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Tobias, Christian

Publications and source records attributed to Tobias, Christian.

Expanding the breeder’s toolbox for perennial grasses

With the increase in world energy demand and the environmental impacts associated with continued fossil fuel use, there is a great need for the development of alternative and sustainable fuel sources. Switchgrass ( Panicum virgatum L. ), a perennial grass native to North America, is poised to become one of the first dedicated biomass crops deployed on a large scale in the United States in large part due to its inherent high yield, adaptability and perennial nature. Current ‘cultivars’ of switchgrass are essentially undomesticated wild selections from natural populations. Thus, there is considerable potential for improvement of this emerging energy crop. Traditional breeding approaches in switchgrass have been hampered by its self-incompatibility and sensitivity to inbreeding, therefore there is a significant need to develop new tools for improved switchgrass breeding. The proposed research uses a systems biology approach to accelerate switchgrass breeding, to develop its biotechnological improvement through the generation of stress-tolerant varieties, and to establish ways to minimize the potential impact of transgenes on ecosystems. In this proposal, we will develop new breeding tools for perennial grasses and apply these tools toward improving switchgrass. Specifically, we will employ two complementary approaches to overcome the difficulty of breeding a large, polyploid, self-incompatible plant. The first approach will accelerate conventional breeding by developing a method to rapidly create doubled haploid (homozygous) lines for breeding. This approach is based on altering CENH3, a centromere-specific histone, leading to the production of haploid offspring when the centromere mutant is crossed to wild type plants. Subsequent genome doubling results in fully homozygous individuals and overcomes one of the major limitations in breeding self-incompatibile crops. These homozygous lines would allow switchgrass breeders to apply a variety of breeding approaches that have been previously impossible. The second approach is to develop tools and reagents to accelerate the biotechnological improvement of switchgrass including: modules for improved stress tolerance, a test bed to evaluate transgenes in a perennial model grass and a robust gene containment system. In order to maintain a favorable energy balance and avoid competition with food crops, dedicated biomass crops must be grown with minimal inputs (fertilizer and water) on marginal lands that are unsuited to the traditional high input agriculture necessary to produce food. Thus, abiotic stress tolerance and efficient water and nutrient use are of paramount importance. Considerable basic research has been directed at these areas and this knowledge has been used to develop transgenic approaches to greatly improve water and nutrient use efficiency. By applying these approaches to switchgrass we will rapidly increase drought tolerance and nutrient use efficiency. This approach can rapidly introduce these traits in different cultivars allowing breeders to add these traits to their elite germplasm. Since biotechnologically improved switchgrass must ultimately be grown in open fields, they must be designed to minimize the potential impact of the transgenes on non-target ecosystems. The best way to do this is to minimize gene flow from the transgenic plants. Thus, we will develop technology to introduce sterility into the transgenic plants. We will then combine this technology with the stress resistance modules to create switchgrass lines with enhanced resistance to abiotic stresses.

09 BIOMASS FUELS↗

Expanding the Breeder’s Toolbox for Perennial Grasses

With the increase in world energy demand and the environmental impacts associated with continued fossil fuel use, there is a great need for the development of alternative and sustainable fuel sources. Switchgrass (Panicum virgatum L.), a perennial grass native to North America, is poised to become one of the first dedicated biomass crops deployed on a large scale in the United States in large part due to its inherent high yield, adaptability and perennial nature. Current ‘cultivars’ of switchgrass are essentially undomesticated wild selections from natural populations. Thus, there is considerable potential for improvement of this emerging energy crop. Traditional breeding approaches in switchgrass have been hampered by its self-incompatibility and sensitivity to inbreeding, therefore there is a significant need to develop new tools for improved switchgrass breeding. The proposed research uses a systems biology approach to accelerate switchgrass breeding, to develop its biotechnological improvement through the generation of stress-tolerant varieties, and to establish ways to minimize the potential impact of transgenes on ecosystems. In this proposal, we will develop new breeding tools for perennial grasses and apply these tools toward improving switchgrass. Specifically, we will employ two complementary approaches to overcome the difficulty of breeding a large, polyploid, self-incompatible plant. The first approach will accelerate conventional breeding by developing a method to rapidly create doubled haploid (homozygous) lines for breeding. This approach is based on altering CENH3, a centromere-specific histone, leading to the production of haploid offspring when the centromere mutant is crossed to wild type plants. Subsequent genome doubling results in fully homozygous individuals and overcomes one of the major limitations in breeding self-incompatibile crops. These homozygous lines would allow switchgrass breeders to apply a variety of breeding approaches that have been previously impossible. The second approach is to develop tools and reagents to accelerate the biotechnological improvement of switchgrass including: modules for improved stress tolerance, a test bed to evaluate transgenes in a perennial model grass and a robust gene containment system. In order to maintain a favorable energy balance and avoid competition with food crops, dedicated biomass crops must be grown with minimal inputs (fertilizer and water) on marginal lands that are unsuited to the traditional high input agriculture necessary to produce food. Thus, abiotic stress tolerance and efficient water and nutrient use are of paramount importance. Considerable basic research has been directed at these areas and this knowledge has been used to develop transgenic approaches to greatly improve water and nutrient use efficiency. By applying these approaches to switchgrass we will rapidly increase drought tolerance and nutrient use efficiency. This approach can rapidly introduce these traits in different cultivars allowing breeders to add these traits to their elite germplasm. Since biotechnologically improved switchgrass must ultimately be grown in open fields, they must be designed to minimize the potential impact of the transgenes on non-target ecosystems. The best way to do this is to minimize gene flow from the transgenic plants. Thus, we will develop technology to introduce sterility into the transgenic plants. We will then combine this technology with the stress resistance modules to create switchgrass lines with enhanced resistance to abiotic stresses.

09 BIOMASS FUELS↗

Using the Model Perennial Grass Brachypodium sylvaticum to Engineer Resistance to Multiple Abiotic Stresses

We are using the perennial model grass Brachypodium sylvaticum to identify combinations of transgenes that enhance tolerance to multiple, simultaneous abiotic stresses. The most successful transgene combinations will ultimately be used to create improved switchgrass (Panicum virgatum L.) cultivars. To further develop B. sylvaticum as a perennial model grass, and facilitate our planned transcriptional profiling, we are sequencing and annotating the genome. We have generated ~40x genome coverage using PacBio sequencing of the largest possible size selected libraries (18, 22, 25 kb). Our initial assembly using only long-read sequence contained 320 Mb of sequence with an N50 contig length of 315 kb and an N95 contig length of 40 kb. This assembly consists of 2,430 contigs, the largest of which was 1.6 Mb. The estimated genome size based on c-values is 340 Mb indicating that about 20 Mb of presumably repetitive DNA remains yet unassembled. Significantly, this assembly is far superior to an assembly created from paired-end short-read sequence, ~100x genome coverage. The short-read-only assembly contained only 226 Mb of sequence in 19k contigs. To aid the assembly of the scaffolds into chromosome-scale assemblies we produced an F2 mapping population and have genotyped 480 individuals using a genotype by sequence approach. One of the reasons for using B. sylvaticum as a model system is to determine if the transgenes adversely affect perenniality and winter hardiness. Toward this goal, we examined the freezing tolerance of wild type B. sylvaticum lines to determine the optimal conditions for testing the freezing tolerance of the transgenics. A survey of seven accessions noted significant natural variation in freezing tolerance. Seedling or adult Ain-1 plants, the line used for transformation, survived an 8 hour challenge down to -6 oC and 50% survived a challenge down to -9 oC. Thus, we will be able to easily determine if the transgenes compromise freezing tolerance. In the effort to develop biotechnological tools for perennial grass improvement, we have completed the transformation of B. sylvaticum with constructs containing 20 genes shown to be associated with enhanced abiotic stress tolerance in monocots. In addition, we have transformed plants with constructs containing a combination of genes (i.e. SARK::IPT- Ubi::HSR1::Ubi::NHX1) in order to simultaneously overexpress genes associated with drought + heat tolerance + salt tolerance. We generated single copy insert T1 lines for all constructs and the generation and bulking of homozygous T2 lines is well underway. In addition to our B. sylvaticum transgenics, we transformed B. distachyon with many of the same genes. Some of the transgenic B. distachyon plants subjected to a combined stress of both drought and salinity were able to produced higher yields than wild type plants. Our results indicate a great potential for the development of grasses with improved performance and yield in water-limited areas.

Gordon, Sean↗

SNP Discovery with EST and NextGen Sequencing in Switchgrass ( Panicum virgatum L. )

Although yield trials for switchgrass (Panicum virgatum L.), a potentially high value biofuel feedstock crop, are currently underway throughout North America, the genetic tools for crop improvement in this species are still in the early stages of development. Identification of high-density molecular markers, such as single nucleotide polymorphisms (SNPs), that are amenable to high-throughput genotyping approaches, is the first step in a quantitative genetics study of this model biofuel crop species. We generated and sequenced expressed sequence tag (EST) libraries from thirteen diverse switchgrass cultivars representing both upland and lowland ecotypes, as well as tetraploid and octoploid genomes. We followed this with reduced genomic library preparation and massively parallel sequencing of the same samples using the Illumina Genome Analyzer technology platform. EST libraries were used to generate unigene clusters and establish a gene-space reference sequence, thus providing a framework for assembly of the short sequence reads. SNPs were identified utilizing these scaffolds. We used a custom software program for alignment and SNP detection and identified over 149,000 SNPs across the 13 short-read sequencing libraries (SRSLs). Approximately 25,000 additional SNPs were identified from the entire EST collection available for the species. This sequencing effort generated data that are suitable for marker development and for estimation of population genetic parameters, such as nucleotide diversity and linkage disequilibrium. Based on these data, we assessed the feasibility of genome wide association mapping and genomic selection applications in switchgrass. Overall, the SNP markers discovered in this study will help facilitate quantitative genetics experiments and greatly enhance breeding efforts that target improvement of key biofuel traits and development of new switchgrass cultivars.

09 BIOMASS FUELS↗

EM Algorithm for Mapping Quantitative Trait Loci in Multivalent Tetraploids

Multivalent tetraploids that include many plant species, such as potato, sugarcane, and rose, are of paramount importance to agricultural production and biological research. Quantitative trait locus (QTL) mapping in multivalent tetraploids is challenged by their unique cytogenetic properties, such as double reduction. We develop a statistical method for mapping multivalent tetraploid QTLs by considering these cytogenetic properties. This method is built in the mixture model-based framework and implemented with the EM algorithm. The method allows the simultaneous estimation of QTL positions, QTL effects, the chromosomal pairing factor, and the degree of double reduction as well as the assessment of the estimation precision of these parameters. We used simulated data to examine the statistical properties of the method and validate its utilization. The new method and its software will provide a useful tool for QTL mapping in multivalent tetraploids that undergo double reduction.

59 BASIC BIOLOGICAL SCIENCES↗