Genome-Wide Association Studies and Transcriptome Changes during Acclimation and Deacclimation in Divergent Brassica napus Varieties.

Information concerning genes and signals regulating cold acclimation processes in plants is abundant; however, less is known about genes and signals regulating the deacclimation process. A population of primarily winter B. napus varieties was used to conduct a genome-wide association study and to compare the transcriptomes from two winter B. napus varieties showing time-dependent differences in response to cold acclimation and deacclimation treatments. These studies helped to identify loci, candidate genes, and signaling processes impacting deacclimation in B. napus. GWAS identified polymorphisms at five different loci associated with freezing tolerance following deacclimation. Local linkage decay rates near these polymorphisms identified 38 possible candidate genes. Several of these genes have been reported as differentially regulated by cold stress in arabidopsis (Arabidopsis thaliana), including a calcium-binding EF-hand family protein (encoded by BnaCnng10250D) that was also differentially expressed during deacclimation in this study. Thousands of other genes differentially expressed during the acclimation and deacclimation treatments implicated processes involving oxidative stress, photosynthesis, light-regulated diurnal responses, and growth regulation. Generally, responses observed during acclimation were reversed within one week of deacclimation. The primary differences between the two winter B. napus varieties with differential deacclimation responses involved protection from oxidative stress and the ability to maintain photosynthesis.

Comparison of phytohormone levels and transcript profiles during seasonal dormancy transitions in underground adventitious buds of leafy spurge.

Leafy spurge (Euphorbia esula L.) is an herbaceous perennial weed that maintains its perennial growth habit through generation of underground adventitious buds (UABs) on the crown and lateral roots. These UABs undergo seasonal phases of dormancy under natural conditions, namely para-, endo-, and ecodormancy in summer, fall, and winter, respectively. These dormancy phases can also be induced in growth chambers by manipulating photoperiod and temperature. In this study, UABs induced into the three phases of dormancy under controlled conditions were used to compare changes in phytohormone and transcriptome profiles. Results indicated that relatively high levels of ABA, the ABA metabolite PA, and IAA were found in paradormant buds. When UABs transitioned from para- to endodormancy, ABA and PA levels decreased, whereas IAA levels were maintained. Additionally, transcript profiles associated with regulation of soluble sugars and ethylene activities were also increased during para- to endodormancy transition, which may play some role in maintaining endodormancy status. When crown buds transitioned from endo- to ecodormancy, the ABA metabolites PA and DPA decreased significantly along with the down-regulation of ABA biosynthesis genes, ABA2 and NCED3. IAA levels were also significantly lower in ecodormant buds than that of endodormant buds. We hypothesize that extended cold treatment may trigger physiological stress in endodormant buds, and that these stress-associated signals induced the endo- to ecodormancy transition and growth competence. The up-regulation of NAD/NADH phosphorylation and dephosphorylation pathway, and MAF3-like and GRFs genes, may be considered as markers of growth competency.

Phytohormone balance and stress-related cellular responses are involved in the transition from bud to shoot growth in leafy spurge.

BACKGROUND: Leafy spurge (Euphorbia esula L.) is an herbaceous weed that maintains a perennial growth pattern through seasonal production of abundant underground adventitious buds (UABs) on the crown and lateral roots. During the normal growing season, differentiation of bud to shoot growth is inhibited by physiological factors external to the affected structure; a phenomenon referred to as paradormancy. Initiation of shoot growth from paradormant UABs can be accomplished through removal of the aerial shoots (hereafter referred to as paradormancy release). RESULTS: In this study, phytohormone abundance and the transcriptomes of paradormant UABs vs. shoot-induced growth at 6, 24, and 72 h after paradormancy release were compared based on hormone profiling and RNA-seq analyses. Results indicated that auxin, abscisic acid (ABA), and flavonoid signaling were involved in maintaining paradormancy in UABs of leafy spurge. However, auxin, ABA, and flavonoid levels/signals decreased by 6 h after paradormancy release, in conjunction with increase in gibberellic acid (GA), cytokinin, jasmonic acid (JA), ethylene, and brassinosteroid (BR) levels/signals. Twenty four h after paradormancy release, auxin and ABA levels/signals increased, in conjunction with increase in GA levels/signals. Major cellular changes were also identified in UABs at 24 h, since both principal component and Venn diagram analysis of transcriptomes clearly set the 24 h shoot-induced growth apart from other time groups. In addition, increase in auxin and ABA levels/signals and the down-regulation of 40 over-represented AraCyc pathways indicated that stress-derived cellular responses may be involved in the activation of stress-induced re-orientation required for initiation of shoot growth. Seventy two h after paradormancy release, auxin, cytokinin, and GA levels/signals were increased, whereas ABA, JA, and ethylene levels/signals were decreased. CONCLUSION: Combined results were consistent with different phytohormone signals acting in concert to direct cellular changes involved in bud differentiation and shoot growth. In addition, shifts in balance of these phytohormones at different time points and stress-related cellular responses after paradormancy release appear to be critical factors driving transition of bud to shoot growth.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no α-carotene was observed, variable amounts of a α-ring derived xanthophyll, lutein, was detected; with greater accumulation of α-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

Glyphosate's impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching.

BACKGROUND: Leafy spurge (Euphorbia esula) is a perennial weed that is considered glyphosate tolerant, which is partially attributed to escape through establishment of new vegetative shoots from an abundance of underground adventitious buds. Leafy spurge plants treated with sub-lethal concentrations of foliar-applied glyphosate produce new vegetative shoots with reduced main stem elongation and increased branching. Processes associated with the glyphosate-induced phenotype were determined by RNAseq using aerial shoots derived from crown buds of glyphosate-treated and -untreated plants. Comparison between transcript abundance and accumulation of shikimate or phytohormones (abscisic acid, auxin, cytokinins, and gibberellins) from these same samples was also done to reveal correlations. RESULTS: Transcriptome assembly and analyses confirmed differential abundance among 12,918 transcripts (FDR ≤ 0.05) and highlighted numerous processes associated with shoot apical meristem maintenance and stem growth, which is consistent with the increased number of actively growing meristems in response to glyphosate. Foliar applied glyphosate increased shikimate abundance in crown buds prior to decapitation of aboveground shoots, which induces growth from these buds, indicating that 5-enolpyruvylshikimate 3-phosphate (EPSPS) the target site of glyphosate was inhibited. However, abundance of shikimate was similar in a subsequent generation of aerial shoots derived from crown buds of treated and untreated plants, suggesting EPSPS is no longer inhibited or abundance of shikimate initially observed in crown buds dissipated over time. Overall, auxins, gibberellins (precursors and catabolites of bioactive gibberellins), and cytokinins (precursors and bioactive cytokinins) were more abundant in the aboveground shoots derived from glyphosate-treated plants. CONCLUSION: Based on the overall data, we propose that the glyphosate-induced phenotype resulted from complex interactions involving shoot apical meristem maintenance, hormone biosynthesis and signaling (auxin, cytokinins, gibberellins, and strigolactones), cellular transport, and detoxification mechanisms.

Dehydration-induced endodormancy in crown buds of leafy spurge highlights involvement of MAF3- and RVE1-like homologs, and hormone signaling cross-talk.

Vegetative shoot growth from underground adventitious buds of leafy spurge is critical for survival of this invasive perennial weed after episodes of severe abiotic stress. To determine the impact that dehydration-stress has on molecular mechanisms associated with vegetative reproduction of leafy spurge, greenhouse plants were exposed to mild- (3-day), intermediate- (7-day), severe- (14-day) and extended- (21-day) dehydration treatments. Aerial tissues of treated plants were then decapitated and soil was rehydrated to determine the growth potential of underground adventitious buds. Compared to well-watered plants, mild-dehydration accelerated new vegetative shoot growth, whereas intermediate- through extended-dehydration treatments both delayed and reduced shoot growth. Results of vegetative regrowth further confirmed that 14 days of dehydration induced a full-state of endodormancy in crown buds, which was correlated with a significant (P < 0.05) change in abundance of 2,124 transcripts. Sub-network enrichment analyses of transcriptome data obtained from the various levels of dehydration treatment also identified central hubs of over-represented genes involved in processes such as hormone signaling (i.e., ABA, auxin, ethylene, GA, and JA), response to abiotic stress (DREB1A/2A, RD22) and light (PIF3), phosphorylation (MPK4/6), circadian regulation (CRY2, PHYA), and flowering (AGL20, AP2, FLC). Further, results from this and previous studies highlight homologs most similar to Arabidopsis HY5, MAF3, RVE1 and RD22 as potential molecular markers for endodormancy in crown buds of leafy spurge. Early response to mild dehydration also highlighted involvement of upstream ethylene and JA-signaling, whereas severe dehydration impacted ABA-signaling. The identification of conserved ABRE- and MYC-consensus, cis-acting elements in the promoter of leafy spurge genomic clones similar to Arabidopsis RVE1 (AT5G17300) implicates a potential role for ABA-signaling in its dehydration-induced expression. Response of these molecular mechanisms to dehydration-stress provides insights on the ability of invasive perennial weeds to adapt and survive under harsh environments, which will be beneficial for addressing future management practices.

The resemblance and disparity of gene expression in dormant and non-dormant seeds and crown buds of leafy spurge (Euphorbia esula).

BACKGROUND: Leafy spurge (Euphorbia esula L.) is a herbaceous perennial weed and dormancy in both buds and seeds is an important survival mechanism. Bud dormancy in leafy spurge exhibits three well-defined phases of para-, endo- and ecodormancy; however, seed dormancy for leafy spurge is classified as physiological dormancy that requires after-ripening and alternating temperature for maximal germination. Overlaps in transcriptome profiles between different phases of bud and seed dormancy have not been determined. Thus, we compared various phases of dormancy between seeds and buds to identify common genes and molecular processes, which should provide new insights about common regulators of dormancy. RESULTS: Cluster analysis of expression profiles for 201 selected genes indicated bud and seed samples clustered separately. Direct comparisons between buds and seeds are additionally complicated since seeds incubated at a constant temperature of 20°C for 21 days (21d C) could be considered paradormant (Para) because seeds may be inhibited by endosperm-generated signals, or ecodormant (Eco) because seeds germinate after being subjected to alternating temperature of 20:30°C. Since direct comparisons in gene expression between buds and seeds were problematic, we instead examined commonalities in differentially-expressed genes associated with different phases of dormancy. Comparison between buds and seeds ('Para to Endo buds' and '21d C to 1d C seeds'), using endodormant buds (Endo) and dormant seeds (1d C) as common baselines, identified transcripts associated with cell cycle (HisH4), stress response/transcription factors (ICE2, ERFB4/ABR1), ABA and auxin response (ABA1, ARF1, IAA7, TFL1), carbohydrate/protein degradation (GAPDH_1), and transport (ABCB2). Comparison of transcript abundance for the 'Eco to Endo buds' and '21d C to 1d C seeds' identified transcripts associated with ABA response (ATEM6), auxin response (ARF1), and cell cycle (HisH4). These results indicate that the physiological state of 21d C seeds is more analogous to paradormant buds than that of ecodormant buds. CONCLUSION: Combined results indicate that common molecular mechanisms associated with dormancy transitions of buds and seeds involve processes associated with ABA and auxin signaling and transport, cell cycle, and AP2/ERF transcription factors or their up-stream regulators.

Genomic prediction for agronomic traits in a diverse Flax (Linum usitatissimum L.) germplasm collection.

Breeding programs require exhaustive phenotyping of germplasms, which is time-demanding and expensive. Genomic prediction helps breeders harness the diversity of any collection to bypass phenotyping. Here, we examined the genomic prediction's potential for seed yield and nine agronomic traits using 26,171 single nucleotide polymorphism (SNP) markers in a set of 337 flax (Linum usitatissimum L.) germplasm, phenotyped in five environments. We evaluated 14 prediction models and several factors affecting predictive ability based on cross-validation schemes. Models yielded significant variation among predictive ability values across traits for the whole marker set. The ridge regression (RR) model covering additive gene action yielded better predictive ability for most of the traits, whereas it was higher for low heritable traits by models capturing epistatic gene action. Marker subsets based on linkage disequilibrium decay distance gave significantly higher predictive abilities to the whole marker set, but for randomly selected markers, it reached a plateau above 3000 markers. Markers having significant association with traits improved predictive abilities compared to the whole marker set when marker selection was made on the whole population instead of the training set indicating a clear overfitting. The correction for population structure did not increase predictive abilities compared to the whole collection. However, stratified sampling by picking representative genotypes from each cluster improved predictive abilities. The indirect predictive ability for a trait was proportionate to its correlation with other traits. These results will help breeders to select the best models, optimum marker set, and suitable genotype set to perform an indirect selection for quantitative traits in this diverse flax germplasm collection.

Induction of endodormancy in crown buds of leafy spurge (Euphorbia esula L.) implicates a role for ethylene and cross-talk between photoperiod and temperature.

Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor facilitating their escape from conventional control measures. A 12-week ramp down in both temperature (27 â†’ 10 °C) and photoperiod (16 â†’ 8 h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks of UABs during this transition, we compared global transcriptome data-sets obtained from leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) versus a ramp down in temperature (RDt) alone. Analysis of data-sets indicated that transcript abundance for genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) preferentially increased in endodormant UABs. Gene-set enrichment analyses also highlighted metabolic pathways involved in endodormancy induction that were associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, miR172A, EINs, DREBs) of molecular networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). We further examined the effects of ethylene by application of 1-aminocyclopropane-1-carboxylate (ACC) to paradormant plants without a ramp down treatment. New vegetative growth from UABs of ACC-treated plants resulted in a dwarfed phenotype that mimicked the growth response in RDtp-induced endodormant UABs. The results of this study provide new insights into dormancy regulation suggesting a short-photoperiod treatment provides an additive cross-talk effect with temperature signals that may impact ethylene's effect on AP2/ERF family members.

Weed-induced crop yield loss: a new paradigm and new challenges.

Direct competition for resources is generally considered the primary mechanism for weed-induced yield loss. A re-evaluation of physiological evidence suggests weeds initially impact crop growth and development through resource-independent interference. We suggest weed perception by crops induce a shift in crop development, before resources become limited, which ultimately reduce crop yield, even if weeds are subsequently removed. We present the mechanisms by which crops perceive and respond to weeds and discuss the technologies used to identify these mechanisms. These data lead to a fundamental paradigm shift in our understanding of how weeds reduce crop yield and suggest new research directions and opportunities to manipulate or engineer crops and cropping systems to reduce weed-induced yield losses.

Weed-induced changes in the maize root transcriptome reveal transcription factors and physiological processes impacted early in crop-weed interactions.

A new paradigm suggests weeds primarily reduce crop yield by altering crop developmental and physiological processes long before the weeds reduce resources through competition. Multiple studies have implicated stress response pathways are activated when crops such as maize are grown in close proximity with weeds during the first 4-8 weeks of growth-the point at which weeds have their greatest impact on subsequent crop yields. To date, these studies have mostly focused on the response of above-ground plant parts and have not examined the early signal transduction processes associated with maize root response to weeds. To investigate the impact of signals from a below-ground competitor on the maize root transcriptome when most vulnerable to weed pressure, a system was designed to expose maize to only below-ground signals. Gene set enrichment analyses identified over-represented ontologies associated with oxidative stress signalling throughout the time of weed exposure, with additional ontologies associated with nitrogen use and transport and abscisic acid (ABA) signalling, and defence responses being enriched at later time points. Enrichment of promoter motifs indicated over-representation of sequences known to bind FAR-RED IMPAIRED RESPONSE 1 (FAR1), several AP2/ERF transcription factors and others. Likewise, co-expression networks were identified using Weighted-Gene Correlation Network Analysis (WGCNA) and Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) algorithms. WGCNA highlighted the potential roles of several transcription factors including a MYB 3r-4, TB1, WRKY65, CONSTANS-like5, ABF3, HOMEOBOX 12, among others. These studies also highlighted the role of several specific proteins involved in ABA signalling as being important for the initiation of the early response of maize to weeds. SC-ION highlighted potential roles for NAC28, LOB37, NAC58 and GATA2 transcription factors, among many others.

Homozygosity mapping identified loci and candidate genes responsible for freezing tolerance in Camelina sativa.

Homozygosity mapping is an effective tool for detecting genomic regions responsible for a given trait when the phenotype is controlled by a limited number of dominant or co-dominant loci. Freezing tolerance is a major attribute in agricultural crops such as camelina. Previous studies indicated that freezing tolerance differences between a tolerant (Joelle) and susceptible (CO46) variety of camelina were controlled by a small number of dominant or co-dominant genes. We performed whole genome homozygosity mapping to identify markers and candidate genes responsible for freezing tolerance difference between these two genotypes. A total of 28 F3 RILs were sequenced to ∼30× coverage, and parental lines were sequenced to >30-40× coverage with Pacific Biosciences high fidelity technology and 60× coverage using Illumina whole genome sequencing. Overall, about 126k homozygous single nucleotide polymorphism markers were identified that differentiate both parents. Moreover, 617 markers were also homozygous in F3 families fixed for freezing tolerance/susceptibility. All these markers mapped to two contigs forming a contiguous stretch of chromosome 11. The homozygosity mapping detected 9 homozygous blocks among the selected markers and 22 candidate genes with strong similarity to regions in or near the homozygous blocks. Two such genes were differentially expressed during cold acclimation in camelina. The largest block contained a cold-regulated plant thionin and a putative rotamase cyclophilin 2 gene previously associated with freezing resistance in arabidopsis (Arabidopsis thaliana). The second largest block contains several cysteine-rich RLK genes and a cold-regulated receptor serine/threonine kinase gene. We hypothesize that one or more of these genes may be primarily responsible for freezing tolerance differences in camelina varieties.

Overwintering Camelina and Canola/Rapeseed Show Promise for Improving Integrated Weed Management Approaches in the Upper Midwestern U.S.

Winter oilseed cash cover crops are gaining popularity in integrated weed management programs for suppressing weeds. A study was conducted at two field sites (Fargo, North Dakota, and Morris, Minnesota) to determine the freezing tolerance and weed-suppressing traits of winter canola/rapeseed (Brassica napus L.) and winter camelina [Camelina sativa (L.) Crantz] in the Upper Midwestern USA. The top 10 freezing tolerant accessions from a phenotyped population of winter canola/rapeseed were bulked and planted at both locations along with winter camelina (cv. Joelle) as a check. To phenotype our entire winter B. napus population (621 accessions) for freezing tolerance, seeds were also bulked and planted at both locations. All B. napus and camelina were no-till seeded at Fargo and Morris at two planting dates, late August (PD1) and mid-September (PD2) 2019. Data for winter survival of oilseed crops (plants m-2) and their corresponding weed suppression (plants m-2 and dry matter m-2) were collected on two sampling dates (SD) in May and June 2020. Crop and SD were significant (p < 0.05) for crop plant density at both locations, and PD in Fargo and crop x PD interaction in Morris were significant for weed dry matter. At Morris and Fargo, PD1 produced greater winter B. napus survival (28% and 5%, respectively) and PD2 produced higher camelina survival (79% and 72%, respectively). Based on coefficient of determination (r2), ~50% of weed density was explained by camelina density, whereas ≤20% was explained by B. napus density at both locations. Camelina from PD2 suppressed weed dry matter by >90% of fallow at both locations, whereas weed dry matter in B. napus was not significantly different from fallow at either PD. Genotyping of overwintering canola/rapeseed under field conditions identified nine accessions that survived at both locations, which also had excellent freezing tolerance under controlled conditions. These accessions are good candidates for improving freezing tolerance in commercial canola cultivars.

Auxin and ABA act as central regulators of developmental networks associated with paradormancy in Canada thistle (Cirsium arvense).

Dormancy in underground vegetative buds of Canada thistle, an herbaceous perennial weed, allows escape from current control methods and contributes to its invasive nature. In this study, ~65 % of root sections obtained from greenhouse propagated Canada thistle produced new vegetative shoots by 14 days post-sectioning. RNA samples obtained from sectioned roots incubated 0, 24, 48, and 72 h at 25°C under 16:8 h light-dark conditions were used to construct four MID-tagged cDNA libraries. Analysis of in silico data obtained using Roche 454 GS-FLX pyrosequencing technologies identified molecular networks associated with paradormancy release in underground vegetative buds of Canada thistle. Sequencing of two replicate plates produced ~2.5 million ESTs with an average read length of 362 bases. These ESTs assembled into 67358 unique sequences (21777 contigs and 45581 singlets) and annotation against the Arabidopsis database identified 15232 unigenes. Among the 15232 unigenes, we identified processes enriched with transcripts involved in plant hormone signaling networks. To follow-up on these results, we examined hormone profiles in roots, which identified changes in abscisic acid (ABA) and ABA metabolites, auxins, and cytokinins post-sectioning. Transcriptome and hormone profiling data suggest that interaction between auxin- and ABA-signaling regulate paradormancy maintenance and release in underground adventitious buds of Canada thistle. Our proposed model shows that sectioning-induced changes in polar auxin transport alters ABA metabolism and signaling, which further impacts gibberellic acid signaling involving interactions between ABA and FUSCA3. Here we report that reduced auxin and ABA-signaling, in conjunction with increased cytokinin biosynthesis post-sectioning supports a model where interactions among hormones drives molecular networks leading to cell division, differentiation, and vegetative outgrowth.

Genomics of Compositae weeds: EST libraries, microarrays, and evidence of introgression.

PREMISE OF STUDY: Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we describe the development of genomic tools and resources for 11 weeds from the Compositae family that will serve as a basis for subsequent population and comparative genomic analyses. Because hybridization has been suggested as a stimulus for the evolution of invasiveness, we also analyze these genomic data for evidence of hybridization. METHODS: We generated 22 expressed sequence tag (EST) libraries for the 11 targeted weeds using Sanger, 454, and Illumina sequencing, compared the coverage and quality of sequence assemblies, and developed NimbleGen microarrays for expression analyses in five taxa. When possible, we also compared the distributions of Ks values between orthologs of congeneric taxa to detect and quantify hybridization and introgression. RESULTS: Gene discovery was enhanced by sequencing from multiple tissues, normalization of cDNA libraries, and especially greater sequencing depth. However, assemblies from short sequence reads sometimes failed to resolve close paralogs. Substantial introgression was detected in Centaurea and Helianthus, but not in Ambrosia and Lactuca. CONCLUSIONS: Transcriptome sequencing using next-generation platforms has greatly reduced the cost of genomic studies of nonmodel organisms, and the ESTs and microarrays reported here will accelerate evolutionary and molecular investigations of Compositae weeds. Our study also shows how ortholog comparisons can be used to approximately estimate the genome-wide extent of introgression and to identify genes that have been exchanged between hybridizing taxa.

Genetic loci associated with freezing tolerance in a European rapeseed (Brassica napus L.) diversity panel identified by genome-wide association mapping.

Winter biotypes of rapeseed (Brassica napus L.) require a vernalization treatment to enter the reproductive phase and generally produce greater yields than spring rapeseed. To find genetic loci associated with freezing tolerance in rapeseed, we first performed genotyping-by-sequencing (GBS) on a diversity panel consisting of 222 rapeseed accessions originating primarily from Europe, which identified 69,554 high-quality single-nucleotide polymorphisms (SNPs). Model-based cluster analysis suggested that there were eight subgroups. The diversity panel was then phenotyped for freezing survival (visual damage and Fv/Fo and Fv/Fm) after 2 months of cold acclimation (5°C) and a freezing treatment (-15°C for 4 h). The genotypic and phenotypic data for each accession in the rapeseed diversity panel was then used to conduct a genome-wide association study (GWAS). GWAS results showed that 14 significant markers were mapped to seven chromosomes for the phenotypes scored. Twenty-four candidate genes located within the mapped loci were identified as previously associated with lipid, photosynthesis, flowering, ubiquitination, and cytochrome P450 in rapeseed or other plant species.

Dehydration and vernalization treatments identify overlapping molecular networks impacting endodormancy maintenance in leafy spurge crown buds.

Leafy spurge (Euphorbia esula L.) is a herbaceous perennial weed that reproduces vegetatively from an abundance of underground adventitious buds (UABs), which undergo well-defined phases of seasonal dormancy (para-, endo-, and ecodormancy). In this study, the effects of dehydration stress on vegetative growth and flowering potential from endodormant UABs of leafy spurge was monitored. Further, microarray analysis was used to identify critical signaling pathways of transcriptome profiles associated with endodormancy maintenance in UABs. Surprisingly, only 3-day of dehydration stress is required to break the endodormant phase in UABs; however, the dehydration-stress treatment did not induce flowering. Previous studies have shown that prolonged cold treatment of UABs breaks endodormancy and induces a vernalization response leading to flowering. Thus, in this study, comparing transcriptome data from UABs exposed to short-term dehydration and vernalization provided a unique approach to identify overlapping molecular mechanisms involved in endodormancy maintenance and floral competence. Analysis of transcriptome data associated with breaking endodormancy by both environmental treatments identified LEC1, PHOTOSYSTEM I RC, and brassinosteroids as common central hubs of upregulated genes, while DREB1A, CBF2, GPA1, MYC2, bHLH, BZIP, and flavonoids were identified as common central hubs of downregulated genes. The majority of over-represented gene sets common to breaking endodormancy by dehydration stress and vernalization were downregulated and included pathways involved in hormone signaling, chromatin modification, and circadian rhythm. Additionally, the over-represented gene sets highlighted pathways involved in starch and sugar degradation and biogenesis of carbon skeletons, suggesting a high metabolic activity is necessary during the endodormant phase. The data presented in this study helped to refine our previous model for dormancy regulation.

Alternating temperature breaks dormancy in leafy spurge seeds and impacts signaling networks associated with HY5.

Non-after-ripened seeds of the herbaceous perennial weed leafy spurge do not germinate when imbibed at a constant temperature (C), but transfer to an alternating temperature (A) induced germination. Changes in the transcriptome of seeds during 1 and 3 days of alternating temperature and germinated seeds were compared with seeds incubated at constant temperature. Statistical analysis revealed that 597, 1,491, and 1,329 genes were differentially expressed (P < 0.05) for the comparisons of 21-day C vs. 21-day C + 1-day A, 21-day C vs. 21-day C + 3-day A, and 21-day C vs. 21-day C + Germ (germination), respectively. Functional classifications based on gene set and sub-network enrichment analysis were performed to identify pathways and gene sub-networks that underlie transcriptome changes in the seeds as they germinate. Sugars, plant hormones, photomorphogenesis, and reactive oxygen species were overrepresented at 21-day C + 1-day A. At 21-day C + 3-day A, an increase in cellular activities was observed as the number of overrepresented pathways greatly increased. Many of the metabolic pathways were involved in the biosynthesis of amino acids, macromolecules, and energy and carbon skeleton production for subsequent germination. The 21-day C + 3-day A and 21-day C + Germ pathways and sub-networks were similar and included an overrepresentation of the amino acid biosynthetic pathways; however, 21-day C + Germ seeds have an even wider array of cellular activities such as translation-related pathways, which are most likely for seedling growth. RT-qPCR analysis indicated that the up- and down-regulation of HISTONE H3, GASA2, DREBIII-1, CHS, AOS, PIF3, PLD α1, and LEA may be germination-related since their expression was dramatically changed only in the 21-day C + Germ seeds. Finally, both short-term alternating temperature and short-term light exposure up-regulated the expression targets of the central hub HY5 in leafy spurge and Arabidopsis, respectively, indicating that a signaling network involving HY5 is important for germination.

Fatty Acid Data and Crop Surveys Indicate Sources of Red Sunflower Seed Weevil (Coleoptera: Curculionidae), Populations and Suggest Strategies for Management.

The red sunflower seed weevil, Smicronyx fulvus L., is a univoltine seed-feeding pest of cultivated sunflower, Helianthus annuus L. Artificial infestations of S. fulvus onto sunflowers with traditional (<25% oleic acid), mid-oleic (55-75%), or high oleic (>80%) fatty acid profiles were used to test if fatty acids could be used as natural markers to estimate the proportion of weevils developing on oilseed sunflowers rather than wild Helianthus spp. and confection (non-oil) types. Oleic acid (%) in S. fulvus confirmed the fatty acid compositions of mature larvae and weevil adults reflected their diets, making primary (oleic or linoleic) fatty acids feasible as natural markers for this crop-insect combination. Oleic acid in wild S. fulvus populations in North Dakota suggests at least 84 and 90% of adults originated from mid-oleic or high oleic sunflower hybrids in 2017 and 2018, respectively. Surveys in 2017 (n = 156 fields) and 2019 (n = 120 fields) extended information provided by S. fulvus fatty acid data; no significant spatial patterns of S. fulvus damage were detected in samples, damage to oilseed sunflowers was greater than confection (non-oil) types, and the majority of damage occurred in ≈10% of surveyed fields. Combined, data suggest a few unmanaged or mismanaged oilseed sunflower fields are responsible for producing most S. fulvus in an area. Improved management seems possible with a combination of grower education and expanded use of non-insecticidal tactics, including cultural practices and S. fulvus-resistant hybrids.

Initial changes in the transcriptome of Euphorbia esula seeds induced to germinate with a combination of constant and diurnal alternating temperatures.

We investigated transcriptome changes in Euphorbia esula (leafy spurge) seeds with a focus on the effect of constant and diurnal fluctuating temperature on dormancy and germination. Leafy spurge seeds do not germinate when incubated for 21 days at 20 degrees C constant temperatures, but nearly 30% germinate after 21 days under fluctuating temperatures 20:30 degrees C (16:8 h). Incubation at 20 degrees C for 21 days followed by 20:30 degrees C resulted in approximately 63% germination in about 10 days. A cDNA microarray representing approximately 22,000 unique sequences was used to profile transcriptome changes in the first day after transfer of seeds from constant to alternating temperature conditions. Functional classification based on MIPS and gene ontology revealed active metabolism including up-regulation of energy, protein synthesis, and signal transduction processes. Down-regulated processes included translation elongation, translation, and some biosynthetic processes. Subnetwork analysis identified genes involved in abscisic acid, sugar, and circadian clock signaling as key regulators of physiological activity in seeds soon after the transfer to alternating conditions.