek, M, Fine, JP, Farrell, PM (2002) Cystic Fibrosis: A worldwide analysis of CFTR mutations-correlation with incidence data and application to screening. Hum. Mut. 19:575-606.

Colwell, RK, Chao, A, Gotelli, NJ, Lin, S-Y, Mao, CX, Chazdon, RL, Longino, JT (2012) Models and estimators linking individual-based and sample-based rarefaction, extrapolation and comparison of assemblages. J. Plant Ecol. 5:3–21.

Colwell, RK. (2013) Estimate S: Statistical estimation of species richness and shared species from samples. v.9.1.0 University of Connecticut, USA.

L21: Identification and characterization of potato long noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection

Stanford Kwenda 1 , Paul Birch2 and Lucy N. Moleleki1

1Forestry and Agricultural Biotechnology Institute, Department of Microbiology and Plant Pathology University of Pretoria

2The James Hutton Institute, University of Dundee, Scotland

Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression, both in mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of lncRNAs in plant defense responses are yet to be fully explored. Here, we used strand-specific RNA sequencing to identify 1649 lncRNAs in potato (Solanum tuberosum) from stem tissues. The lncRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lncRNAs (86%) are transcribed from intergenic regions and possess single exons. A time-course RNA-seq analysis between a tolerant and susceptible potato cultivar challenged with Pectobacterium carotovorum subsp. brasilience revealed that 227 of these lncRNAs could be associated with response to this pathogen. These results suggest that lncRNAs have potential functional roles in potato defense responses. This work provides the foundation for further functional studies in understanding potato defense mechanisms.

References:Ma, L, Bajic, VB, Zhang, Z (2013) On the classification of long non-coding RNAs. RNA biol. 10, 924-933.

Zhu, B, Yang, Y, Li, R, Fu, D, Wen, L, Luo, Y, Zhu, H (2015) RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening. J of Exp Bot. erv203.

L22: Detection of Grapevine Leafroll Associated Virus 3, Viti- and Foveaviruses in Vitis Rootstocks 1

Megan Harris and 1,2Gerhard Pietersen

1Department of Microbiology and Plant Pathology , University of Pretoria, , Pretoria 0002, South Africa 2ARC-Plant Protection Research Institute, , Pretoria 0121, South Africa

Grapevine-leafroll disease (GLD), associated with Grapevine-leafroll associated virus 3 (GLRaV-3) in South Africa, is one of the most economically important viral diseases, impacting both vine health and quality of the grape. Vine health is also affected by a range of other grapevine viruses including Viti- and Foveaviruses. The symptoms of GLRaV-3 infection vary among Vitis vinifera cultivars but shows no discernible symptoms in Vitis rootstocks. Little is known about the presence in rootstocks of GLRaV-3 and Vitiviruses. ELISA tests detect GLRaV-3 poorly in Vitis rootstocks. In this study we determine the population of GLRaV-3 variants, Viti- and Foveaviruses in rootstocks and their respective scions, using primers to conserved regions of the viral genera flanking variable regions to allow virus identification through high throughput sequencing (HTS). Thirty-six samples of both the scion and the rootstock were collected and tested. While clear differences in detection of GLRaV-3 in rootstocks as opposed to scions were observed this was not the case with Viti- and Foveaviruses. Differences in the GLRaV-3 variant populations between rootstocks and their respective scions were observed but no consistent trend was identified. These results will be presented in greater detail. Information generated by this study will lead to the eventual improvement in detection of these viruses in rootstocks.

L23: Virus diversity and biogeography in Namib Desert soils

Olivier Zablocki 1, 2 , Evelien M. Adriaenssens1, 3, Jean-Baptiste Ramond1, 3, Aline Frossard1, 3, Surendra Vikram1, 3, Vincent Scola1, 2, Mary Seely4, 5 and Don Cowan1, 3

1Centre for Microbial Ecology and Genomics, University of Pretoria, South Africa

2Department of Microbiology and Plant Pathology, University of Pretoria, South Africa3Department of Genetics, University of Pretoria, South Africa

4Gobabeb Research and Training Centre, Walvis Bay, Namibia.5School of Animal, Plant and Environmental Sciences (AP&ES), University of the Witwatersrand,

Johannesburg, South Africa

The taxonomic composition of soil viruses along the Namib Desert aridity gradient was assessed using deep sequencing of metavirome libraries extracted from surface soils. Soil physicochemical data were also used in bivariate correlations to determine influences, if any, on the observed soil-specific taxonomic compositions of virus communities. Within the low proportion of identified viruses (~20% of the dataset), less than 1% of the total virus diversity was shared across the soil samples. The spatial distributions of Namib Desert soil virus communities were significantly correlated to soil texture (sand particle size) and soil chemistry. Relative virus abundance showed a positive correlation with microbial activity and increased as a function of distance from the coast. However, we could not identify any significant correlation between viral abundance and mean annual soil relative humidity. The ssDNA virus fraction was spatially restricted within inland soils, and sequences identified as Microviridae spanned three sub-families. From the low virus species overlap between samples, we suggest that limited virus dispersal occurs across the Namib Desert. We propose that the biogeographical patterns of the extracellular virus fraction are primarily determined by host distribution, and is consistent with previous studies on the factors which determine microbial community structure in the Namib Desert. Increased viral abundance with microbial activity is the first indirect indication of active viral predation, and the possibility that soil viruses may contribute to nutrient cycling (via viral lysis) in a hot hyperarid desert soil environment.

L24: Specificity of gene regulation in Bacillus atrophaeus UCMB5137 during plant colonization compared to Bacillus amyloliquefaciens FZB42, the paradigm of plant growth promoting Bacillus

Liberata Mwita 1 , Wai Yin Chan2, Oleg N. Reva1

1Centre for Bioinformatics and Computational Biology, University of Pretoria

2Department of Microbiology and Plant Pathology, University of Pretoria

Plant Growth Promoting Rhizobacteria (PGPR) freely live in the soil rhizosphere. They interact with plants through root exudates. They are potential ecological safe alternative of chemical fertilizers and pesticides. Root exudate collected from maize root was used to stimulate chemical signals affecting Bacillus atrophaeus UCMB5137 in the rhizosphere. RNA sequences were obtained by MiSeq Illumina 500 and analyzed by CLC Genomics Workbench 7. Gene regulation in UCMB5137 during plant colonization was afterwards studied. Bacillus atrophaeus UCMB5137 and Bacillus amyloliquefaciens FZB42 are both Gram positive PGPR belonging to Bacillus subtilis taxonomic group. Comparison of gene regulation patterns to a paradigm Bacillus PGPR -Bacillus amyloliquefaciens FZB42 unravel differences in plant colonization strategies between two strains.

POSTERS

Transcriptome sequencing of the Zea mays-Exserohilum turcicum interaction

MP Human 1 , DK Berger2 & BG Crampton1

1 Cereal Foliar Pathogen Research Programme, Plant Science Department, Forestry and Agricultural Biotechnology Institute, University of Pretoria

2 Molecular Plant-Pathogen Interactions, Plant Science Department, Forestry and Agricultural Biotechnology Institute, University of Pretoria

Exserohilum turcicum is the causal agent of Northern corn leaf blight (NCLB), a yield-limiting foliar disease of maize, sorghum and related grass species. The resistance of maize to NCLB is mediated by four major resistance (R)-genes; namely Ht1, Ht2, Ht3 and HtN. Exserohilum turcicum is classified into races based on the ability of the pathogen to overcome these R-genes. Fungal effectors play an important role in mediating susceptibility of the host to disease. The aim of this study is therefore to identify specific effectors interacting with the Ht-genes in maize. Maize seedlings at the trifoliate leaf stage were infected with either a race 13N or a race 23N E. turcicum isolate. Plants were collected prior to inoculation as well as at two, five, seven and thirteen days post inoculation. RNA was extracted from infected leaf material and sent to the Beijing Genomics Institute in Hong Kong for paired-end, strand-specific transcriptome sequencing. Transcripts were assessed for quality and were mapped to the available E. turcicum and Zea mays genomes. The results from this study will be used to identify the effectors that play an important role in the pathogenicity of E. turcicum on maize.

Genetic architecture of the sex determination system in two invasive Hymenoptera, Sirex noctlio and Leptocybe invasa

G. Barnard 1 , A. Postma2, G. Dittrich-Schröder3, B. Slippers1

1Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI) and Genomics Research Institute, University of Pretoria, South Africa

2 Department of Biochemistry, Forestry and Agricultural Biotchnology Institute (FABI),

University of Pretoria, South Africa

3 Department of Zoology and Entomology, Forestry and Agricultural Biotchnology Institute (FABI), University of Pretoria, South Africa

Invasive pests are responsible for substantial damage to both native and plantation forests. Two of the most globally important invasive pests of plantation forests are the Hymenoptera Sirex noctilio and Leptocybe invasa, which affect pine and eucalypts respectively. These wasps, as other Hymenoptera, have a unique, haplodiploid sexual reproductive system, whereby the females arise from fertilised eggs and are diploid, whilst males are haploid and develop from unfertilised eggs. As the sex determination system of an insect is critical to their invasion success, understanding the genetic mechanisms of this pathway would provide invaluable insight into the invasion process. In this study the sex determination loci of both S. noctilio and L. invasa were identifid and annotated in the recently assembled genomes of these species, using the honeybee genome as a reference. Furthermore, primers were designed for the complementary sex determination (csd) gene, which is the primary signal governing sexual development in S. noctilio. Amplicons of the gene were sequenced and two allele variants appear to be present within the population studied. Additionally, variant calling using Illumina paired end sequences was performed on the feminizer gene within the sex determination system, upstream to csd. This gene was shown to be highly conserved in both S. noctilio and L. invasa. These results provide the first insight into the genetic basis of sex determination in these invasive wasps and lays the foundation for future work aimed at targeting the sex determination system as a means of pest control.

Insight into three putative Cercospora zeina effector genes and the role they play in virulence.

Brigitte Langenhoven 1,2 , Dave Berger1,2 and Bridget Crampton1,2

1Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria; 2 Genomics Research Institute, University of Pretoria

Grey leaf spot (GLS) disease is an economically important foliar disease of maize caused by Cercospora zeina in southern Africa. However, little is known about the molecular mechanisms underlying C. zeina infection. Fungal effectors are a pathogen’s strategic method to evade host detection or to be able to suppress or interfere with the host defence mechanisms for successful infection. Most fungal effector proteins identified have been species-specific, and shared no sequence similarity with any other known protein sequences. Recently, fungal effectors Avr4, Ecp2, and Ecp6 from Cladosporium fulvum have been shown to have homologs in other fungal species belonging to the Dothideomycete class. The aim of this study was to identify whether Avr4, Ecp2, and Ecp6 effector homologs were present in C. zeina. Homologs of the effector genes Avr4, Ecp2, and Ecp6 were identified and annotated in the draft C. zeina genome. The presence of the Avr4, Ecp2, and Ecp6 effectors in the C. zeina genome, together with their putative conserved domains, provide insight into the possible roles that these proteins might play during maize infection. The in planta expression profiles of C. zeina Avr4, Ecp2, and Ecp6 were analysed by reverse transcriptase quantitative PCR (RT-qPCR). The study identified two C. zeina reference genes suitable for in planta gene expression normalisation, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytochrome c oxidase subunit III (Cyt III). GAPDH and Cyt III showed constant expression across all inoculation time points analysed making them suitable reference genes for expression normalisation. It was shown that C. zeina Avr4 and Ecp6 were expressed at constant levels during infection, while Ecp2 was expressed at very low levels at all time points analysed. Determination of C. zeina genomic DNA content by means of an optimised qPCR method at each time point enabled correlation studies between fungal quantity and effector gene expression. Avr4 and Ecp6 expression showed a weak positive correlation to fungal quantity. Phytotron inoculations of maize with C. zeina were established in this study which facilitated experiments independent of the maize growing season. In planta expression analysis of the effector genes Avr4, Ecp2, and Ecp6 therefore has yielded further insight into the molecular mechanisms of maize infection by C. zeina.

In Planta Expression of Novel Antarctic Bacterial Stress-Response Protein

Jasmin Mertens 1 , Eloy Ferreras1, Bilal A Mir1,2, Don A Cowan1

1 Centre for Microbial Ecology and Genomics (CMEG), Genomics Research Institute, University of

Pretoria

2 Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute, University of

Pretoria

Background: A novel bacterial gene, homologous to Water Hypersensitive domain (WHy) which is a typical component of Late Embryogenesis Abundant (LEA) proteins, was identified in an Antarctic desert metagenomic library. The LEA proteins occur widely in prokaryotes as well as in eukaryotes (e.g. bacteria, archaea and plants) and are expressed under stress conditions. A previous study showed significant protection of an E. coli recombinant, expressing the novel WHy protein, against freeze and cold stress. Objective: The aim of this study is to address the question of whether this novel WHy protein can be functionally expressed in Arabidopsis and whether it will confer cold- and freeze-protection in planta. Materials/Method: Two different WHy gene constructs were created; one carrying an additional signal peptide sequence that is thought to be beneficial for protein expression and a truncated construct without this sequence (WHy and ΔWHy). WHy gene expression in Arabidopsis thaliana used Agrobacterium-mediated transformation of the host plant. First and second generation (T0 and T1) recombinant plants were screened for the successful integration of the WHy gene sequence in the host genome and WHy protein expression. Results/Outlook: Both WHy and ΔWHy recombinant plants have showed a successful integration of the novel gene into the Arabidopsis genome, and positive WHy protein expression was observed in both recombinants. For stress tolerance tests, plant candidates showing (I) a successful integration of the WHy gene into the Arabidopsis genome, (II) constitutive expression of the WHy protein and (III) the capacity to develop seeds to produce healthy T1 offspring have been selected. Stress tolerance tests are now underway.

Unexpected size polymorphism in the mitochondrial genomes of closely related Chrysoporthe species

Aquillah M. Kanzi1, Brenda D. Wingfield1, Emma T. Steenkamp2, Sanushka Naidoo1, Michael J. Wingfield1, and Nicolaas A. van der Merwe1

1Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private bag x20, Pretoria 0028, South Africa

2Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private bag x20, Pretoria 0028, South Africa

Chrysoporthe austroafricana, Chrysoporthe cubensis and Chrysoporthe deuterocubensis (family Cryphonectriaceae) are canker causing pathogens of Eucalyptus spp. and other members of Myrtales. The complete mitochondrial (mt) genomes of C. austroafricana (190,834 bp), C. cubensis (89,084 bp) and C. deuterocubensis (124,412 bp) were determined in this study. The mt genome of Cryphonectria parasitica (158,902 bp), another member of the Cryphonectriaceae, was retrieved and annotated for comparative purposes. High levels of synteny were observed, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNA (rRNAs) and transfer RNAs (tRNAs), as well as intron positions in these genomes. Comparative analyses revealed signatures of gene duplication, intron number and length variation, and diverse intron encoded Homig endonuclease genes (HEGs), which highlighted the genetic diversity of mt genomes among Cryphonectriaceae. An ORF encoding a reverse transcriptase domain within a Group II intron was identified in the C. austroafricana genome, emphasizing the diversity of mt genomes. The large size variations in the mt genomes of these closely related Chrysoporthe species can be attributed to the varying number and length of introns, and expanded intergenic sequences. The C. austroafricana mt genome, is the second largest compared to all publicly available mt genomes of Ascomycetes thus far.

ReferencesGryzenhout M, Myburg H, van der Merwe NA, Wingfield BD and Wingfield MJ (2004) Chrysoporthe, a new genus to accommodate Cryphonectria cubensis. Studies in Mycology 50: 119–142.

Van der Merwe NA, Gryzenhout M, Steenkamp ET, Wingfield BD and Wingfield MJ (2010) Multigene phylogenetic and population differentiation data confirm the existence of a cryptic species within Chrysoporthe cubensis. Fungal Biology 114: 966-979.

Wingfield et al. (2015) IMA Genome-F 4. Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata. IMA Fungus 6: 233-248.

Investigating mitochondrial CI disorders in a Southern African cohort

Maryke Schoonen 1 , Izelle Smuts2, Richard Rodenburg3, Etresia van Dyk1, Roan Louw1, Francois van der Westhuizen1

1Centre for Human Metabonomics, North-West University, Potchefstroom, South Africa 2Department of Paediatrics and Child Health, Steve Biko Academic Hospital, University of

Pretoria, South Africa3Nijmegen Center for Mitochondrial Disorders at the Department of Paediatrics, Radboud

University Medical Center (Radboudumc), Nijmegen, The Netherlands

Defects in Complex I (CI) of the mitochondrial respiratory chain are the cause of most common mitochondrial diseases. Information about, and detailed understanding of, the aetiology of mitochondrial diseases in African populations is still lacking to a great extent. [1]

Disease diagnosis is mostly based on criteria and information from other populations and is compounded by issues such as lack of awareness and inadequate diagnostic procedures at the major academic medical institutions. In a South African paediatric patient cohort of ~200 cases with a predominantly muscle phenotype we have previously found a very low prevalence of common and other mtDNA mutations. [2]

Complex I-associated nuclear gene mutations were investigated using a targeted enrichment sequencing approach. For this study, 32 patients, diagnosed with a CI deficiency, were included.

Target enrichment of 92 CI-associated nuclear genes (coding sequences) was done using Agilents’ Haloplex target enrichment system and next-generation sequencing by an Ion Torrent PGM. Data analysis was done using an in-house bioinformatics pipeline. Following data analysis and validation of CI nuclear genes, an average of 750 variants were identified. Of these variants an average of 5 novel variants and 4 variants (2014) classified as possibly pathogenic were identified per patient. These variants are of interest for further evaluation.

As these variants have varied potential to be disease-causing, and thus would require further investigation, we give here an overview of these variants and key parameters for an initial estimation of pathogenicity. At this time we successfully sequenced and evaluated CI nuclear genes and can conclude that, as is the case for mtDNA, there is a general lack of known mutations that cause complex I deficiency in African patients. The pathogenicity of novel variants and interactions between variants remain to be further evaluated to better understand the genetic basis of the disease in this population.

Bacterial phylogenetic diversity in Sub-Antarctic peatlands from Tierra del Fuego, Argentina

Felix Oloo 1 , Angel Valverde1, María Victoria Quiroga2, Gabriela Mataloni2 and Don Cowan1

1Centre for Microbial Ecology and Genomics (CMEG), Department of Genetics, Natural Sciences 2, University of Pretoria, Hatfield, Pretoria, 0028

2Instituto de Investigación e Ingeniería Ambiental (3iA), Universidad Nacional de San Martín, Buenos Aires, Argentina.

Bacteria play critical roles in peatland ecosystems. However, very little is known on how habitat heterogeneity affects the structure of the bacterial communities thriving in these ecosystems. This investigation focused on bacterial communities of two Sub-Antarctic peatlands in Tierra del Fuego, Argentina. We used next generation sequencing of the 16S rRNA gene and measured several environmental parameters (e.g., pH and nutrients) to investigate phylogenetic diversity and bacterial community composition in three different habitat types: water from the Sphagnum moss matrix, water from vegetated pools and water from clear fresh water pools. We found these bacterial communities to be phylogenetically diverse across all three habitats. Sphagnum-associated bacterial communities differed significantly from communities in clear and vegetated pools, which clustered together. Furthermore, Sphagnum-associated bacterial communities were more similar in bacterial community composition, and more species rich, than those from the two pools habitats. Environmental conditions and nutrient status also showed a clear separation between Sphagnum and pool water samples, with Sphagnum-associated samples presenting a more heterogeneous environment. The clear divergence of Sphagnum-associated samples from the clear and vegetated pool samples, both in terms of bacterial community structure and abiotic factors, reflects the inextricable link of community structure and diversity to localized environmental conditions.

References

Limpens, J., et al. (2008). Peatlands and the carbon cycle: from local processes to global implications–a synthesis. Biogeosciences 5: 1475-1491.

Quiroga, M. V., Valverde, A., Mataloni, G. & Cowan, D. Understanding diversity patterns in bacterioplankton communities from a sub Antarctic peatland. ‐ Environmental Microbiology Reports (2015)..

Developing a successful method for DNA extraction from archival formalin-fixed, paraffin-embedded myocardial tissue blocks for post mortem genetic analysis of the SCN5A gene in SIDS

babies

Van Deventer, BS., DuToit-Prinsloo, L. and van Niekerk, C.

Introduction: Research has shown that the majority of cardiac disorders leading to sudden death in the young are often caused by genetic defects. At most large medico-legal death investigation centres, forensic pathologists have established large archives of formalin-fixed, paraffin-embedded (FFPE) tissue blocks, which can serve as an excellent - and sometimes the only - source of study material for genetic testing in such cases. However, the quality of genetic material contained in and extracted from these specimens - and the reliability of analyses and results thus obtained - has compromised diagnostic progress and the possible clinical benefit for survivors and siblings. The aim of this study was to develop and establish a successful DNA extraction method from archived FFPE tissue which is suitable for post mortem mutational analysis.

Methods: DNA extracted from FFPE myocardial tissue blocks was used to test for mutations in the SCN5A gene linked to Long QT Syndrome (LQTS) in sudden infant death syndrome (SIDS) cases by using real-time polymerase chain reaction (PCR) and sequencing. These tissue samples were obtained from material which has been systematically archived over the past ten years (as part of the routine retention of tissue for medico-legal autopsy / diagnostic purposes) from SIDS cases in Pretoria. In addition, the efficiency, quantity and quality of DNA extracted from FFPE tissue for successful DNA amplification was compared to that of DNA extracted from blood samples.

Results: DNA was successfully extracted from archived FFPE myocardial tissue blocks using the QIAamp DNA FFPE Tissue Kit. Small adjustments to the protocol yielded a better quantity and quality of DNA. Extracted DNA yielded concentrations ranging from 150 – 900 ng/µl with 260/280 ratios between 1.7 and 2.1. To prevent inhibition and ensure successful PCR amplification, the high concentrations of DNA were diluted to concentrations in the range of 50-75 ng/µl. Thus far two exons of the SCN5A gene have been successfully amplified by PCR, with the correct PCR product visualized on 2% agarose gels.

Discussion: The successful DNA extraction from FFPE tissue blocks has proven the unique potential and advantage of post mortem archived tissue samples and may have greatly enhanced the prospects of future research studies in this and other fields.

Keywords: Sudden death, sudden infant death syndrome (SIDS), formalin-fixed paraffin-embedded (FFPE) tissue, deoxyribonucleic acid (DNA), polymerase chain reaction (PCR)

Cloning, purification and crystallisation of a bifunctional Paenibacillus mucilaginosus exoglucanase

Leticia Mosina 1 , Wolf-Dieter Schubert2 and Don Cowan1

1Centre for Microbial Ecology and Genomics, University of Pretoria2Department of Biochemistry, University of Pretoria

The production of various bio-products including bioethanol is initiated through the conversion of cellulose to fermentable sugars. The bioconversion of cellulose is a complex process and requires the synergistic action of the endo-β-1,4-glucanses, exo-β-1,4-glucanases and β-glucosidases. Enzymatic hydrolysis of cellulose proves to be a challenging technological and economical step in bioethanol production which demands a robust cellulase with a high hydrolytic efficiency. The aim of the present study is to understand the structural and functional properties of a novel bifunctional (displaying both exo-/endo-glucanase) Paenibacillus mucilaginosus exoglucanase. The exoglucanase gene was cloned into different pET plasmid vectors and transformed into chemically competent Escherichia coli BL21 cells. E. coli clones displaying cellulase activity were used in protein expression trials: optimal protein expression was at 42°C with an induction period of 8 hours. The his-tagged exoglucanase was purified by immobilised metal affinity chromatography and gel filtration chromatography. The 127 kDa exoglucanase displayed activity on both carboxymethyl cellulose and avicel. Using 7 mg/ml protein, crystallisation experiments were set up using commercially available screens by the hanging drop and sitting drop method. Rhombohedral-like and needle-like crystals were among the different types of protein crystals obtained. Optimisation of the crystallisation conditions will yield larger three-dimensional crystals suitable for X-ray diffraction.

ReferencesKellerman, SJ, Rentmeister, A (2014) Current developments in cellulose engineering. ChemBioEng Reviews, 1: 6-13.

Kumar, R, Singh, S, Singh, O (2008) Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. Journal of Industrial Microbiology and Biotechnology. 35: 377 - 391.

Somatic mutations in South African breast cancer samples

Shaza Fadlalla ¹ , Fourie Joubert ¹ , Elizabeth Jansen van Rensburg ²

¹Centre for Bioinformatics and Computational Biology, University of Pretoria

2 Department of Genetics , University of Pretoria

Breast cancer has a high prevalence in South Africa, being the second most common form of cancer in women. Screening and early diagnosis of breast cancer is critically important for the successful treatment of this disease. The most important risk factor for developing cancer of the breast is a family history of the disease. Two high penetrance breast cancer susceptibility genes, BRCA1 and BRCA2 are clinically the most important genes associated with hereditary breast cancer. It has previously been shown that 67% of high-risk South African breast cancer families (multiple affected women) carry a BRCA1 or BRCA2 germ-line mutation. We have also found that 5,4% of a hospital-based cohort of Black women, diagnosed with breast cancer aged < 55 years, carried a BRCA1 / BRCA2 mutation

This project compares tumor-normal samples in selected South African patients from blood and FFPE samples. Initial sequencing was performed using the Ion Torrent Comprehensive Cancer Panel and a range of somatic mutation-calling methods were employed, including the Torrent Variant Caller, MuTect, JointSNVMix, Strelka,Varscan and SomaticIndelDetectors. Due to problems encountered in analyzing the results, the samples were subsequently sequenced again using whole exome sequencing, and reanalyzed. The results from the two sequencing platforms are compared, and discussed.

In silico analysis of horizontal gene transfer in Deladenus siricidicola

Frederick Clasen1, Alisa Postma1, Rian Pierneef1, Oleg Reva1 and Bernard Slippers2

1 Centre for Bioinformatics and Computational Biology, University of Pretoria2 Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of

Pretoria

Horizontal or Lateral Gene Transfer (HGT or LGT), the asexual movement of genetic material between different species, is believed to play an important role in the evolution of symbioses. Here we explore the role of this process in the evolution of the Sirex (woodwasp) – Amylostereum (fungus) – Deladenus (nematode) symbiosis by studying the genome of D. siricidicola with an in silico approach. For this purpose we compared the utility and accuracy of SeqWord Genomic Island Sniffer, SigHunt and AlienHunter as Genomic Island prediction tools. Combined results from these analyses suggested that approximately 2% of the genome show variance in oligonucleotide usage patterns, possibly indicating foreign origin. Using a compositional similarity search against the Pre_GI database, which contains known bacterial genomic islands, it was shown that these loci were commonly linked to genes from Bacillus, but also a range of other bacteria. The function of genes of potential foreign origin were determined using InterProScan, Blast2GO and other eukaryotic annotation tools. These genes were associated with a diverse range of functions, but were found to be mostly involved in transmembrane transport, metabolism and immunity. Finally, we could show that the identified loci often contained known genes associated with HGT mechanisms in bacteria, suggesting that a similar mechanism drives this process in nematodes. The study gives important insight into the evolution and adaptation of D. siricidicola, but also has relevance for understanding the role and mechanism of HGT in eukaryotic genome evolution more broadly. The results and the pipeline that has been developed can now be applied to the genomes of the wasp and fungal symbionts, as well as to other eukaryotes.

References

Andersson, J. O. (2005). "Lateral gene transfer in eukaryotes." Cellular and Molecular Life Sciences CMLS 62(11): 1182-1197.

Slippers, B., B. P. Hurley and M. J. Wingfield (2015). "Sirex Woodwasp: A Model for Evolving Management Paradigms of Invasive Forest Pests." Annual review of entomology 60: 601-619.

Pierneef, R., L. Cronje, O. Bezuidt and O. N. Reva (2015). "Pre_GI: a global map of ontological links between horizontally transferred genomic islands in bacterial and archaeal genomes." Database 2015: bav058

Towards understanding human leukocyte antigen (HLA) diversity in southern African populations.

Mqondisi Tshabalala 1, Juanita Mellet 1 and Michael Sean Pepper 1

1Department of Immunology, and the Institute for Cellular and Molecular Medicine,University of Pretoria, South Africa; South African Medical Research Council Extramural Unit for

Stem Cell Research and Therapy

Despite the increasingly well-documented evidence of high genetic and ethno-linguistic diversity amongst African populations, little is known about their HLA diversity. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system, and also plays a key role in ‘self’ recognition which is important in transplantation. With the high disease burden in southern Africa, HLA diversity data is becoming increasingly important in the design of population specific vaccines and improvement of therapeutic transplantation outcomes. HLA diversity data was retrieved from a Pubmed literature search and from the publicly available Allele Frequency Database (AFDN) to highlight the paucity of data from these populations which have high disease burden. We identified 10 studies giving a total of about 1137 individuals with publicly available HLA data. Amongst southern African countries, South Africa had the highest number of publicly available HLA data (no data for Angola, Namibia, Lesotho, Swaziland, Malawi). HLA-A*30 (A*30:01, A*30:02) and HLA-A*02:01:01 were most common in blacks and South African Caucasians (similar to European and American Caucasians) respectively. HLA B and HLA C allele frequencies were generally low (< 0.2) across the black populations. Globally, North Africa had the highest reported HLA alleles in AFND, with southern Africa in the top five. More than 50 % of the reported sub Saharan alleles were from southern African populations highlighting diversity in this region. Interestingly five new class I alleles (A*30:01:02, A*30:02:02, A*68:27, B*42:06, and B*45:07) were reported in a recent South African study. Generally, HLA diversity amongst southern Africa populations is poorly understood as represented by a low number of studies with publicly available data. This highlights the need for collaborative southern African data submission to centralized databases like the AFND. It is currently difficult to find donor-recipient matches for most Black Africans, largely due to the lack of donors and unknown HLA diversity in these populations. In-depth studies on HLA diversity in southern Africans will further guide HLA-disease association studies, will result in improvements in population specific vaccine development, and improve donor-recipient matching.

References 1. Disotell, TR (2012). Archaic human genomics. Am J Phys Anthropol. 55: p. 24-39 2. http://www.allelefrequencies.net 3. Paximadis M, Mathebula TY, Gentle NL, Vardas E, Colvin M, Gray CM, Tiemessen C, Puren A (2012). Human leukocyte antigen class I (A, B, C) and II (DRB1) diversity in the black and Caucasian South African population. Human Immunol. 73: 80-92. 4. Tshabalala M, Mellet J, Pepper MS (2015). Human leukocyte antigen diversity: A southern African perspective J. Immunology Research. http://dx.doi.org/10.1155/2015/746151

Mapping of quantitative trait loci (QTLs) for caffeine and catechins content in tea

(Camellia sinensis)

Koech Robert 1,3 , Malebe Pelly1, Mose Richard2, Kamunya Samsom3, Myburg Zander4, Apostolides Zeno1

1CAM Research Group, Department of Biochemistry, University of Pretoria, Pretoria, South Africa2James Finlays Limited, Kericho, Kenya

3Tea Research Institute, Kenya Agricultural Livestock Research Organization, Kericho, Kenya4Department of Genetics, University of Pretoria, Pretoria, South Africa

Tea contains a number of compounds such as flavonoids and caffeine, which contribute to tea quality. These compounds have also been demonstrated to have pharmacological activities. To identify the quantitative trait loci (QTL) for catechins and caffeine a pseudo-testcross population of 250 F1 progenies was derived from an intra-specific cross between clones Gw Ejulu and TRFK 303/577, which have diverse catechins and caffeine content. There were high levels of variation in caffeine and individual catechins content between the parental lines and progenies. A large number of progeny values fell outside those of the parent values pointing high transgressive segregation. Caffeine, EC, ECg, EGC, EGCg F1 mean fell between two parental mean whereas catechin (C) F1 mean was lower as compared to parental mean. Of the 17000 informative markers identified in the cross, 6588 DNA markers were mapped. The map consisted of 15 linkages groups (LGs), corresponding to the haploid chromosome number of tea plant (2n = 2x = 30). A total of 20 putative QTL associated with catechin contents were identified. Putative QTLs for C, EC, ECg, EGC and EGCg were detected in LG 4, while C, ECg and EGC were only detected LG 7 and LG 8. The ability to assess tea quality traits by integrating UPLC and DNA markers will provide a foundation for identification of catechins and caffeine QTLs for improving tea breeding.

References

Kamunya, SM, Wachira, FN, Pathak, RS, Korir, R, Sharma, V, Kumar, R, Bhardwaj, P, Muoki, RC, Ahuja, PS and Sharma, RK (2010) Genomic mapping and testing for quantitative trait loci in tea (Camellia sinensis (L.) O. Kuntze). Tree Genet. Genomes 6: 915- 929.

Ma, JQ, Yao, MZ, Ma, CL, Wang, XC, Jin, JQ, Wang, XM, Chen, L (2014) Construction of a SSR-based genetic map and identification of QTLs for catechins content in tea plant (Camellia sinensis). PLoS ONE 9 (3): e93131.

Cool viruses from Antarctic Soils

Rolf Kramer, Evelien Adriaenssens, and Don Cowan

Centre for Microbial Ecology and Genomics, Genomics Research Institute, University of Pretoria

Antarctica is an extreme environment; it is arguably the driest, coldest continent on earth and is mostly covered with permanent ice. However, it is certainly not a ‘dead’ continent: communities representing all three domains of life inhabit Antarctica – especially in the ice-free regions and in the McMurdo Dry Valleys (DV). Wherever life is found, viruses constitute a major component in each biosphere. Their influence in cellular life drives microbial adaption to extreme environments, and viruses may therefore be considered key players in cellular evolution. Viruses infecting bacteria (bacteriophages) are the most abundant biological entities on earth, yet very few environments have been well characterized and virus ecology remains to be fully understood. Here, we describe a metagenomic survey designed to elucidate virus communities in soils from 14 geographically distinct Antarctic DV regions. Virus-like particles were extracted from soils and metaviromic DNA extracts were generated by sequence-independent, single-primer amplification. Metavirome sequencing was performed using the Ion Proton™ Sequencer technology. Preliminary analyses revealed between 17x106 and 21.5x106 sequences, assembling into a median of 17,000 contigs per sample. Homologs to known phage proteins are found in all metaviromes and further phylogenetic analyses are currently applied to determine phylotypes. Virus community profiles will be generated for each sample and these profiles will be compared in respect to the individual conditions of each sampling side. Together with a parallel project analysing the broad microbiome of each side, the dynamic relationships between hosts and viruses will be elucidated. Thus, we aim to gain insights into virus ecology as well as into the role of phages in adaptation of extremophiles and cellular evolution.

ReferencesWeynberg KD, Wood-Charlson EM, Suttle CA, van Oppen MJH (2014) Generating viral metagenomes from the coral holobiont. Front. Microbiol. 5:206.

Zablocki O, van Zyl L, Adriaenssens EM, Rubagotti E, Tuffin M, Cary C, Cowan D (2014) High diversity of tailed phages, eukaryotic viruses and virophage-like elements in the metaviromes of Antarctic soils. Appl. Environ. Microbiol. 80(22):6888-97.

Adriaenssens EM, Van Zyl L, De Maayer P, Rubagotti E, Rybicki E, Tuffin M, Cowan DA (2015) Metagenomic analysis of the viral community in Namib Desert hypoliths. Environ. Microbiol. 17:480–495.

Microbial communities in the Namib Desert Fairy Circles

Van der Walt, A. J. 1 , Johnson, R. M.1, Cowan, D. A.1, Ramond, J-B.1

1Centre for Microbial Ecology and Genomics (CMEG), Genomics Research Institute, Department of Genetics, University of Pretoria,

Fairy Circles (FCs) are mysterious endemic features of the coastal Namib Desert observed both in the gravel plains and in the dunes. These circular patches of soil are devoid of vegetation but surrounded by a fringe of longer grass. Since first reported in the 1970’s, a striking number of hypotheses attempting to explain their origin, development or sustainability have been developed; for example, micro-faunal activity (ants, termites or rodents), soil physics, gas seepages and vegetative self-organization. Nevertheless, none has to date been able to adequately explain this phenomenon. In this study, we therefore investigated the hypothesis that FC formation is due to microbial phytopathogenic activities. Surface soils from five gravel plain and five dune FCs as well as control soils were collected in April 2014 (n=20). Pyrosequencing of bacterial, archaeal (16S rRNA gene) and fungal (ITS region) phylogenetic markers in combination with multivariate analyses showed that gravel plain and dune FC microbial communities are phylogenetically distinct from the control vegetated soils and that soil physicochemical properties had significant influences on their community structure. Furthermore, 9 bacterial, 1 archaeal and 57 fungal taxa were found to be FC-specific; i.e. were detected solely within the gravel plain and dune FC centres. These included fungi of the genera Culvaria, Periconia and Aspergillus, members of which have been found to exhibit phytopathogenicity. While we cannot as yet affirm whether microbial communities are responsible for the appearance and development of FCs, these results suggest their involvement in the FC phenomenon.

Transcriptional Analysis of Cysteine Proteases in Soybean Root Nodules Experiencing Premature Senescence Due to Water Deficit

Magdeleen Cilliers 1 , Stefan van Wyk1, Riekert van Heerden1,2, and Barend Vorster 1

1University of Pretoria, Department of Plant Production and Soil Science, Pretoria 0002;2 South African Sugarcane Research Institute, Mount Edgecombe 4300

Contact information: magdeleen.duplessis@fabi.up.ac.za

Cysteine proteases found in soybean’s (Glycine Max) crown nodules, are involved in premature senescence due to water deficit conditions. Water deficit stress in soybean affects the plant growth, development as well as the lifespan and nitrogen fixation ability of these root nodules. The expression of papain-like and legumain-like genes have been investigated under different water stress conditions ranging from 60%, 40% and 30% vermiculite water content respectively. The severity of the stress conditions were evaluated by measuring leaf and nodule water potential indicating a significant difference in the water content of these two organs at a 30% vermiculite water content level. A transcriptome analysis, which was validated by quantitative PCR, was used as a gene discovery technique. Twenty nine papain-like cysteine proteases were found to be expressed in crown nodule tissue. Five of these papain-like proteases showed to be induced by water deficit over the increasing stress treatments and seven showed an increase in expression until 40% vermiculite water content but showed a decrease in expression from 40% to 30%. There were also two papain-like proteases that were only expressed in plants affected by water deficit conditions. Eight of the above mentioned proteases showed a 2-fold and higher increase in expression from control plants to water deficit stressed plants. Eight legumain-like proteases were expressed in the root nodules. Legumain-like cysteine proteases showed to be induced by senescence as well as water deficit stress. Compared to natural senescence, the expression of three legumain-like cysteine proteases in water deficit conditions did show a fold change of above 2 over the different treatment. While legumain-like cysteine proteases involvement during premature senescence is clear, they seem not to be the main protease group responsible for the initiation of premature senescence.

ReferencesKardailsky, I.V. and Brewin, N.J. (1996) Expression of cysteine protease genes in pea nodule development and senescence. Molecular Plant-Microbe Interactions, 9: 689-695.

Puppo, A., Groten, K., Bastian, F., Carzaniga, R., Soussi, M., Lucas, M.M., De Felipe, M.R., Harrison, J., Vanacker, H. and Foyer, C.H. (2005) Legume nodule senescence: roles for redox and hormone signalling in the orchestration of the natural aging process. New Phytologist, 165: 683-701.

Genomics of Antarctic polyextremophile Nesterenkonia sp. AN1

Habibu Aliyu1, 2, Pieter De Maayer1, 3, Don Cowan1, 2

1 Center for Microbial Ecology and Genomics, University of Pretoria, 2 Department of Genetics, University of Pretoria, 3 Department of Microbiology, University of Pretoria

Microorganisms in Antarctic dry soils have evolved features which compensate for the physicochemical constraints imposed on their cellular function by extreme cold, alkalinity, aridity, salinity, starvation and UV irradiation. Nesterenkonia sp. AN1 is the first member of the genus Nesterenkonia isolated from Antarctic desert soils. We have used genomic and comparative genomic analyses to elucidate the genetic determinants underlying its survival strategies. To highlight psychrotolerance mechanisms we also compared, via RNA-seq analyses, the transcriptome of the strain grown at 5 ºC and 21 ºC. The Nesterenkonia sp. AN1 genome encodes a large number of proteins putatively involved in cold shock, cold acclimation, osmotic and oxidative stress responses as well as the modulation of membrane fluidity. The majority of these are shared with the mesophilic strains of Nesterenkonia, implying that members of the genus are naturally resilient. Transcriptome data of Nesterenkonia sp. AN1 revealed that ~ 97% of the predicted genes were expressed under the experimental conditions. Analyses of the transcriptome showed that there was significant induction of transcripts that code for antioxidants at 5 ºC, demonstrated by the upregulation of sodA, bcp and bpoA2. There was also overexpression of universal stress protein genes related to uspA, along with genes encoding other characterised cold stress features. Genes encoding the two key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (AceB) were induced at 5 ºC, suggesting possible adaptation strategies for energy metabolism in cold habitats. These genomic features may contribute to the survival of Nesterenkonia sp. AN1 in arid Antarctic soils.

References

Aliyu H, De Maayer P, Rees J, Tuffin M, Cowan Da (2014). Genome Announc, 2.

De Maayer P, Anderson D, Cary C, Cowan DA (2014). EMBO Rep, 15(5): 508-517.

Nel AJM, Tuffin IM, Sewell BT, Cowan DA (2011). Appl Environ Microbiol, 77(11):3696–702.

Garnier M, Matamoros S, Chevret D, Pilet MF, Leroi F, Tresse O (2010). Appl Environ Microbiol, 76(24):8011–8.

Kunze M, Pracharoenwattana I, Smith SM, Hartig A (2006). Biochim. Biophys. Acta, 1763, 1441-1452.

Variance in common cancer genes from African populations

Greg Milner and Fourie Joubert

Bioinformatics and Computational Biology Unit, Department of Biochemistry ,University of Pretoria

The effort to determine the role played by specific genes in the clustering of cancer susceptibility within family groups has been bolstered by rapid advances in various sequencing technologies, as well as variant detection and analysis software. This has resulted in the production of a number of cancer panels, each consisting of multiple genes known or suspected to play a role in disease formation. These panels have been built using predominantly European population genomic data however. Considering the high degree of genetic diversity within and between African populations, there is a potentially large degree of unknown variation contributing to disease formation in these groups. This project attempts to make a comparison of several African and European populations against a combination of cancer panels, using the GRCh37 reference genome in order to determine any differences in the roles that certain variants may play in increasing disease risk in African populations.

1000 Genomes data relating to 5 African and 5 European populations is collected in conjunction with data from two South African populations (Xhosa and Sotho) from the South African Human Genome Project. The chromosome data, in variant call file format, for all individuals from each population are generated by aligning to a custom built reference genome (hs37d5), using a bed file comprised of the exonic sequences for all cancer panel genes to specify gene regions of interest. These vcf files are then aligned against one another and analysed using phylogenetic software (BEAST). The alignments are used to calculate allele-frequencies and measure gene distances (Cavalli- Sforza and Edwards), using both R packages and Gendist (PHYLIP) software.

We hypothesis that the relative frequencies of disease alleles that act to increase susceptibility in African populations will show deviations from the more derived and less heterogenous European populations.

References

Parka J-H, Gaila MH, Weinberg CR, Carrollc RJ, Chung CC, Wang Z, et al. Distribution of allele frequencies and effect sizes and their interrelationships for common genetic susceptibility variants. Proceedings of the National Academy of Sciences. 2011.

Comparative mitogenomics of members of the superfamilies Spiruroidea and Thelazioidea (Phylum Nematoda)

1 Janine Burger , 1Willem J.S. Pretorius, 2Sarah J. Clift, 1Jaco M. Greeff and 1Pamela J. de Waal

1Department of Genetics, University of Pretoria.2Section Pathology, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of

Pretoria.

Spirocerca lupi is a nematode parasite that causes spirocercosis in canids, especially dogs. The nematode forms nodules in the oesophagous of the dog which become cancerous and may be fatal. Recent evidence suggests a substantial increase in the reported incidence of the disease over the last ten years. Spirocerca lupi is classified within two families, Spirocercidae (superfamily Spiruroidea) and Thelaziidae (superfamily Thelazioidea) with little evidence to back up why one classification should be favoured over the other. Molecular data to support the correct classification of S. lupi may help to identify suitable drug targets by comparison to other closely related parasitic nematodes. We have amplified the mitogenome of a South African Spirocerca lupi sample. We have sequenced and annotated the genome. We are in the process of generating sequence datasets for the nematode parasites Cylicospirura spp and Philonema Oncchorynchis.

Niche-partitioning of edaphic microbial communities in the central Namib Desert

Riegardt Johnson 1 , Jean-Baptiste Ramond1, Eoin Gunnigle1, Mary Seely2 and Don A. Cowan1

1Centre for Microbial Ecology and Genomics, Genomics Research Institute, University of Pretoria, South Africa

2Gobabeb Research and Training Centre, Namibia

The Namib Desert, which is considered the oldest dryland environment on Earth, has been arid for the last 43 million years. It presents a diverse range of soil biotopes, which include sand dunes, gravel plains, ephemeral riverbeds and salt pans. Understanding the drivers of microbial diversity and adaptation in specific desert biotopes, where extreme environmental conditions already result in limited microbial diversity, will lead to an increased understanding of the dynamics of microbial community assembly.

In this study, we assessed bacterial, archaeal and fungal community structures within nine distinct soil biotopes of the central Namib Desert by Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis. Four replicate surface (0- 5cm) soil samples were collected from dune tops, dune slopes and interdunes, the wet and dry portions of a salt pan and a riverbed, as well as gravel plain soils from the arid and hyperarid zones (n=36).

TRFLP data indicated that bacterial, fungal and archaeal community structures were each significantly different in the biotopes studied (ANOSIM global R= 0.609; 0.723; 0.659, p≤0.001 respectively), with the majority of bacterial, fungal and archaeal operational taxonomic units (OTUs) being specific to each biotope. Redundancy analysis further indicated that bacterial, fungal and archaeal communities exhibited different adaptive responses to environmental parameters within their respective edaphic biotopes.

Our results suggest that abiotic drivers exert strong influences on the structure of desert bacterial, fungal and archaeal communities, resulting in biotope-specific communities; i.e., that niche partitioning plays an important role in the assembly of desert edaphic microbial communities.

Aggressive prostate cancer presentation within an African setting – are bacterial pathogens contributing to exacerbated disease course?

Pieter H. Bouwman 1 , Elizabeth A. Tindall2, Angel Valverde Portal1, Don A. Cowan1, Riana M.S. Bornman3, Vanessa M. Hayes4

1Centre for Microbial Ecology and Genomics, University of Pretoria, South Africa2J. Craig Venter Institute, La Lolla, CA, USA

3School of Health Systems and Public Health, University of Pretoria, South Africa4Garvan Institute of Medical Research, Sydney, Australia

Global disparities in prostate cancer (PCa) are well documented. Australia and the United States have the highest incidence rates. Within the United States, incidence and mortality rates are disproportionally high within African-American men. Figures for African countries, however, are largely lacking. It has been proposed that infection and inflammation are drivers of PCa, as infectious agents are known to play a role in the development of cancers such as gastric cancer, cervical cancer and vaginal cancer.

The Southern African Prostate Cancer Study (SAPCS) was established in 2008, with th e objective of establishing a ‘first-of-its-kind’ pure African PCa study. This unique resource is being used to investigate clinical presentation, epidemiological risk factors, and associated microbial pathogenic contributions to PCa status within Black South Africans from rural and urban localities.

Over 1,300 men have enrolled to date. When compared with African-Americans, the SAPCS patients presented significantly more aggressive PCa defined by a Gleason score >7 (17% and 36%, respectively) and PSA ≥20mg/L (17.2% and 83.2%, respectively). Additionally, we found disease aggression to be significantly exacerbated in men from rural localities (p<0.0001). We investigated 24 possible contributing demographic and lifestyle measures. We found current sexual activity and erectile dysfunction as significant risk factors (p<0.0001), leading to our hypothesis that lack of pathogenic shedding may be contributing to aggressive disease within the region. Using T-RFLP analysis, we profile for the first time bacterial communities within prostate tissue of Black South African men. These communities were found to be diverse, but no significant difference was observed between PCa and control patients. We propose that the majority of the bacteria represent the core microbiome of the prostate.

ReferencesSfanos, KS, De Marzo, AM (2012) Prostate Cancer and Inflammation: The Evidence. Histopathology. 60:199-215.

Tindall, EA, Monare, LR, Petersen, DC, Van Zyl, S, Hardie, R, Segone, AM, Venter, PA, Bornman, MS, Hayes, VM (2014) Clinical Presentation of Prostate Cancer in Black South Africans. The Prostate, 74: 880-891.

The times they are a-changing: ddRAD sequencing is a powerful approach to evolutionary studies of non-model species

Connor Stobie 1 , Kerry Reid1, Samantha Mynhardt1, Carel Oosthuizen1, Arrie Klopper1, Thierry Hoareau1, Michael Cunningham1 and Paulette Bloomer1

1Molecular Ecology & Evolution Programme, Department of Genetics, University of Pretoria, Pretoria, South Africa

Restriction-site Associated DNA (RAD) sequencing is a reduced-representation Genotyping-by-Sequencing (GBS) approach to sampling genome-wide diversity. Here we present results from several exploratory studies using double-digest RAD (ddRAD) sequencing across a range of vertebrate species. This method reduces the fraction of the genome under analysis, to fragments of 300-500 bp each flanked by two different restriction sites (specifically, we used: NlaIII, CATG/, and MluCI, /AATT). This approach aims to improve accuracy of SNP calls by increasing the coverage and specificity of sequenced fragments, within and among individuals. Our initial data processing identified a number of pitfalls in ddRAD sequence analysis. We have designed our pipeline to address these issues while retaining as much data as possible for downstream analyses. We showcase analyses of processed datasets exploring evolutionary questions that one can tackle with RAD sequencing data. These include species delimitation, identifying conservation units, and understanding population divergence.

Diversity of edaphic bacterial communities in the Namib Desert Sand Sea

Sandra Ronca 1 , Jean-Baptiste Ramond1, Brian E. Jones1, 2, Mary Seely3, 4 and Don A. Cowan1

1 Department of Genetics, Centre for Microbial Ecology and Genomics, University of Pretoria, Pretoria, South Africa,

2 DuPont Industrial Biosciences, Leiden, Netherlands,3 Gobabeb Research and Training Centre, Walvis Bay, Namibia,

4 School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa

The hyper-arid Namib Desert Sand Sea represents heterogeneous soil habitats. As little is known about their indigenous edaphic bacterial communities, we aimed to evaluate their diversity and factors of assembly in a single and in multiple dune systems. We performed at the local scale 5 parallel dune/interdune transects collecting a total of 125 samples and characterized 21 physico-chemical edaphic parameters coupled with 16S rRNA gene bacterial community fingerprinting using T-RFLP and 454 pyrosequencing. Bacterial community diversity at the landscape was investigated by performing 60 km long longitudinal transect from the cost inland, characterized by a salt and iron gradient and an inverse fog-rainfall gradient. We collected open soil samples every 7 km for 8 dune systems and analysed their bacterial and archaea communities using Illumina sequencing. Multivariate analyses of T-RFLP data on dune/interdune showed significantly different bacterial communities related to physico-chemical gradients, in four distinct dune habitats: the dune top, slope, base and interdune. Pyrosequencing of 16S rRNA gene amplicon set showed that each dune zone presented a unique phylogenetic profile, suggesting a high degree of environmental selection. Habitat stability, soil texture and mineral and nutrient contents are the main environmental drivers of bacterial community structures in Namib Desert dune systems. The preliminary analysis of the bacterial and archaea community structure of the 16S rRNA illumina sequences showed different phylogenetic microbial community profiles across the Sand Sea exhibiting a fog, iron and salt-related distribution as indicated by the significant East–West clustering. The combined results strongly imply that habitat filtering is a major driver of bacteria community structure in the Namib Sand Sea.

References

Makhalanyane, TP, Valverde, A, Gunnigle, E, Frossard, A, Ramond, JB, Cowan, DA (2015) Microbial ecology of hot desert edaphic systems. FEMS microbiology reviews, 39(2), 203-221.

Stomeo, F, Valverde, A, Pointing, SB, McKay, CP, Warren-Rhodes, KA, Tuffin, IM, Seely, M, Cowan, DA

(2013) Hypolithic and soil microbial community assembly along an aridity gradient in the Namib

Desert. Extremophiles, 17: 329-337.

Comparison of Bacillus endophyticus with B. anthracis isolated from anthrax outbreaks in South Africa

Kgaugelo Edward Lekota 1,2,3 , Joseph Mafofo 1, Jasper Rees

1, Farai Catherine Muchadeyi1, Evelyn

Madoroba3 and Henriette van Heerden2

1 The Biotechnology Platform, Agricultural Research Council, Private Bag X5, Onderstepoort 0110,

South Africa, E-mail: LekotaE@gmail.comb University of Pretoria, Department of Veterinary Tropical Diseases, Private bag X4, Onderstepoort,

0110, South Africa,c Agricultural Research Council-Onderstepoort Veterinary Institute, Private bag X5, Onderstepoort

0110, South Africa

Bacillus anthracis, the causal agent of anthrax identified as a Gram positive rod shaped, endospore forming, non-motile, non-hemolytic, penicillin and gamma-phage sensitive. The very closely morphologically related B. endophyticus is a Gram positive, rod shaped, endospore forming, non-motile, non-hemolytic, Penicillin sensitive but gamma-phage resistant bacterium. Bacillus endophyticus strains were isolated along with B. anthracis strains from animals that died of anthrax in 2009, in Northern Cape Province, South Africa. Bacillus endophyticus strains were differentiated from B. anthracis using 16S rRNA gene sequences. This study characterized B. endophyticus strains using morphological, biochemical characteristics and whole genome sequencing. Whole genome sequencing was carried out using illumina platform with 100 bp insert size paired end. The polyglutamate (PGA) biosynthesis genes were compared between B. anthracis and B. endophyticus using genome sequences. PGA genes were characterized by screening on the shotgun genome sequences and the presence or absence of a capsule was determined. The presence of PGA genes encoded by pgs BCAD genes of B. endophyticus and cap BCADE genes of B. anthracis indicated that the subunits BCAD were found in the B. endophyticus strains and the available sequenced B. endophyticus 2102 strain. However, the presence of a capsule was not observed in B. endophyticus strains. Sequence nucleotide variations on the PGA subunits BCAD were observed in the B. endophyticus strains when aligned with the B. anthracis. The PGA genes identified in the B. endophyticus strains suggest that they might have a biological role of increasing the resistance to adverse environment rather as a virulent capsule in B. anthracis. The study highlights the importance of distinguishing the B. anthracis from the B. endophyticus strains for diagnostic purposes.

References

Candela, T, Fouet, A (2006) Poly-gamma-glutamate in bacteria. Mol. Microbiol. 60, 1091-1098.

Reva, ON, Smirnov, VV, Petterson, B, Priest, FG (2002) Bacillus endophyticus sp. Nov., isolated from the inner tissues of cotton plants (Gossypium sp.). Int. J. Syst. Evol. Microbiol. 52, 101-107.

Next-generation Sequencing tag based genetic linkage map of a tea reciprocal cross population and the Identification of Putative Molecular Markers

Masixolise P Malebe 1 , Hastings Nyirenda2, Samson Kamunya3 Alexander A Myburg4 and Zeno Apostolides1

1Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa2Tea Research Foundation of Central Africa, Mulanje, Malawi

3Tea Research Institute, Kericho, Kenya4Department of Genetics, University of Pretoria, Pretoria 0002, South Africa

Several linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, there has been no documentation of dominant marker systems developed from current technologies such as next-generation sequencing in tea. The aim of this study was to fill this gap in our knowledge. We developed a genetic linkage map for tea using a next-generation sequencing platform from Diversity Arrays Technology (DArT). A population of 261 F1

progeny derived from a reciprocal cross between GW Ejulu and TRFK 303/577 was used for the linkage analysis. The two parental maps contain 15 linkage groups, which corresponds to the haploid chromosome number of tea (2n=2x=30). The total length of the parental maps was 1026 cM and 1056 cM with an average locus spacing of 1.5 cM and 1.9 cM, respectively. The second portion of the study describes the integration of next-generation sequencing (NGS) to develop cost-effective markers for breeding crops with unsequenced genomes. This research improved a putative marker from Random Amplified Polymorphic DNA (RAPD) that segregated with drought tolerance. RAPD analysis was used to screen 42 cultivars from Malawi. The newly developed SCAR467 primers produced a marker that segregated with 43% (10/23) drought tolerant cultivars and 0% (0/19) of the drought susceptible cultivars. A population from Kenya verified the association of the SCAR467 marker to drought tolerance. This SCAR467 marker was present in 25% (5/20) of the drought tolerant cultivars and 6% (1/17) of drought susceptible cultivars from the Kenyan validation population. The potential impact of this study lies in drought tolerance breeding in tea and as an approach that can be adopted similarly to unsequenced crop genomes as well as by breeding laboratories lacking expensive genomic resources.

ReferencesGrattapaglia, D, Ronald, S (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics 137(4): 1121-1137.

Deciphering the Effect of Elevated Temperatures on Antarctic Soil Microbial Community Structure and Function.

de Scally, S.Z.1, Makhalanyane, T.P.1, Frossard, A.1, and Cowan, D.A.1

1Centre for Microbial Ecology and Genomics (CMEG), Department of Genetics, Natural Sciences 2, University of Pretoria, Hatfield, Pretoria, 0028

Terrestrial systems are important carbon reservoirs and are currently undergoing extensive changes due to elevated greenhouse gas emissions. The effects of this change may have consequences on the relationship between biodiversity and ecosystem function (biogeochemical cycling), particularly on microbial communities who are major drivers of these cycles. Antarctica soils lack higher life forms (i.e. plants), are microbially-driven, and may be sensitive model systems for understanding the effects of global change processes (such as temperature fluctuations) on biogeochemical cycles. To clarify the relationship between biodiversity and ecosystem function, we constructed Antarctic soil microcosms and applied temperature fluctuations over a 30 day period. The applied temperatures included two stable controls (0°C, 15°C) and one test increasing by 1.5°C increments per day until a plateau of 15°C was reached. We used 16S rRNA gene amplicon sequencing and enzymatic assays to assess microbial community structure and function. The extracellular enzyme activities assessed included those functioning in carbon, nitrogen and phosphorus acquisition, as well as lignin degradation. Through these analyses, we have shown that microbial community composition and potential functional capacity does not significantly vary due to increased temperature on a short time scale. We have found that these communities retain their functional capacity at elevated temperatures, and that a few dominant phyla that are consistently present may be responsible for driving functional processes in Antarctic soils.

References:

Cowan, DA, Makhalanyane, TP, Dennis, PG, Hopkins, DW (2014) Microbial ecology and biogeochemistry of continental Antarctic soils. Frontiers in Microbiology, 5:154.

Tiao, G, Lee, CK, McDonald, IR, Cowan, DA, Cary, SC (2012) Rapid microbial responses to the presence of an ancient relic in the Antarctic Dry Valleys. Nat. Commun. 3:660