A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Genetic architecture and QTL selection response for Kernza perennial grain domestication traits.

KEY MESSAGE: Analysis of multi-year breeding program data revealed that the genetic architecture of an intermediate wheatgrass population was highly polygenic for both domestication and agronomic traits, supporting the use of genomic selection for new crop domestication. Perennial grains have the potential to provide food for humans and decrease the negative impacts of annual agriculture. Intermediate wheatgrass (IWG, Thinopyrum intermedium, Kernza®) is a promising perennial grain candidate that The Land Institute has been breeding since 2003. We evaluated four consecutive breeding cycles of IWG from 2016 to 2020 with each cycle containing approximately 1100 unique genets. Using genotyping-by-sequencing markers, quantitative trait loci (QTL) were mapped for 34 different traits using genome-wide association analysis. Combining data across cycles and years, we found 93 marker-trait associations for 16 different traits, with each association explaining 0.8-5.2% of the observed phenotypic variance. Across the four cycles, only three QTL showed an FST differentiation > 0.15 with two corresponding to a decrease in floret shattering. Additionally, one marker associated with brittle rachis was 216 bp from an ortholog of the btr2 gene. Power analysis and quantitative genetic theory were used to estimate the effective number of QTL, which ranged from a minimum of 33 up to 558 QTL for individual traits. This study suggests that key agronomic and domestication traits are under polygenic control and that molecular methods like genomic selection are needed to accelerate domestication and improvement of this new crop.

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.

Nested association mapping reveals the genetic architecture of spike emergence and anthesis timing in intermediate wheatgrass.

Intermediate wheatgrass (Thinopyrum intermedium) is an outcrossing, cool season grass species currently undergoing direct domestication as a perennial grain crop. Though many traits are selection targets, understanding the genetic architecture of those important for local adaptation may accelerate the domestication process. Nested association mapping (NAM) has proven useful in dissecting the genetic control of agronomic traits many crop species, but its utility in primarily outcrossing, perennial species has yet to be demonstrated. Here, we introduce an intermediate wheatgrass NAM population developed by crossing ten phenotypically divergent donor parents to an adapted common parent in a reciprocal manner, yielding 1,168 F1 progeny from 10 families. Using genotyping by sequencing, we identified 8,003 SNP markers and developed a population-specific consensus genetic map with 3,144 markers across 21 linkage groups. Using both genomewide association mapping and linkage mapping combined across and within families, we characterized the genetic control of flowering time. In the analysis of two measures of maturity across four separate environments, we detected as many as 75 significant QTL, many of which correspond to the same regions in both analysis methods across 11 chromosomes. The results demonstrate a complex genetic control that is variable across years, locations, traits, and within families. The methods were effective at detecting previously identified QTL, as well as new QTL that align closely to the well-characterized flowering time orthologs from barley, including Ppd-H1 and Constans. Our results demonstrate the utility of the NAM population for understanding the genetic control of flowering time and its potential for application to other traits of interest.

A needle in a seedstack: an improved method for detection of rare alleles in bulk seed testing through KASP.

BACKGROUND: Amaranthus palmeri is an aggressive and prolific weed species with major impact on agricultural yield and is a prohibited noxious weed across the Midwest. Morphological identification of A. palmeri from other Amaranthus species is extremely difficult in seeds, which has led to genetic testing for seed identification in commercial seed lots. RESULTS: We created an inexpensive and reliable genetic test based on novel, species-specific, single nucleotide polymorphisms (SNPs) from GBS (Genotyping by Sequencing) data. We report three SNP-based genetic tests for identifying A. palmeri alone or in a mixed pool of Amaranthus spp. Sensitivity ranged from 99.8 to 100%, specificity from 99.59 to 100%. Accuracy for all three tests is > 99.7%. All three are capable of reliably detecting one A. palmeri seed in a pool of 200 Amaranthus spp. seeds. The test was validated across 20 populations of A. palmeri, along with eight other Amaranthus species, the largest and most genetically diverse panel of Amaranthus samples to date. CONCLUSION: Our work represents a marked improvement over existing commercial assays resulting in an identification assay that is (i) accurate, (ii) robust, (iii) easy to interpret and (iv) applicable to both leaf tissue and pools of up to 200 seeds. Included is a data transformation method for calling of closely grouped competitive fluorescence assays. We also present a comprehensive GBS dataset from the largest geographic panel of Amaranthus populations sequenced. Our approach serves as a model for developing markers for other difficult to identify species. © 2021 Society of Chemical Industry.

Patterns of genetic variation in a prairie wildflower, Silphium integrifolium, suggest a non-prairie origin and locally adaptive variation.

PREMISE: Understanding the relationship between genetic structure and geography provides information about a species' history and can be used for breeding and conservation goals. The North American prairie is interesting because of its recent origin and subsequent fragmentation. Silphium integrifolium, an iconic perennial American prairie wildflower, is targeted for domestication, having undergone a few generations of improvement. We present the first application of population genetic data in this species to address the following goals: (1) improve breeding by characterizing genetic structure and (2) identify the species geographic origin and potential targets and drivers of selection during range expansion. METHODS: We developed a reference transcriptome as a genotyping reference for samples from throughout the species range. Population genetic analyses were used to describe patterns of genetic variation, and demographic modeling was used to characterize potential processes that shaped variation. Outlier scans for selection and associations with environmental variables were used to identify loci linked to putative targets and drivers of selection. RESULTS: Genetic variation partitioned samples into three geographic clusters. Patterns of variation and demographic modeling suggest that the species origin is in the American Southeast. Breeding program accessions are from the region with lowest observed genetic variation. CONCLUSIONS: This prairie species did not originate within the prairie. Breeding may be improved by including accessions from outside of the germplasm founding region. The geographic structuring and the identified targets and drivers of adaptation can guide collecting efforts toward populations with beneficial agronomic traits.

The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding.

KEY MESSAGE: The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Nucleic acid damage and DNA repair are affected by freezing stress in annual wheat (Triticum aestivum) and by plant age and freezing in its perennial relative (Thinopyrum intermedium).

PREMISE: Nucleic acid integrity can be compromised under many abiotic stresses. To date, however, few studies have considered whether nucleic acid damage and damage repair play a role in cold-stress adaptation. A further insufficiently explored question concerns how age affects cold stress adaptation among mature perennials. As a plant ages, the optimal trade-off between growth and stress tolerance may shift. METHODS: Oxidative damage to RNA and expression of genes involved in DNA repair were compared in multiple mature cohorts of Thinopyrum intermedium (an emerging perennial cereal) and in wheat and barley under intermittent freezing stress and under nonfreezing conditions. Activity of glutathione peroxidase (GPX) and four other antioxidative enzymes was also measured under these conditions. DNA repair genes included photolyases involved in repairing ultraviolet-induced damage and two genes involved in repairing oxidatively induced damage (ERCC1, RAD23). RESULTS: Freezing stress was accompanied by large increases in photolyase expression and ERCC1 expression (in wheat and Thinopyrum) and in GPX and GR activity (particularly in Thinopyrum). This is the first report of DNA photolyases being overexpressed under freezing stress. Older Thinopyrum had lower photolyase expression and less freezing-induced overexpression of ERCC1. Younger Thinopyrum plants sustained more oxidative damage to RNA. CONCLUSIONS: Overexpression of DNA repair genes is an important aspect of cold acclimation. When comparing adult cohorts, aging was associated with changes in the freezing stress response, but not with overall increases or decreases in stress tolerance.

Sequenced-based paternity analysis to improve breeding and identify self-incompatibility loci in intermediate wheatgrass (Thinopyrum intermedium).

KEY MESSAGE: Paternity assignment and genome-wide association analyses for fertility were applied to a Thinopyrum intermedium breeding program. A lack of progeny between combinations of parents was associated with loci near self-incompatibility genes. In outcrossing species such as intermediate wheatgrass (IWG, Thinopyrum intermedium), polycrossing is often used to generate novel recombinants through each cycle of selection, but it cannot track pollen-parent pedigrees and it is unknown how self-incompatibility (SI) genes may limit the number of unique crosses obtained. This study investigated the potential of using next-generation sequencing to assign paternity and identify putative SI loci in IWG. Using a reference population of 380 individuals made from controlled crosses of 64 parents, paternity was assigned with 92% agreement using Cervus software. Using this approach, 80% of 4158 progeny (n = 3342) from a polycross of 89 parents were assigned paternity. Of the 89 pollen parents, 82 (92%) were represented with 1633 unique full-sib families representing 42% of all potential crosses. The number of progeny per successful pollen parent ranged from 1 to 123, with number of inflorescences per pollen parent significantly correlated to the number of progeny (r = 0.54, p < 0.001). Shannon's diversity index, assessing the total number and representation of families, was 7.33 compared to a theoretical maximum of 8.98. To test our hypothesis on the impact of SI genes, a genome-wide association study of the number of progeny observed from the 89 parents identified genetic effects related to non-random mating, including marker loci located near putative SI genes. Paternity testing of polycross progeny can impact future breeding gains by being incorporated in breeding programs to optimize polycross methodology, maintain genetic diversity, and reveal genetic architecture of mating patterns.

Transcriptional reprogramming and enhanced photosynthesis drive inducible salt tolerance in sugarcane mutant line M4209.

Sugarcane (Saccharum officinarum) is a globally cultivated cash crop whose yield is negatively affected by soil salinity. In this study, we investigated the molecular basis of inducible salt tolerance in M4209, a sugarcane mutant line generated through radiation-induced mutagenesis. Under salt-contaminated field conditions, M4209 exhibited 32% higher cane yield as compared with its salt-sensitive parent, Co86032. In pot experiments, post-sprouting phenotyping indicated that M4209 had significantly greater leaf biomass compared with Co86032 under treatment with 50 mM and 200 mM NaCl. This was concomitant with M4209 having 1.9-fold and 1.6-fold higher K+/Na+ ratios, and 4-fold and 40-fold higher glutathione reductase activities in 50 mM and 200 mM NaCl, respectively, which suggested that it had better ionic and redox homeostasis than Co86032. Transcriptome profiling using RNA-seq indicated an extensive reprograming of stress-responsive modules associated with photosynthesis, transmembrane transport, and metabolic processes in M4209 under 50 mM NaCl stress. Using ranking analysis, we identified Phenylalanine Ammonia Lyase (PAL), Acyl-Transferase Like (ATL), and Salt-Activated Transcriptional Activator (SATA) as the genes most associated with salt tolerance in M4209. M4209 also exhibited photosynthetic rates that were 3-4-fold higher than those of Co86032 under NaCl stress conditions. Our results highlight the significance of transcriptional reprogramming coupled with improved photosynthetic efficiency in determining salt tolerance in sugarcane.

Genome mapping of quantitative trait loci (QTL) controlling domestication traits of intermediate wheatgrass (Thinopyrum intermedium).

Allohexaploid (2n = 6x = 42) intermediate wheatgrass (Thinopyrum intermedium), abbreviated IWG, is an outcrossing perennial grass belonging to the tertiary gene pool of wheat. Perenniality would be valuable option for grain production, but attempts to introgress this complex trait from wheat-Thinopyrum hybrids have not been commercially successful. Efforts to breed IWG itself as a dual-purpose forage and grain crop have demonstrated useful progress and applications, but grain yields are significantly less than wheat. Therefore, genetic and physical maps have been developed to accelerate domestication of IWG. Herein, these maps were used to identify quantitative trait loci (QTLs) and candidate genes associated with IWG grain production traits in a family of 266 full-sib progenies derived from two heterozygous parents, M26 and M35. Transgressive segregation was observed for 17 traits related to seed size, shattering, threshing, inflorescence capacity, fertility, stem size, and flowering time. A total of 111 QTLs were detected in 36 different regions using 3826 genotype-by-sequence markers in 21 linkage groups. The most prominent QTL had a LOD score of 15 with synergistic effects of 29% and 22% over the family means for seed retention and percentage of naked seeds, respectively. Many QTLs aligned with one or more IWG gene models corresponding to 42 possible domestication orthogenes including the wheat Q and RHT genes. A cluster of seed-size and fertility QTLs showed possible alignment to a putative Z self-incompatibility gene, which could have detrimental grain-yield effects when genetic variability is low. These findings elucidate pathways and possible hurdles in the domestication of IWG.

Genes associated with chloroplasts and hormone-signaling, and transcription factors other than CBFs are associated with differential survival after low temperature treatments of Camelina sativa biotypes.

Winter annual biotypes of Camelina sativa regularly survive after winter conditions experienced in northern regions of the U.S., whereas summer annual biotypes do not. To determine potential molecular mechanisms associated with these biotype differences in survival after low temperature treatments, we examined genetic and transcript variations in both a winter- (Joelle) and a summer- (CO46) biotype. It was determined that as few as one or two dominant genes may control differential survival after low temperature treatments. Of the 1797 genes that were differentially expressed in response to cold in both the winter and summer biotypes many COR genes were identified, indicating that the CBF regulon is functional in both. However, only 153 and 76 genes from Joelle and CO46, respectively, were either differentially expressed or not expressed at all in one biotype versus the other following cold acclimation. We hypothesize that these 229 genes play a significant role in, or are primarily responsive to, differences in survival after freezing between these two biotypes. Promoter analysis provided few clues as to the regulation or these genes; however, genes that were down-regulated specifically in the winter biotype Joelle were enriched with the sequence TGGCCCTCGCTCAC, which is over-represented among genes associated with chloroplasts in Arabidopsis. Additionally, several genes involved in auxin signaling were down-regulated specifically in Joelle. A transcription factor with strong similarity to MYB47, known to be up-regulated by salt, drought, and jasmonic acid, but not cold in Arabidopsis, was essentially off in the freezing sensitive biotype CO46, but was cold-induced in the winter biotype Joelle. Several other transcription factors genes including three with similarity to WRKY70, that may be involved in SA/JA-dependent responses, a HOMEOBOX 6 gene involved in ABA signaling, and two others (NUCLEAR FACTOR Y and CONSTANS-like 2) known to be implicated in photoperiodic flowering were also differentially expressed between the two biotypes.

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.

Translational genomics using Arabidopsis as a model enables the characterization of pennycress genes through forward and reverse genetics.

Thlaspi arvense (pennycress) has the potential for domestication as a new oilseed crop. Information from an extensive body of research on the related plant species Arabidopsis can be used to greatly speed this process. Genome-scale comparisons in this paper documented that pennycress and Arabidopsis share similar gene duplication. This finding led to the hypothesis that it should be possible to isolate Arabidopsis-like mutants in pennycress. This proved to be true, as forward genetic screens identified floral and vegetative pennycress mutants that were similar to mutants found in Arabidopsis. Extending this approach, it was shown that most of the pennycress genes responsible for the formation of oxidized tannins could be rapidly identified. The causative mutations in the pennycress mutants could be identified either by PCR amplification of candidate genes or through whole-genome sequencing (WGS) analysis. In all, WGS was used to characterize 95 ethyl methane sulfonate mutants, which revealed a mutation rate of 4.09 mutations per megabase. A sufficient number of non-synonymous mutations were identified to create a mutant gene index that could be used for reverse genetic approaches to identify pennycress mutants of interest. As proof of concept, a Ta-max3-like dwarf mutant and Ta-kcs5/cer60-like wax mutants deficient in the biosynthesis of long chain fatty acids were identified. Overall, these studies demonstrate that translational genomics can be used to promote the domestication of pennycress. Furthermore, the ease with which important findings could be made in pennycress makes this species a new potential model plant.

Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times.

Field pennycress (Thlaspi arvense L.) is currently being developed as a new cold-tolerant oilseed crop. In natural populations, pennycress, like many Brassicaceae relatives, can exhibit either a winter or spring annual phenotype. Pennycress is a diploid relative of Arabidopsis thaliana, a model species that has been used to study many adaptive phenotypes, including flowering time and developmental timing. In Arabidopsis and other Brassicaceae species, mutations in negative regulators of flowering, including FLOWERING LOCUS C and FRIGIDA can cause the transition to a spring annual habit. The genetics underlying the difference between spring and winter annual pennycress lines are currently unknown. Here, we report the identification of four natural alleles of FLC in pennycress that confer a spring annual growth habit identified through whole genome sequencing, cosegregation analyses, and comparative genomics. The global distribution of these spring annual alleles of FLC suggests that the spring annual growth habit has arisen on several independent occasions. The two spring annual FLC alleles present in European accessions were only identified in North American accessions collected in southern Montana, which indicates accessions harboring these two alleles were introduced to North America, likely after pennycress became a widespread species on the continent. These findings provide new information on the natural history of the introduction and spread of spring annual pennycress accessions from Europe into North America. At the molecular level, these findings are important for the ongoing development of pennycress as a winter annual crop. An enhanced understanding of the regulation of flowering in this species should allow for the fine-tuning of flowering in commercial varieties.

Transcriptome assembly and annotation of johnsongrass (Sorghum halepense) rhizomes identify candidate rhizome-specific genes.

Rhizomes facilitate the wintering and vegetative propagation of many perennial grasses. Sorghum halepense (johnsongrass) is an aggressive perennial grass that relies on a robust rhizome system to persist through winters and reproduce asexually from its rootstock nodes. This study aimed to sequence and assemble expressed transcripts within the johnsongrass rhizome. A de novo transcriptome assembly was generated from a single johnsongrass rhizome meristem tissue sample. A total of 141,176 probable protein-coding sequences from the assembly were identified and assigned gene ontology terms using Blast2GO. Estimated expression analysis and BLAST results were used to reduce the assembly to 64,447 high-confidence sequences. The johnsongrass assembly was compared to Sorghum bicolor, a related nonrhizomatous species, along with an assembly of similar rhizome tissue from the perennial grain crop Thinopyrum intermedium. The presence/absence analysis yielded a set of 98 expressed johnsongrass contigs that are likely associated with rhizome development.

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.

The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization.

BACKGROUND: Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. RESULTS: Pennycress flowers contain four equivalent nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from 'immature' (pre-secretory) and 'mature' (secretory) nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR. CONCLUSIONS: Pennycress nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.

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.