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.

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.

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.

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.

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.

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.

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.