Evaluation of tetraploid switchgrass (Poales: Poaceae) populations for host suitability and differential resistance to four cereal aphids.

Switchgrass, Panicum virgatum L., is being developed as a bioenergy feedstock. The potential for large-scale production has encouraged its evaluation as a host for important grass pests. Eight no-choice studies were performed for two developmental stages of two switchgrass cultivars ('Kanlow' and'Summer') and two experimental strains, K x S, and S x K produced by reciprocal mating of these cultivars followed by selection for high yield. Plants were evaluated for host suitability and damage differences to herbivory by four important cereal aphids, Sipha flava (Forbes), Schizaphis graminum (Rondani) (biotype I), Rhopalosiphum padi (L.), and Diuraphis noxia (Mordvilko). All switchgrasses were found to be unsuitable feeding and reproductive hosts to R. padi and D. noxia, which were unable to establish on the plants. However, both S. flava and S. graminum were able to establish on all switchgrasses tested. Differential levels of resistance to S. flava and S. graminum were detected among the switchgrasses by both cumulative aphid days (CAD) and plant damage ratings. Kanlow was consistently rated as highly resistant based on CAD and damage ratings for both aphid species, while Summer was consistently among the most susceptible to both aphids at both developmental stages, with relatively high damage ratings. The resistance of the K x S and S x K populations in relationship to their Summer and Kanlow parents indicted that they inherited some resistance to S. graminum and S. flava from their Kanlow parent. These studies provide valuable baseline information concerning the host suitability of switchgrass to four cereal aphids and the plant-insect interactions within a system that has been largely overlooked and indicate that there are genetic differences among switchgrass populations for resistance to some insects.

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Using genetic comparisons of populations from Arizona, Mexico, and Texas to investigate fall armyworm migration in the American southwest.

Fall armyworm (FAW) is a global agricultural pest, causing substantial economic losses in corn and many other crops. Complicating efforts to control this pest is its capacity for long distance flights, which has been described in greatest detail for the central and eastern sections of the United States. FAW infestations are also routinely found in agricultural areas in southern Arizona, which lie beyond the western limits of the mapped migratory pathways. Climate suitability analysis found that the affected Arizona locations cannot support permanent FAW populations, indicating that these FAW most likely arise from annual migrations. A better understanding of this migration would provide insights into how large moth populations can move across desert habitats as well as the degree of gene flow occurring between FAW populations across the North American continent. In this study the Arizona populations were genetically characterized and compared to a selection of permanent and migratory FAW from multiple sites in the United States and Mexico. The results are consistent with migratory contributions from permanent populations in the states of Texas (United States) and Sinaloa (Mexico), while also providing evidence of significant barriers to gene flow between populations within Mexico. An unexpected finding was that two genetically distinct FAW subpopulations known as "host strains" have a differential distribution in the southwest that may indicate significant differences in their migration behavior in this region. These findings indicate that the combination of mitochondrial and Z-linked markers have advantages in comparing FAW populations that can complement and extend the findings from other methods.

Native Solitary Bees Provide Economically Significant Pollination Services to Confection Sunflowers (Helianthus annuus L.) (Asterales: Asteraceae) Grown Across the Northern Great Plains.

The benefits of insect pollination to crop yields depend on genetic and environmental factors including plant self-fertility, pollinator visitation rates, and pollinator efficacy. While many crops benefit from insect pollination, such variation in pollinator benefits across both plant cultivars and growing regions is not well documented. In this study, across three states in the northern Great Plains, United States, from 2016 to 2017, we evaluated the pollinator-mediated yield increases for 10 varieties of confection sunflowers, Helianthus annuus L. (Asterales: Asteraceae), a plant that is naturally pollinator-dependent but was bred for self-fertility. We additionally measured pollinator visitation rates and compared per-visit seed set across pollinator taxa in order to determine the most efficacious sunflower pollinators. Across all locations and hybrids, insect pollination increased sunflower yields by 45%, which is a regional economic value of over $40 million and a national value of over $56 million. There was, however, some variation in the extent of pollinator benefits across locations and plant genotypes, and such variation was significantly related to pollinator visitation rates, further highlighting the value of pollinators for confection sunflowers. Female Andrena helianthi Robertson (Hymenoptera: Andrenidae) and Melissodes spp. (Hymenoptera: Apidae) were the most common and effective pollinators, while other bees including managed honey bees (Hymenoptera: Halictidae), Apis mellifera L. (Hymenoptera: Apidae), small-bodied sweat bees (Hymenoptera: Halictidae), bumble bees Bombus spp. (Hymenoptera: Apidae), and male bees were either infrequent or less effective on a per-visit basis. Our results illustrate that wild bees, in particular the sunflower specialists A. helianthi and Melissodes spp., provide significant economic benefits to confection sunflower production.

Dung Beetles Increase Greenhouse Gas Fluxes from Dung Pats in a North Temperate Grassland.

Soil fauna plays a critical role in various ecosystem processes, but empirical data measuring its impact on greenhouse gas (GHG) emissions from rangelands are limited. We quantified the effects of dung beetles on in situ CO, CH, and NO emissions from simulated cattle dung deposits. Soil in meadows of the semiarid Nebraska Sandhills was treated with three treatments (dung pats with exposure and without exposure to dung beetles, and a no dung control). A closed-chamber method was used to measure GHG fluxes at 0, 1, 2, 3, 7, 10, 14, 21, 28, and 56 d after dung placement in the early season (June-August) and late season (July-September) in 2014 and 2015. The greatest dung beetle abundance was 6 ± 2 beetles per quarter pat on Day 7; the abundance decreased to <2 ± 0.6 on Day 14 and 28 and zero on Day 56. Dung beetles increased fluxes of CO by 0.2 g C d m, NO by 0.4 mg N d m (only in late season 2015), and CH by 0.2 mg C d m. These increases were due to beetle-made macropores that facilitated gas transport in wet dung (initial moisture = 4.6 g g on a dry-weight basis) within 7 d after dung placement. Seasonal environmental differences resulted in greater CO, NO, and CH fluxes in the early season than in the late season. This study concluded that dung beetles increased GHG fluxes from early- and late-season dung deposits on meadows of the semiarid Nebraska Sandhills.

Towards uncovering the roles of switchgrass peroxidases in plant processes.

Herbaceous perennial plants selected as potential biofuel feedstocks had been understudied at the genomic and functional genomic levels. Recent investments, primarily by the U.S. Department of Energy, have led to the development of a number of molecular resources for bioenergy grasses, such as the partially annotated genome for switchgrass (Panicum virgatum L.), and some related diploid species. In its current version, the switchgrass genome contains 65,878 gene models arising from the A and B genomes of this tetraploid grass. The availability of these gene sequences provides a framework to exploit transcriptomic data obtained from next-generation sequencing platforms to address questions of biological importance. One such question pertains to discovery of genes and proteins important for biotic and abiotic stress responses, and how these components might affect biomass quality and stress response in plants engineered for a specific end purpose. It can be expected that production of switchgrass on marginal lands will expose plants to diverse stresses, including herbivory by insects. Class III plant peroxidases have been implicated in many developmental responses such as lignification and in the adaptive responses of plants to insect feeding. Here, we have analyzed the class III peroxidases encoded by the switchgrass genome, and have mined available transcriptomic datasets to develop a first understanding of the expression profiles of the class III peroxidases in different plant tissues. Lastly, we have identified switchgrass peroxidases that appear to be orthologs of enzymes shown to play key roles in lignification and plant defense responses to hemipterans.

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.