Skip to main content Skip to navigation

Past Poster Abstracts

2019: Foundations for the Future: Embracing New Agricultural Technology

Hybrid wheat: a promising technology for feeding the future

Anil Adhikari

Annual wheat yield gains have not met the required 2.3% increase to meet the global demand and are currently leveling off at ~1%. Hybrid wheat is a promising approach to break the yield stagnation since it has potential to offer higher yield due to heterosis, yield stability and higher tolerance to biotic and abiotic stresses. To test this hypothesis, elite winter wheat lines from the wheat breeding programs of the University of Nebraska-Lincoln and Texas A&M University were crossed in a 25 x 25 full diallel design; using a chemical hybridizing agent to produce experimental hybrids in 2015 and 2016. These hybrids were planted in a modified augmented design with commercial checks and parents at McGregor, TX in 2016 (n = 612); Greenville and Bushland, TX in 2017 (n = 470) to evaluate for yield heterosis and combining ability. The grain yields of these trials were spatially corrected using spatial correction models in ASReml-R for downstream analysis. Commercial heterosis which is defined as hybrid performance compared to the best check, ranged between -78.3 to 20.4% in 2016 and -11.3% to 33.7% in 2017. High-parent heterosis ranged from -74.4% to 54.3% in 2016 and -24.3% to 29.4% in 2017. The yield of the best performing hybrid exceeded the yield of the best commercial check in 2016 by 0.6 ton ha-1 whereas the best performing hybrid in 2017 yielded 1.6 ton ha-1 higher than the best check. Yield components analysis from Greenville via path co-efficient analysis revealed the phenotypic basis of heterosis as higher number of effective tillers per unit area. General combining ability (GCA) variance was significantly higher than zero whereas specific combining ability (SCA) variance was not. This indicates that most of the heterosis is due to additive rather than dominance effect. These results highlight the possibility of exploiting GCA for higher yield whereas also underscore the need for development of heterotic pools to maximize SCA and dominance effects of heterosis.

Characterization of Brassica rapa RAP2.4-related Proteins in Stress Response and as CUL3-Dependent E3 Ligase Substrates Authors & Affiliations

Authors: Raed Al-Saharin, Sutton L. Mooney, Kyle Tucker, Chase Beathard, Christina M. Choi, Hanjo A. Hellmann

Turnip (Brassica rapa) has important economic value and represents a good model system to study gene function in crop plants. ERF/AP2 transcription factors are a major group of proteins that are often involved in regulating stress-responses and developmental programs. Some ERF/AP2 proteins are targets of CULLIN3-based E3 ligases that use BTB/POZ-MATH proteins as substrate receptors. These receptors bind the transcription factor and facilitate their ubiquitylation and subsequent degradation via the 26S proteasome. Here we show tissue and stress-dependent expression patterns for three Brassica rapa ERF/AP2 proteins that are closely related to Arabidopsis thaliana AtRAP2.4. Cloning of the Brassica genes showed that the corresponding proteins can assemble with a BPM protein and CULLIN3, and that they are instable in a 26S proteasome dependent manner. This work demonstrates the conserved nature of the ERF/AP2-CULLIN3-based E3 ligase interplay, and represents a first step to analyze their function in a commercially relevant crop plant.

Efficacy and Economic Viability of Organic Herbicides

Authors: Aaron Appleby and Lynne Carpenter-Boggs.

Organic agriculture suffers from a dearth of cost-effective weed management methods and materials. The difficulty and expense of managing weeds is cited as a major barrier to transitioning into organic and for successful organic production. Newly registered organic herbicides have entered the market and may help organic producers effectively and economically manage weeds. Very little research has been done on those newly registered organic herbicides, and previous research shows organic herbicides to not be an effective tool at removing above ground biomass of weeds. Without proper research on the topic, organic farmers are hesitant to spend money on new products that may or may not work as intended. We hypothesize that newly registered organic herbicides are as effective at removing aboveground biomass as mechanical weed management techniques. Each of the herbicide treatments, contains a different active ingredient: d-limonene, eugenol, acetic acid, and a mix of capric and caprylic acid. Based on two years of field research managing Canada thistle (Cirsium arvense) and field bindweed (Convolvulus arvensis) in a certified organic apple orchard, we found that all treatments had significantly less weed cover than the do-nothing control. However, Suppress®, with an active ingredient composed of the combination of capric and caprylic acid, was as effective at removing aboveground biomass of weeds compared to hand weeding. By adding an effective organic herbicide to existing weed management strategies, organic producers will be able to reduce weed pressure, till less often, decrease greenhouse gas emissions, and improve production and profitability.

Perturbation of Photosynthetic Performance Via Chloroplastic Expression of the Soluble N-Terminal Domain of Albino3

Authors: Ryan Christian, Scott Schaeffer, and Amit Dhingra

Albino3 (Alb3) is a membrane translocase and a homolog of bacterial YidC. It is required for biogenesis of the thylakoid membrane, and assembly of the photosystems and electron transport chain protein machinery. preAlb3 is a substrate of a heterodimer complex of Alb3 and cpSecYEG, the union of which permits transient permeability to ions during translocation of soluble loop domains across the membrane. Alb3 mutants and overexpression lines are affected in plant development, photosynthesis, and ionomics. We tested the hypothesis that this phenotype is dependent on membrane depolarization caused by Alb3/cpSecY interaction. A truncation series of the N-terminal soluble domain of Alb3 was generated and used to observe subcellular localization via confocal microscopy of transiently expressed GFP chimeras. Stable chloroplast transformations of the truncated series and full proteins were evaluated for physiological changes with pulse amplitude fluorometry, chlorophyll quantification, TEM, and ICP-MS elemental analysis. Truncation mutants corresponding to the N-terminal 1st and 2nd conserved motifs of Alb3 were determined to comprise the primary chloroplast transit peptide and to have no effect on physiology. In contrast, the 3rd conserved motif and the full N-terminal soluble domain were determined to generate effects on chloroplast morphology and photosynthetic efficiency independent of chlorophyll levels. Furthermore, overexpression of both the full Alb3 protein and 3rd and 4th N-terminal truncations showed changes to chloroplast ultrastructure and whole tissue K+, Mg2+, and Ca2+ concentrations. These data suggest that Alb3 truncation lines do not disrupt native translocase activity, but rather affect chloroplasts via perturbation of ion homeostasis.

Toward a new use for Carbon Isotope Discrimination in Plant Breeding

Authors: Liam Dixon and Arron H. Carter

Drought stress is the greatest threat to wheat (Triticum aestivum L.) productivity worldwide. The development of tolerant varieties is complicated by the variability associated with drought stress, including timing of onset and severity. A successful breeding approach will combine knowledge of the specific drought impacting a region with knowledge of the genetic traits appropriately suited to mitigate the yield losses. Many regions suffer from terminal drought. A number of traits are associated with improved yield in a terminal drought environment, most notably water use behavior, deep roots, and reliance on stem reserves. These traits are challenging to screen for, however, ultimately slowing progress toward tolerant cultivars. Carbon isotope discrimination (∆) analysis of mature grains may serve as a relatively high-throughput approach to identify lines exhibiting these traits. This study analyzed grain ∆ of 480 advanced winter wheat varieties in five distinct terminal drought environments. Considering all environments, 30 high yielding, low ∆ and 52 high yielding, high ∆ genotypes were identified. Based on preliminary evidence, the high yield, low ∆ genotypes from Pullman 2015—the most severe drought environment—are suspected of conservative water use, whereas the high yield, high ∆ genotypes from Pullman 2016—the least severe drought environment—are suspected of high water use. In the event that future studies can confirm the water use behavior of these lines, grain ∆ may be recommended as an initial screening tool for more specific drought tolerance traits—representing an entirely new use for grain ∆ to plant breeding.

Identification of resistance QTL to Fusarium Crown Rot, and Identification of the Role of Lignin in Resistance

Authors: Aichatou Djibo Waziri, Karen Sanguinet, Kimberly Garland-Campbell

Fusarium crown rot caused by a complex of fusarium species is an understudied disease. The pathogen attacks the roots and stem base, kills the plant tissue and prevent water and nutrient transport towards the upper parts of the plant. The disease causes yield reduction up to 35% in the pacific Northwest (Smiley et al, 2005) of the United States and 58% in Australia (Murray and Brennan. 2009, Nicol et al. 2001). Any agricultural management sole or in combinations has been able to completely control for disease occurrence and spread. Additionally, there does not exist any fully resistant cultivars. Therefore, breeding for resistant cultivar is considered as the most efficient alternative
Our research is focusing on determining novel quantitative trait loci associated with disease resistance, or at least confirm in our population those that had already been found in other genetic background. We are screening a population derived from a susceptible parent and an Iranian landrace resistant to multiple diseases.
Another difficulty associated with breeding for resistant cultivars to the disease is a limited knowledge of the physiological mechanisms triggered in resistant plants (Bhandari et al. 2018, Powell et al. 2016). However, few studies have suggested a sudden synthesis of cell-wall strengthening chemical compounds ensuing infection by the pathogen (Bhandari et al. 2018). Along that line, we are also investigating the role of lignin total content in resistance to pathogen penetration.
Preliminary analysis of the root-total-lignin content in the two parents showed as expected a higher concentration of lignin in ‘IWA8608077’ the resistant parent than in Louise the susceptible parent.
We are currently working on data for QTL mapping from field and greenhouse phenotyping.

Genotyping by Multiplexed Sequencing in Barley

Authors: Jonathan Eagle, Travis Ruff, Marcus Hooker, Sajal Sthapit, K. Marlowe, Dolores Covarrubias, Daniel Skinner, and Deven See

Currently genotyping by sequencing (GBS) and single nucleotide polymorphism (SNP) chip technologies are the primary genotyping technologies for genetic selection (1,2). The GBS protocol is difficult to analyze because it produces random pieces of ligated DNA which are used for SNP discovery. SNP chip technology is based on a hybridization technique which results in a dot plot that may misrepresent true allelic states. We have developed a robust protocol that uses informative markers from 90k SNP chip. Results from this protocol are more informative than GBS or SNP chip technologies because they are derived from direct sequence reads. This PCR based direct sequencing protocol provides a more streamlined and accurate analysis process, by multiplexing thousands of markers into a single sequencing run. This protocol is highly adaptable allowing marker sets to be customized to the species of interest and the objective of the study. We have identified more than 1,055 markers that work well in barley with this protocol, 651 of which are from barley SNP discovery efforts and 404 are from wheat 90k SNP chip (2,3). This map has a total of 1,055 markers; 150 map to chromosome 1H, 151 to 2H, 133 to 3H, 130 to 4H, 211 to 5H, 112 to 6H, and 168 to 7H.

The Transcription Factor SlBSD1 Is a Substrate of the SlSINA1 Ubiquitin Ligase

Authors: Youhong Fan and Fangming Xiao

Transcription factors (TFs) play key roles in plant development by temporarily and spatially regulating the transcription of their target genes. As master regulators, these TFs themselves are subjected to various transcriptional and post-transcriptional regulations, as well as post-translational modifications, including ubiquitination. The E3 ubiquitin-ligases, as the major component in the ubiquitination pathway, confer specificity of substrate recognition. Previously, we reported that SlSINA1, a tomato homologue of the Drosophila SINA RING-finger protein, possesses RING-dependent E3 ubiquitin ligase activity. In this work, we demonstrate that a novel nucleus-localized BSD domain-containing transcription factor, designated as SlBSD1, is a direct substrate of SlSINA1. SlBSD1 was identified based on a yeast two-hybrid screening using SlSINA1 as bait. Further yeast two-hybrid and pull-down assays confirmed that SlBSD1 interacts with SlSINA1. Significantly, in vitro ubiquitination and in vivo transient expression suggested that SlSINA1 ubiquitinates SlBSD1 promoting its degradation. Overexpression of SlBSD1 causes male sterile in tomato. Together, these results suggest that the SlBSD1 transcription factor plays a role in pollen development and is subjected to ubiquitination-mediated degradation by SlSINA1 ubiquitin ligase.

Effects of Overexpression of AtSeipin and CeFAT5 on oil composition and recovery in CeFAT5 expressing Camelina sativa

Authors: Nick Fisher, Jesse Bengsston and John Browse

Camelina sativa is an up and coming oil seed crop which contains saturated fatty acids. The nutritional value of the Camelina sativa seeds can be improved by decreasing the amount of saturated fatty acids and increasing the amount of unsaturated fatty acids. Past work in Arabidopsis has shown that the Caenorhabditis elegans desaturase CeFAT5 reduces the amount of 16:0 (Palmitic acid) while increasing the amount of 16:1 (Palmitoleic acid) but at the cost of reducing total seed oil content. The Seipin1 gene in Arabidopsis has been shown to effectively recover oil in oil deficient plants. When both CeFAT5 and Seipin1 were expressed in Camelina, out of the 30 seed lines analyzed, each transgenic line contained more unsaturated fatty acids than the wild type average, thus improving both the nutritional and industrial value. There appeared to be no significant correlation between total oil content and expression of the transgenes using CeFAT5 activity as a proxy for expression, indicating that the loss of total oil due to CeFAT5 may have been recovered due to Seipin.

Identification of Molecular Strategies to Combat Pale Cyst Nematode in Potato

Authors: Rachel Gross, Allan Caplan, Joseph Kuhl, Louise-Marie Dandurand, and Fangming Xiao

Potatoes are a significant crop in America, bringing in 4 billion dollars nationally in 2017(NASS, 2017). Therefore, it is very important to stay one step ahead of pests and diseases. One of those pests is Globodera pallida, Pale Cyst nematode (PCN). PCN was not a concern in the US until 2006 when several fields in Eastern Idaho were quarantined. PCN is very damaging and can cause up to 80% yield loss to potatoes (Greco, 1988). Cysts can remain in the soil for up to 30 years and are highly resistant to pesticides. One approach to fighting PCN is to generate resistant potato cultivars by finding resistance gene(s) and introducing such gene(s) into potatoes. Litchi tomato, a relative of potato, is resistant to PCN, presumably by recognition of PCN effector proteins by litchi tomato R gene(s). We have found a specific PCN effector, termed Gp0400, that can elicit HR-cell death in Nicotiana benthamiana and tomato leaves. Gp0400 is localized in the subventral glands of the nematode, and contains two enzymatically active thioredoxin domains. By creating deletion mutants, we discovered that the N-terminal thioredoxin domain is required to trigger cell death. However, the ability to trigger cell death is not dependent on the enzymatic activity of this domain. Using a combination of immunoprecipitation and mass spectrometry, Gp0400-interacting proteins from potato, tomato and litchi tomato were identified. We hope to determine how Gp0400 interacts with host plant proteins to elicit a defense response which could be exploited for resistance engineering in potato plants.

The cyst nematode effector GpRHA1B ubiquitinates the CcR4-NOT deadenylase complex to promote its degradation

Authors: Li Huang, Joanna Kud and Fangming Xiao

Globodera pallida, a plant endoparasites cyst nematode, significantly threats global agronomically important crops. It uses a hollow mouth spear to puncture effectors into host cells to modify the root cell and form a feeding site to complete its lifecycle. These manipulations are accompanied by transcriptomic and proteomic changes in host plants. However, the mechanisms behind such changes are poorly understood. Here we focus on a novel nematode effector GpRHA1B, a Ring-finger E3 ligase that plays a significant role in nematode parasitism. We found GpRHA1B can interact and ubiquitinate the host CcR4-NOT deadenylase complex, which can shorten targets mRNA poly-A tails and suppress their translation. GpRHA1B can degrade at least two subunits of the CcR4-NOT, CcR4-NOT10 and CAF1, of which the latter is a putative deadenylase. We concluded that nematode secretes effector GpRHA1B to modulate the host CcR4-NOT deadenylase complex to manipulate the translation process of host cell.

Weathering Heights: Weathering Heights: Comparison of mating behavior in Apis mellifera utilizing RFID

Authors: Melanie M. Kirby

The improvement of honey bee stocks through selective breeding represents one sustainable approach to assure future pollination services for food production and security, and to maintain and enhance the queen production industry, which struggles to meet market demand in the early spring. The goal of this research is to improve our understanding of honey bee (Apis mellifera) mating flight behavior of various strains utilizing RFID (radio frequency identification). The findings could improve breeding protocols to enhance production and minimize losses. Inadequate mating of queens is known to limit quality and affect the capacity of producers to provide quality stock. RFID technology will permit evaluation of a larger set of genotype x environment combinations and number of mating events than has been possible using direct observation. Field testing of the initial RFID units designed by WSU engineering undergraduates occurred this past 2018 spring and summer. This project is in continuation with modification of RFID readers to solar power which will allow data collection in sites that do not have power access. Results will provide a fundamental improvement of our understanding of honey bee mating behavior and enhance profitability of queen producers and beekeepers. With this information, producers will be better able to select, integrate, and propagate appropriate honey bee stocks relative to seasonal weather conditions.
1) Are there observable differences in virgin queen and drone mating flight periods and duration among Apis mellifera subspecies/strains?
2) Are there significant interactions between genotype and environment in mating behavior in honey bee subspecies/strains?

Peanut Seed Imaging Using the Opticount Seed Counter

Authors: Roshan Kulkarni, Paul Otyama, Ethalinda Cannon, Alan Gaul, Greagory MacDonald, and Steven B. Cannon

In-depth phenotypic characterization is desirable for extracting extensive phenotypic variations and for accurate associations of phenotype to a genotype. In this study we demonstrate the use of the OptiCount seed counter for the high quality and high throughput imaging of peanut seeds. Seeds from around 800 accessions, representing the U.S. peanut core collection, were imaged using an OptiCount machine (Process Vision LLC), calibrated and programmed for taking measurements on peanut seeds. Seed phenotypic characters including seed length, seed thickness, seed volume, percentage splits, percentage oblongs, seed weight, thousand seed weight and seed color were obtained for all the accessions. Some parameters such as seed shape were calculated from the axis ratios obtained from the OptiCount. Seed color and color variation were captured, using red, green, and blue pixel values. We plan to use phenotypic data obtained from this study to complement genotyping for association studies such as GWAS.

Correlating Near-Infrared Spectra of Kernels to Grain Yield in Maize

Authors: Holly M. Lane, Seth C. Murray, Ivan D. Barrero Farfan, David Rooney, and Cristine L.S. Morgan

Conducting field evaluations to select best yielding varieties in breeding programs is time-consuming and expensive. Reducing replications or improving data accuracy has potential to save resources and improve selection ability. High-throughput phenotyping tools offer potential for inexpensive, fixed-cost methods of indirectly evaluating best yielding varieties. One potential method is Near-Infrared Reflectance Spectroscopy (NIRS), commonly used to estimate characteristics of plants including protein, starch, and oil content, and successful at differentiating between species and even genotypes. This project evaluates the ability of Fourier Transformed NIR spectra (FT-NIRS), of maize (Zea mays L.) grain samples, to predict yield as a phenotype. Samples were grown over two years in College Station, Texas as part of a structured genome wide association study (GWAS) of 346 lines crossed to Tx714. The dataset consists of approximately 2300 scans of whole kernel samples, collected with corresponding yield data on a plot basis. PLSR models were built with the first derivative of the entire spectra to predict yield. After eliminating samples below 25 bu ac-1, the average yield for the data set was 94 (range 25 to 233) bu ac-1. Preliminary results show root mean standard error of prediction (RMSEP) of less than 25 bu ac-1 on a spectrally representative validation set of 750 samples; achieved using both 2011 and 2012 spectral data to train the model. When training the model on 2011 spectral data alone, the model predicts all 2012 samples with a RMSEP of 36.5 bu ac-1. Ultimately, NIRS offers a low-cost method of analysis for breeders, and its ability to be evaluated for phenotypes beyond traditional grain composition, such as yield, could save money by reducing field testing. Potential uses include improving accuracy of combine yield data, allowing single ear hand-harvested yield estimates, or for eliminating varieties predicted to do poorly under a certain threshold level.

Seed Specific Expression of Engineered Synechococcus elongates DES9 desaturase in Camelina sativa reduces saturated fatty acids.

Authors: Anh Le, Jesse Bengtsson, Shuangyi Bai, and John Browse

Camelina sativa (false flax), even though is not a prominent plant seed, but has been used in Europe as an oilseed since the middle ages. Camelina seed is known for its high amount of omega-3 fatty acids, which makes it suitable for use as a nutritional supplement and as an industrial supplement. This project was proceeded to find out a potential source of unsaturated and polyunsaturated fatty acids using Cyanobacterial D9 fatty acid desaturase from Synechococcus elongates. Directed evolution was used to identify mutations that result in favorable amino acid change. One such change, Q240R was found to give a dramatic increase in total desaturation activity. DES9* variants expressed in Camelina and driven by the seed-specific phaseolin promoter resulted in a reduced proportion of saturated fatty acids in the seed oil along with a generally more favorable fatty acids profile.

Characterization of the SEVEN IN ABSENTIA (SINA) Ubiquitin Ligase Family in Tomato

Authors: Cheng Li, Wenjie Wang and Fangming Xiao

Seven in absentia (SINA) protein is one subgroup of ubiquitin ligases possessing an N-terminal cysteine-rich Really Interesting New Gene (RING) domain, two zinc-finger motifs and a C-terminal domain responsible for substrate-binding and dimerization. In tomato (Solanum Lycopersicum), the SINA family has six members possessing RING-dependent E3 ubiquitin ligase activity, exhibiting similar specificity towards the E2 ubiquitin-conjugating enzyme. The functionality of SlSINA proteins has been investigated. SlSINA4 plays a positive role in defense signaling, as manifested by elicitation of E3-dependent hypersensitive response (HR)-like cell death; the other SlSINAs are negative regulator and capable to suppress HR cell death. In particular, SINA1 can interact with the tomato resistance protein Prf and trigger its degradation, suggesting SINA1 regulates the stability of Prf via ubiquitination-mediated degradation. Transgenic tomato plants overexpressing SlSINA2 exhibit pale-green leaf phenotype, suggesting SlSINA2 regulates chlorophyll level in plant cells; whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma, implicating SlSINA5 plays a role in flower development.

Phenotyping Aphanomyces Root Rot Resistance in Peas using Volatile Organic Compounds

Authors: Afef Marzougui, Abirami Rajendran, Yu Ma, Scott D. Mattinson, Rebecca J. McGee, and Sindhuja Sankaran

Plants produce a wide range of biogenic volatile organic compounds (BVOCs) in response to environmental factors. The response can be manifested as a change in the profile of BVOCs, either by stimulating or suppressing the emissions or as an induction of de-novo synthesis of new compounds. Recent insights on high-throughput phenotyping have led to hypothesize that monitoring the change of BVOCs profiles could provide a non-destructive means to phenotype traits such as disease resistance. In this study, we investigated the role of a soil-borne pathogen Aphanomyces euteiches on the pea plant infection process that contributed to the changes in BVOCs released using a resistant and a susceptible pea cultivar. We have used analytical chromatographic techniques for monitoring BVOCs emitted from a total of 12 pea plants grown under two treatments (control and inoculated) for about one month. We collected volatiles through head-space Solid Phase Micro-Extraction (SPME) dynamic sampling in combination with Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectroscopy (GC-MS) for compound identification. At the end of the experiment, we analyzed the profile of pea root and shoot samples using (i) solvent extraction coupled with GC-FID and GC-MS, and (ii) Pyrolysis-GC-MS (Py-GC-MS). We established a method for peak analysis of the resulting chromatograms. Three major compounds were found from the analysis of volatile profiles. The BVOC data is further being analyzed along with comparison to ground-truth data (visual scores, plant biomass, etc.). We believe that identifying BVOCs associated with Aphanomyces root rot resistance could help plant breeders in accurately and objectively phenotype plants for disease resistance.

Does disease-resistance tradeoff with symbiotic function in domesticated Soybean?

Authors: Jordan Schinke, Zoie Lopez, Angeliqua Montoya, Emily Helliwell, Miles Roberts, Kyle Nguyen, Niall Millar, the WSUV 2018 Ecology 372 class, and Stephanie Porter

The process of domesticating crop plants improves many qualities of staple food crops, such as crop yield and pathogen resistance. Crop species can undergo evolutionary tradeoffs during domestication, where certain traits are artificially selected for result in the degradation of other functions. Artificial selection on agriculturally beneficial traits can result in crops that shunt resources to early, large yield traits, while sacrificing allocation to costly symbiosis traits. Pathogen defense traits also represent an important class of favored traits that can tradeoff with symbiotic function, as both pathogen resistance and symbiotic functionality with soil microbes utilize similar molecular pathways. We performed a greenhouse experiment to test 1) whether domesticated soybean, Glycine max, is less responsive to rhizobial inoculation than its wild progenitor species, Glycine soja, and 2) whether susceptibility to the pathogen Xanthomonas axonopodis pv. glycines (Bacterial Pustule Disease) tradesoff with responsiveness to symbiotic soil bacteria among G. max cultivars. Undergraduates from the 2018 Ecology 357 class at Washington State University Vancouver, grew 460 plants of 22 different Glycine genotypes in a randomized block design, with selected genotypes of G.max varying in the degree of susceptibility to Bacterial Pustle. Plants were either inoculated with Bradyrhizobium japonicum USDA 110 symbionts, or left uninoculated to measure the growth response to symbiosis. Students harvested plants five weeks after planting and measured shoot dry weight and nodule biomass. Our results confirm that growth in G.max is less responsive to the rhizobial symbiosis than its wild progenitor. However, we found that resistance to Bacterial Pustule in G. max is positively correlated with the responsiveness of growth to rhizobial inoculation. While symbiotic function appears degraded in G. max, this does not appear to be due to a tradeoff due to selection for disease resistance.

Effects of Management on Dynamic Soil Health Properties & Enzymatic Activities

Authors: Katherine Naasko, Haiying Tao, William Pan, Isaac Madsen, and David Huggins

Our goal was to assess the varying effects of conventional till and no-till practices (till, no-till and native grassland) on dynamic soil health properties in the Palouse. Enzymatic activity of p-nitrophenyl were quantified using a colorimetric enzyme assay. We studied management practice effects on carbon, nitrogen and phosphorus cycling enzyme activities including β-glucosidase, β-glucosaminidase, phosphatase, phosphosdiesterase and arylsulfatase. We compared these results to carbon and nitrogen concentrations to analyze the impact of management on enzyme productivity and nutrient cycling.

The Genetics of Exploitation in Mutualism

Authors: Kyle Nguyen, Miles Roberts, Emily Helliwell, Joel Griffitts, Paul Price, Maren Friesen, Zoie Lopez, and Stephanie Porter

Plants have long adapted to coexist with soil microbes. Legumes can form beneficial symbioses with microbial partners, establishing a mutualism. However, a dilemma in mutualism occurs because selection can favor uncooperative symbionts that obtain benefits from a plant but confer inadequate resources in return (ie cheaters). Hosts and symbionts may be intertwined in a race of mutual exploitation driven by fitness conflicts over net benefits acquired by cooperation and cost of being a cooperator. We are interested in how cooperation between mutualists evolve in a biological invasion, specifically between an invasive weed (Medicago polymorpha) and its rhizobial symbiont (Ensifer medicae). Host range restriction peptidase (hrrP) is a plasmid-borne peptidase found in some E. medicae lineages that can render a strain capable of cheater behavior by cleaving cysteine-rich host peptides that enforce cooperation. We conducted a greenhouse experiment to measure impact of variation in the hrrP locus on symbiotic nitrogen fixation using 12 wild hrrP+ strains of E. medicae and isogenic knockout counterparts. With one M. polymorpha genotype (RTM) as the host, a randomized block design was used with 425 plants, each inoculated with a unique strain of E. medicae. Knocking out hrrP did not impact symbiotic phenotypes for many strains but had large impacts in others. In RTM196, functional hrrP decreases plant shoot mass, however shoot mass was increased in PEA63. HrrP alleles may differ in their impact on host fitness or variation could be due to other genes present in the genome. Our findings suggest hrrP can play a large role in modulating symbiotic outcomes however its impacts are variable. This presents a new hypothesis for the evolution of cooperation whereby an optimal level of host peptides results in a cooperative symbiosis and altering symbiotic outcomes by decreasing the abundance of cystine-rich peptides in the nodule can have positive or negative effects on cooperation.

Genetic Estimation of Straw Residue Decomposition in Winter Wheat

Authors: Nathan Nielsen, Arron Carter, and Tami Stubbs

No-till farming is an excellent option to minimize soil erosion. However, adoption of no-till farming in Eastern Washington has been slow due to difficulties of managing straw residue in no-till systems. We hypothesize that a genome-wide association study (GWAS) will identify single nucleotide polymorphisms (SNPs) that can assist in understanding the genetic loci associated with straw decomposition. The straw from a panel of 468 soft white winter wheat cultivars in the Pacific Northwest was harvested over two years at three locations throughout Eastern Washington. The samples were analyzed for several decomposition constituents, including NDF, ADF, ADL, cellulose, and hemicellulose using a wet chemistry procedure whereas carbon and nitrogen were determined using dry combustion. This panel was genotyped using the 90K Illumina SNP chip and a GWAS was conducted using FarmCPU implemented in the statistical program R. SNPs significantly associated with straw breakdown characteristics were identified on chromosomes 1B, 2A, 2B, 3B, 4A, 4B, 5B, 6A, 6B, and 7D. ADF was the most useful trait for identifying chromosomal loci of interest. IWB43355 on chromosome 4B was associated with both ADF and NDF, whereas IWB80987 on chromosome 5B was associated with both ADF and cellulose. On chromosome 6B, IWB28178 (associated with ADF) and IWB9377 (associated with hemicellulose) were located within 4 cM of each other. Nevertheless, the distribution of the SNPs across chromosomes demonstrates the genetic complexity of these straw breakdown constituents. Selection for straw breakdown characteristics may be better served using genomic selection rather than a GWAS approach.

Gaining a functional understanding of the nuclear-encoded genetic space of the chloroplast using a gene family targeting amiRNA library in Arabidopsis thaliana

Authors: Anna Pratta, Felix Hauserb, and Hans-Henning Kunza

The chloroplast plays a central role in plant cell function; despite this, the majority of proteins active in the chloroplast have yet to be studied. A high-throughput screening method that efficiently targets the entire chloroplast proteome would allow for work that elucidates the complex network of gene expression and physiological function within the chloroplast. Here, I describe a mechanism for this methodology using an artificial microRNA (amiRNA) library in a large-scale mutagenesis screen, coupled with phenotypic analysis, which allows for rapid generation of mutants with identifiable phenotypes within the first generation. This amiRNA library causes efficient and simultaneous knock-down of entire gene families from a single expression construct, bypassing the problem of functional redundancy, while generating phenotypes with a single transgenic event. Further, we propose the use of this amiRNA library to identify genes responsible for Ca2+ flux within the chloroplast.

Visualization and Modeling of Population Structure for GWAS using Autoencoders

Authors: Samuel Revolinski, Chunpeng James Chen, and Zhiwu Zhang

Principal Components (PCs) derived from genetic markers are commonly used to reveal population structure and used as cofactors for testing marker-trait association at the expense of diluting the association due to highly confounding between testing markers and PCs. For examples, for simulated traits with population structure confounded with environmental effects, it is necessary to include PCs as cofactors in Genome-Wide Association Studies (GWAS) to reduce false positives by using FarmCPU (Fixed and random model Circulating Probability Unification) method. On the other hand, fitting PCs as cofactors decreases statistical power when environmental effects do not confound with population structure. It is in critical need to find alternatives that can reduce spurious associations and increase true positives. We investigated the impact of fitting as cofactors in FarmCPU method. The autoencoder was created using a neural network with seven layers, including the input layer, 2 hidden encoding layers, a middle latent layer, two decoding layers, and the output layer. Keras, a Python library was used for optimization (ADAM) with 20-30 epochs. Datasets from multiple species were examined, including cattle, pig, and wheat. These populations include both association populations that were collected from diverse individuals (cattle and pig) and the joint linkage population with a common parent and a limited number of families (wheat). Visually, autoencoders demonstrated population structures that were not able to be seen using PCs. For simulated traits in populations without environmental effects confounded with structure, PCs decreased and autoencoders increased statistical power for GWAS.

Changes in lag time underlie the trade-off between nickel tolerance and growth in Mesorhizobium

Authors: Miles Roberts, Angeliqua Montoya, Zoie Lopez, and Stephanie Porter

Although most of life’s genetic diversity resides in bacteria, biologists have not fully explained why. It is often assumed that trade-offs, where improving one trait comes at a cost to another trait, are the main drivers of bacterial diversity. Yet trade-offs and their underlying molecular mechanisms have not been described in many lineages of wild bacteria. It was previously found that, in one clade of wild Mesorhizobium bacteria, nickel tolerance trades-off with growth in the absence of nickel. However, it is not known which aspect of growth, growth rate or lag time, contributes most to this trade-off. To investigate this question, we grew Mesorhizobium strains of varying nickel tolerance in nickel-rich and nickel-poor media and estimated their maximum growth rates and lag times using the R package GrowthRates. We found that the growth of almost all strains significantly differed between the two mediums. For growth in the presence of nickel relative to the absence of nickel, about the same number of strains had lower intrinsic growth rates as the number that had increased lag time, and some strains had both. Among the strains with higher lag times, their increase in lag time was more dramatic than any changes in their growth rate. Furthermore, the proportional differences in lag time between nickel-rich and nickel-poor media negatively correlated with nickel tolerance, whereas the proportional differences in growth rate did not correlate with nickel tolerance. This trend is consistent with the mechanism of an exclusion-nutrient access trade-off, where the exclusion of nickel from a cell also excludes nutrients that are required for growth to begin. Conducting more experiments like this one may narrow down the mechanism further and contribute to a molecular-level understanding of how trade-offs impact diversity. Specifically understanding trade-offs in Mesorhizobia, a genus that contributes to plant productivity, could also yield useful agricultural applications.

Diverse microbial mutualists benefit plant growth

Authors: Katelyn Sedig, Zoie Lopez, Miles Roberts, Kyle Nguyen, Christian Love, Kathleen Ibarreta, Jordan Schinke, Cameron Green, and Stephanie Porter

A diversity of soil microbes associate with plants. Those that are mutualists can provide critical limiting resources or services to host species in exchange for host resources. Experiments demonstrate that individual strains of mutualistic microbes vary dramatically in the fitness benefits they confer to their hosts. However, most hosts associate with many strains of microbes simultaneously and we lack a systematic assessment of how multiple symbiont strains impact host growth compared to each strain singly. We ask, are the effects of multiple symbiont strains on plant fitness equal to the mean of individual symbiont effects, or could there be positive or negative synergies among symbionts on plant fitness? Positive synergies could occur if hosts preferentially allocate resources to more beneficial strains or if strains provide complementary benefits to a host. Negative synergies could occur if competition among symbionts is detrimental to symbiotic outcomes or if less beneficial strains are more competitive for colonizing a host. To determine which scenario is broadly supported in the literature, we used a meta-analysis of 28 published studies on the legume-rhizobium symbiosis, spanning 18 plant species, and 26 published studies on the plant-mycorrhizal symbiosis, spanning 33 plant species. We show that inoculation with multiple microbe strains tends to provide a greater benefit to host fitness than the strains singly, though this pattern is stronger for rhizobia than for mycorrhizae. Consistent with their designation as mutualists, the fitness benefit of inoculation with these symbiotic bacteria and fungi is positive, with the benefit of rhizobia being greater for plants than the benefit of mycorrhizae. These results suggest that symbiont diversity has positive impacts on plant fitness, and that the benefit of diversity is different in the rhizobial and mycorrhizal mutualism.

Improving preharvest sprouting tolerance without sacrificing emergence: a genomic prediction approach

Authors: Stephanie Sjoberg, Camille Steber, and Arron Carter

Preharvest sprouting (PHS) in wheat is the germination of matured grains on the mother plant causing significant price reduction and profit loss. The major obstacle to increasing PHS tolerance through seed dormancy is reduced rates of fall seedling emergence and associated decreased yield. The objective of this study was to develop wheat varieties that have tolerance to PHS and vigorous and quick emergence from deep planting. PHS tolerance and emergence in 720 Pacific Northwest-adapted winter wheat lines were recorded using field and lab methods across three years. Best linear unbiased estimates per line were calculated for each trait, within and across environments, using the ASreml package in R. Genomic data for 35,910 markers collected from all lines was used in a genome-wide association mapping study to detect genetic loci associated with PHS tolerance and field emergence with the GAPIT package. The same data was used to calculate breeding values and validate genomic prediction models with the rrBLUP package. Association mapping revealed three significant loci related to emergence on chromosomes 3B, 5A, and 7A and four significant loci related to PHS on chromosomes 1B, 2A, 3B, and 4D. The accuracies of prediction models ranged from 0.34 to 0.47 depending on the trait. Our results demonstrate that a combination of association mapping and genomic prediction modeling could break the inverse correlation between both traits, allowing improvement of PHS tolerance without compromising emergence in winter wheat varieties.

Genetic diversity in historical and modern wheat varieties of the Pacific Northwest

Authors: S.R. Sthapit, K. Marlowe, J. Eagle, T.M. Ruff, M.A. Hooker, D.Z. Skinner, and D.R. See

The Pacific Northwest (PNW) is a major wheat producing region that accounts for over 15% of the United States’ annual wheat production. More than 300 public sector hexaploid wheat varieties have been grown in the region since 1900. We explore how genetic diversity in wheat cultivated in the PNW states of Idaho, Oregon and Washington has varied over time. Genotyping by Multiplexed Sequencing (GMS) was used to genotype 263 wheat varieties released for cultivation in the PNW region between 1904 and 2018. The varieties were categorized by decade of release and gene diversity was calculated using 712 polymorphic SNP markers with mean distance of 6.41 cM between them. Markers on chromosomes 1D, 2B, 5A, 7A and 7B were the most informative with a median PIC value of 0.40 or higher, while markers on chromosome 4D were the least informative (median PIC < 0.01). Gene diversity, defined as the probability of two randomly sampled alleles being different, has stayed stable over the century for both spring (between 27-29%) and winter varieties (between 24-27%). Among market classes, only hard red spring has seen a decline in gene diversity from 26% pre-1970 to 18% today. Principal coordinate analysis indicates that PNW wheat varieties have genetically diverged from each other based on growth habit (spring vs. winter) and market classes over time while maintaining similar gene diversity in most cases.

Effectors from Different Pathogens Target the Same Host Plant SINA Ubiquitin Ligase Family to Promote Pathogenicity

Authors: Yulin Yuan and Fangming Xiao

The Ubiquitin-26S proteasome degradation system (UPS), which mainly promotes proteins’ degradation, plays a vital role in host immune responses to invading pathogens. Tomato seven in absentia ubiquitin ligase (SINA) proteins carry a structure homologous to RING-finger/U-box E3 Ub ligases. Two unrelated effectors AvrptoB and RHA1B, respectively secreted from the bacterial pathogen Pseudomonas syringae and nematode pathogen Globodera pallida, are functional ubiquitin ligase. We found both AvrPtoB and RHA1B interact with tomato SINA proteins in plant cells, suggesting different pathogens may use a similar strategy to interfere with host immune system for their benefits, which, in this case, may be achieved through manipulation of the host UPS. Consistent with this hypothesis, we found both AvrPtoB and RHA1B can trigger degradation of endogenous targets of SINA proteins, implying that P. syringae and G. pallida use AvrPtoB and RHA1B, respectively, to hijack SINAs’ substrates to subvert host immunity and/or exploit host resources for colonization.

Monitoring flowering in cool-season crops using proximal and aerial remote sensing techniques

Authors: Chongyuan Zhang, Wilson Craine, Rebecca McGee, George Vandemark, James B. Davis, Jack Brown, Scot H. Hulbert, and Sindhuja Sankaran

Time and duration of flowering period are two of the important selection criteria in plant breeding, as these traits describe the robustness of the variety in escaping stress (drought) and contribute to high yield potential. Visual assessment is a standard protocol used for phenotyping flowering, which can be low-throughput and subjective, and limit the frequency of data acquisition. To address these limitations, high-throughput phenotyping technologies for flower detection was developed and applied to four cool-season crops, including pea, chickpea, canola, and camelina, in this study. Two sensing methods (proximal and aerial sensing) were used to collect data with visible, near infrared, and multispectral cameras. The results showed that lower flying altitude is needed to image small flowers (⁓15 – 30 m above the ground level), compared to assessment of other traits (e.g. canopy area and plant vigor). Results from canola data acquired using RGB camera (2017) demonstrated that positive linear correlations were found between proximal and aerial data, as well as between sensing data and visual ratings (P < 0.0001). Currently, more comprehensive data analysis on all crops are being performed to assess the feasibility of phenotyping flowers using sensing techniques. With the right sensor selection, data acquisition protocol, and image processing protocol, high-throughput phenotyping techniques can improve the throughput and objectivity during phenotyping in plant breeding programs.

The New Compact Plant Phenomics Center at WSU

Authors: Dominik Schneider, Kiwamu Tanaka, Henning Kunz, John Peters, Kim Campbell, Maren Friesen, Scot Hulbert, Helmut Kirchoff, Karen Sanguinet, Sindhuja Sankaran, and Zhiwu Zhang

Recent adverse environmental changes, increasing food demand, and predicted population growth has led to an urgent need for more stress-resistant crop species. High-throughput phenotyping will accelerate plant research by overcoming the current technical limitations in phenomcs. A new high-throughput phenotyping service is now operational at WSU for compact plant and other model organisms. The fully automated phenomics platform from LemnaTec is built into a light/temperature/humidity/CO2-controlled growth chamber. The platform carries a suite of cameras for visible RGB, photon counting, pulsed chlorophyll fluorescence, and shortwave-infrared imaging systems, as well as a 3D laser scanner. It also includes a 42-tray hotel for growing plants with automatic watering. Understanding genome-phenome relationships accurately and in detail will help scientists unveil molecular mechanisms involved in plant-environment interactions through genomic-based prediction of plant performance under multiple stress conditions. Thus, the complex genetic architecture of heritable phenotypic traits can be addressed with this system. We are currently in an operational testing stage and accepting proposals for free-of-charge pilot studies. Our goal is to establish this phenomics platform as a paid-use resource for the WSU community and beyond.


2018: Diversity in Agriculture

Fine Mapping and Candidate Gene Discovery of the Bean Golden Yellow Mosaic Virus Resistance Gene, bgm-1

Authors: Alvaro Soler-Garzon, Eliana Macea,  Juan D. Lobaton,  Sthephen Beebe, Phil N. Miklas, Bodo Raatz

Bean Golden Yellow Mosaic Virus (BGYMV) is a severe disease of common bean (Phaseolus vulgaris L.) that causes important yield losses. BGYMV is a geminivirus (family Geminiviridae) transmitted by Whitefly in tropical and sub-tropical countries of Latin America and the Caribbean. Breeding for resistance to the virus has been the most effective strategy for controlling the disease. One resistance source, A429, derived from the Durango landrace Garrapato (G2402), and subsequent DOR lines developed at CIAT, were shown to contain a single recessive gene, bgm-1, which reduces mosaic and yellowing symptoms. The marker SR2 has been used in marker-assisted selection programs because it is linked to the bgm-1 gene for resistance to BGYMV. In this study, SNP markers were developed from Genotyping by Sequencing (GBS) data on the region associated with the bgm-1 gene. These SNPs markers could constitute an important tool for marker-assisted selection programs for improvement of common bean cultivars with resistance to BGYMV.

Investigating the effects of Soybeans on Verticillium dahliae populations.

Authors: Daniel Farber, Dennis Johnson

Verticillium dahliae is a vascular wilt plant pathogen, causing Verticillium wilt, a disease with a wide host range, reducing yields on many important agricultural crops, including on potatoes. Growing soybeans and potatoes in rotation may be a good option for field managers, as soybeans harbor nitrogen-fixing bacteria, improving soil fertility. While soybeans have not been shown to express Verticillium wilt symptoms, it is important to investigate if V. dahliae populations can survive or increase either within the plant tissue or in soil. To examine this potential, soybeans planted in potting mix in the greenhouse were inoculated with seven strains of V. dahliae. After senescence of aboveground tissue, severities from 0.5 g of potting mix and two 5 cm cross sections of the stem centered at the soil line and at 15 cm aboveground were visually assessed. In the lower stem sections, six of the seven strains of V. dahliae did not significantly differ from the control, while one strain, V493, exhibited significantly increased severity, with a mean of 16.9 % severity (p = 0.029). No Verticillium was observed in the soil samples, and the severities in the upper sections of stem were not significant.

iPat: Intelligent Prediction and Association Tool for Genomic Research

Authors: Chunpeng (James) Chen and Zhiwu Zhang

The ultimate goal of genomic research is to effectively predict phenotypes from genotypes so that medical management can improve human health and molecular breeding can increase agricultural production. Genomic prediction or selection (GS) plays a complementary role to genome-wide association studies (GWAS), which is the primary method to identify genes underlying phenotypes. Unfortunately, most computing tools cannot perform data analyses for both GWAS and GS. Furthermore, the majority of these tools are executed through a command-line interface (CLI), which requires programming skills. Non-programmers struggle to use them efficiently because of the steep learning curves and zero tolerance for data formats and mistakes when inputting keywords and parameters. To address these problems, this study developed a software package, named the Intelligent Prediction and Association Tool (iPat), with a user-friendly graphical user interface (GUI). With iPat, GWAS and GS can be performed using a pointing device to simply drag and/or click on graphical elements to specify input data files, choose input parameters, and select analytical models.


2017: Innovations in Agriculture

Improved Functionality of Combined Pea-Rice Protein Isolates by Direct Steam Injection

Authors: E Pietrysiak, GM Ganjyal, B Smith and DM Smith

Demand for functional pea and rice proteins is increasing due to consumer interest in plant protein ingredients. When combined, pea and rice protein isolates create a complete protein, containing sufficient quantities of all essential amino acids. However, pea and rice isolates exhibit poor functionality in comparison to other proteins. This study investigated high pressure direct steam injection (DSI) to create modified pea-rice isolate blends (PR) with enhanced protein functional properties.

A DSI system was used to modify 5% (w/w) slurries of commercial isolates at pea:rice ratios of 1:2, 1:1, 2:1 (w/w) across a range of steam temperatures (66-107°C) and pH values (2, 3, 9, 10, 11). After DSI, protein blends were cooled, adjusted to pH 7, and freeze-dried. Compositional, biochemical, and protein functional properties of the DSI 2:1 PR blends were evaluated.

Based on protein solubility profiles, optimal DSI conditions were pea:rice ratio 2:1, pH 11, and steam injection at 107°C. The essential amino acid composition of 2:1 PR blends after DSI was confirmed by amino acid analysis. When the 2:1 DSI-PR blend was compared to the untreated PR, solubility (from 3 to 41%, at pH 7), emulsifying activity index (from 5.9 to 52.5 m2/g), foam stability (from 68.2 to 82.8 %), foam half-life (from 68 to 123 min), and oil holding capacity (from 1.8 to 4.9 g/g) values increased (P<0.05). DSI decreased (P<0.05) water holding capacity (from 3.4 to 2.5 g/g).

DSI can be used to alter the protein functional properties of blended PR isolates without affecting the essential amino acid composition and may allow for use of these ingredients in wider food processing applications, especially in the rapidly expanding gluten-free, vegan and nutritional beverage markets.


In Situ Imaging of Root System Architecture to Improve Drought Tolerance and Yield in Spring Wheat

Authors: Bikash Ghimire, Raul Arroyo & Karen A. Sanguinet
Department of Crop & Soil Sciences, Washington State University

Plant growth and development is largely dependent on the activity of the root, which acts to scavenge water and essential nutrients. The root system displays unique phenotypic plasticity that modulates growth over time and space and in response to environmental stresses in a reiterative process giving rise to overall root system architecture (RSA). The understanding of RSA in wheat is an untapped source for crop improvement. However, the meager knowledge of mature root systems in field environments has left a knowledge gap due to the inaccessibility of roots in soil. Thus, researchers are left to extrapolate adult root architectural traits from young seedling traits, which is still hotly debated. Our research focuses on the study of root system traits of young seedlings as well as mature roots of spring wheat parental lines and recombinant inbred lines (RILs) in both controlled conditions and dryland field conditions. We are using a minirhizotron system to conduct in situ studies of RSA traits. We hypothesize that the root system traits of the parental lines differ from one another irrespective of growth condition and the yield of RILs correlates with the specific root traits in dryland conditions. We will also describe the correlation of seedling root traits with mature root traits from field environments to validate past findings. Here we detail preliminary analyses of RSA traits in spring wheat lines. The overall goal of this study is to identify specific root traits which can either be directly considered for varietal screening through ideotype breeding or further exploited to breed drought tolerant wheat cultivars with enhanced yield for growers in the Pacific Northwest (PNW). The knowledge gained from these studies can then be leveraged to improve root systems of agronomic crops to feed the increasing world population in the face of climate change, particularly with the increasing occurrence of drought.

Engineering Virus Resistance in Cotton through Gene Stacking

Authors: Sabin Aslam1, Sultan Habibullah Khan2 and Aftab Ahmad2
1Center of Agricultural Biochemistry and Biotechnology,
2U.S.-Pakistan Center for Advanced Studies in Agriculture and Food Security (USPCAS-AFS)
University of Agriculture, 38040, Faisalabad, PAKISTAN.
Corresponding author:

Viral diseases are posing a serious threat to crop production worldwide. Quick breakdown of plant resistance to viruses like CLCuV demands application of a proficient technology to engineer durable resistance. Gene stacking has recently emerged as a potential approach for integrating multiple genes in crop plants. In present study, we are using recombinase technology for site specific gene stacking in cotton. pGRec plasmid was constructed for recombination by inserting Rec cassette in pGreen-0029 plasmid. Resulting plasmid pGRec was of 4.8Kb with npt-II as an antibiotic resistance gene. pGRec was transformed in cotton using bioloistic in cotton varieties Z-33 and Z-231. Embryos were excised from germinating seeds and shot with particles coated with PG-Rec. After transformation, kanamycin resistant embryos were selected to generate a target line for gene stacking. PCR was performed for confirmation of target line and seeds have been collected from transgenic plants. The developed target line will be used for further transformation of genes through site specific gene integration by using Bxb1 and Cre recombinase. In the next step we will stepwise stack transcription activator like effector nucleases (TALENs), CRISPR/Cas9 and guided RNAs (for Rep gene of CLCuV) and removal of marker gene. Consequently, transgenic marker free plants will be produced with one or two genes stacked at the specific site. These transgenic plants will be tested for resistance against various strains of cotton leaf curl virus (CLCuV).

Assessment of Consumers’ Willingness to Pay for Biodegradable Plastic Mulches

Authors: Chen Kuan-Ju Chen and Thomas L. Marsh
School of Economic Sciences, Washington State University

Consumer awareness about environmentally friendly products as they have realized that they could have a direct impact on ecological issues from their purchasing behavior. To evaluate consumers’ willingness to pay (WTP) for strawberries grown on biodegradable plastic mulches (hereinafter defined as “BDMs”), we conduct a survey with 1,500 consumers to gauge their shopping habits, environmental friendliness and perception on different types of strawberries. Using Contingent Valuation (CV) method, we estimate consumers’ premium on strawberries grown on BDMs versus the more conventional form of strawberries grown on non-biodegradable mulches. Our results show that survey respondents with higher income, more favorable attitude towards environmental friendliness, and lower shopping frequency are more likely to purchase strawberries grown on BDMs, and the effects are revealed to be significantly positive. From a research standpoint, it is interesting to understand how food products grown in a more environmental sustainable manner affect consumers’ willingness to pay. It is also interesting to understand how green technologies affect strawberries prices when they introduce their products to the market. This is true for both policymakers and industry participants as well. Finally, we discuss the effect on consumers willingness to pay from information regarding the biodegradable plastic mulches and environmentally-friendly disposal processes.

The Arabidopsis thaliana Cellulose Synthase Mutant rsw1 Root Swelling Phenotype is Rescued by a Panel of Hormones

Authors: Thiel A. Lehman & Karen A. Sanguinet
Washington State University, Crop and Soil Sciences Department

Plant cells are sheathed in a complex network of polysaccharides forming an integral matrix known as the cell wall. Primary cell wall biosynthesis occurs throughout cellular growth and is responsible for turgor-driven directional anisotropic cell expansion caused by the loosening of the cell wall matrix perpendicular to the axis of cell growth. Crosstalk between phytohormones and the primary cell wall plays a pivotal role in regulating cell growth and expansion, which is crucial for the overall development of the plant. Cellulose microfibrils are synthesized by CELLULOSE SYNTHASE A (CESA) proteins that arrange in a hexameric rosette forming a cellulose synthase complex (CSC). The activation and regulation of the CESA genes are of fundamental importance to plant morphogenesis and it has been hypothesized that hormone signaling plays key regulatory roles in the activation and biosynthesis of cellulose and ipso facto cell morphogenesis and whole plant growth. We used the conditional rsw1 mutant, which harbors a single amino acid change in AtCESA1 to investigate how hormones affect or contribute to changes in isotropic verses anisotropic growth. We found certain hormones can rescue growth and reduce isotropic expansion in rsw1 mutant roots. To support these data, we have introduced a series of hormone marker lines into the rsw1 mutant background to examine in vivo changes in hormone fluxes during the shift from anisotropic growth to isotropic growth. In addition, we have uncovered some subtle phenotypes of the rsw1 mutant. Taken together these data will shed light on the link between hormone signaling and gene regulation in the context of cell wall biosynthesis and overall morphology.

Characterizing the Genomic Variation of Wireworms Across the Inland Northwest

Authors: Alida T Gerritsen and Samuel S Hunter
Genomics Resources Core, Institute for Bioinformatics and Evolutionary Studies, University of Idaho

The Genomics Resources Core at the University of Idaho is an advanced next-generation sequencing facility and analysis resource for PIs both internal and external. Herein, we document a project in partnership with the Idaho Wheat Commission on characterizing the sugarbeet wireworm (Limonius californicus), a common agricultural pest in both Washington and Idaho that is commonly found in wheat fields. This project highlights the ability of the GRC to perform advanced molecular techniques such as Restriction Site Associated DNA marker preparation (RAD), de novo genome assembly, population genomics, and phylogenetic mapping, along with advanced bioinformatic analyses that can be scaled to fit any project.

Biodiversity of Native Yeast in Vineyards and Spontaneous Alcoholic Fermentation in Washington State

Authors: Xuefei Wang1, Dan Schlatter2, Charles Edwards3, Tim Paulitz2, David Weller2, Patricia Okubara2
1Department of Plant Pathology, Washington State University, Pullman, WA;
2USDA-ARS, Wheat Health, Genetics and Quality Research Unit, Pullman, WA;
3Department of Food Science, Washington State University, Pullman, WA

Spontaneous alcoholic fermentation is carried out by native microflora, which can improve wine quality. However, the variation of native microflora across viticultural areas can lead to unpredictable quality. In Washington state, little is known about the diversity of yeasts originating from vineyards and their contributions to alcoholic fermentation. The objectives of this study were to characterize native yeast communities found on Cabernet Sauvignon from two different vineyards in two Washington viticultural areas (Walla Walla and Horse Heaven Hills) and to track yeast populations in flask-scale fermentations. Intact grape berries were collected at five sites across each vineyard at veraison and just before commercial harvest in 2015. Spontaneous fermentations (250 mL) were performed in 500 mL fermenters with/without 80 ppm SO2 and weights of a subset of fermenters were recorded over 21 days. Residual sugar utilization, quantified for each flask, varied between fermentations from different vineyards, and indicated that 3, 7 and 21 days represented early, mid- and late-stage fermentation. Sluggish and stuck fermentation occurred in samples of both vineyards, which lacked a commercial starter yeast. Total DNA was extracted from veraison and harvest berry and early, mid- and late-stage fermentation samples. The fungal internal transcribed spacer1 (PfITS1) and D2 domain of large subunit rDNA regions were amplified and sequenced using the paired-end Illumina MiSeq platform. The numbers of yeast and non-yeast fungal operational taxonomic units (OTUs) indicated a high degree of yeast community diversity, especially on the berries. Many non-yeast fungi were identified but most declined during fermentation; Aureobasidium pullulans and Mycosphaerella tassiana tended to persist through mid-fermentation. The annotated OTUs of ITS1 and D2 showed that over 80% of the yeast species were common in both of the samples regardless of location and fermentation conditions. However, the populations of shared OTUs varied with the vineyard. Our findings suggest that a subset of common yeasts is present in both vineyards, but site-specific species could also be identified.

From Phenotype to Transcriptome: Changes in Plant and Soil Microbiome Associated with Brassica Seed Meal Amendment

Authors: Likun Wang1 and Mark Mazzola2
1 Washington State University; 2 USDA/ARS Tree Fruit Research Lab

Pre-pant application of Brassica seed meal (SM) formulations can provide fumigant level control of apple replant disease. Apple trees planted in SM treated soil exhibit a significantly different phenotype with trees planted in no treatment control soil, which indicating a different gene regulation event may occur in apple planted in SM treated soil. Transcriptomic changes associated with apple root response to soil treatment was identified using transcriptome sequencing technology. A total number of 72, 249 and 265 transcripts were exclusively differentially expressed in apple rootstock G.210 that harvested at 48 hr, 72 hr and 7 d, respectively, in SM treated soil when compare to control. In addition, a total number of 12, 247 and 47 transcripts were significantly differentially expressed in apple rootstock M.26 that harvested at 48 hr, 72 hr and 7 d, respectively. The findings suggesting that gene regulations are not only different between soil treatments but also different between rootstock genotypes.

Efficacy and Economic Viability of Organic Herbicides in a Young High-Density Apple Orchard

Authors: Aaron Appleby
Washington State University; Undergraduate Senior studying Organic Agriculture

Organic agriculture is responsible for less greenhouse gases, less erosion, less groundwater contamination, increased biodiversity, and increased soil organic matter than conventional agriculture. Organic agriculture, however, has been criticized as having very few cost-effective ways to deal with weeds. While many farmers are attracted to organic agriculture due to its low environmental impact, this lack of weed control is a major factor for people considering transitioning into organic production. There has been very little research done on organic herbicides, and those herbicides that are available for organic production are not cheap. Without proper research on the topic, organic farmers are hesitant to spend money on new products. I tested the efficacy and economic viability of four different organic herbicides. On a per acre basis all of the organic herbicides were less expensive than paying laborers to hand weed the plots; except that only Suppress, composed of capric and caprylic acid was as effective as hand weeding. Because all of the herbicides were less effective than hand weeding, they had to be applied twice as often, resulting in all of the herbicides except Suppress being more expensive to control weeds than paying laborers. This study did not take into account the monetary value of soil structure nor the effects of hand weeding on soil aggregates; however, this is a major concern of organic agriculture. Lastly, organic herbicides are contact herbicides and do not translocate within the plant, making it difficult to control perennial weeds. This was particularly troublesome due to Canada thistle and field bindweed being the main problem weeds in the high-density apple orchard, where the study took place. However, there is some evidence that caprylic acid may have some systemic abilities, thus more research needs to be done on the potential for organic herbicides to translocate to perennial structures.

Evaluation Study of Using Different Protectants to Produce Freeze-dried Enterococcus faecium NRRL B-2354 as a Dry Inoculum for Microbial Thermal Inactivation in Wheat Flour

Authors: Jie Xu1, Juming Tang 1, Meijun Zhu 2
1 Department of Biological Systems Engineering, Washington State University, Pullman
2 School of Food Science, Washington State University, Pullman

Dry-inoculation has several potential advantages over traditional wet-inoculation method to validate a thermal pasteurization process for low moisture foods. The objective of this study is to develop a dry-inoculation method using freeze-dried Enterococcus faecium NRRL B-2354, a potential surrogate of Salmonella, and apply the dry inoculum to validate Radio Frequency (RF) pasteurization processes for bulk wheat flour.
E. faecium mixed with 10% skim milk solution were freeze-dried. The protective effect of skim milk was studied by examining changes in microstructure and by evaluating survivors after the drying. Decimal reduction time (D value) of E. faecium in wheat flour with a water activity of 0.45 (at room temperature) was determined using thermal inactivation tests at 75, 80, 85°C. Thermal inactivation in RF pasteurization processes was validated by inoculated pack tests using a 12 kW 27.14 MHz pilot RF system. The samples were heated to three different end point temperatures (75, 80, and 85°C).
Skim milk formed slim layers around bacterial cells and yielded freeze-dried inoculum with a high survival probability. D-values of freeze dried E. faecium in wheat flour (water activity 0.45) were determined as 13.82±1.76 min, 5.92±0.39 min,  2.83±0.26 min. RF pasteurization could achieve 4.93 log CFU/mL reduction of after the inoculated samples were heated to 85°C and held 10 min. This study suggests that freeze drying of E. faecium NRRL B-2354 in skim milk produces high quality dry inoculants that can be successfully used to validate a RF process.

Mapping Leaf Rust Resistance of Wheat

Authors: Suraj Sapkota

Leaf rust, caused by the fungal pathogen Puccinia tritcina, is the most destructive foliar disease of wheat worldwide. Genetic resistance is the most effective economic and environmentally safe method to reduce losses caused by leaf rust. Over 70 leaf rust resistance genes have been reported thus so far; however, only few are effective to a wide range of pathogen populations. In this study, an F3 population was developed by crossing a resistant cultivar (AGS 2038) and a susceptible (UGA 111729) cultivar to characterize the leaf rust resistance genes in AGS 2038. While both genotypes were susceptible to leaf rust in seedling stage, AGS 2038 was resistant at adult plant stage. Phenotypic evaluation of the F3 population and parents was conducted in the Plains, GA in 2015. The disease severity (DS) of the two parents, AGS 2038 and UGA 111729 were 20% and 70%, respectively. The distribution of the F3 population DS showed a transgressive segregation with a mean of 33% and a range of 5 to 90%. Bulk segregant analysis (BSA) was done using 504 SSR markers, and our preliminary result indicated that 2 markers were most likely linked to leaf rust resistance. We are advancing this population to develop recombinant inbred lines (RILs), which will be ultimately genotyped using 90K SNPs. A combination of SSR and SNP markers will be used for linkage map construction and QTL mapping.

Investigating the Link Between Ion Flux Across the Inner Plastid Envelope Membrane and Photosynthesis

Authors: Carsten Völkner and Hans-Henning Kunz
School of Biological Sciences, Washington State University

Increased temperatures have globally led to more drought stress and higher rates of irrigation. This is usually followed by elevated soil salinity which plants experience as salt stress in their habitat. The overwhelming majority of plants are so-called glycophytes, i.e. plants that are not well-adapted to salt stress. In glycophytes, salt stress exerts a highly detrimental effect on photosynthesis and plant performance which diminishes global crop yield and biomass production. Plant function relies heavily on high levels of internal potassium (K+) and low levels of toxic sodium (Na+) ions. Under salt stress, Na+ accumulates in the plant body, the cells and in cell organelles resulting in a drain of K+. Loss of K+ in the chloroplasts was shown many times to negatively impact photosynthesis.
The recent discovery of envelope ion carriers and ion channels may open new avenues to suppress plastid loss of K+ during salt stress. Interestingly, loss-of-function mutants for both systems are strongly compromised under control conditions. However, the dramatic phenotypes can be rescued by exposing mutants to salt stress.
The goal of my work is to study the functional linkage between the so far only characterized plastid ion transport mechanisms across the envelope membrane; K+/H+ efflux antiporters (KEA) and mechanosensitive ion channels MSL. I will further explore the physiological significance of plastid KEAs and MSL members for photosynthesis under control and abiotic stress conditions. On my poster, I will present some exciting early data from my PhD research and will discuss their relevance and my future project directions.

Molecular characterization of Rubisco activase genes in cereals for heat tolerance

Authors: Ragupathi Nagarajan

Plants adapted to different environmental conditions during evolution mostly by altering its physiological process through gene mutations and modifications. Understanding these evolutionary changes are important as this knowledge can be used to engineer future plants with better and wider adaptability. This study shows the evolution of Rubisco activase (Rca) gene in cereal crops and its implications on heat tolerance. During photosynthesis, RCA activates Rubisco enzyme by removing the sugar phosphates from the CO2 and metal binding sites. The inability of RCA to keep pace with faster Rubisco deactivation during heat stress constrains photosynthesis. Rubisco activation and CO2 fixation rate of wheat start to decline even at 30°C and complete photosynthesis inhibition occurs at temperatures above 38°C. Among cereal crops, sorghum, maize and rice have comparatively more thermal tolerant RCA enzymes than wheat. Characterization of Rca genes by cloning in cereal crops showed that a tandem duplication of Rca occurred in a common grass ancestor, probably before the divergence of Poaceae subfamilies. Different insertions and mutations in the promoter and coding sequences of one of the Rca gene are appeared to be responsible for its heat induced expression pattern and thermal stability in these heat tolerant cereal crops. Improving the thermal stability of wheat RCA by using the knowledge from closely related heat adapted crops may help us to engineer heat tolerant wheat cultivars.

Analysis of nutritional diversity among selected accessions of dry beans and nuña beans (Phaseolus vulgaris L.) from the USDA-ARS National Plant Germplasm System.

Authors: Girish Ganjyal1, Cristen Frieszell1, Amit Dhingra2, and Theodore Kisha3
1Washington State University, Department of Food Science and Human Nutrition, 2Washington State University Department of Horticulture, 3USDA-ARS Western Regional Plant Introduction Station

Beans (Phaseolus spp.) are one of the most economically and nutritionally important crops world-wide, with a value of over $17 billion harvested annually (FAO statistics, 2012). They are one of the most ancient crops of the New World, having been cultivated for thousands of years (Kaplan and Lynch, 1999). They are an environmentally diverse crop, growing in temperate and sub-tropical environments from sea level to more than 3000m above sea level (Broughton et al., 2003), and are consumed as either fresh pods or as a dry bean, making them an ideal nutritional food legume in areas where storage without refrigeration is necessary. They are the most important legume for direct human consumption with more than 23 million metric tons produced in 2013; more than twice that of the next most important legume, chickpea (Cicer arietinum L.) with just over 11 million tons ( A recent review on the nutritional aspects of bean consumption (Hayat et al., 2014) lists numerous qualities, such as high protein (Siddiq et al, 2010), high amounts of starch, dietary fiber, minerals, and vitamins (Kutos et al., 2003; Costa et al., 2006), an array of healthy phytochemicals (Beninger and Hosfield, 2003; Choung et al., 2003; Aparicio-Fernandez et al., 2005; Granito et al., 2008; Lin et al., 2008), and an association with health benefits such as reduced cardiovascular disease, the prevention of diabetes, and even the prevention of cancer (Jenkins, 2007; Chung et al., 2008)
Growing consumer awareness regarding the health benefit of beans in general will likely increase demand, perhaps especially among those who frequent farmers markets and health food stores. Among the many accessions of Phaseolus vulgaris in the Western Regional Plant Introduction Station (WRPIS), 90 of these are classified as nuña beans, or the Peruvian “popping” bean. These beans have been selected and raised among the Andean natives in the high mountains for millennia (Kaplan and Lynch, 1999), and have the unique characteristic of bursting when subjected to heat, much like popcorn, making them a high protein food in conditions where boiling would consume scarce fuel. This characteristic is also ideal for the development of a highly nutritious snack food to complement the goals under the Pulse Health Initiative, which include fighting obesity and its related chronic diseases as well as indirect healthcare costs and loss of productivity, improved sustainable agricultural production systems, and increased employment opportunities.

GAPIT3: New Architecture to Quickly Adapt Third Party Packages for Genomic Association and Prediction

Authors: Jiabo Wang

Maintaining and development of software packages for genome wide association studies (GWAS) are challenging not only because the demands of problem-free application, but also because that computing speed must be boosted for euphonically growing data size and statistical models must be advanced to control both false positives and false negatives. GAPIT is one of the software packages widely used for GWAS and genomic prediction. Several handy features in GAPIT were widely used, including Principal Component Analyses (PCA), kinship, data visualization (e.g. population structure in 3D) and results interpretation (e.g. Manhattan plot and QQ plot). The package implemented multiple statistical methods to improve speed and statistical power, including the compressed mixed linear model (CMLM) developed in 2010, enriched CMLM and SUPER methods developed in 2014. Multiple methods and packages were developed to produce better GWAS results, including MLMM and FarmCPU. There is critical need to develop a package that can quickly integrate existing and new developments. We redesign GAPIT to fit the need. The new version of GAPIT (version 3) is capable to adapt third party packages easily. Here we describe the design and implementations for adapting original version of GAPIT and the most recently developed packages, MLMM and FarmCPU. The advantages and the performances of using these packages in GAPIT3 were compared to using them along. GAPIT3 has been released to public and GAPIT users has started to use the new version of GAPIT substantially.

Ion gradients

Authors: Rachael DeTar

Ion gradients across the chloroplast envelope and thylakoid membranes are crucial for organelle function and photosynthesis. These functions include regulating osmotic balance, maintaining chloroplast shape and architecture, regulating key photosynthetic enzymes, and finally the maintenance and regulation of the proton motive force (PMF) to generate ATP. While it is known that ion and pH gradients are important for the regulation of photosynthetic proteins and membranes, little is known about how ion gradients may impact gene expression, and more specifically, retrograde signaling by disruption of photosynthesis. To better understand how chloroplast ion gradients impact gene transcription, we ran an RNA sequencing experiment on kea1kea2 double mutants which lack two main K+ /H+ antiporters in the chloroplast
inner envelope membrane. Simultaneous loss of function in kea1 and kea2 results in reduced photosynthetic capacity, and altered PMF partitioning.
The primary objective of our study is to examine to what extent the phenotype corresponds to downregulation of nucleus encoded photosynthetic genes. Our bioinformatics analysis on RNA sequencing data reveal a slew of deregulated genes in the kea1kea2 double mutants compared to wildtype controls. This includes downregulation of genes related to chlorophyll and heme
biosynthesis, a pathway that plays a key role in retrograde signaling. Additionally, genes encoding proteins associated with circadian rhythm, and chloroplast rRNA processing where found to be differentially expressed in kea1kea2 mutants. Interestingly, kea1kea2 double mutant treated with 75 mM NaCl which to triggers visual phenotypic rescuing led to more wildtype-like gene expression
for genes in these aforementioned pathways. Our experiment provides global insights into the wideranging importance of intact plastid ion flux for transcriptional control of photosynthesis,
chloroplast development and signaling processes that involve the plant chloroplast. On my poster, I will present more detailed transcriptional network and pathway analysis and discuss these data along with future research directions.

Targeted Amplicon Sequencing

Authors: Matthew Fagnan, Dan New
University of Idaho

If you have DNA of interest that can be PCR amplified, we can help you sequence that with free consulting, phone support, and custom barcodes that allow you to sample over 1000 samples per Illumina sequencing run. Some current applications are microbial community sequencing, phylogenetics, genotyping by sequencing, microsatellite typing, transposon insertion detection and localization, and CRISPR editing validation by sequencing. If you can PCR it we can sequence it.

Corrosion Behavior of C1010 Carbon Steel in the Presence of an Apple Pomace Derived Green Inhibitor

Authors: Mehdi Honarvar Nazari

This work evaluated an apple pomace-derived inhibitor which was prepared through a novel chemical/biological process for mitigating chloride/corrosion impact on the environment. The corrosion inhibition efficiency of this solution on C1010 carbon steel in 3.5 wt.% NaCl was investigated over time using electrochemical measurements. Chemical analyses were performed to understand the main compounds of inhibitor. Surface analyses were employed to study the characteristics of corrosion product layer. The corrosion inhibition efficiency increased with increasing the concentration of apple pomace extract. Used at 3 vol.%, this green inhibitor exhibited an excellent inhibition efficiency of 100% after 7 days of steel immersion time. The adsorption of organic molecules followed the Langmuir adsorption isotherm through the physical adsorption in which electrostatic interaction occurs between inhibitor molecules and metal surface. The apple pomace extract was an anodic inhibitor that blocked the anodic active sites on the surface of carbon steel. The corrosion inhibition by apple pomace extract was mainly due to the adsorption of C26H50NO7P molecules, as active ingredient, which formed a barrier phosphate layer on the surface of steel coupons and changed the characteristics of passive layer.

Rapid, accurate, and on-site molecular detection of soilborne potato pathogens

Joseph DeShields

On-site diagnosis for plant disease is pivotal for growers as timely decisions regarding early-stage treatment are often crucial and can thereby reduce secondary spread of disease in the field. Polymerase chain reaction (PCR) is currently the most sensitive and accurate method for plant pathogen detection. However, standard PCR requires expensive laboratory equipment and skilled personnel. Here, we propose a rapid and simple method for on-site detection of soilborne potato pathogens comprised of magnetic bead-based nucleic acid extraction, portable real-time PCR equipment and data analysis that can be done remotely on a laptop computer. The capabilities of a portable real-time PCR thermocycler were compared with a standard real-time PCR thermocycler for the detection of the soilborne potato pathogens. Our conclusion is that the method we developed enables highly sensitive and rapid, on-site detection of important soilborne pathogens in the field, which can serve as an alternative to conventional diagnostic methods.

Drought stress

Authors: Avjinder Kaler

Drought stress is a major global constraint for crop production. Cooler canopy temperature (CT), slow-canopy wilting (CW), faster establishment of a closed canopy (CC) are the promising traits associated for improving drought tolerance. The objective of this study was to identify the SNPs and genes associated with CT, CW, and CC. A panel of 373 MG IV soybean genotypes was grown in multiple environments to evaluate CT, CW, and CC. Statistical analysis of phenotype indicated wide variation of the trait, with significant effects of genotype (G), environment (E) and G x E interaction. Over 42,000 SNP markers were obtained from the Illumina Infinium SoySNP50K iSelect SNP Beadchip. After filtration for quality control, 31,260 SNPs with a minor allele frequency (MAF) ≥ 5% were used for association mapping using the FarmCPU model, which reduces the both false positives and negatives. Analysis with the FarmCPU model identified several environment-specific SNPs, and SNPs that were present in more than one environment, which were significantly associated with these drought related traits. Most significant SNPs were located within a gene or very close to genes that may have some association with CT, CW, and CC. Based on the favorable alleles, accessions were identified from whole soybean germplasm that have coolest canopy temperature, slowest canopy wilting, and fastest establishment of closed canopy, which can be used as breeding parental materials to improve drought tolerance.

An NGS approach to identify a cell division kinase involved in root growth in B. distachyon

Authors: Rachel Dannay1, Ying Wu1,2, Tobias Baskin3, and Karen Sanguinet1
1. Washington State University, Department of Crop and Soil Sciences, Pullman, WA 99164
2. Northeast Normal University, Institute of Genetics & Cytology, Changchun, China 130024
3. University of Massachusetts, Department of Biology, Amherst, MA 01003

During the green revolution, breeders successfully improved aboveground biomass and seed yield in agriculturally important crop species; however, root systems have predominantly been ignored due to their inaccessibility. Roots have become a major focus for current crop improvement, and a better understanding of root systems is needed when addressing agronomic problems that root systems encounter in the soil. It has been shown that crop varieties with deeper or larger root systems perform better when water is limiting, and further investigation of this enhanced performance can be applied to agriculturally important grasses to increase crop performance under abiotic stress. Brachpodium distachyon has emerged as a model species for temperate grasses due to its relatedness to economically important crops like wheat and barley. Through an EMS chemical mutagenesis screen, a novel root-hairless mutant named buzz was identified that exhibits a two-fold increase in root growth rate with no impact on above-ground biomass. This mutant phenotype cannot be rescued by exogenous auxin or ethylene, two hormones that play an integral role in root and root hair growth. Through whole genome resequencing and differential single nucleotide polymorphism analysis, we have identified the putative causal mutation to be in the active site of an uncharacterized cell division kinase. Upon further molecular and genetic investigation of this kinase, buzz will be an ideal mutant line to better understand the interplay between root hair growth and root growth rate and will provide an excellent tool for future studies of abiotic stress in grasses.

A targeted amplicon solution for charactizing CRISPR/Cas9 induced mutations

Authors: Samuel Hunter
University of Idaho IBEST Genomics Resources Core

The CRISPR/Cas9 targeted genome editing system has rapidly grown in popularity and has seen wide spread adoption across multiple disciplines including agriculture. Although greatly improving the ease, efficiency, and expense associated with gene knockout and editing, the double strand break produced by Cas9 results in random mutations when repaired through non-homologous end joining. When the homology-directed strategy is used, repair occurs at low frequencies. This uncertainty in outcome necessitates screening to detect whether a mutation has been induced, and characterization to determine if the mutation is of the desired type. Somatic mosaicism is also a common outcome, further complicating the characterization of induced mutations. Screening and characterization is often done through techniques such as T7E1/Surveyor mismatch digestion or Sanger sequencing, however these strategies are laborious, low-throughput and provide limited information about the induced mutation. We present a newly developed service being offered by the IBEST Genomics Resources Core which addresses these limitations through a targeted amplicon based strategy. This approach allows any researcher to leverage deep sequencing technology in order to rapidly screen and characterize hundreds or thousands of individual samples at one or more loci cheaply and efficiently.

Pythium damping-off of chickpea is an important disease

Authors: Moying Wang

Pythium damping-off of chickpea is an important disease, and has been traditionally managed through seed treatment using the fungicide metalaxyl. However, Pythium populations in the US Pacific Northwest have developed resistance to this fungicide and metalaxyl is no longer effective. Conventional approach to managing fungicide resistance is to stop using the fungicide to let the resistant population disappear due to resistance-associated fitness cost. To evaluate this approach for managing the metalaxyl-resistant Pythium, we investigated the stability and saprophytic fitness of metalaxyl-resistant Pythium isolates. The stability of the metalaxyl-resistance trait was studied over 10 generations, and the relative competitiveness of the metalaxyl-resistant isolates was compared with that of metalaxyl-sensitive isolates for growth rate. Results showed that metalaxyl-resistant isolates consistently grew faster in terms of colony diameter (4.9 cm/day) and mycelia dry weight (24 mg/day) than metalaxyl-sensitive isolates (3.6 cm and 19 mg/day, respectively). The metalaxyl-resistant isolates remained resistant after 6 generations of sub-culturing in the absence of metalaxyl, as indicated by ED50 values. Because of the fast growth rate and stable resistance trait of the resistant isolates, simply stopping using metalaxyl is not a viable solution. More active management approaches such as using alternative fungicides are required to manage the metalaxyl-resistant Pythium.

Long Read Sequencing with Oxford MinION

Authors: Matthew Fagnan
University of Idaho

Oxford Nanopore Technologies is developing cheap, efficient, and novel pore sensing technologies for DNA sequencing. The field is rapidly advancing, faster than most researchers realize. The IBEST Genomics Core at University of Idaho has acquired Oxford’s MinION sequencer and is exploring how the small, USB-powered device produces long read sequences to potentially change the paradigm of the sequencing world.

Diversity and Evolution of Plastid Transit Peptides in Higher Plants

Authors: Ryan Christian, Scott Schaeffer, and Amit Dhingra.
Horticulture Department, Washington State University

The expanding availability of sequenced plant genomes and the development of high-throughput targeting prediction algorithms has made it possible to do large-scale genetic comparisons between species. Though much work has been done in gene annotation and genome-wide comparisons, the evolution of subcellular localization functions has received little attention. In plants, the vast majority of chloroplast proteins are nuclear-encoded, but there has been little insight into how much this pool of nuclear-encoded proteins varies between species. We approached this knowledge gap by combinatorial use of the subcellular localization program TargetP and the clustering algorithms UCLUST and ReMark to identify predicted shared and unique plastid-targeted peptides in seven sequenced plant genomes. This analysis has identified 741 chloroplast proteins shared between all species, while individual species had between 631 to 9185 proteins unique to their plastid proteome. Peptides which are plastid-localized in single species may exhibit novel traits even if their non-plastid targeted function is well known. Apple (Malus x domestica) and switchgrass (Panicum virgatum) were found to have an extreme richness of unique plastid-targeted genes, with 3,647 and 9,185 unique clusters respectively. Species-specific plastid-targeted peptides may be a rich source of novel biological functions due to their abnormal localization signal. For high-value crops such as apple, or emerging bioenergy crops such as switchgrass, gaining a higher understanding of how these crops differ genetically from other species presents opportunities to improve crop management for maximum yield and minimal loss to physiological diseases. Furthermore, this work offers an opportunity to shed light on the evolution of plastid-nuclear trafficking and on the evolutionary mechanisms through which proteins become plastid-targeted. And finally, our analysis will provide guidance for how to interpret genetics data from one species and apply the results to homologs in other species which may or may not have similar transit peptides.

Effectors from Puccinia suppress plant defense responses

Authors: Sowmya Ramachandran

Rust fungi of the genus Puccinia are an important class of plant pathogens that cause severe yield losses and are a serious threat to global food security. Upon infection, the biotrophic fungus secretes a number of effector proteins inside the host which interfere with many plant host processes. In this study, we mined the transcriptome of Puccinia graminis and P. striiformis for putative effector candidates up-regulated during infection, for their ability to suppress plant programmed cell death. Twenty putative effector proteins were selected and expressed in N. benthamiana using Agrobacterium- mediated transient transformation. Nine proteins Shr1 – Shr9 (Suppressors of Hypersensitive Response) were found to suppress HR triggered by Pto (Y207D), GPA/RBP-1 and ATR13/RPP13. Expression analysis of these nine effectors by RTq-PCR at different time-points during infection revealed no set pattern of expression. In addition to ETI, Shr7 also suppressed production of reactive oxygen species, indicating its ability to interfere with host innate immunity. Delivery of Shr7 into wheat plants via bacterial type-III secretion system caused partial reduction in ETI induced by Pseudomonas syringae DC3000. This study provides the first evidence for the presence of plant defense suppressors in Puccinia, which can be further exploited for creating transgenic lines more resistant to wheat rusts.

Plants utilize a variety of hormones to modulate defense responses to pests and pathogens.

Authors: Paul Chisholm

Plants utilize a variety of hormones to modulate defense responses to pests and pathogens. Artificial induction of these defense pathways through genetic or chemical means has been used to increase plant resistance, but phytohormone-induced resistance often comes with tradeoffs relating to yield and vulnerability to other types of pests. In order to determine how phytohormones influence pest and disease dynamics over the course of a growing season, we applied salicylic acid and methyl jasmonate to open-air experimental plots. Untreated and neonicotinoid-treated plots served as controls. Performance metrics such as pest abundances, defoliation, disease incidence, and yield, were recorded. Application of methyl jasmonate resulted in reductions in aphid pests similar to that achieved by insecticide application. However, none of the treatments reduced viral incidence. Despite this, yield was significantly higher in plots treated with salicylic acid. Because rates of viral infection were very high across all treatments, we believe that salicylic acid could be increasing plant tolerance to viruses. Many pathogens overcome plant defenses by suppressing the production of salicylic acid, so supplying salicylic acid exogenously may activate these suppressed defensive pathways and allow the plant to adequately defend itself. Despite its demonstrated effectiveness, foliar application of phytohormones remains a crude method of delivery, and genetic approaches may provide a more robust solution for achieving phytohormone-mediated resistance in plants.