Multiomics analysis reveals the molecular mechanisms underlying virulence in Rhizoctonia and jasmonic acid-mediated resistance in Tartary buckwheat (Fagopyrum tataricum).

Rhizoctonia solani is a devastating soil-borne pathogen that seriously threatens the cultivation of economically important crops. Multiple strains with a very broad host range have been identified, but only 1 (AG1-IA, which causes rice sheath blight disease) has been examined in detail. Here, we analyzed AG4-HGI 3 originally isolated from Tartary buckwheat (Fagopyrum tataricum), but with a host range comparable to AG1-IA. Genome comparison reveals abundant pathogenicity genes in this strain. We used multiomic approaches to improve the efficiency of screening for disease resistance genes. Transcriptomes of the plant-fungi interaction identified differentially expressed genes associated with virulence in Rhizoctonia and resistance in Tartary buckwheat. Integration with jasmonate-mediated transcriptome and metabolome changes revealed a negative regulator of jasmonate signaling, cytochrome P450 (FtCYP94C1), as increasing disease resistance probably via accumulation of resistance-related flavonoids. The integration of resistance data for 320 Tartary buckwheat accessions identified a gene homolog to aspartic proteinase (FtASP), with peak expression following R. solani inoculation. FtASP exhibits no proteinase activity but functions as an antibacterial peptide that slows fungal growth. This work reveals a potential mechanism behind pathogen virulence and host resistance, which should accelerate the molecular breeding of resistant varieties in economically essential crops.

DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers.

BACKGROUND: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications. RESULTS: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases. CONCLUSIONS: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

Genome-wide investigation of UDP-Glycosyltransferase family in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) belongs to Polygonaceae family and has attracted increasing attention owing to its high nutritional value. UDP-glycosyltransferases (UGTs) glycosylate a variety of plant secondary metabolites to control many metabolic processes during plant growth and development. However, there have been no systematic reports of UGT superfamily in F. tataricum. RESULTS: We identified 173 FtUGTs in F. tataricum based on their conserved UDPGT domain. Phylogenetic analysis of FtUGTs with 73 Arabidopsis UGTs clustered them into 21 families. FtUGTs from the same family usually had similar gene structure and motif compositions. Most of FtUGTs did not contain introns or had only one intron. Tandem repeats contributed more to FtUGTs amplification than segmental duplications. Expression analysis indicates that FtUGTs are widely expressed in various tissues and likely play important roles in plant growth and development. The gene expression analysis response to different abiotic stresses showed that some FtUGTs were involved in response to drought and cadmium stress. Our study provides useful information on the UGTs in F. tataricum, and will facilitate their further study to better understand their function. CONCLUSIONS: Our results provide a theoretical basis for further exploration of the functional characteristics of FtUGTs and for understanding the growth, development, and metabolic model in F. tataricum.

Strigolactones affect the yield of Tartary buckwheat by regulating endogenous hormone levels.

BACKGROUND: As a newly class of endogenous phytohormones, strigolactones (SLs) regulate crop growth and yield formation by interacting with other hormones. However, the physiological mechanism of SLs affect the yield by regulating the balance of endogenous hormones of Tartary buckwheat is still unclear. RESULTS: In this study, a 2-year field experiment was conducted on Tartary buckwheat (Jinqiao 2) to study the effects of different concentrations (0, 10, and 20 µmol/L) of artificial synthetic analogs of SLs (rac-GR24) and inhibitor of SL synthesis (Tis-108) on the growth, endogenous-hormone content, and yield of Tartary buckwheat. The main-stem branch number, grain number per plant, grain weight per plant, and yield of Tartary buckwheat continuously decreased with increased rac-GR24 concentration, whereas the main-stem diameter and plant height initially increased and then decreased. Rac-GR24 treatment significantly increased the content of SLs and abscisic acid (ABA) in grains, and it decreased the content of Zeatin (Z) + Zeatin nucleoside (ZR). Conversely, Tis-108 treatment decreased the content of SLs and ABA but increased the content of Z + ZR. Results of correlation analysis showed that the content of ABA and SLs, the ratio of SLs/(Z + ZR), SLs/ABA, and ABA/(Z + ZR) were significantly negatively correlated with the yield of Tartary buckwheat, and that Z + ZR content was significantly positively correlated with the yield. Regression analysis further showed that ABA/ (Z + ZR) can explain 58.4% of the variation in yield. CONCLUSIONS: In summary, by adjusting the level of endogenous SLs in Tartary buckwheat, the balance of endogenous hormones in grains can be changed, thereby exerting the effect on yield. The results can provide a new agronomic method for the high-yield cultivation of Tartary buckwheat.

Growth performance, lipid metabolism, and systemic immunity of weaned piglets were altered by buckwheat protein through the modulation of gut microbiota.

Tartary buckwheat protein (BWP) is well known for the wide-spectrum antibacterial activity and the lipid metabolism- regulating property; therefore, BWP can be applied as feed additives to improve the animal's nutritional supply. With the aim to investigate the bioactive actions of the BWP, growth performance, lipid metabolism and systemic immunity of the weaned piglets were measured, and the alterations of pig gut microbiota were also analyzed. According to the results, the growth performances of the weaned piglets which were calculated as the average daily gain (ADG) and the average daily feed intake (ADFI) were significantly increased when compared to the control group. Simultaneously, the serum levels of the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were decreased, while the levels of high-density lipoprotein cholesterol (HDL-C) were increased in the BWP group. Moreover, the relative abundances of Lactobacillus, Prevotella_9, Subdoligranulum, Blautia, and other potential probiotics in the gut microbiota of weaned piglets were obviously increased in the BWP group. However, the relative abundances of Escherichia-Shigella, Campylobacter, Rikenellaceae_RC9_gut_group and other opportunistic pathogens were obviously decreased in the BWP group. In all, BWP was proved to be able to significantly improve the growth performance, lipid metabolism, and systemic immunity of the weaned piglets, and the specific mechanism might relate to the alterations of the gut microbiota. Therefore, BWP could be explored as a prospective antibiotic alternative for pig feed additives.

Multi-omics identification of a key glycosyl hydrolase gene FtGH1 involved in rutin hydrolysis in Tartary buckwheat (Fagopyrum tataricum).

Rutin, a flavonoid rich in buckwheat, is important for human health and plant resistance to external stresses. The hydrolysis of rutin to quercetin underlies the bitter taste of Tartary buckwheat. In order to identify rutin hydrolysis genes, a 200 genotypes mini-core Tartary buckwheat germplasm resource was re-sequenced with 30-fold coverage depth. By combining the content of the intermediate metabolites of rutin metabolism with genome resequencing data, metabolite genome-wide association analyses (GWAS) eventually identified a glycosyl hydrolase gene FtGH1, which could hydrolyse rutin to quercetin. This function was validated both in Tartary buckwheat overexpression hairy roots and in vitro enzyme activity assays. Mutation of the two key active sites, which were determined by molecular docking and experimentally verified via overexpression in hairy roots and transient expression in tobacco leaves, exhibited abnormal subcellular localization, suggesting functional changes. Sequence analysis revealed that mutation of the FtGH1 promoter in accessions of two haplotypes might be necessary for enzymatic activity. Co-expression analysis and GWAS revealed that FtbHLH165 not only repressed FtGH1 expression, but also increased seed length. This work reveals a potential mechanism behind rutin metabolism, which should provide both theoretical support in the study of flavonoid metabolism and in the molecular breeding of Tartary buckwheat.

Fagopyrins from Buckwheat Flowers: Structural and Stereochemical Characterization Through Combined NMR/CD Spectroscopy and Theoretical Calculations.

Fagopyrins are phenantroperylenequinones present in the flowers of Fagopyrum esculentum (buckwheat) endowed with photodynamic activity. It has been reported that fagopyrin extracts actually contain a complex mixture of closely related compounds, differing only on the nature of the perylenequinone substituents. We report our systematic and detailed study on the chemical composition of fagopyrin extracts by a combination of preparative and analytical techniques. The combined use of 1H-NMR and CD spectroscopy was found to be particularly suited to fully characterize all stereochemical aspects of the extracted fagopyrins. For the first time nine isomers have been structurally characterized and their stereochemistry fully elucidated. The presence of two different heterocyclic ring substituents, two stereogenic centers and the inherent axial chirality of the aromatic system provides a complex stereochemical relationships among isomers, thus giving account of the high level of molecular multiplicity found in the extract.

Fagopyrin F fraction from Fagopyrum tataricum demonstrates photodynamic inactivation of skin infecting bacterium and squamous cell carcinoma (A431) cells.

Photodynamic therapy (PDT) stands out as a noteworthy development as an alternative targeted treatment against skin ailments. While PDT has advanced significantly, research into photo-activatable "Green drugs" derived from plants which are less toxic than the synthetic drugs has not kept pace. This study investigates the potential of Fagopyrin F Containing Fraction (FCF) derived from Fagopyrum tataricum in mediating PDT against Staphylococcus aureus and skin cancer cells (A431). FCF was isolated from the plant extract using thin-layer chromatography, followed by identification of the compound through high-performance liquid chromatography and high-resolution liquid chromatography-mass spectrometry. FCF was tested to determine its antibacterial and anticancer efficacy. Results revealed that FCF-mediated PDT exhibited potent action against S. aureus, significantly reducing bacterial viability (MIC 19.5 µg/100 µL). Moreover, FCF-mediated PDT showed good efficacy against A431 cells, resulting in a notable reduction in cell viability (IC50 29.08 µg/mL). Given the known association between S. aureus and squamous cell carcinoma (SCC), FCF shows the potential to effectively target and eradicate both SCC and the related S. aureus present within the lesions. In silico study reveals that Fagopyrin F effectively binds with the epidermal growth factor (EGFR), one among the highly expressed proteins in the A431 cells, with a binding energy of - 9.6 kcal/mol. The affinity of Fagopyrin F for EGFR on A431 cancer cells along with its cytotoxicity against skin cancer cells while safeguarding the normal cells (L929) plays a major part in the way it targets cancer cells. However, its safety, efficacy, and long-term advantages in treating skin conditions require more investigation, including in vivo investigations and clinical trials.

Anticancer effects of rutin from Fagopyrum tataricum (tartary buckwheat) against osteosarcoma cell line.

BACKGROUND: The present study is analysisof the seeds of buckwheat (Fagopyrum sp.),member of the Polygonaceae family for isolation of rutin and its anticancer property againstOsteosarcoma celllines (SAOS2). The selected plant is traditionally used for diabetes and cancer. It has several biological properties such as antibacterial, antioxidant and anti-aging. PURPOSE: Thirty-five buckwheat cultivars were obtained from Nepal Agriculture Genetic Resources Centre (NAGRC) Khumaltar, Kathmandu, Nepal, and Kumrek Sikkim. These plant varieties are scientifically evaluated their biological properties. METHODS: Rutin wasfractionated from buckwheat seeds using methanol fraction and analysed for quality by HPLC method. The rutin fraction of the cultivar NGRC03731 a tartary buck wheat and standard rutin was used against Osteosarcoma cell lines (SAOS2) and human gingival fibroblast cells (hGFs) for anticancer activity. The cell viability using rutin fraction and standard rutin treated with SAOS2 cells were assessed by MTT assay. For further research, the best doses (IC-50: 20 g/ml) were applied. By using AO/EtBr dual staining, the effects of Rutin fraction on SAOS2 cell death were analysed. The scratch wound healing assay was used to analyse cell migration. Real-time PCR was used to analyse the pro-/anti-apoptotic gene expression. RESULTS: The seeds with the highest rutin content, NGRC03731 seeds, had 433 mg/100 g of rutin.The rutin fraction treatment and standard rutin significantly reduced cell viability in the MTT assay, and osteosarcoma cells were observed on sensitive to the IC-50 dose at a concentration of 20 g/ml after 24 h.The SAOS2 cells exposed to rutin fraction at 20 g/ml and standard rutin at 10 g/ml exhibited significant morphological alterations, cell shrinkage and decreased cell density, which indicate apoptotic cells.Rutin-fraction treated cells stained with acridine orange/ethidium bromide (AO/EtBr) dual staining cells turned yellow, orange, and red which indicatesto measure apoptosis.The anti-migration potential of rutin fraction, results prevented the migration of SAOS2 cancer cells.Rutin-fraction significantly increased the expression of pro-apoptotic proteinsBad, using real-time PCR analysis (mRNA for Bcl-2 family proteins) resulted Bcl-2's expression is negatively regulated. CONCLUSION: Osteosarcoma (SAOS2) cell lines' proliferation, migration, and ability to proliferate were reduced markedly by rutin fraction and it also causes apoptosis of Osteosarcoma cell lines (SAOS2).

Unravelling effects of phytochemicals from buckwheat on cholesterol metabolism and lipid accumulation in HepG2 cells and its validation through gene expression analysis.

BACKGROUND: The objective of this research was to elucidate the hypocholesterolemic effects of a bioactive compound extracted from buckwheat, and to delineate its influence on the regulatory mechanisms of cholesterol metabolism. The compound under investigation was identified as quercetin. MATERIAL AND RESULTS: In vitro experiments conducted on HepG2 cells treated with quercetin revealed a significant reduction in intracellular cholesterol accumulation. This phenomenon was rigorously quantified by assessing the transcriptional activity of key genes involved in the biosynthesis and metabolism of cholesterol. A statistically significant reduction in the expression of HMG-CoA reductase (HMGCR) was observed, indicating a decrease in endogenous cholesterol synthesis. Conversely, an upregulation in the expression of cholesterol 7 alpha-hydroxylase (CYP7A1) was also observed, suggesting an enhanced catabolism of cholesterol to bile acids. Furthermore, the study explored the combinatory effects of quercetin and simvastatin, a clinically utilized statin, revealing a synergistic action in modulating cholesterol levels at various dosages. CONCLUSIONS: The findings from this research provide a comprehensive insight into the mechanistic pathways through which quercetin, a phytochemical derived from buckwheat, exerts its hypocholesterolemic effects. Additionally, the observed synergistic interaction between quercetin and simvastatin opens up new avenues for the development of combined therapeutic strategies to manage hyperlipidemia.

Fate characteristics and risk identification of thifluzamide in buckwheat across China: Analytical method development, occurrence, and health assessment.

Elaborating on the fate tendency of thifluzamide (thiazole-amide fungicide) in buckwheat based on nationwide application is vital for grain security and human health based on nationwide application. A rapid and sensitive analytical method was developed to trace thifluzamide in buckwheat matrices using an ultrahigh-performance liquid chromatography-tandem triple quadrupole mass spectrometer (UHPLC-MS/MS), with a retention time of 2.90 min and limit of quantitation (LOQ) of 0.001 mg/kg. Thifluzamide could be stably stored for 84 d in buckwheat matrices under -20 °C under dark condition. The occurrence, dissipation and terminal magnitudes of thifluzamide were reflected by the primary deposition of 0.02-0.55 mg/kg, half-lives of 12-14 d, and highest residues of 0.41 mg/kg. The long-term risks (ADI%) of thifluzamide were 37.268 %-131.658 % in registered crops, and the risks for the rural population were significantly higher than those of the urban population. The unacceptable dietary risks of thifluzamide should be continuously emphasized for children aged 2-7 with an ADI% values of 100.750 %-131.658 %. A probabilistic model was further introduced to evaluate the risk discrepancy of thifluzamide in buckwheat, showing the risks in Tartary buckwheat (Fagopyrum tararicum Gaerth) were 1.5-75.4 times than that in sweet buckwheat (Fagopyrum esculentum Moench). Despite the low risks for dietary buckwheat, the high-potential health hazards of thifluzamide should be pay more attention given the increasing applications and cumulative effects.

Haplotype-resolved genomes of two buckwheat crops provide insights into their contrasted rutin concentrations and reproductive systems.

BACKGROUND: Two widely cultivated annual buckwheat crops, Fagopyrum esculentum and F. tataricum, differ from each other in both rutin concentration and reproductive system. However, the underlying genetic mechanisms remain poorly elucidated. RESULTS: Here, we report the first haplotype-resolved chromosome-level genome assemblies of the two species. Two haplotype genomes of F. esculentum were assembled as 1.23 and 1.19 Gb with N50 = 9.8 and 12.4 Mb, respectively; the two haplotype genomes of F. tataricum were 453.7 and 446.2 Mb with N50 = 50 and 30 Mb, respectively. We further annotated protein-coding genes of each haplotype genome based on available gene sets and 48 newly sequenced transcriptomes. We found that more repetitive sequences, especially expansion of long terminal repeat retrotransposons (LTR-RTs), contributed to the large genome size of F. esculentum. Based on the well-annotated sequences, gene expressions, and luciferase experiments, we identified the sequence mutations of the promoter regions of two key genes that are likely to have greatly contributed to the high rutin concentration and selfing reproduction in F. tartaricum. CONCLUSIONS: Our results highlight the importance of high-quality genomes to identify genetic mutations underlying phenotypic differences between closely related species. F. tataricum may have been experienced stronger selection than F. esculentum through choosing these two non-coding alleles for the desired cultivation traits. These findings further suggest that genetic manipulation of the non-coding promoter regions could be widely employed for breeding buckwheat and other crops.

Water extract of Fagopyrum dibotrys (D. Don) Hara straw increases selenium accumulation in peach seedlings under selenium-contaminated soil.

To promote the selenium (Se) uptakes in fruit trees under Se-contaminated soil, the effects of water extract of Fagopyrum dibotrys (D. Don) Hara straw on the Se accumulation in peach seedlings under selenium-contaminated soil were studied. The results showed that the root biomass, chlorophyll content, activities of antioxidant enzymes, and soluble protein content of peach seedlings were increased by the F. dibotrys straw extract. The different forms of Se (total Se, inorganic Se, and organic Se) were also increased in peach seedlings following treatment with the F. dibotrys straw extract. The highest total shoot Se content was treated by the 300-fold dilution of F. dibotrys straw, which was 30.87% higher than the control. The F. dibotrys straw extract also increased the activities of adenosine triphosphate sulfurase (ATPS), and adenosine 5'-phosphosulfate reductase (APR) in peach seedlings, but decreased the activity of serine acetyltransferase (SAT). Additionally, correlation and grey relational analyses revealed that chlorophyll a content, APR activity, and root biomass were closely associated with the total shoot Se content. Overall, this study shows that the water extract of F. dibotrys straw can promote Se uptake in peach seedlings, and 300-fold dilution is the most suitable concentration.

The water extract of Fagopyrum dibotrys (D. Don) Hara straw promoted the selenium (Se) uptake in peach seedlings under selenium-contaminated soil. The concentration of F. dibotrys straw extract showed a quadratic polynomial regression relationship with the total root and shoot Se. Furthermore, chlorophyll a content, APR activity, and root biomass were closely associated with the total shoot Se. This study shows that water extract of F. dibotrys straw can promote Se uptake in peach seedlings, and 300-fold dilution is the most suitable concentration.

Variations in chemical composition of common buckwheat (Fagopyrum esculentum Moench) as a result of different environmental conditions.

BACKGROUND: Common buckwheat (Fagopyrum esculentum Moench) is a pseudo cereal that is gaining interest in the world. The chemical profile of common buckwheat determines its high nutritional and health-promoting value. The accumulation of these valuable ingredients depends on many factors, such as: variety, location of cultivation and related weather and agrotechnical conditions. Due to the growing interest in common buckwheat as a natural plant material for food production, it is important to know the factors affecting the quantitative and qualitative composition of its grains. The aim of the research was to determine the effect of the genotype (G), environment (E) and G × E interaction on the content of nutrients (protein, starch, ash, lipids) and bioactive components [dietary fiber (DF), total phenolic content (TPC)] in the common buckwheat grains. The study covered four cultivars grown in three locations for three consecutive vegetation seasons (2016/2017, 2017/2018, 2018/2019). RESULTS: Based on the obtained results, a significant influence of the environment and G × E interaction on the content of the studied parameters was found. The greatest impact on the diversity of the content of nutrients had environmental conditions, which in the case of protein and ash determined these features in more than 80%, and in the case of starch, 70%. With regard to bioactive compounds, the greatest influence of the environment was observed for the amount of TPC (78%), lignin (51%) and the DF complex (56%). CONCLUSION: The obtained results are useful for breeders working on expanding the pool of common buckwheat genotypes, stable in changing environmental conditions. © 2023 Society of Chemical Industry.

Comparative study of dietary phenols with Tartary buckwheat protein (2S/13S): impact on structure, binding sites and functionality of protein.

BACKGROUND: This research was to investigate the interaction mechanism between 2S albumin and 13S globulin (2S and 13S, the most important storage proteins in Tartary buckwheat seeds) and three phenols (rutin, quercetin and myricetin) regarding the structural and antioxidant properties of their complexes. RESULTS: There are differences in the binding affinity of phenols for 2S and 13S. Rutin had a higher binding affinity for 2S, myricetin had a higher binding affinity for 13S, and 13S exhibited a higher affinity toward phenols than did 2S. Binding with phenols significantly changed the secondary and tertiary structures of 2S and 13S, decreased the surface hydrophobic value and enhanced the antioxidant capacity. Molecular docking and isothermal titration calorimetry showed that the binding processes were spontaneous and that there were hydrogen bonds, hydrophobic bonds and van der Waals force interactions between phenols and proteins. CONCLUSION: These findings could provide meaningful guidance for the further application of buckwheat protein complex. © 2023 Society of Chemical Industry.

JA-induced FtBPM3 accumulation promotes FtERF-EAR3 degradation and rutin biosynthesis in Tartary buckwheat.

Buckwheat accumulates abundant flavonoids, which exhibit excellent health-promoting value. Flavonoids biosynthesis is mediated by a variety of phytohormones, among which jasmonates (JAs) induce numerous transcription factors, taking part in regulation of flavonoids biosynthesis genes. However, some transcriptional repressors appeared also induced by JAs. How these transcriptional repressors coordinately participate in JA signaling remains unclear. Here, we found that the disruption of the GCC-box in FtF3H promoter was associated with flavonoids accumulation in Tartary buckwheat. Further, our study illustrated that the nucleus-localized FtERF-EAR3 could inhibit FtF3H expression and flavonoids biosynthesis through binding the GCC-box in the promoter of FtF3H. The JA induced FtERF-EAR3 gene expression while facilitating FtERF-EAR3 protein degradation via the FtBPM3-dependent 26S proteasome pathway. Overall, these results illustrate a precise modulation mechanism of JA-responsive transcription suppressor participating in flavonoid biosynthesis, and will further help to improve the efficiency of flavonoids biosynthesis in Tartary buckwheat.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn.

BACKGROUND: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. RESULTS: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. CONCLUSIONS: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.

Metabolic-GWAS provides insights into genetic architecture of seed metabolome in buckwheat.

BACKGROUND: Buckwheat (Fagopyrum spp.), belonging to the Polygonaceae family, is an ancient pseudo-cereal with high nutritional and nutraceutical properties. Buckwheat proteins are gluten-free and show balanced amino acid and micronutrient profiles, with higher content of health-promoting bioactive flavonoids that make it a golden crop of the future. Plant metabolome is increasingly gaining importance as a crucial component to understand the connection between plant physiology and environment and as a potential link between the genome and phenome. However, the genetic architecture governing the metabolome and thus, the phenome is not well understood. Here, we aim to obtain a deeper insight into the genetic architecture of seed metabolome in buckwheat by integrating high throughput metabolomics and genotyping-by-sequencing applying an array of bioinformatics tools for data analysis. RESULTS: High throughput metabolomic analysis identified 24 metabolites in seed endosperm of 130 diverse buckwheat genotypes. The genotyping-by-sequencing (GBS) of these genotypes revealed 3,728,028 SNPs. The Genome Association and Prediction Integrated Tool (GAPIT) assisted in the identification of 27 SNPs/QTLs linked to 18 metabolites. Candidate genes were identified near 100 Kb of QTLs, providing insights into several metabolic and biosynthetic pathways. CONCLUSIONS: We established the metabolome inventory of 130 germplasm lines of buckwheat, identified QTLs through marker trait association and positions of potential candidate genes. This will pave the way for future dissection of complex economic traits in buckwheat.

QTL mapping and candidate gene analysis for yield and grain weight/size in Tartary buckwheat.

BACKGROUND: Grain weight/size influences not only grain yield (GY) but also nutritional and appearance quality and consumer preference in Tartary buckwheat. The identification of quantitative trait loci (QTLs)/genes for grain weight/size is an important objective of Tartary buckwheat genetic research and breeding programs. RESULTS: Herein, we mapped the QTLs for GY, 1000-grain weight (TGW), grain length (GL), grain width (GW) and grain length-width ratio (L/W) in four environments using 221 recombinant inbred lines (XJ-RILs) derived from a cross of 'Xiaomiqiao × Jinqiaomai 2'. In total, 32 QTLs, including 7 for GY, 5 for TGW, 6 for GL, 11 for GW and 3 for L/W, were detected and distributed in 24 genomic regions. Two QTL clusters, qClu-1-3 and qClu-1-5, located on chromosome Ft1, were revealed to harbour 7 stable major QTLs for GY (qGY1.2), TGW (qTGW1.2), GL (qGL1.1 and qGL1.4), GW (qGW1.7 and qGW1.10) and L/W (qL/W1.2) repeatedly detected in three and above environments. A total of 59 homologues of 27 known plant grain weight/size genes were found within the physical intervals of qClu-1-3 and qClu-1-5. Six homologues, FtBRI1, FtAGB1, FtTGW6, FtMADS1, FtMKK4 and FtANT, were identified with both non-synonymous SNP/InDel variations and significantly differential expression levels between the two parents, which may play important roles in Tatary buckwheat grain weight/size control and were chosen as core candidate genes for further investigation. CONCLUSIONS: Two stable major QTL clusters related to grain weight/size and six potential key candidate genes were identified by homology comparison, SNP/InDel variations and qRT‒qPCR analysis between the two parents. Our research provides valuable information for improving grain weight/size and yield in Tartary buckwheat breeding.

The pan-plastome of tartary buckwheat (fagopyrum tataricum): key insights into genetic diversity and the history of lineage divergence.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is an important food and medicine crop plant, which has been cultivated for 4000 years. A nuclear genome has been generated for this species, while an intraspecific pan-plastome has yet to be produced. As such a detailed understanding of the maternal genealogy of Tartary buckwheat has not been thoroughly investigated. RESULTS: In this study, we de novo assembled 513 complete plastomes of Fagopyrum and compared with 8 complete plastomes of Fagopyrum downloaded from the NCBI database to construct a pan-plastome for F. tartaricum and resolve genomic variation. The complete plastomes of the 513 newly assembled Fagopyrum plastome sizes ranged from 159,253 bp to 159,576 bp with total GC contents ranged from 37.76 to 37.97%. These plastomes all maintained the typical quadripartite structure, consisting of a pair of inverted repeat regions (IRA and IRB) separated by a large single copy region (LSC) and a small single copy region (SSC). Although the structure and gene content of the Fagopyrum plastomes are conserved, numerous nucleotide variations were detected from which population structure could be resolved. The nucleotide variants were most abundant in the non-coding regions of the genome and of those the intergenic regions had the most. Mutational hotspots were primarily found in the LSC regions. The complete 521 Fagopyrum plastomes were divided into five genetic clusters, among which 509 Tartary buckwheat plastomes were divided into three genetic clusters (Ft-I/Ft-II/Ft-III). The genetic diversity in the Tartary buckwheat genetic clusters was the greatest in Ft-III, and the genetic distance between Ft-I and Ft-II was the largest. Based on the results of population structure and genetic diversity analysis, Ft-III was further subdivided into three subgroups Ft-IIIa, Ft-IIIb, and Ft-IIIc. Divergence time estimation indicated that the genera Fagopyrum and Rheum (rhubarb) shared a common ancestor about 48 million years ago (mya) and that intraspecies divergence in Tartary buckwheat began around 0.42 mya. CONCLUSIONS: The resolution of pan-plastome diversity in Tartary buckwheat provides an important resource for future projects such as marker-assisted breeding and germplasm preservation.