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 deeptranscriptome 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
1
Dept. 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 genomewide gene expression studies of human adipogenesis
Melvin Ambele1, Carla Dessels1, and Michael Pepper1
1
Department 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 upregulated 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 Pinard1, 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 Adriaenssens1, Lonnie van Zyl2, Marla Tuffin2 and Don Cowan1
1
Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria
2
Institute 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, sedimentfilled 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
1
Department 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 Swart1, 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 Peloakgosi1, Kgomotso Sibeko², Andrew Leisewitz3 and Tshepo Matjila 2
¹Department of Life and Consumer Sciences, University of South Africa
²Department of Veterinary and Tropical Diseases, University of Pretoria
3
Department 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.
References
Jacobson, 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
1
Department of Genetics, University of Pretoria , South Africa.
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
2
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 entkaurene 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 Cowan 1, 2 *
*Corresponding author: [email protected]
Centre for Microbial Ecology and Genomics, Department of Genetics, University of Pretoria,
Private Bag X20, Pretoria, 0028, South Africa
2
Department of Genetics, University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa
3
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag
X20, Pretoria, 0028, South Africa
4
Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028,
South Africa
1
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 Stewart1 and Michael Pepper1
1
Institute 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
1
Department 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. Wierzbicki1 , 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 Alessandrini1 and Michael S. Pepper1
1
Department 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. Behrens1, A.A. Myburg1, E. Mizrachi1
1
Department 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 posttranscriptionally 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 Christie1, Karen van der Merwe1, Dave Berger2, Zander Myburg1
1
Department 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 highthroughput computational and bioinformatics capacity. We have developed bioinformatics
resources for the identification, analysis and visualisation of eQTLs as well as for the colocalisation 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 coexpression 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.
References
1. 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 Makhalanyane
Centre 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.
2.
Mayewski, P.A., et al., State of the Antarctic and Southern Ocean climate system.
Reviews of Geophysics, 2009. 47(1).
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 Molemane1, Frans Jongejan1,2, Nicola Collins1, Kgomotso Sibeko-Matjila1
1
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of
Pretoria, South Africa
2
Utrecht 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 Biljon1, Lindsey Altenhofen2, Jandeli Niemand1, Manuel Llinas2, Lyn-Marié Birkholtz1
1
Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria,
Private bag x20, Hatfield, Pretoria, South Africa, 0028;
2
Pennsylvania 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 Kwenda1, Paul Birch2 and Lucy N. Moleleki1
1
Forestry and Agricultural Biotechnology Institute, Department of Microbiology and Plant
Pathology University of Pretoria
2
The 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 proteincoding 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
1
Department 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 (GLRaV3) 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 Zablocki1, 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
1
Centre for Microbial Ecology and Genomics, University of Pretoria, South Africa
Department of Microbiology and Plant Pathology, University of Pretoria, South Africa
3
Department of Genetics, University of Pretoria, South Africa
4
Gobabeb Research and Training Centre, Walvis Bay, Namibia.
5
School of Animal, Plant and Environmental Sciences (AP&ES), University of the
Witwatersrand, Johannesburg, South Africa
2
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 Mwita1, Wai Yin Chan2, Oleg N. Reva1
1
Centre for Bioinformatics and Computational Biology, University of Pretoria
2
Department 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 Human1, DK Berger2 & BG Crampton1
1
2
Cereal Foliar Pathogen Research Programme, Plant Science Department, Forestry and
Agricultural Biotechnology Institute, University of Pretoria
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 Rgenes. 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 Htgenes 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. Barnard1, A. Postma2, G. Dittrich-Schröder3, B. Slippers1
1
Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI) and
Genomics Research Institute, University of Pretoria, South Africa
2
3
Department of Biochemistry, Forestry and Agricultural Biotchnology Institute (FABI),
University of Pretoria, South Africa
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 Langenhoven1,2, Dave Berger1,2 and Bridget Crampton1,2
1
Department 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 Mertens1, 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 freezeprotection 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
1
Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University
of Pretoria, Private bag x20, Pretoria 0028, South Africa
2
Department 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.
References
Gryzenhout 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 Schoonen1, Izelle Smuts2, Richard Rodenburg3, Etresia van Dyk1, Roan Louw1, Francois
van der Westhuizen1
1
Centre for Human Metabonomics, North-West University, Potchefstroom, South Africa
Department of Paediatrics and Child Health, Steve Biko Academic Hospital, University of
Pretoria, South Africa
3
Nijmegen Center for Mitochondrial Disorders at the Department of Paediatrics, Radboud
University Medical Center (Radboudumc), Nijmegen, The Netherlands
2
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 Oloo1, Angel Valverde1, María Victoria Quiroga2, Gabriela Mataloni2 and Don Cowan1
1
Centre for Microbial Ecology and Genomics (CMEG), Department of Genetics, Natural Sciences
2, University of Pretoria, Hatfield, Pretoria, 0028
2
Instituto 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, paraffinembedded 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 Mosina1, Wolf-Dieter Schubert2 and Don Cowan1
1
Centre for Microbial Ecology and Genomics, University of Pretoria
2
Department 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.
References
Kellerman, 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 hospitalbased 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
2
Centre for Bioinformatics and Computational Biology, University of Pretoria
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 Robert1,3, Malebe Pelly1, Mose Richard2, Kamunya Samsom3, Myburg Zander4, Apostolides
Zeno1
1
CAM Research Group, Department of Biochemistry, University of Pretoria, Pretoria, South Africa
2
James Finlays Limited, Kericho, Kenya
3
Tea Research Institute, Kenya Agricultural Livestock Research Organization, Kericho, Kenya
4
Department 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.
References
Weynberg 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
1
Centre 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 FCspecific; 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 Cilliers1, Stefan van Wyk1, Riekert van Heerden1,2, and Barend Vorster 1
1
University of Pretoria, Department of Plant Production and Soil Science, Pretoria 0002;
2
South African Sugarcane Research Institute, Mount Edgecombe 4300
Contact information: [email protected]
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.
References
Kardailsky, 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
1
Department of Genetics, University of Pretoria.
Section Pathology, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of
Pretoria.
2
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 Johnson1, Jean-Baptiste Ramond1, Eoin Gunnigle1, Mary Seely2 and Don A. Cowan1
1
Centre for Microbial Ecology and Genomics, Genomics Research Institute, University of Pretoria,
South Africa
2
Gobabeb 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. Bouwman1, Elizabeth A. Tindall2, Angel Valverde Portal1, Don A. Cowan1,
Riana M.S. Bornman3, Vanessa M. Hayes4
1
Centre for Microbial Ecology and Genomics, University of Pretoria, South Africa
2
J. Craig Venter Institute, La Lolla, CA, USA
3
School of Health Systems and Public Health, University of Pretoria, South Africa
4
Garvan 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 the 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.
References
Sfanos, 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 Stobie1, Kerry Reid1, Samantha Mynhardt1, Carel Oosthuizen1, Arrie Klopper1, Thierry
Hoareau1, Michael Cunningham1 and Paulette Bloomer1
1
Molecular Ecology & Evolution Programme, Department of Genetics, University of Pretoria,
Pretoria, South Africa
Restriction-site Associated DNA (RAD) sequencing is a reduced-representation Genotyping-bySequencing (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 Ronca1, 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 physicochemical 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: [email protected]
b
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, nonmotile, 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 Malebe1, Hastings Nyirenda2, Samson Kamunya3 Alexander A Myburg4 and Zeno
Apostolides1
1
Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa
2
Tea Research Foundation of Central Africa, Mulanje, Malawi
3
Tea Research Institute, Kericho, Kenya
4
Department 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 nextgeneration sequencing platform from Diversity Arrays Technology (DArT). A population of 261 F 1
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.
References
Grattapaglia, 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): 11211137.
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
1
Centre 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
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