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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue

Jin-Ho Kang; ; Starla Zemelis-Durfee; Jameel M. Al-Haddad; ; Frank W. Telewski; Federica Brandizzi; Gregg A. Howe

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2016

Trichomes are epidermal structures that provide a first line of defense against arthropod herbivores. The recessive hairless (hl) mutation in tomato (Solanum lycopersicum L.) causes severe distortion of trichomes on all aerial tissues, impairs the accumulation of sesquiterpene and polyphenolic compounds in glandular trichomes, and compromises resistance to the specialist herbivore Manduca sexta Here, we demonstrate that the tomato Hl gene encodes a subunit (SRA1) of the highly conserved WAVE regulatory complex that controls nucleation of actin filaments in a wide range of eukaryotic cells. The tomato SRA1 gene spans a 42-kb region containing both Solyc11g013280 and Solyc11g013290 The hl mutation corresponds to a complex 3-kb deletion that removes the last exon of the gene. Expression of a wild-type SRA1 cDNA in the hl mutant background restored normal trichome development, accumulation of glandular trichome-derived metabolites, and resistance to insect herbivory. These findings establish a role for SRA1 in the development of tomato trichomes and also implicate the actin-cytoskeleton network in cytosolic control of specialized metabolism for plant defense. We also show that the brittleness of hl mutant stems is associated with altered mechanical and cell morphological properties of stem tissue, and demonstrate that this defect is directly linked to the mutation in SRA1.

Monolignol-ferulate conjugates are naturally incorporated into plant lignins

Steven D. Karlen; Chengcheng Zhang; Matthew L. Peck; Rebecca A. Smith; Dharshana Padmakshan; Kate E. Helmich; Heather C.A. Free; Seonghee Lee; Bronwen G. Smith; Fachuang Lu; John C. Sedbrook; Richard Sibout; John H. Grabber; Troy M. Runge; Kirankumar S. Mysore; Philip J. Harris; Laura E. Bartley; John Ralph

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2016

Multiple resource use efficiency (mRUE): a new concept for ecosystem production

Juanjuan Han; Jiquan Chen; Yuan Miao; Shiqiang Wan

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2016

Nanoscale structure of biomass

Shi-You Ding

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2016

Plant biomass is a renewable source that can be processed to produce biofuels and biomaterials. The plant cell walls are the major material in biomass. Depending on plant species and the time of harvest, biomass varies in its anatomical structure and chemical composition. This chapter summarizes general structure of the plant cell walls in different plant tissues and plant species, and updates are given from new findings in in situ imaging at nanoscale resolution and real-time changes during biomass deconstruction processes. The physicochemical properties of biomass that affect the efficiency of thermochemical pretreatment and enzymatic hydrolysis are also discussed.

Next-generation ammonia pretreatment enhances cellulosic biofuel production

Leonardo da Costa Sousa; Mingjie Jin; Shishir P.S. Chundawat; Vijay Bokade; Xiaoyu Tang; Ali Azarpira; Fachuang Lu; Utku Avci; James Humpula; Nirmal Uppugundla; Christa Gunawan; Sivakumar Pattathil; Albert M. Cheh; Ninad Kothari; Rajeev Kumar; John Ralph; Michael G. Hahn; Charles E. Wyman; Singh. Seema; Blake A. Simmons; Bruce E. Dale; Venkatesh Balan

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2016

Nitrogen fertilization challenges the climate benefit of cellulosic biofuels

Leilei Ruan; Ajay K. Bhardwaj; Stephen K. Hamilton; Philip Robertson

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2016

Nitrogen fertilization effects on productivity and nitrogen loss in three grass-based perennial bioenergy cropping systems

Brianna E.L. Duran; David S. Duncan; Lawrence G. Oates; Christopher J. Kucharik; Randall D. Jackson

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2016

Nitrogen (N) fertilization can greatly improve plant productivity but needs to be carefully managed to avoid harmful environmental impacts. Nutrient management guidelines aimed at reducing harmful forms of N loss such as nitrous oxide (N2O) emissions and nitrate (NO3-) leaching have been tailored for many cropping systems. The developing bioenergy industry is likely to make use of novel cropping systems, such as polycultures of perennial species, for which we have limited nutrient management experience. We studied how a switchgrass (Panicum virgatum) monoculture, a 5-species native grass mixture and an 18-species restored prairie responded to annual fertilizer applications of 56 kg N ha-1 in a field-scale agronomic trial in south-central Wisconsin over a 2-year period. We observed greater fertilizer-induced N2O emissions and sub-rooting zone NO3- concentrations in the switchgrass monoculture than in either polyculture. Fertilization increased aboveground net primary productivity in the polycultures, but not in the switchgrass monoculture. Switchgrass was generally more productive, while the two polycultures did not differ from each other in productivity or N loss. Our results highlight differences between polycultures and a switchgrass monoculture in responding to N fertilization.

Nitrous oxide emissions during establishment of eight alternative cellulosic bioenergy cropping systems in the North Central United States

Lawrence G. Oates; David S. Duncan; Ilya Gelfand; Neville Millar; Philip Robertson; Randall D. Jackson

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2016

Greenhouse gas (GHG) emissions from soils are a key sustainability metric of cropping systems. During crop establishment, disruptive land-use change is known to be a critical, but under reported period, for determining GHG emissions. We measured soil N2O emissions and potential environmental drivers of these fluxes from a three-year establishment-phase bioenergy cropping systems experiment replicated in southcentral Wisconsin (ARL) and southwestern Michigan (KBS). Cropping systems treatments were annual monocultures (continuous corn, corn–soybean–canola rotation), perennial monocultures (switchgrass, miscanthus, and poplar), and perennial polycultures (native grass mixture, early successional community, and restored prairie) all grown using best management practices specific to the system. Cumulative three-year N2O emissions from annuals were 142% higher than from perennials, with fertilized perennials 190% higher than unfertilized perennials. Emissions ranged from 3.1 to 19.1 kg N2O-N ha−1 yr−1 for the annuals with continuous corn > corn–soybean–canola rotation and 1.1 to 6.3 kg N2O-N ha−1 yr−1 for perennials. Nitrous oxide peak fluxes typically were associated with precipitation events that closely followed fertilization. Bayesian modeling of N2O fluxes based on measured environmental factors explained 33% of variability across all systems. Models trained on single systems performed well in most monocultures (e.g., R2 = 0.52 for poplar) but notably worse in polycultures (e.g., R2 = 0.17 for early successional, R2 = 0.06 for restored prairie), indicating that simulation models that include N2O emissions should be parameterized specific to particular plant communities. Our results indicate that perennial bioenergy crops in their establishment phase emit less N2O than annual crops, especially when not fertilized. These findings should be considered further alongside yield and other metrics contributing to important ecosystem services.

Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network

Meihua Christina Kuang; Paul D. Hutchins; Jason D. Russell; Joshua J. Coon; Chris Todd Hittinger

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2016

The evolutionary mechanisms leading to duplicate gene retention are well understood, but the long-term impacts of paralog differentiation on the regulation of metabolism remain underappreciated. Here we experimentally dissect the functions of two pairs of ancient paralogs of the GALactose sugar utilization network in two yeast species. We show that the Saccharomyces uvarum network is more active, even as over-induction is prevented by a second co-repressor that the model yeast Saccharomyces cerevisiae lacks. Surprisingly, removal of this repression system leads to a strong growth arrest, likely due to overly rapid galactose catabolism and metabolic overload. Alternative sugars, such as fructose, circumvent metabolic control systems and exacerbate this phenotype. We further show that S. cerevisiae experiences homologous metabolic constraints that are subtler due to how the paralogs have diversified. These results show how the functional differentiation of paralogs continues to s

OptSSeq: high-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

Indro N. Ghosh; Robert Landick

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2016

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression signals (promoters and ribosome-binding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression signals and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. In contrast to current dogma, we discovered that the Fe2+-dependent AdhB is preferable to Zn2+-dependent AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L

Dan Luo; Roberta Callari; Britta Hamberger; Sileshi Gizachew Wubshet; Morten T. Nielsen; Johan Andersen-Ranberg; Björn M. Hallstrom; Federico Cozzi; Harald Heider; ; Björn Hamberger

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2016

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Paving the way for lignin valorisation: Recent advances in bioengineering, biorefining and catalysis

Roberto Rinaldi; Robin Jastrzebski; Matthew T. Clough; John Ralph; Marco Kennema; Pieter C.A. Bruijnincx; Bert M. Weckhuysen

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2016

Lignin is an abundant biopolymer with a high carbon content and high aromaticity. Despite its potential as a raw material for the fuel and chemical industries, lignin remains the most poorly utilised of the lignocellulosic biopolymers. Effective valorisation of lignin requires careful fine-tuning of multiple "upstream" (i.e., lignin bioengineering, lignin isolation and "early-stage catalytic conversion of lignin") and "downstream" (i.e., lignin depolymerisation and upgrading) process stages, demanding input and understanding from a broad array of scientific disciplines. This review provides a "beginning-to-end" analysis of the recent advances reported in lignin valorisation. Particular emphasis is placed on the improved understanding of lignin's biosynthesis and structure, differences in structure and chemical bonding between native and technical lignins, emerging catalytic valorisation strategies, and the relationships between lignin structure and catalyst performance.

Pectin methylesterification impacts the relationship between photosynthesis and plant growth

Sarathi Maheshika Weraduwage; Sang-Jin Kim; Luciana Renna; Fransisca Chidinma Anozie; Thomas David Sharkey; Federica Brandizzi

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2016

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth is not known. To fill this gap, we analyzed Arabidopsis thaliana plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line overexpressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit area (LMA); however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and LMA.

Predicting plant attractiveness to pollinators with passive crowdsourcing

Christie A. Bahlai; Douglas A. Landis

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2016

Global concern regarding pollinator decline has intensified interest in enhancing pollinator resources in managed landscapes. These efforts frequently emphasize restoration or planting of flowering plants to provide pollen and nectar resources that are highly attractive to the desired pollinators. However, determining exactly which plant species should be used to enhance a landscape is difficult. Empirical screening of plants for such purposes is logistically daunting, but could be streamlined by crowdsourcing data to create lists of plants most probable to attract the desired pollinator taxa. People frequently photograph plants in bloom and the Internet has become a vast repository of such images. A proportion of these images also capture floral visitation by arthropods. Here, we test the hypothesis that the abundance of floral images containing identifiable pollinator and other beneficial insects is positively associated with the observed attractiveness of the same species in controlled field trials from previously published studies. We used Google Image searches to determine the correlation of pollinator visitation captured by photographs on the Internet relative to the attractiveness of the same species in common-garden field trials for 43 plant species. From the first 30 photographs, which successfully identified the plant, we recorded the number of Apis (managed honeybees), non-Apis (exclusively wild bees) and the number of bee-mimicking syrphid flies. We used these observations from search hits as well as bloom period (BP) as predictor variables in Generalized Linear Models (GLMs) for field-observed abundances of each of these groups. We found that non-Apis bees observed in controlled field trials were positively associated with observations of these taxa in Google Image searches (pseudo-R2 of 0.668). Syrphid fly observations in the field were also associated with the frequency they were observed in images, but this relationship was weak. Apis bee observations were not associated with Internet images, but were slightly associated with BP. Our results suggest that passively crowdsourced image data can potentially be a useful screening tool to identify candidate plants for pollinator habitat restoration efforts directed at wild bee conservation. Increasing our understanding of the attractiveness of a greater diversity of plants increases the potential for more rapid and efficient research in creating pollinator-supportive landscapes.

Prediction of cell wall properties and response to deconstruction using alkaline pretreatment in diverse maize genotypes using Py-MBMS and NIR

Muyang Li; Daniel L. Williams; Marlies Heckwolf; Natalia de Leon; Shawn Kaeppler; Robert W. Sykes; David Hodge

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2016

PtMo bimetallic catalysts synthesized by controlled surface reactions for water gas shift

Canan Sener; Thejas S. Wesley; Ana C. Alba-Rubio; Mrunmayi D. Kumbhalkar; Sikander H. Hakim; Fabio H. Riberio; Jeffrey T. Miller; James A. Dumesic

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2016

Supported PtMo bimetallic catalysts were prepared by controlled surface reactions (CSR) and studied for water gas shift (WGS) at 543 K. Carbon and silica supports were used for the preparation of monometallic Pt catalysts, and Mo was deposited onto these catalysts by reaction with cycloheptatriene molybdenum tricarbonyl ((C7H8)Mo(CO)3). Catalysts were characterized by CO chemisorption, inductively coupled plasma-atomic emission spectroscopy (ICP-AES), STEM/EDS, and XAS analysis. We report that carbon-supported Pt nanoparticles are saturated with Mo species at a Mo:Pt atomic ratio of 0.32. Molybdenum has a strong promotional effect in these catalysts, increasing the TOF by up to a factor of more than 4000. Silica-supported catalysts were found to be more active, but the TOF promotional effect of Mo was smaller than for the carbon-supported catalysts at 15. EDS analyses and activity studies showed that the formation of bimetallic catalysts was therefore more efficient using the carbon support. The active sites for WGS are suggested to be at the interface between Pt atoms and Mo moieties that are possibly in an oxidized form.

Quantifying pretreatment degradation compounds in solution and accumulated by cells during solids and yeast recycling in the Rapid Bioconversion with Integrated recycling Technology process using AFEX™ corn stover

Cory Sarks; Alan Higbee; Jeff Piotrowski; Saisi Xue; Joshua J. Coon; Trey K. Sato; Mingjie Jin; Venkatesh Balan; Bruce E. Dale

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2016

Effects of degradation products (low molecular weight compounds produced during pretreatment) on the microbes used in the RaBIT (Rapid Bioconversion with Integrated recycling Technology) process that reduces enzyme usage up to 40% by efficient enzyme recycling were studied. Chemical genomic profiling was performed, showing no yeast response differences in hydrolysates produced during RaBIT enzymatic hydrolysis. Concentrations of degradation products in solution were quantified after different enzymatic hydrolysis cycles and fermentation cycles. Intracellular degradation product concentrations were also measured following fermentation. Degradation product concentrations in hydrolysate did not change between RaBIT enzymatic hydrolysis cycles; the cell population retained its ability to oxidize/reduce (detoxify) aldehydes over five RaBIT fermentation cycles; and degradation products accumulated within or on the cells as RaBIT fermentation cycles increased. Synthetic hydrolysate was used to confirm that pretreatment degradation products are the sole cause of decreased xylose consumption during RaBIT fermentations.

Quantitative analysis of lignin monomers by a thioacidolysis method tailored for higher-throughput analysis

Anne E. Harman-Ware; Cliff Foster; Renee M. Happs; Crissa Doeppke; Kristoffer Meunier; Jackson Gehan; Fengxia Yue; Fachuang Lu; Mark Davis

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2016

We report on a quantitative thioacidolysis method that is accessible with general laboratory equipment and uses a non-chlorinated organic solvent and is tailored for higher-throughput analysis. The method utilizes lignin arylglycerol monomer standards for calibration, requires 1-2 mg of biomass per assay and has been quantified using fast-GC techniques including a Low Thermal Mass Modular Accelerated Column Heater (LTM MACH). Cumbersome steps, including standard purification, sample concentrating and drying have been eliminated to help aid in consecutive day-to-day analyses needed to sustain a high sample throughput for large screening experiments without the loss of quantitation accuracy. The method reported in this manuscript has been validated against a commonly used thioacidolysis method and across two different research sites with three common biomass varieties to represent hardwoods, softwoods, and grasses.

Rapid Py-GC/MS assessment of the structural alterations of lignins in genetically modified plants

Jorge Rencoret; José Carlos Del Rio; Klaas G.J. Nierop; Ana Gutiérrez; John Ralph

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2016

Genetic modifications for perturbing the lignin pathway in three different species of angiosperm plants,including non-woody (Arabidopsis and alfalfa) and woody (poplar) plants, were readily evaluated byanalytical pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS). Pyrolysis showedthat the composition of Arabidopsis plants was severely altered when the expression of the geneencoding the enzyme caffeic acid O-methyltransferase (COMT) was downregulated, resulting in a ligninlargely enriched in guaiacyl (G) units (88%). Alfalfa plants in which lignin biosynthesis was modified bydown-regulation of the p-coumarate 3-hydroxylase (C3H) gene, showed extremely high proportions ofp-hydroxyphenyl (H) units (71%) relative to the naturally prevailing guaiacyl (G) and syringyl (S) units.Finally, Py-GC/MS analyses indicated that overexpression in poplar of the gene that encodes the enzymeferulate 5-hydroxylase (F5H) resulted in a lignin with a higher content of syringyl lignin units (88%)compared to the wild-type control (71%). In conclusion, Py-GC/MS is a useful and convenient tool for therapid evaluation of compositional changes in lignin from genetically modified plants.

Reconstructing the backbone of the Saccharomycotina yeast phylogeny using genome-scale data

Xing-Xing Shen; Xiaofan Zhou; Jacek Kominek; Cletus P. Kurtzman; Chris T. Hittinger; Antonis Rokas

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2016

Understanding the phylogenetic relationships among the yeasts of the subphylum Saccharomycotina is a prerequisite for understanding the evolution of their metabolisms and ecological lifestyles. In the last two decades, the use of rDNA and multilocus data sets has greatly advanced our understanding of the yeast phylogeny, but many deep relationships remain unsupported. In contrast, phylogenomic analyses have involved relatively few taxa and lineages that were often selected with limited considerations for covering the breadth of yeast biodiversity. Here we used genome sequence data from 86 publicly available yeast genomes representing nine of the 11 known major lineages and 10 nonyeast fungal outgroups to generate a 1233-gene, 96-taxon data matrix. Species phylogenies reconstructed using two different methods (concatenation and coalescence) and two data matrices (amino acids or the first two codon positions) yielded identical and highly supported relationships between the nine major lineages. Aside from the lineage comprised by the family Pichiaceae, all other lineages were monophyletic. Most interrelationships among yeast species were robust across the two methods and data matrices. However, eight of the 93 internodes conflicted between analyses or data sets, including the placements of: the clade defined by species that have reassigned the CUG codon to encode serine, instead of leucine; the clade defined by a whole genome duplication; and the species Ascoidea rubescens These phylogenomic analyses provide a robust roadmap for future comparative work across the yeast subphylum in the disciplines of taxonomy, molecular genetics, evolutionary biology, ecology, and biotechnology. To further this end, we have also provided a BLAST server to query the 86 Saccharomycotina genomes, which can be found at http://y1000plus.org/blast.

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