Dave K. Berger

High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

Cercospora zeina is the causal agent of grey leaf spot disease of maize in southern Africa

The aim of our study was to identify the causal agent of grey leaf spot disease of maize in southern Africa. Single-conidial cultures were recovered from maize leaves with typical disease symptoms sampled from several fields in South Africa, Zambia and Zimbabwe. Morphology, cultural characteristics, and a PCR-based test using Cercospora zeae-maydis and C. zeina-specific primer sets identified all single-conidial cultures as C. zeina. In addition, sequence alignment of DNA fragments of the internal transcribed spacer region (ITS1, ITS2, and the 5.8S gene) and elongation factor 1-α grouped all cultures in the same clade as the C. zeina ex-type culture CBS 118820. To by-pass cultivation of the slow-growing fungus, a rapid method to isolate DNA directly from lesions was successfully applied for PCR identification of C. zeina with species-specific ITS and histone primers. Koch’s postulates were fulfilled for C. zeina by artificially inoculating maize plants in a greenhouse, re-isolating conidia emerging from lesions and verifying pathogen identity with molecular techniques. These results provide evidence that confirms the presence of C. zeina and absence of C. zeae-maydis in commercial maize plantations in southern Africa.

Salicylic acid confers resistance to a biotrophic rust pathogen, Puccinia substriata, in pearl millet (Pennisetum glaucum)

Studies were undertaken to assess the induction of defence response pathways in pearl millet (Pennisetum glaucum) in response to infection with the leaf rust fungus Puccinia substriata. Pretreatment of pearl millet with salicylic acid (SA) conferred resistance to a virulent isolate of the rust fungus, whereas methyl jasmonate (MeJA) did not significantly reduce infection levels. These results suggest that the SA defence pathway is involved in rust resistance. In order to identify pearl millet genes that are specifically regulated in response to SA and not MeJA, and thus could play a role in resistance to P. substriata, gene expression profiling was performed. Substantial overlap in gene expression responses between the treatments was observed, with MeJA and SA treatments exhibiting 17% co-regulated transcripts. However, 34% of transcripts were differentially expressed in response to SA treatment, but not in response to MeJA treatment. SA-responsive transcripts represented genes involved in SA metabolism, defence response, signal transduction, protection from oxidative stress and photosynthesis. The expression profiles of pearl millet plants after treatment with SA or MeJA were more similar to one another than to the response during a compatible infection with P. substriata. However, some SA-responsive genes were repressed during P. substriata infection, indicating possible manipulation of host responses by the pathogen.

Bin mapping of tomato diversity array (DArT) markers to genomic regions of Solanum lycopersicum × Solanum pennellii introgression lines

Marker-trait association studies in tomato have progressed rapidly due to the availability of several populations developed between wild species and domesticated tomato. However, in the absence of whole genome sequences for each wild species, molecular marker methods for whole genome comparisons and fine mapping are required. We describe the development and validation of a diversity arrays technology (DArT) platform for tomato using an introgression line (IL) population consisting of wild Solanumpennellii introgressed into Solanumlycopersicum (cv. M82). A tomato diversity array consisting of 6,912 clones from domesticated tomato and twelve wild tomato/Solanaceous species was constructed. We successfully bin-mapped 990 polymorphic DArT markers together with 108 RFLP markers across the IL population, increasing the number of markers available for each S.pennellii introgression by tenfold on average. A subset of DArT markers from ILs previously associated with increased levels of lycopene and carotene were sequenced, and 44% matched protein coding genes. The bin-map position and order of sequenced DArT markers correlated well with their physical position on scaffolds of the draft tomato genome sequence (SL2.40). The utility of sequenced DArT markers was illustrated by converting several markers in both the S.pennellii and S.lycopersicum phases to cleaved amplified polymorphic sequence (CAPS) markers. Genotype scores from the CAPS markers confirmed the genotype scores from the DArT hybridizations used to construct the bin map. The tomato diversity array provides additional “sequence-characterized” markers for fine mapping of QTLs in S.pennellii ILs and wild tomato species.

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.

Draft genome of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens and Ophiostoma ips

The genomes of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens, and Ophiostoma ips are presented in this genome announcement. Three of these genomes are from plant pathogens and otherwise economically important fungal species. Fusarium pininemorale and H. decipiens are not known to cause significant disease but are closely related to species of economic importance. The genome sizes range from 25.99 Mb in the case of O. ips to 4.82 Mb for H. lignivorus. These genomes include the first reports of a genome from the genus Hawksworthiomyces. The availability of these genome data will allow the resolution of longstanding questions regarding the taxonomy of these species. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these species or close relatives cause disease.

Expression profiling of putative Eucalyptus grandis defence marker genes in response to treatment with methyl jasmonate and salicylic acid

Background Eucalyptus species and their hybrids encompass approximately 40% of forestry plantation area in South Africa and contribute significantly to the paper pulp industry due to their favourable wood fibre properties. Eucalypt plantation trees are affected by numerous pathogens during their lifetime, some of which can cause severe losses such as Phytophtora spp and Chrysoporthe spp. Plant defence mechanisms against pathogens is currently better understood in the model plant Arabidopsis thaliana where it has been shown that the salicylic acid (SA) and jasmonic acid (JA) signalling pathways enhance resistance against biotrophic and necrotrophic pathogens, respectively [1] . This process involves the up-regulation of specific defence genes which are considered to be marker (diagnostic) genes for the two signalling pathways [2,3].

Opportunities for unlocking the potential of genomics for African trees

Trees (or their absence) represent one of the most defining features of landscapes on the African continent. However, they face major threats including habitat loss and degradation, invasive alien species, disturbance from frequent fire, over-harvesting, pollution, changes in pollinators or dispersers populations, and climate change (Balmford et al., 2001; Davies et al., 2011). Understanding how trees respond to these impacts would require an integrative approach of which genomic science has a potentially major role to play (Plomion et al., 2016). Since the advent of genomic science, its investigative power has been exploited for trees in temperate regions, particularly involving members ofPinus,Picea,Pseudotsuga,Populus,Eucalyptus,Quercus, Castanea, Malus, Prunus, and Fraxinus (Neale & Kremer, 2011; Neale et al., 2013). These species serve as models for exploring various processes in molecular genetics, functional biology, evolutionary biology, phenotypic and genotypic adaptation, physiology and organismal development (Tuskan et al., 2006; Plomion et al., 2016). Although the tropics have exceptionally high tree diversity –with Africa alone having c. 50 timesmore native tree species than temperate Europe (Slik et al., 2015) – tree genomic research in this region lags behind that of temperate ones. Limited funding and the lack of reference genomes for tropical trees have limited the progress of genomic science on the African continent. With over 6000 tree species on the African continent (Slik et al., 2015), there is a need to establish reference genomes for the major tree families. First, we discuss ways to exploit next generation sequencing (NGS) technologies including genotyping by sequencing (GBS), de novo transcriptome assembly and whole genome sequencing to generate genomic resources for nonmodel tree species on the African continent. Second, we discuss landscape genomics, an emerging field in genomic science and discuss research areas in which the genomic resources of trees inAfrica can be used to inform research on landscape genomics and to improve food production.