2019: Foundations for the Future: Embracing New Agricultural Technology
Hybrid wheat: a promising technology for feeding the future
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.