BIOEN WORKSHOP ON SUGARCANE IMPROVEMENT
São Paulo - Brazil
March 18th to 19th, 2009
The objective of the workshop is to explore key issues behind a roadmap to the goal of sugarcane improvement as a sustainable bioenergy and biomaterial feedstock. The Workshop has been divided in four sessions followed by a debate and preceded by an introductory conference.
Opening Conferece - Sucrose metabolism and sugar physiology: An introductory, historical perspective on sugarcane research focusing on the physiological aspects of growth and development of this crop.
Section 1: Gene Discovery and Sugarcane Genomics: We are all thinking about how to best exploit the amazing new capacity for fast genomic sequencing - does it make sense as an international effort? What tools are available for sugarcane Gene Discovery efforts? What agronomic traits of interest should be targeted? Yield, sucrose content, drough tolerance, insect and disease resistance? How successful have we been in using the knowledge already available (ESTs data) for the improvement of sugarcane? What are the strategies in Functional Genomics most suited to address sugar metabolism and allow for a systemic understanding of physiology and then how to engineer the energy cane?
Section 2: Transgenics and Controlled Transgene Expression: Gene transfer into sugarcane has been routine for more than 15 years, but can we deliver commercial lines for 'next generation' traits? What are the hurdles to efficient production of lines with stable transgene expression in the required developmental patterns?
Section 3: Photosynthesis, sucrose metabolism, drought and sugar physiology: What are the key gaps in knowledge of sugar accumulation, cell wall metabolism, source-sink control mechanisms, water usage, carbon partitioning and ways to 'redesign' sugarcane as a sustainable bioenergy feedstock. Which questions must we answer in sugarcane, and which ones can we answer faster with help from other model systems?
Section 4: Breeding and Statistical Genetics: How can molecular biotech best interface with conventional breeding approaches? How much progress can be expected using non-GM approaches with better selection methods? How can molecular markers be efficiently identified and effectively used for the improvement of sugarcane? Do we have efficient trial designs to detect elite transgenics against a large background of lines with unwanted adverse effects on other commercial traits? How quickly can favourable transgene events be moved into commercial lines through crossing?
March 18th Wednesday
Opening Conference: Paul Moore (Sucrose metabolism and sugar physiology) (USDA, USA)
Coffee 10:00 – 10:30 am
Section 1: Gene Discovery and Sugarcane Genomics
10:30 h Rosanne Casu (CSIRO, Australia)
11:00 h Derek Watt (SASRI, South Africa)
11:30 h Glaucia Souza (IQ-USP)
12:00 h Debate
reference term: Glaucia Souza
Debate leaders: Marie-Anne Van-Sluys (IB-USP), Manuel Sainz (Syngenta), Paulo Arruda (Allelyx)
Section 2: Transgenics and Controlled Transgene Expression
14:00 h Helaine Carrer (ESALQ)
14:30 h Robert Birch (University of Queensland, Australia)
15:00 h João Carlos Bespalhok (UFPR, Brazil)
15:30 h Debate
Debate reference terms: Helaine Carrer and Robert Birch
Debate leaders: Marcelo Menossi (IB-UNICAMP), Eduardo Romano (EMBRAPA), Eugenio Ulian (Monsanto)
Day 3 March 19th Thursday
9:00 h coffee
Section 3: Photosynthesis, sucrose metabolism, drought and sugar physiology
9:30 h Rowan Sage (University of Toronto)
10:00 h Marcos Buckeridge (IB-USP)
10:30 h Graham Bonnett (CSIRO, Australia)
11:00 h Laurício Endres (UFAL)
11:30 h Debate
Debate reference terms: Buckeridge
Debate leaders: Rejane Mansur (UFRPE) , Paul Moore (USDA), Katia Scortecci (UFRN)
12:30 h Lunch
Section 4: Breeding and Statistical Genetics
14:30 h Marcos Sanches (UFSCAR)
15:00 h Augusto Garcia (ESALQ)
15:30 h Robert Henry (Southern Cross University)
16:00 h Debate
Debate reference term: Anete de Souza
Debate leaders: Jorge da Silva (TAMU), Walter Maccheroni (Canavialis), Marcos Landell (IAC)
Sugarcane Biology and Yield
Paul H. Moore
USDA, ARS, US Pacific Basin Agriculture Research Center (retired)
Hawaii Agriculture Research Center (current)
Aiea, HI, USA
Although we have a working knowledge of the general biology of sugarcane and are fairly efficient in crop production, we are just beginning to develop the tools that will be needed to break the yield ceiling that limits sugarcane productivity and uses. The last 50 years of research has identified and characterized a suite of physiological processes and enzymes involved in various aspects of sugarcane productivity. More recently, genes encoding these enzymes have been isolated, cloned, and used to transform plants to increase or decrease expression of specific enzymes with the goal of altering traits to enable higher productivity. However, results of this reductionism approach toward crop improvement have fallen short of expectations, apparently because of failure to control the complex interactions among the multitude of simultaneous processes affecting the plant.
It is becoming obvious that increases in yields and yield potential will not arise from purely intuitive assaults, nor from traditional physiology experiments, that try to explain observable phenomena by reducing them to an interplay of elementary processes that are investigated independently of each other. Instead, insights into the complex interactions among processes involved in crop growth and development such as carbon/water/mineral relations, leaf energy budgets, material allocations, growth, life cycles, biological interactions, ecological biochemistry, and ecosystem reactions will require integrated systems-level approaches that recognize the importance of problems of organization and phenomena not resolvable into local events. A deeper understanding of the negative impacts of biotic and abiotic stresses, coupled with the factors involved in carbon fixation, partitioning and storage will suggest systems level approaches for overcoming the myriad of forces constraining crop growth and development
Systems-level approaches involve evaluating the dynamic interactions seen in the different behavior of parts of the system when they are isolated versus when they are in higher configurations to predict behavior of the whole system and its parts. Currently, systems-level approaches towards understanding various aspects of biology are gaining momentum primarily because of the rapid progress that is being made in technologies for genome sequencing, proteomics, and high-throughput measurements of gene expression. Large-scale sequencing projects have not only provided complete sequence information for a number of genomes, they are also facilitating the development of integrated pathway-genome databases that provide organism-specific connectivity maps of metabolic and other cellular networks. To date, the processes that generate mass, energy, information transfer, and cell-fate specification have been analyzed primarily at the microorganism and cell levels where they are shown to be seamlessly integrated through a complex network of cellular constituents and reactions. Current systems-level research on human health have revealed genes and pathways that can be perturbed for prevention or treatment of critical diseases.
Recent rapid expansion of sugarcane molecular datasets and the beginning of a systems approach to metabolic modeling of sucrose accumulation point the way for future research efforts to integrate processes from gene to crop performance.
Gene Discovery: Approaches, Developments and Applications to Sugarcane Improvement at the South African Sugarcane Research Institute
Crop Biology Resource Centre, South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, 4300, South Africa; School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa
Knowledge of the identity of genes participating in the delivery of important traits or displaying tissue- or organ-specific expression is advantageous in the development of rational molecular breeding strategies. Over the past fifteen years, the South African Sugarcane Research Institute (SASRI) has enjoyed considerable success in the discovery of such genes, which have been used within marker-assisted breeding and genetic engineering strategies. For example, identified genes have facilitated the development of genetic markers depicting smut resistance and susceptibility, as well as in the isolation of gene promoter sequences that have the potential to target transgene expression to specific organs. Over the years, several technologies have been used at SASRI for gene discovery, including Expressed Sequence Tag Analysis, cDNA Differential Display, Suppression Subtractive Hybridisation and Affymetrix® Sugarcane Genome GeneChip analysis. While the application of these technologies has been successful, the recent advent of the next-generation DNA sequencing and gene expression analysis technologies and the release of the Sorghum and Brachypodium genome sequences heralds a new era for gene discovery in sugarcane; one which presents SASRI scientists with new challenges and opportunities. Presented here is an overview of gene discovery strategies at SASRI and the impact that they have had on sugarcane improvement research. Also described is the manner in which recent technological and bioinformatical advances are being embraced within this area of research.
Keywords: gene isolation, gene expression, transgenesis, promoter, DNA markers, molecular breeding.
The SUCEST-FUN Project: identifying genes associated to agronomic traits of interest in sugarcane
Glaucia Mendes Souza – Instituto de Química – Universidade de São Paulo
MODERN sugarcane cultivars are complex hybrids resulting from crosses among several species of the Saccharum genus. Traditional breeding methods have been extensively employed in different countries over past decades to develop varieties with increased sucrose yield, and resistance to pests and diseases. Conventional variety improvement, however, may be limited by the narrow pool of suitable genes. In this sense, molecular genetics is seen as a promising tool to assist in the process of developing improved varieties. The SUCEST-FUN Project (http://sucest-fun.org) aims to associate function to sugarcane genes using a variety of tools, in particular through the study of the sugarcane transcriptome. An extensive analysis has been conducted to characterize phenotipically sugarcane genotypes in regards to their sucrose content, biomass and drought responses. Through the analysis of progenies and cultivars we identified genes associated with sucrose content, yield, lignin and drought. We are currently developing tools to determine signaling and regulatory networks in grasses and to identify sugarcane promoters. This is being done through the implementation of the SUCEST-FUN (http://sucest-fun.org) and GRASSIUS databases (http://grassius.org), the cloning of sugarcane promoters, the identification of CREs using ChIP-HTS and the generation of a comprehensive Signal Transduction and Transcription gene catalogue (SUCAST Catalogue). Most recently we have designed oligonucleotide arrays containing 21901 oligos represented twice totalling 43802 features using the Agilent array technology. These include sense and anti-sense oligos for SUCEST SAS. The arrays have been validated using qPCR on samples derived from sugarcane plants submitted to drought conditions. Anti-sense expression of sugarcane genes was observed that will aid in understanding gene expression regulation and promoter identification in this plant.
Can we deliver controlled transgene expression in sugarcane?
Robert G Birch, Botany Department, The University of Queensland, Brisbane 4072, Australia.
Sugarcane has perhaps the greatest potential of any crop to make a global-scale contribution to sustainable biofuel production. But that development needs a quantum increase in productivity. Otherwise there is simply not enough land for the global task. Metabolic engineering approaches hold great promise, but they depend on ability to deliver reliable patterns of transgene expression at the right developmental stages over successive crop generations. Sugarcane transformation has been routine for 20 years. Field trials of transgenic sugarcane have been conducted on at least four continents. But how close are we to the delivery of reliable transgene expression in developmental patterns likely to be needed for metabolic engineering? There is little evidence for sustained, high-level transgene expression over generations in the field; and no evidence for reliable transgene expression in any other developmentally-regulated pattern. Sugarcane is very efficient at silencing most introduced transgene constructs, driven by sugarcane-derived or heterologous promoters. We are just beginning to understand the mechanisms – and how to avoid them. Very little has been elucidated about the possibility of differential silencing or developmental regulation of endogenous promoter alleles in the polyploid sugarcane genome. We are just assembling the tools to gain these insights. Nevertheless, having ‘admitted the problem’, there is a possibility of rapid progress and it will be very exciting to see the results of the first practical field tests in the coming 1-2 years.
Genomics based approaches to genetic improvement in sugarcane
Robert J Henry
Centre for Plant Conservation Genetics - Southern Cross University
The genetic improvement of sugarcane will be improved by greater understanding of the genome. Transcriptome and genome analysis should help define how traits are determined by specific alleles in this complex polypoid species. We are now able to apply next generation sequencing to these questions. Genetic markers can be discovered very efficiently by sequencing. A range of platforms are also available for the analysis of desirable alleles in sugarcane populations to support breeding selection. These analyses need to be quantitative to allow assessment of allele dose.
We have demonstrated some of these approaches. A draft whole genome sequence and detailed sequencing of gene regions will define the variation in sugarcane germplasm. The sequencing of the whole genome, gene rich fractions, and cDNA s will all make a contribution. Comparisons will be made with progress in other crop species.
Genetic Architecture of Quantitative Traits in Sugarcane
Antonio Augusto Franco Garcia
ESALQ - Universidade de São Paulo
QTL mapping is useful to help understanding the genetic architecture of quantitative traits in sugarcane, such as yield, fiber and sucrose content, providing the basis to marker assisted selection. However, it is necessary to develop new approaches, since the ones currently used are just adaptations of methods developed for inbred-based populations, such as F2 and backcrosses. For species without available inbred lines QTL mapping is in general conducted using full-sib families, where each locus can have different segregation patterns and linkage phases. This seminar will have two parts. First, we will show a new statistical approach for QTL mapping in full-sibs, based on integrated multipoint maps and composite interval mapping. Three orthogonal contrasts were defined and used to develop the model. Second, we will show results using the new model to map QTL for traits related to sugarcane productivity. The new approach provided better results then currently ones available in the literature, having more statistical power and precision and allowing the identification of more QTL across the genome. It was also possible to identify QTL with different segregation patterns, as well as their additive and dominance effects. Applications of the results will be discussed.
Gene discovery as an aid to the understanding of agronomically important metabolic processes of sugarcane
CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, QLD, 4067, Australia and CRC Sugar Industry Innovation through Biotechnology, Level 5, John Hines Building, The University of Queensland, St Lucia, QLD, 4072, Australia
Sugarcane produces a very large biomass and, unlike other grass crops such as rice or wheat, accumulates large amounts of sucrose in the stem. These two characteristics have greatly increased its attractiveness as a bioenergy crop since the sucrose can be fermented to produce ethanol directly and the large biomass could be also converted to ethanol once efficient, cost-effective conversion of cellulose to ethanol is achieved. Traditional breeding has concentrated on improvements in cane yield with an associated increase in sucrose production and resistance to endemic pests and diseases.
Historically, very little was known about the sequence of sugarcane transcripts or the sugarcane genome prior to the commencement of the gene discovery programs approximately 10 years ago. In Australia, gene discovery targeting the maturing stem followed by expression profiling using custom microarrays based on the generated EST collection resulted in the identification of a number of genes involved in carbohydrate metabolism, fibre metabolism as well as growth and development of the stem. Greater utility was achieved when all ESTs generated by the four gene discovery programs were made public, allowing for the first public clustering of transcript sequences to form a sugarcane transcriptome that probably contains most of the expressed genes. This allowed for the development of the Affymetrix® Sugarcane Genome Array which was the first commercial large-scale expression profiling tool, the first tool that could potentially allow for the generation of a bank of comparable expression profiles for further data-mining.
Candidate genes determined by bioinformatic analysis of EST collections and by large-scale expression profiling require further testing in order to prove their utility for variety improvement. Currently, two avenues for testing are being exploited: (1) transgenic testing in planta; and (2) marker development. Transgenic testing via RNAi-mediated gene silencing offers opportunities to explore whether (a) it is possible to modify gene expression in a complex polyploid, (b) genes involved in a core metabolic function can be manipulated while maintaining a viable plant and (c) a suitable phenotype can be linked directly to the gene being tested. Exploitation of candidate genes for single nucleotide polymorphism (SNP) development also has the potential to greatly enrich the marker landscape for variety improvement since SNPs would appear to be relatively common and can be linked to QTL and traits of interest. Therefore, the initial process of gene discovery has greatly enriched our choices for genomic approaches to sugarcane variety improvement.
Environmental and genetic manipulation of carbon partitioning between growth and storage in sugarcane
Graham Bonnett1,2, Geoff Inman-Bamber3, Barrie Fong Chong2,4 and Annathurai Gnanasambandam2,4
1CSIRO Plant Industry, QBP, 306 Carmody Road, St Lucia, QLD 4067, Australia; 2Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Level 5, John Hines Building, The University of Queensland, St Lucia, QLD 4072, Australia; 3CSIRO Sustainable Ecosystems, Davies Laboratory, PMB, PO Aitkenvale, QLD 4814, Australia; 4David North Plant Research Centre, BSES Limited, 50, Meiers Road, Indooroopilly, QLD 4068, Australia.
Increasing sucrose content (sugarcane’s harvest index) has been an aim of both genetic improvement programs and agronomic research. A higher proportion of sucrose in the stem at the seasonal peak (maximum sucrose content) is the main target but has been an elusive goal. Selection of sugarcane varieties that have high sucrose content early in the harvest season has been more successful. Together with agronomic treatments such as drying off and ripener application, this can be used to increase overall sucrose content of a region’s crop.
The success of these approaches relies on a combination of genetics and a response to the environment. Understanding the physiological basis to these changes (i) may provide useful insights for genetic improvement of sugarcane; (ii) identify any useful genetic by environment interactions that can be exploited through agronomic practice and (iii) give insights into carbon partitioning that may be exploited in future biomass systems.
A series of experiments using controlled conditions and treatments has demonstrated (i) how different genotypes partition carbon at the plant level and how that is altered under water stress; (ii) that variation in sucrose content occurs at a very early stage of development. This research has also shown that we do not yet fully understand sugarcane’s response to environmental factors.
One outcome from these experiments has been an experimental system that allows measurement of whole chamber photosynthesis. This is being used to explore more recent reports of sugarcane’s photosynthetic response to sucrose accumulation and in the future, elevated CO2. These are important responses to understand in order to drive future sugarcane improvement in new ways to utilise both new product opportunities and altered environments (through climate change) to their potential.
The role of giberellins in the control of carbohydrate metabolism in seedlings of sugarcane: a possible relationship between cell wall expansion and sucrose accumulation
Department of Botany, Institute of Biosciences, University of São Paulo
Using sugarcane seedlings as model system, we performed experiments to evaluate the effect of GA on carbohydrate metabolism, including sucrose, glucose, fructose and the cell wall.
Increasing concentrations (from 3 to 60uM) of gibberellic acid (GA3) induced proportional extension of the epicotyls of sugarcane seedlings from zero to the 21st day after germination. The concentration of sugars (sucrose, glucose and fructose) in the shoots increased with the addition of GA3.
However, these effects were reverted under addition of paclobutrazol, an inhibitor of GA biosynthesis. This indicated that GA synthesis is necessary for the sugar accumulation to occur. The microscopy analyses of the tissues revealed that vacuolization has intensely increased under addition of GA3 and that it was reverted by addition of the inhibitor. When the composition of cell walls was examined in the same tissues, we found that the mixed-linkage-beta-glucan (MLG) decreased in the absence of GA synthesis and that an increase in xylose (from the arabinoxylan, the main hemicellulose in sugarcane) occurred more intensely under the presence of GA3. As it is thought that the MLG functions as a scaffold for the building of the wall, directing the interaction of the hemicellulose (arabinoxylan) to the cellulose microfibrils and consequently inducing cell wall loosening and cell expansion, it is reasonable to suggest that GA plays a role as a connector of the network of sucrose and cell wall metabolism, possibly opening “space” in the cells of the developing meristem for increasing vacuolization so that more sucrose accumulates in the tissue.
It is not yet possible to know for sure whether such a network connection occurs in the stem of adult sugarcane plants, but the fact that GA is used in some places in the word as a means to increase sucrose concentration in sugarcane, one can expect that a similar system exists in its stem. We have already analysed the anatomical structure of the developing stem and found that the external layer of it is compatible with a rapidly developing system that produces vascular bundles by cell division and expansion.
As a perspective, we intend to perform a series of observations and experiments that will increase our understanding the role of GA and other plant hormones (ABA, ethylene, auxin and cytokinins) in the control of the carbon flux that leads to the changes in cell walls and soluble carbohydrates metabolism in sugarcane. We intend to approach the developing stem tissues morphologically, biochemically and molecularly (gene expression) so that we may be able to understand how the networks at
different levels of organization correlate to each other, generating the emergent pattern that is the sucrose accumulation dependent on cell wall expansion in the stems of sugarcane.