Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates.

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high-quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe non-coding RNAs, pseudogenes and transposable elements, and highlight tissue-specific expression and methylation patterns. Resequencing of forty wild accessions provided insights into genome-wide genetic variation, and QTL regions were identified for a seedling colour phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

Genetic mapping and phenotypic analysis of shotH.3.2 in Drosophila melanogaster.

Genetic screens are used to identify genes involved in specific biological processes. An EMS mutagenesis screen in Drosophila melanogaster identified growth control phenotypes in the developing eye. One mutant line from this screen, H.3.2, was phenotypically characterized using the FLP/FRT system and genetically mapped by complementation analysis and genomic sequencing by undergraduate students participating in the multi-institution Fly-CURE consortium. H.3.2 was found to have a nonsense mutation in short stop (shot), anortholog of the mammalian spectraplakin dystonin (DST). shot and DST are involved in cytoskeletal organization and play roles during cell growth and proliferation.

CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.).

Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

Bioinformatic analysis suggests potential mechanisms underlying parasitoid venom evolution and function.

Hymenopteran parasitoid wasps are a diverse collection of species that infect arthropod hosts and use factors found in their venoms to manipulate host immune responses, physiology, and behaviour. Whole parasitoid venoms have been profiled using proteomic approaches, and here we present a bioinformatic characterization of the venom protein content from Ganaspis sp. 1, a parasitoid that infects flies of the genus Drosophila. We find evidence that diverse evolutionary processes including multifunctionalization, co-option, gene duplication, and horizontal gene transfer may be acting in concert to drive venom gene evolution in Ganaspis sp.1. One major role of parasitoid wasp venom is host immune evasion. We previously demonstrated that Ganaspis sp. 1 venom inhibits immune cell activation in infected Drosophila melanogaster hosts, and our current analysis has uncovered additional predicted virulence functions. Overall, this analysis represents an important step towards understanding the composition and activity of parasitoid wasp venoms.

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyl-triacylglycerol-containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress' rapid domestication to meet the growing sustainable food and fuel demands.