SBIR-STTR Award

Hybrid poplar plantations will play a large role in the supply of cellulosic feedstock, in meeting the Nation's 30x30 goals for the renewable transportation fuels industry. The success of the poplar plantation industry will be contingent on elite plant material that has undergone hybridization for improved agronomic characteristics. Hybridization between the cottonwoods and the poplars has been the preferred method of generating elite varieties. We propose to integrate molecular tools into a traditional hybridization program to breed a new class of varieties that are capable of bio-fuels production with improved conversion economics. To accomplish this, we are currently conducting an association genetics study to identify genes controlling cellulose production in black cottonwood, based on single nucleotide polymorphisms (SNP's). We now propose a comparative study with black poplar to complete our understanding of how best to re-design a hybridization program using molecular markers and bioinformatics. A large black poplar collection will be cloned and established at two contrasting locations in the Pacific Northwest. We will sequence the same lignin and cellulose biosynthetic pathway genes identified in the black cottonwood study to discover new SNPs that can be used in a Phase II association study. The comparative study will advance our understanding of the manner in which reciprocal parental populations should be managed in a marker-assisted breeding program for improved first-generation hybridization for enhanced bio-fuels application. OBJECTIVES: Phase I research includes two objectives. Firstly, we will clonally propagate a large collection of P. nigra germplasm and establish in replicated field trials at two locations. The collection will form the experimental population for discovery of single nucleotide polymorphisms that control cellulose and lignin production. The technical design of the field trials will answer the question as to what extent genotype-by-environment interactions may have on the conservation of the marker-trait association across sections of the genus. Secondly, we will search for single nucleotide polymorphism in selected genes chosen partially on the basis of those identified as controlling cellulose and lignin production in our current P. trichocarpa association genetics project. This acquired knowledge of single nucleotide polymorphims will not only be used in the Phase II association study but will be used in deciding which P. nigra genotypes to include in a GreenWood breeding orchard, a decision based to maximize genetic diversity within P. nigra as well as between P. trichocarpa and P. nigra. APPROACH: Clonal propagation and testing of P. nigra. GreenWood currently manages three seedling populations of P. nigra. The populations were assembled from open-pollinated collections throughout all of western Europe. Sixty-six open pollinated families are represented in the collection. The collection will be re-propagated by vegetative means in a glasshouse to produce a uniform clonal population free of C effects. Each selected genotype will be represented by six clonal ramets. Ramets will be propagated as containerized rooted stock from stem cuttings. The clonal population will be established during the spring of 2008 in replicated field trials in two contrasting sites in Oregon where production poplar plantations are currently being managed. One site will be located along the lower Columbia River floodplain near the ocean coast. The environment at Westport is a mild climate with ample rainfall of up to 2,000 mm per year. The soil is low in pH and high in organic matter content. The second site will be located in the arid mid-Columbia River basin. This site is a high desert shrub-steppe environment with a continental climate and a coarse, low-organic soil. Rainfall is less than 250 mm per year and poplar is grown under drip irrigation with good results. The stock will be planted using a randomized complete block design with three replications. Discovery of single nucleotide polymprphisms (SNPs). Genetic variation in genes determining lignin and cellulose components of xylem must be discovered before the genetic variation can be associated with phenotypic variation. The contemporary and simplest way to discover genetic variation is to sequence genes of the lignin and cellulose biosynthetic pathways in a small number of unrelated P. nigra clones. Individual nucleotide differences among clones are known as single nucleotide polymorphisms (SNPs). We have built a high-throughput SNP discovery pipeline in collaboration with Agencourt Biosciences in Beverly, MA. Agencourt performs the DNA sequencing reactions using standard Sanger sequencing technology. The sequencing trace files are piped to servers at the University of California, Davis where bioinformaticians in the Neale lab identify the SNPs. We propose to sequence 2-3 fragments from each of the same 40 lignin and cellulose biosynthetic pathway genes as are under study in P. trichocarpa. Many of the custom sequencing reagents (PCR primers) that were designed and synthesized for P. trichocarpa can be used directly with P. nigra because of the high degree of sequence conservation between these species, thus significantly reducing the cost of the study. SNPs will be called using a modified PHRED-PHRAP-POLYPRED bioinformatics pipeline at University of California, Davis that has been used extensively for pine, Douglas-fir and poplar. Based on a predicted SNP frequency in P. nigra of 1/100 nucleotides, we expect to discover 500 new SNPs in these important genes that can be used in association studies in Phase II.

Highly productive, third-rotation commercial plantations have been successfully managed in the United States by the pulp and paper, timber, and the environmental-remediation industries for more than 40 years with great success in terms of wood yields and job creation. The success of the poplar plantation industry has consistently been shown to be dependent the propagation of elite varieties that have undergone hybridization and intense selection for improved agronomic characteristics. Hybrid poplar will similarly play a large role in the future supply of cellulosic energy feedstock as a key component of the Nation's 30x30 goals for the renewable transportation fuels industry. However a new class of poplar varieties of improved chemistry composition will be needed that are capable of bio-fuels manufacture with improved conversion economics. The breeding of such varieties will require the development and integration of novel molecular tools into existing traditional hybridization and varietal selection program. To accomplish this, GreenWood Resources currently conducting an association genetics study to identify genes controlling cellulose production in black cottonwood (Populus trichocarpa) based on single nucleotide polymorphisms (SNP's). We are now completing a comparative study with European black poplar (P. nigra) so as to augment GreenWood's conventional hybridization program for these two important poplar species using molecular markers and bioinformatics to improve biomass composition and the economics of liquid fuels conversion. (The goal is a re-designed reciprocal recurrent selection program for inter-sectional hybridization of P. trichocarpa and P. nigra. ) A large 612-genotype collection of P. nigra has been cloned and established at two contrasting locations in the Pacific Northwest. We have sequenced the same lignin and cellulose biosynthetic pathway genes identified in the P. trichocarpa study to discover new SNPs. We propose to: 1) Phenotype the 612 clones for an array of chemical and structural traits at each of the two locations, 2) Genotype the collection for important SNPs, and 3) Study the genotype-phenotype associations and the effect to which they interact with planting site. The comparative study in P. nigra will also provide insight into the pattern of genetic diversity between the two species from distinct sections of the genus. The comparison will also advance our understanding of the manner in which reciprocal parental populations should be managed as a marker-assisted breeding program for improved first-generation hybridization for enhanced bio-fuels application. OBJECTIVES: Objective 1 - Clonal testing and phenotyping of P. nigra for biomass production and compositional traits (GreenWood Resources). -Determine the magnitude of genetic variation in biomass compositional traits in P. nigra (lignin content, syringyl-to-guaicyl lignin ratio, glucose content, xylose content, specific gravity, etc.). -Determine how this variation is partitioned between the family and clone-within-family levels. -Determine the importance of genotype-by-environment interactions for biomass compositional traits. Objective 2 - SNP genotyping in an association population of 612 P. nigra clones (University of California, Davis). -SNP genotype 612 P. nigra clones for ~1536 SNPs (Illumina Golden Gate assay) at the UC Davis Genome Center. Objective 3 - Association testing (GreenWood Resources and University of California, Davis). -Perform association genetics analyses to identify genes controlling lignin content, and syringyl/guaiacyl lignin ratio, cellulose quantity, wood specific gravity. -Compare results with those derived for P. trichocarpa. -Determine stability of marker-trait associations within population tested at two contrasting sites. APPROACH: Replicated clonal fields trials at GreenWood Resources' Westport and Boardman test sites will be grown using optimal practices for weed control, irrigation, fertilization, and pest control through the 2009 - 2011 growing seasons. Data will be collected following the second growing season (2009); this is coincident with the expected rotation length for biomass energy feedstock. Both agronomic and biomass-compositional data will be collected. GreenWood Resources will also develop family-specific yield equations to estimate dry weight on an individual tree basis. These will be expanded to a 36-tree plot basis for an area yield estimate for each family. Biomass compositional data will include wood specific gravity, total lignin content, ratio of syringyl-to-guaicyl (S/G) lignin forms, and glucose and xylose contents as determined by near infrared spectroscopy (Brimrose Luminar 5030 spectrometer). DNA samples from P. nigra leaf tissue will be isolated in the Neale lab at UC Davis. SNP genotyping (1,536 SNPs) will be performed at the UC Davis Genome Center DNA Technologies Core using the Illumina Golden Gate SNP genotyping platform. SNP genotype calls will be piped directly to the bioinformatics servers in the Neale lab for data analysis. Association tests with chemical and physical wood property phenotypes (lignin content, syringyl/guiacyl lignin ratio, cellulose quantity and wood specific gravity) will be performed using methods described in Gonzalez-Martinez et al.(2007).* Correction will be made for family (K matrix) and population (Q matrix) structure as described in Gonzalez-Martinez et al. (2007). Correction for multiple testing will also be made using the false discovery rate (FDR) method (Gonzalez-Martinez et al. (2007)). Tests will address the conservation of associations both across environments (i.e. Westport versus Boardman) and species (i. e. P. nigra versus P. trichocarpa). * Gonzalez-Martinez, S. C., Wheeler, N. C., Ersoz, E., Nelson, C. D., and Neale, D. B. 2007. Association genetics in Pinus taeda L. I. wood property traits. Genetics 175: 399-409