Forest Molecular Genetics

Research Areas

Principal Investigators

Prof Sanushka Naidoo

The United Nations General Assembly declared 2020, the international year of plant health. This is an opportunity to raise awareness of plant health and reflect on the developments that have contributed to improving crop health and increasing crop production over the past few decades. One such approach, is precision breeding, which refers to tools to produce new varieties with desirable traits such as pest and disease resistance more rapidly and precisely. The challenge is that pests and pathogens are not easily predicted and incidences may rise due to new conducive niches emerging under climate change. Plant monocultures are at risk of being overcome by single pathogens or pests. What of forest trees? Eucalyptus and pine production are affected by various pathogens and pests. We have developed a molecular model of Eucalyptus defence to various biotic stress interactions: the gall-inducing pest, Leptocybe invasa , the stem canker pathogen, Chrysoporthe austroafricana , the myrtle rust pathogen Austropuccinia psidii and the root rot pathogen, Phytophthora cinnamomi . We can now determine signature resistant responses and signature susceptible responses and identify tailored mechanisms to the different biotic stresses. In this manner, we can identify the most important genes that could confer resistance against a biotic stress. We are working on extending this model to include a mild drought stress response and my PhD student is investigating whether a mild drought stress aggravates disease symptoms and suppresses the defence responses. We are also extending our understanding of pine defences by interrogating the responses of several species: P. greggii, P. patula, P. tecunumanii and P. oocarpa to Fusarium circinatum challenge. We hypothesize that the resistant species share a similar molecular response in rapid activation of defences compared to the susceptible species. In addition, we are adopting new approaches to rapidly detect resistant clones by developing a Fourier Transform Infra-Red model for Eucalyptus . The model is being trained on the stem canker phenotypes of various Eucalyptus clones. The idea is that we can rapidly detect a resistant clone early on in a breeding cycle. While we are at the early stages in forestry, we believe our research is contributing to precision breeding for disease and pest resistance in the future.

Technology Platforms

Synthetic Biology

Eucalyptus s ynthetic biology resources for re-engineering woody biomass for biomaterials and bioenergy The ability to assemble standardized biological (DNA) parts idempotently is a core feature of synthetic biology. Recently, the Phytobrick standard (Patron et al. , 2015) incorporating a universal syntax enabling one-step assembly of level 0 eukaryotic gene elements (i.e. promoters, 5' UTRs, coding sequences, etc) into transcriptional units has gained acceptance in the plant synthetic biology community. These standards are compatible with modular cloning (MoClo), Golden Gate, GoldenBraid, Gibson and LOOP DNA assembly methods (Engler et al. 2008, 2014; Gibson et al. 2009; Pollak et al. 2018; Sarrion-Perdigones et al. 2011, 2013; Weber et al. 2011), allowing scarless, versatile, hierarchical and one-pot assembly of large multigene constructs. The US Department of Energy Joint Genome Institute (DOE-JGI), through a DNA Synthesis Science Program grant (CSP-503083), has funded the synthesis of 221 secondary cell wall-related E. grandis transcription factors and 65 promoter sequences to enable bioenergy and biomaterials-focused research. Specifically, we designed the synthetic panel for high-throughput protein-DNA interaction screening (e.g. DAP-seq; eY1H) (Bartlett et al ., 2017; Gaudinier et al ., 2011), in addition to functional genomics applications. Moreover, the majority of the synthetic constructs were designed as standardized Phytobricks, with additional GATEWAY functionality (Fig. 1; Hussey et al. , in preparation). The full list of DNA constructs can be downloaded here . The University of Pretoria (as Recipient) has a Materials Transfer Agreement (MTA) in place with the Regents of the University of California through the DOE-JGI (Contractor) regarding the use and transfer of constructs to third parties. The materials are freely available to nonprofit universities and institutions, subject to an MTA between that institution and the University of Pretoria. For any queries regarding the constructs or requests for material transfer, please contact Dr Steven Hussey . Figure 1. Design of standardized synthetic E. grandis SCW-related transcription factors and promoters. Hammers indicate domesticated sequences, dotted lines indicate restriction cleavage sites, coloured bases indicate standard syntax sequences and lowercase sequences indicate spacer nucleotides. (A) Transcription factor Phytobricks contain att L GATEWAY recombination sites (green), fifty-one basepair chewback linkers, BsaI Type IIS recognition sites and standard syntax sequences (purple text). The start codon of the domesticated coding sequence (green) remains in frame with N-terminal tags in GATEWAY destination vectors, while being primarily intended for Golden Gate, MoClo and Golden Braid assembly. (B) The DNA Affinity Purification Sequencing (DAP-seq) panel of transcription factors is available as a C-terminal fusion to the HALO purification tag, intended for in vitro transcription and translation via the SP6 phage promoter. While not standardized by a universal syntax, the coding sequences are fully domesticated and can thus be subcloned as standardized parts into a universal acceptor plasmid. (C) The secondary cell wall promoter panel features semi-domesticated 2 kb promoter sequences (including 5’ UTRs) compatible with GATEWAY and AarI-mediated Golden Gate cloning. Standard prefix and suffix syntax sequences allow for two-step Type IIS assembly to any Phytobrick panel CDS in (A). AmpR, ampicillin resistance gene; CDS, coding sequence; SpecR, spectinomycin resistance gene. References Bartlett, A., O'Malley, R.C., Huang, S.C., Galli, M., Nery, J.R., Gallavotti, A. and Ecker, J.R. (2017) Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc 12 , 1659-1672. Engler, C., Kandzia, R. and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3 , e3647. Engler, C., Youles, M., Gruetzner, R., Ehnert, T.M., Werner, S., Jones, J.D., Patron, N.J. and Marillonnet, S. (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol 3 , 839-843. Gaudinier, A., Zhang, L., Reece-Hoyes, J.S., Taylor-Teeples, M., Pu, L., Liu, Z., Breton, G., Pruneda-Paz, J.L., Kim, D., Kay, S.A., Walhout, A.J., Ware, D. and Brady, S.M. (2011) Enhanced Y1H assays for Arabidopsis. Nat Methods 8 , 1053-1055. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6 , 343-345. Patron, N.J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., Raitskin, O., Leveau, A., Farré, G., Rogers, C., Smith, A., Hibberd, J., Webb, A.A., Locke, J., Schornack, S., Ajioka, J., Baulcombe, D.C., Zipfel, C., Kamoun, S., Jones, J.D., Kuhn, H., Robatzek, S., Van Esse, H.P., Sanders, D., Oldroyd, G., Martin, C., Field, R., O'Connor, S., Fox, S., Wulff, B., Miller, B., Breakspear, A., Radhakrishnan, G., Delaux, P.M., Loqué, D., Granell, A., Tissier, A., Shih, P., Brutnell, T.P., Quick, W.P., Rischer, H., Fraser, P.D., Aharoni, A., Raines, C., South, P.F., Ané, J.M., Hamberger, B.R., Langdale, J., Stougaard, J., Bouwmeester, H., Udvardi, M., Murray, J.A., Ntoukakis, V., Schäfer, P., Denby, K., Edwards, K.J., Osbourn, A. and Haseloff, J. (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol 208 , 13-19. Pollak, B., Cerda, A., Delmans, M., Álamos, S., Moyano, T., West, A., Gutiérrez, R.A., Patron, N., Federici, F. and Haseloff, J. (2018) Loop Assembly: a simple and open system for recursive fabrication of DNA circuits. bioRxiv , 247593. Sarrion-Perdigones, A., Falconi, E.E., Zandalinas, S.I., Juarez, P., Fernandez-del-Carmen, A., Granell, A. and Orzaez, D. (2011) GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6 , e21622. Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A. and Orzaez, D. (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162 , 1618-1631. Weber, E., Engler, C., Gruetzner, R., Werner, S. and Marillonnet, S. (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6 , e16765.

The QuantStudio 12K Flex Real-Time PCR Facility

Quantitative Real-Time PCR is an extensively used analytical method in molecular biology research. With a grant from the NRF National Equipment Platform (NEP) the QuantStudio™12K Flex Real-Time PCR Facility was established in 2014, aimed towards the provision of a user-friendly real-time PCR platform to researchers throughout South Africa and to promote realtime PCR-based research. The QuantStudio System allows for small to large-scale functional genomics studies, with two main functions, gene expression analysis or genotyping assays, being used in either 384 well format or high throughput OpenArray® technology. As such the QuantStudio™12K Flex Real-Time PCR Facility has become an indispensable tool used by staff and students in FMG, the university and surrounding universities across South Africa. Specifically in FMG, functional genomic characterization of wood development and disease resistance projects benefit from the facility. These projects include the expression analysis of hundreds of genes under varying conditions and treatments, as well as SNP marker genotyping of up to 256 SNP markers across thousands of individuals. In the past, FMG as a group has used custom designed assays for SNP marker genotyping, and analysis of gene expression of pathogen defence related genes in Eucalyptus trees. Currently FMG uses the platform to investigate genes involved in secondary cell wall development in Populus, understanding gene expression of nuclear vs plastid genomes, and several 384 well studies looking at genes of interest. Since the establishment of the facility the QuantStudio™12K Flex Platform has encouraged collaboration and mentorship across a wide range of research groups, universities and institutes and has been used by researchers in many different fields whether they are studying the metabolism of humans or researching drought tolerance in cassava.

Wood Chemistry Platform

The wood chemistry platform was started as a collaborative initiative between Sappi, the University of Pretoria (FMG Programme) and the University of British Columbia (Prof Shawn Mansfield) to support FMG’s functional genetics research, as well as tree improvement for commercially grown eucalypt and pine species in South Africa. The platform, including an FMG owned HPLC instrument, is currently housed at the Sappi Technology Centre (STC) where the wood chemistry team are implementing new protocols and using established analytical protocols to chemically characterise the major constituents of wood (cellulose, hemicellulose and lignin). The technology is not only used to supply phenotypic data in functional genetic studies and breeding programmes, but also supplies valuable data to generate and update near-infrared analysis (NIRA)models.

Tissue Culture and Transformation Platform

The long generation time of trees and the prolonged period required for evaluation of mature traits are strong limitations for classical breeding and selection. The development of methods for in vitro culture and genetic engineering has increased the possibility of producing Eucalyptus genotypes improved in insect pest resistance, herbicide tolerance, growth rate and wood quality. However, transformation efficiency for Eucalyptus trees in particular has been very low and requires efficient in vitro protocols for plant induction, regeneration and selection that allow the production of transgenic plants from the transformed cell groups. Poplar has become the model tree species for genetic transformation due to the high susceptibility to Agrobacterium and high regeneration rates from transformed cells. Production of transgenic poplar trees has been perceived as a possible approach for the control of diseases, and improvement of plant quality. The development of genome engineering technologies, based on the CRISPR-associated RNA-guided endonuclease Cas9 systems, has broadened the agricultural research area, and brought in new opportunities to develop novel plant varieties. The genetic engineering of poplar trees becomes a powerful approach to test traits of high commercial value such as cellulose and hemi-cellulose biosynthesis in trees. We have established capacity for the development of transgenic Arabidopsis and poplar plants and gene testing in the laboratory and greenhouse. We can transfer the plants from the laboratory to the growth facilities for comparative growth studies. We also have the capacity for routine tissue culture and in vitro propagation of a variety of different Eucalyptus genotypes. FMG has also set up collaborations with Prof Steve Strauss at Oregon State University (OSU) towards developing a Eucalyptus transgenic platform at the University of Pretoria.

Bioinformatics Platform

The Bioinformatics and Data Science Platform is essential for meeting the programme's data storage, analysis and visualization needs. Established to support large-scale systems biology and genome diversity research, this platform enables researchers to effectively analyse complex data, driving innovative discoveries in the field of forest genetics.

DNA Fingerprinting Platform

Led by Ms Melissa Reynolds, the PTB platform, originally known as the "DNA Marker Platform" has made significant strides in DNA fingerprinting since its inception in 2008. This platform has successfully generated DNA marker profiles for 100,000 trees in South African trees breeding programmes, using microsatellite DNA markers and more advance SNP DNA marker arrays for pines and eucalypts. The team conducts comprehensive DNA fingerprinting, parentage and genomic relationship analyses. Expanding its focus, the platform now also encompasses macadamia and wattle species, supported by a highly skilled technical team and cutting-edge high throughput DNA analysis equipment.