Genetic dissection of seed characteristics in field pennycress via genome-wide association mapping studies.

Field pennycress (Thlaspi arvense L.) is a new winter annual cash cover crop with high oil content and seed yield, excellent winter hardiness, early maturation, and resistance to most pests and diseases. It provides living cover on fallow croplands between summer seasons, and in doing so reduces nutrient leaching into water sources, mitigates soil erosion, and suppresses weed growth. The first ever genome-wide association study (GWAS) was conducted on a pennycress diversity panel to identify marker trait associations with important seed size and composition related traits. The entire population was phenotyped in three total environments over 2 yr, and seed area, length, width, thousand grain weight, total oil, and total protein were measured post-harvest with specialized high-throughput imaging and near-infrared spectroscopy. Basic unbiased linear prediction values were calculated for each trait. Seed size traits tended to have higher entry mean reliabilities (0.76-0.79) compared with oil content (0.51) and protein content (0.37). Genotyping-by-sequencing identified 33,606 high quality genome-wide single nucleotide polymorphism (SNPs) that were coupled with phenotypic data to perform GWAS for seed area, length, width, thousand grain weight, total oil, and total protein content. Fifty-nine total marker-trait associations were identified revealing genomic regions controlling each trait. The significant SNPs explained 0.06-0.18% of the total variance for that trait in our population. A list of candidate genes was identified based on their functional annotations and characterization in other species. Our results confirm that GWAS is an efficient strategy to identify significant marker-trait associations that can be incorporated into marker-assisted selection pipelines to accelerate pennycress breeding progress.

Field pennycress is an excellent winter annual oilseed that can serve as a cash cover crop. Genotyping-by-sequencing is an effective strategy to genotype pennycress affordably for high-quality genome-wide single nucleotide polymorphisms. First-ever mapping study in a field pennycress association mapping population was conducted. GWAS identified 59 significant marker-trait associations for important quantitative traits. Seed size traits had a higher reliability estimate compared with seed composition traits.

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.

Technologies enabling rapid crop improvements for sustainable agriculture: example pennycress (Thlaspi arvense L.).

Growing concerns over food insecurity and ecosystems health related to population growth and climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In this review, we discuss the domestication of the oilseed-producing cover crop pennycress, which along with the development of other new crops and improvements to farming practices can provide sustainable solutions to address malnutrition and environmental impacts of production agriculture. We highlight some of the new technologies such as bioinformatics-enabled next-generation sequencing and CRISPR genome editing in combination with traditional mutation breeding that has accelerated pennycress development as a new crop and a potential model system. Furthermore, we provide a brief overview of the technologies that can be integrated for improving pennycress and other crops and the status of pennycress development using these technologies.

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.

Expression of FLOWERING LOCUS C and a frameshift mutation of this gene on chromosome 20 differentiate a summer and winter annual biotype of Camelina sativa.

The nature of the vegetative to reproductive transition in the shoot apical meristem of Camelina sativa summer annual cultivar CO46 and winter annual cultivar Joelle was confirmed by treating seedlings with or without 8 weeks of vernalization. True to their life cycle classification, Joelle required a vernalization treatment to induce bolting and flowering, whereas CO46 did not. In this study, whole genome sequence, RNAseq, and resequencing of PCR-amplified transcripts for a key floral repressor were used to better understand factors involved in the flowering habit of summer and winter biotypes at the molecular level. Analysis of transcriptome data indicated that abundance for one of the three genes encoding the floral repressor FLOWERING LOCUS C (FLC; Csa20 g015400) was 16-fold greater in Joelle compared to CO46 prior to vernalization. Abundance of this transcript decreased only slightly in CO46 postvernalization, compared to a substantial decrease in Joelle. The results observed in the winter annual biotype Joelle are consistent with repression of FLC by vernalization. Further characterization of FLC at both the genome and transcriptome levels identified a one base deletion in the 5th exon coding for a keratin-binding domain in chromosome 20 of CO46 and Joelle. The one base deletion detected in chromosome 20 FLC is predicted to result in a frameshift that would produce a nonfunctional protein. Analysis of whole genome sequence indicated that the one base deletion in chromosome 20 FLC occurred at a greater ratio in the summer biotype CO46 (2:1) compared to the winter biotype Joelle (1:4); similar trends were also observed for RNAseq and cDNA transcripts mapping to chromosome 20 FLC of CO46 and Joelle.

Perennial Grain and Oilseed Crops.

Historically, agroecosystems have been designed to produce food. Modern societies now demand more from food systems-not only food, fuel, and fiber, but also a variety of ecosystem services. And although today's farming practices are producing unprecedented yields, they are also contributing to ecosystem problems such as soil erosion, greenhouse gas emissions, and water pollution. This review highlights the potential benefits of perennial grains and oilseeds and discusses recent progress in their development. Because of perennials' extended growing season and deep root systems, they may require less fertilizer, help prevent runoff, and be more drought tolerant than annuals. Their production is expected to reduce tillage, which could positively affect biodiversity. End-use possibilities involve food, feed, fuel, and nonfood bioproducts. Fostering multidisciplinary collaborations will be essential for the successful integration of perennials into commercial cropping and food-processing systems.

Gene duplication and divergence affecting drug content in Cannabis sativa.

Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop.

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress.

New approaches to facilitate rapid domestication of a wild plant to an oilseed crop: example pennycress (Thlaspi arvense L.).

Oilseed crops are sources of oils and seed meal having a multitude of uses. While the domestication of soybean and rapeseed took extended periods of time, new genome-based techniques have ushered in an era where crop domestication can occur rapidly. One attractive target for rapid domestication is the winter annual plant Field Pennycress (Thlaspi arvense L.; pennycress; Brassicaceae). Pennycress grows widespread throughout temperate regions of the world and could serve as a winter oilseed-producing cover crop. If grown throughout the USA Midwest Corn Belt, for example, pennycress could produce as much as 840L/ha oils and 1470kg/ha press-cake annually on 16 million hectares of farmland currently left fallow during the fall through spring months. However, wild pennycress strains have inconsistent germination and stand establishment, un-optimized maturity for a given growth zone, suboptimal oils and meal quality for biofuels and food production, and significant harvest loss due to pod shatter. In this review, we describe the virtues and current shortcomings of pennycress and discuss how knowledge from studying Arabidopsis thaliana and other Brassicas, in combination with the advent of affordable next generation sequencing, can bring about the rapid domestication and improvement of pennycress and other crops.

De novo assembly of the pennycress (Thlaspi arvense) transcriptome provides tools for the development of a winter cover crop and biodiesel feedstock.

Field pennycress (Thlaspi arvense L.) has potential as an oilseed crop that may be grown during fall (autumn) and winter months in the Midwestern United States and harvested in the early spring as a biodiesel feedstock. There has been little agronomic improvement in pennycress through traditional breeding. Recent advances in genomic technologies allow for the development of genomic tools to enable rapid improvements to be made through genomic assisted breeding. Here we report an annotated transcriptome assembly for pennycress. RNA was isolated from representative plant tissues, and 203 million unique Illumina RNA-seq reads were produced and used in the transcriptome assembly. The draft transcriptome assembly consists of 33 873 contigs with a mean length of 1242 bp. A global comparison of homology between the pennycress and Arabidopsis transcriptomes, along with four other Brassicaceae species, revealed a high level of global sequence conservation within the family. The final assembly was functionally annotated, allowing for the identification of putative genes controlling important agronomic traits such as flowering and glucosinolate metabolism. Identification of these genes leads to testable hypotheses concerning their conserved function and to rational strategies to improve agronomic properties in pennycress. Future work to characterize isoform variation between diverse pennycress lines and develop a draft genome sequence for pennycress will further direct trait improvement.

Analysis of purified glabra3-shapeshifter trichomes reveals a role for NOECK in regulating early trichome morphogenic events.

Transcriptome analysis using the Affymetrix ATH1 platform has been completed on purified trichomes from the gl3-sst mutant. These trichomes display immature features, such as glassy cell walls and blunted branches. The gl3-sst trichome transcriptome was greatly enriched for genes involved in lipid biosynthesis, including those mediating the synthesis of fatty acids and wax. In addition, gl3-sst trichomes displayed reduced expression of the R3 MYBs TRY and CPC, which normally function to limit trichome development. The expression of the MIXTA-like MYB gene NOK was elevated. Members of the MIXTA-like family promote conical cell outgrowth, and in some cases, trichome initiation in diverse plant species. In contrast, NOK limits trichome outgrowth in wild-type Arabidopsis plants. Similar to other MIXTA-like genes, NOK was required for the expansion of gl3-sst trichomes, as the gl3-sst nok double mutant trichomes were greatly reduced in size. Expression of NOK in nok mutants reduced branch formation, whereas in gl3-sst nok, NOK expression promoted trichome cell outgrowth, illustrating duel roles for NOK in both promoting and limiting trichome development. MIXTA-like genes from phylogenetically diverse plant species could substitute for NOK in both nok and gl3-sst nok backgrounds. These findings suggest that certain aspects of NOK and MIXTA-like gene function have been conserved.

Phylogenetically distinct cellulose synthase genes support secondary wall thickening in arabidopsis shoot trichomes and cotton fiber.

Abstract Through exploring potential analogies between cotton seed trichomes (or cotton fiber) and arabidopsis shoot trichomes we discovered that CesAs from either the primary or secondary wall phylogenetic clades can support secondary wall thickening. CesA genes that typically support primary wall synthesis, AtCesA1,2,3,5, and 6, underpin expansion and secondary wall thickening of arabidopsis shoot trichomes. In contrast, apparent orthologs of CesA genes that support secondary wall synthesis in arabidopsis xylem, AtCesA4,7, and 8, are up-regulated for cotton fiber secondary wall deposition. These conclusions arose from: (a) analyzing the expression of CesA genes in arabidopsis shoot trichomes; (b) observing birefringent secondary walls in arabidopsis shoot trichomes with mutations in AtCesA4, 7, or 8; (c) assaying up-regulated genes during different stages of cotton fiber development; and (d) comparing genes that were co-expressed with primary or secondary wall CesAs in arabidopsis with genes up-regulated in arabidopsis trichomes, arabidopsis secondary xylem, or cotton fiber during primary or secondary wall deposition. Cumulatively, the data show that: (a) the xylem of arabidopsis provides the best model for secondary wall cellulose synthesis in cotton fiber; and (b) CesA genes within a "cell wall toolbox" are used in diverse ways for the construction of particular specialized cell walls.

TrichOME: a comparative omics database for plant trichomes.

Plant secretory trichomes have a unique capacity for chemical synthesis and secretion and have been described as biofactories for the production of natural products. However, until recently, most trichome-specific metabolic pathways and genes involved in various trichome developmental stages have remained unknown. Furthermore, only a very limited amount of plant trichome genomics information is available in scattered databases. We present an integrated "omics" database, TrichOME, to facilitate the study of plant trichomes. The database hosts a large volume of functional omics data, including expressed sequence tag/unigene sequences, microarray hybridizations from both trichome and control tissues, mass spectrometry-based trichome metabolite profiles, and trichome-related genes curated from published literature. The expressed sequence tag/unigene sequences have been annotated based upon sequence similarity with popular databases (e.g. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Transporter Classification Database). The unigenes, metabolites, curated genes, and probe sets have been mapped against each other to enable comparative analysis. The database also integrates bioinformatics tools with a focus on the mining of trichome-specific genes in unigenes and microarray-based gene expression profiles. TrichOME is a valuable and unique resource for plant trichome research, since the genes and metabolites expressed in trichomes are often underrepresented in regular non-tissue-targeted cDNA libraries. TrichOME is freely available at http://www.planttrichome.org/.

Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry.

Trichomes are specialized epidermal structures that function as physical and chemical deterrents against arthropod herbivores. Aerial tissues of cultivated tomato (Solanum lycopersicum) are populated by several morphologically distinct trichome types, the most abundant of which is the type VI glandular trichome that produces various specialized metabolites. Here, the effect of the hairless (hl) mutation on trichome density and morphology, chemical composition, and resistance to a natural insect herbivore of tomato was investigated. The results show that the major effect of hl on pubescence results from structural distortion (bending and swelling) of all trichome types in aerial tissues. Leaf surface extracts and isolated type VI glands from hl plants contained wild-type levels of monoterpenes, glycoalkaloids, and acyl sugars, but were deficient in sesquiterpene and polyphenolic compounds implicated in anti-insect defence. No-choice bioassays showed that hl plants are compromised in resistance to the specialist herbivore Manduca sexta. These results establish a link between the morphology and chemical composition of glandular trichomes in cultivated tomato, and show that hl-mediated changes in these leaf surface traits correlate with decreased resistance to insect herbivory.

A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development.

WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins (PAs), and mucilage in the seed and the development of trichomes and root hairs. We have cloned and characterized a WD40 repeat protein gene from Medicago truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in the seed, reduces epicatechin levels without corresponding effects on other flavonoids in flowers, reduces isoflavone levels in roots, but does not impair trichome or root hair development. MtWD40-1 is expressed constitutively, with highest expression in the seed coat, where its transcript profile temporally parallels those of PA biosynthetic genes. Transcript profile analysis revealed that many genes of flavonoid biosynthesis were down-regulated in a tissue-specific manner in M. truncatula lines harboring retrotransposon insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa glabrous1 mutant. We discuss the function of MtWD40-1 in natural product formation in M. truncatula and the potential use of the gene for engineering PAs in the forage legume alfalfa (Medicago sativa).

Transcriptome analysis of Arabidopsis wild-type and gl3-sst sim trichomes identifies four additional genes required for trichome development.

Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3-sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3-sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3-sst sim trichomes. The analysis of genes preferentially expressed in gl3-sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD-ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

Identification of candidate genes affecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa.

RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses.

A new procedure has been developed for the isolation of wild-type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23-fold ratio of pectin-related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.

E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes.

This is an addendum to our recent paper published in The Plant Journal (52:352-61). The major findings were: (1) trichomes on the leaves of gl3-sst sim double mutants developed as large multi-cellular clusters whereas wild type trichomes are composed of single cells; (2) ectopic CYCD3;1 expression in gl3-sst trichomes also resulted in trichome cluster formation; and (3) that GL1 expression is prolonged in the gl3-sst sim trichome clusters. This addendum shows that ectopic CYCD3;1 expression in gl3-sst also enhanced GL1 expression. An analysis of the GL1 promoter found two overlapping potential E2F binding sites in a region of the promoter known to be essential for GL1 function. This finding indicates that GL1 may be directly regulated by the activity of a CYCD3/CDKA complex that phosphorylates E2F-RB bound to the GL1 promoter.

Genetic interaction between glabra3-shapeshifter and siamese in Arabidopsis thaliana converts trichome precursors into cells with meristematic activity.

The identity of many genes required for trichome differentiation is known. This paper describes a novel interaction between mutant alleles of two such genes. One of the alleles, called gl3-sst, is derived from the GL3 locus, which encodes a basic helix-loop-helix type transcription factor. The mutation in the gl3-sst protein modifies its ability to form a complex with the GL1 protein (a MYB transcription factor required for trichome formation), leading to changes in gene expression compared with wild type during gl3-sst mutant trichome development. The other mutant allele, sim, is a likely loss of function allele derived from the SIM locus, which is predicted to encode a negative regulator of D-type cyclin activity. The gl3-sst sim double mutant exhibits mounds of cells derived from the proliferation of single trichome precursors. The ectopic expression of a D-type cyclin gene in gl3-sst mimics the double mutant phenotype. Thus, an interaction between altered trichome gene expression caused by the gl3-sst mutation and relaxed regulation of D-type cyclin activity in the double mutant converted a non-dividing cell into a novel highly proliferating cell type.