Corrigendum: The genome of Chenopodium quinoa.

This corrects the article DOI: 10.1038/nature21370.

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Genetic variation for tolerance to the downy mildew pathogen Peronospora variabilis in genetic resources of quinoa (Chenopodium quinoa).

BACKGROUND: Quinoa (Chenopodium quinoa Willd.) is an ancient grain crop that is tolerant to abiotic stress and has favorable nutritional properties. Downy mildew is the main disease of quinoa and is caused by infections of the biotrophic oomycete Peronospora variabilis Gaüm. Since the disease causes major yield losses, identifying sources of downy mildew tolerance in genetic resources and understanding its genetic basis are important goals in quinoa breeding. RESULTS: We infected 132 South American genotypes, three Danish cultivars and the weedy relative C. album with a single isolate of P. variabilis under greenhouse conditions and observed a large variation in disease traits like severity of infection, which ranged from 5 to 83%. Linear mixed models revealed a significant effect of genotypes on disease traits with high heritabilities (0.72 to 0.81). Factors like altitude at site of origin or seed saponin content did not correlate with mildew tolerance, but stomatal width was weakly correlated with severity of infection. Despite the strong genotypic effects on mildew tolerance, genome-wide association mapping with 88 genotypes failed to identify significant marker-trait associations indicating a polygenic architecture of mildew tolerance. CONCLUSIONS: The strong genetic effects on mildew tolerance allow to identify genetic resources, which are valuable sources of resistance in future quinoa breeding.

Diverse Traits Contribute to Salinity Tolerance of Wild Tomato Seedlings from the Galapagos Islands.

Traits of modern crops have been heavily selected in agriculture, leaving commercial lines often more susceptible to harsh conditions compared with their wild relatives. Understanding the mechanisms of stress tolerance in wild relatives can enhance crop performance under stress conditions such as high salinity. In this study, we investigated salinity tolerance of two species of wild tomato endemic to the Galapagos Islands, Solanum cheesmaniae and Solanum galapagense Since these tomatoes grow well despite being constantly splashed with seawater, they represent a valuable genetic resource for improving salinity tolerance in commercial tomatoes. To explore their potential, we recorded over 20 traits reflecting plant growth, physiology, and ion content in 67 accessions and two commercial tomato lines of Solanum lycopersicum. Salt treatments were applied for 10 d using supported hydroponics. The Galapagos tomatoes displayed greater tolerance to salt stress than the commercial lines and showed substantial natural variation in their responses. The accessions LA0317, LA1449, and LA1403 showed particularly high salinity tolerance based on growth under salinity stress. Therefore, Galapagos tomatoes should be further explored to identify the genes underlying their high tolerance and be used as a resource for increasing the salinity tolerance of commercial tomatoes. The generated data, along with useful analysis tools, have been packaged and made publicly available via an interactive online application (https://mmjulkowska.shinyapps.io/La_isla_de_tomato/) to facilitate trait selection and the use of Galapagos tomatoes for the development of salt-tolerant commercial tomatoes.

Characterization of epidermal bladder cells in Chenopodium quinoa.

Chenopodium quinoa (quinoa) is considered a superfood with its favourable nutrient composition and being gluten free. Quinoa has high tolerance to abiotic stresses, such as salinity, water deficit (drought) and cold. The tolerance mechanisms are yet to be elucidated. Quinoa has epidermal bladder cells (EBCs) that densely cover the shoot surface, particularly the younger parts of the plant. Here, we report on the EBC's primary and secondary metabolomes, as well as the lipidome in control conditions and in response to abiotic stresses. EBCs were isolated from plants after cold, heat, high-light, water deficit and salt treatments. We used untargeted gas chromatography-mass spectrometry (GC-MS) to analyse metabolites and untargeted and targeted liquid chromatography-MS (LC-MS) for lipids and secondary metabolite analyses. We identified 64 primary metabolites, including sugars, organic acids and amino acids, 19 secondary metabolites, including phenolic compounds, betanin and saponins and 240 lipids categorized in five groups including glycerolipids and phospholipids. We found only few changes in the metabolic composition of EBCs in response to abiotic stresses; these were metabolites related with heat, cold and high-light treatments but not salt stress. Na+ concentrations were low in EBCs with all treatments and approximately two orders of magnitude lower than K+ concentrations.

SOS1 tonoplast neo-localization and the RGG protein SALTY are important in the extreme salinity tolerance of Salicornia bigelovii.

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

A comprehensive characterization of agronomic and end-use quality phenotypes across a quinoa world core collection.

Quinoa (Chenopodium quinoa Willd.), a pseudocereal with high protein quality originating from the Andean region of South America, has broad genetic variation and adaptability to diverse agroecological conditions, contributing to the potential to serve as a global keystone protein crop in a changing climate. However, the germplasm resources currently available to facilitate quinoa expansion worldwide are restricted to a small portion of quinoa's total genetic diversity, in part because of day-length sensitivity and issues related to seed sovereignty. This study aimed to characterize phenotypic relationships and variation within a quinoa world core collection. The 360 accessions were planted in a randomized complete block design with four replicates in each of two greenhouses in Pullman, WA during the summer of 2018. Phenological stages, plant height, and inflorescence characteristics were recorded. Seed yield, composition, thousand seed weight, nutritional composition, shape, size, and color were measured using a high-throughput phenotyping pipeline. Considerable variation existed among the germplasm. Crude protein content ranged from 11.24% to 17.81% (fixed at 14% moisture). We found that protein content was negatively correlated with yield and positively correlated with total amino acid content and days to harvest. Mean essential amino acids values met adult daily requirements but not leucine and lysine infant requirements. Yield was positively correlated with thousand seed weight and seed area, and negatively correlated with ash content and days to harvest. The accessions clustered into four groups, with one-group representing useful accessions for long-day breeding programs. The results of this study establish a practical resource for plant breeders to leverage as they strategically develop germplasm in support of the global expansion of quinoa.

A chromosome-scale assembly of the quinoa genome provides insights into the structure and dynamics of its subgenomes.

Quinoa (Chenopodium quinoa Willd.) is an allotetraploid seed crop with the potential to help address global food security concerns. Genomes have been assembled for four accessions of quinoa; however, all assemblies are fragmented and do not reflect known chromosome biology. Here, we use in vitro and in vivo Hi-C data to produce a chromosome-scale assembly of the Chilean accession PI 614886 (QQ74). The final assembly spans 1.326 Gb, of which 90.5% is assembled into 18 chromosome-scale scaffolds. The genome is annotated with 54,499 protein-coding genes, 96.9% of which are located on the 18 largest scaffolds. We also report an updated genome assembly for the B-genome diploid C. suecicum and use it, together with the A-genome diploid C. pallidicaule, to identify genomic rearrangements within the quinoa genome, including a large pericentromeric inversion representing 71.7% of chromosome Cq3B. Repetitive sequences comprise 65.2%, 48.6%, and 57.9% of the quinoa, C. pallidicaule, and C. suecicum genomes, respectively. Evidence suggests that the B subgenome is more dynamic and has expanded more than the A subgenome. These genomic resources will enable more accurate assessments of genome evolution within the Amaranthaceae and will facilitate future efforts to identify variation in genes underlying important agronomic traits in quinoa.

Lipid remodeling of contrasting maize (Zea mays L.) hybrids under repeated drought.

The role of recovery after drought has been proposed to play a more prominent role during the whole drought-adaption process than previously thought. Two maize hybrids with comparable growth but contrasting physiological responses were investigated using physiological, metabolic, and lipidomic tools to understand the plants' strategies of lipid remodeling in response to repeated drought stimuli. Profound differences in adaptation between hybrids were discovered during the recovery phase, which likely gave rise to different degrees of lipid adaptability to the subsequent drought event. These differences in adaptability are visible in galactolipid metabolism and fatty acid saturation patterns during recovery and may lead to a membrane dysregulation in the sensitive maize hybrid. Moreover, the more drought-tolerant hybrid displays more changes of metabolite and lipid abundance with a higher number of differences within individual lipids, despite a lower physiological response, while the responses in the sensitive hybrid are higher in magnitude but lower in significance on the level of individual lipids and metabolites. This study suggests that lipid remodeling during recovery plays a key role in the drought response of plants.

Quantitation of 20 Phytosterols in 34 Different Accessions of Quinoa (Chenopodium quinoa).

Phytosterols were analyzed in 34 different quinoa accessions, which were obtained from the same field trial. Twenty different sterols were detected, and 17 could be structurally assigned by means of gas chromatography with mass spectrometry. Sterols were quantitated in selected ion monitoring mode (GC/MS-SIM) with the novel internal standard 3-O-tert-butyldimethylsilyl-cholestanol (cholestanyl-TBDMS). GC/MS-SIM response factors of minor sterols were determined after enrichment by countercurrent chromatography. The total sterol contents varied from 120 to 180 mg/100 g of seeds, which is higher than has been described in quinoa before. This was due to the fact that Δ7-sterols (e.g., Δ7-sitosterol, spinasterol, and Δ7-avenasterol) were quantitated for the first time in quinoa and contributed ∼64% to the total sterol content. Clustering allowed distributing of the 34 different quinoa accessions into four distinct groups on the basis of the different sterol patterns.

Genome-wide association study in quinoa reveals selection pattern typical for crops with a short breeding history.

Quinoa germplasm preserves useful and substantial genetic variation, yet it remains untapped due to a lack of implementation of modern breeding tools. We have integrated field and sequence data to characterize a large diversity panel of quinoa. Whole-genome sequencing of 310 accessions revealed 2.9 million polymorphic high confidence single nucleotide polymorphism (SNP) loci. Highland and Lowland quinoa were clustered into two main groups, with FST divergence of 0.36 and linkage disequilibrium (LD) decay of 6.5 and 49.8 kb, respectively. A genome-wide association study using multi-year phenotyping trials uncovered 600 SNPs stably associated with 17 traits. Two candidate genes are associated with thousand seed weight, and a resistance gene analog is associated with downy mildew resistance. We also identified pleiotropically acting loci for four agronomic traits important for adaptation. This work demonstrates the use of re-sequencing data of an orphan crop, which is partially domesticated to rapidly identify marker-trait association and provides the underpinning elements for genomics-enabled quinoa breeding.

As human populations grow and climate change tightens its grip, developing nutritious crops which can thrive on poor soil and under difficult conditions will become a priority. Quinoa, a harvest currently overlooked by agricultural research, could be an interesting candidate in this effort. With its high nutritional value and its ability to tolerate drought, frost and high concentrations of salt in the soil, this hardy crop has been cultivated in the Andes for the last 5,000 to 7,000 years. Today its commercial production is mainly limited to Peru, Bolivia, and Ecuador. Pinpointing the genetic regions that control traits such as yields or flowering time would help agronomists to create new varieties better suited to life under northern latitudes and mechanical farming. To identify these genes, Patiranage et al. grew 310 varieties of quinoa from all over the world under the same conditions; the genomes of these plants were also examined in great detail. Analyses were then performed to link specific genetic variations with traits relevant to agriculture, helping to pinpoint changes in the genetic code linked to differences in how the plants grew, resisted disease, or produced seeds of varying quality. Candidate genes likely to control these traits were then put forward. The study by Patiranage et al. provides a genetic map where genes of agronomical importance have been precisely located and their effects measured. This resource will help to select genetic profiles which could be used to create new quinoa breeds better adapted to a changing world.

The diversity of quinoa morphological traits and seed metabolic composition.

Quinoa (Chenopodium quinoa Willd.) is an herbaceous annual crop of the amaranth family (Amaranthaceae). It is increasingly cultivated for its nutritious grains, which are rich in protein and essential amino acids, lipids, and minerals. Quinoa exhibits a high tolerance towards various abiotic stresses including drought and salinity, which supports its agricultural cultivation under climate change conditions. The use of quinoa grains is compromised by anti-nutritional saponins, a terpenoid class of secondary metabolites deposited in the seed coat; their removal before consumption requires extensive washing, an economically and environmentally unfavorable process; or their accumulation can be reduced through breeding. In this study, we analyzed the seed metabolomes, including amino acids, fatty acids, and saponins, from 471 quinoa cultivars, including two related species, by liquid chromatography - mass spectrometry. Additionally, we determined a large number of agronomic traits including biomass, flowering time, and seed yield. The results revealed considerable diversity between genotypes and provide a knowledge base for future breeding or genome editing of quinoa.

NAC transcription factors ATAF1 and ANAC055 affect the heat stress response in Arabidopsis.

Pre-exposing (priming) plants to mild, non-lethal elevated temperature improves their tolerance to a later higher-temperature stress (triggering stimulus), which is of great ecological importance. 'Thermomemory' is maintaining this tolerance for an extended period of time. NAM/ATAF1/2/CUC2 (NAC) proteins are plant-specific transcription factors (TFs) that modulate responses to abiotic stresses, including heat stress (HS). Here, we investigated the potential role of NACs for thermomemory. We determined the expression of 104 Arabidopsis NAC genes after priming and triggering heat stimuli, and found ATAF1 expression is strongly induced right after priming and declines below control levels thereafter during thermorecovery. Knockout mutants of ATAF1 show better thermomemory than wild type, revealing a negative regulatory role. Differential expression analyses of RNA-seq data from ATAF1 overexpressor, ataf1 mutant and wild-type plants after heat priming revealed five genes that might be priming-associated direct targets of ATAF1: AT2G31260 (ATG9), AT2G41640 (GT61), AT3G44990 (XTH31), AT4G27720 and AT3G23540. Based on co-expression analyses applied to the aforementioned RNA-seq profiles, we identified ANAC055 to be transcriptionally co-regulated with ATAF1. Like ataf1, anac055 mutants show improved thermomemory, revealing a potential co-control of both NAC TFs over thermomemory. Our data reveals a core importance of two NAC transcription factors, ATAF1 and ANAC055, for thermomemory.

Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae.

With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L. tarentolae (2 µg/mL culture medium) for PCh.

Validation of suitable genes for normalization of diurnal gene expression studies in Chenopodium quinoa.

Quinoa depicts high nutritional quality and abiotic stress resistance, attracting strong interest in the last years. To unravel the function of candidate genes for agronomically relevant traits, studying their transcriptional activities by RT-qPCR is an important experimental approach. The accuracy of such experiments strongly depends on precise data normalization. To date, validation of potential candidate genes for normalization of diurnal expression studies has not been performed in C. quinoa. We selected eight candidate genes based on transcriptome data and literature survey, including conventionally used reference genes. We used three statistical algorithms (BestKeeper, geNorm and NormFinder) to test their stability and added further validation by a simulation-based strategy. We demonstrated that using different reference genes, including those top ranked by stability, causes significant differences among the resulting diurnal expression patterns. Our results show that isocitrate dehydrogenase enzyme (IDH-A) and polypyrimidine tract-binding protein (PTB) are suitable genes to normalize diurnal expression data of two different quinoa accessions. Moreover, we validated our reference genes by normalizing two known diurnally regulated genes, BTC1 and BBX19. The validated reference genes obtained in this study will improve the accuracy of RT-qPCR data normalization and facilitate gene expression studies in quinoa.

Quinoa Phenotyping Methodologies: An International Consensus.

Quinoa is a crop originating in the Andes but grown more widely and with the genetic potential for significant further expansion. Due to the phenotypic plasticity of quinoa, varieties need to be assessed across years and multiple locations. To improve comparability among field trials across the globe and to facilitate collaborations, components of the trials need to be kept consistent, including the type and methods of data collected. Here, an internationally open-access framework for phenotyping a wide range of quinoa features is proposed to facilitate the systematic agronomic, physiological and genetic characterization of quinoa for crop adaptation and improvement. Mature plant phenotyping is a central aspect of this paper, including detailed descriptions and the provision of phenotyping cards to facilitate consistency in data collection. High-throughput methods for multi-temporal phenotyping based on remote sensing technologies are described. Tools for higher-throughput post-harvest phenotyping of seeds are presented. A guideline for approaching quinoa field trials including the collection of environmental data and designing layouts with statistical robustness is suggested. To move towards developing resources for quinoa in line with major cereal crops, a database was created. The Quinoa Germinate Platform will serve as a central repository of data for quinoa researchers globally.

Overexpression of the NAC transcription factor JUNGBRUNNEN1 (JUB1) increases salinity tolerance in tomato.

Soil salinity is a major abiotic stress affecting plant growth and yield, due to both osmotic and ionic stresses. JUBGBRUNNEN1 (JUB1) is a NAC family transcription factor that has been shown to be involved in responses to abiotic stresses, such as water deficit, osmotic, salinity, heat and oxidative stress. In Arabidopsis thaliana (Arabidopsis), JUB1 has been shown to improve plant stress tolerance by regulating H2O2 levels. In the horticultural crop, Solanum lycopersicum cv. Moneymaker (tomato), overexpression of AtJUB1 has been shown to partially alleviate water deficit stress at the vegetative stage. In this study, we investigated the effect of Arabidopsis JUB1 overexpression in salinity tolerance in tomato. In hydroponically grown tomato seedlings, AtJUB1 overexpression results in higher prolines levels and improves the maintenance of water content in the plant under salinity stress. The transgenic tomato plants are more tolerant to salinity stress compared to control lines based on plant biomass. However, at the reproductive stage, we found that overexpression of AtJUB1 only provided marginal improvements in yield-related parameters, in the conditions used for the current work. The combination of improved water deficit and salinity stress tolerance conferred by AtJUB1 overexpression may be beneficial when tomato plants are grown in the field under marginal environments.

The Arabidopsis thaliana K+-Uptake Permease 5 (AtKUP5) Contains a Functional Cytosolic Adenylate Cyclase Essential for K+ Transport.

Potassium (K+) is the most abundant cation in plants, and its uptake and transport are key to growth, development and responses to the environment. Here, we report that Arabidopsis thaliana K+ uptake permease 5 (AtKUP5) contains an adenylate cyclase (AC) catalytic center embedded in its N-terminal cytosolic domain. The purified recombinant AC domain generates cAMP in vitro; and when expressed in Escherichia coli, increases cAMP levels in vivo. Both the AC domain and full length AtKUP5 rescue an AC-deficient E. coli mutant, cyaA, and together these data provide evidence that AtKUP5 functions as an AC. Furthermore, full length AtKUP5 complements the Saccharomyces cerevisiae K+ transport impaired mutant, trk1 trk2, demonstrating its function as a K+ transporter. Surprisingly, a point mutation in the AC center that impairs AC activity, also abolishes complementation of trk1 trk2, suggesting that a functional catalytic AC domain is essential for K+ uptake. AtKUP5-mediated K+ uptake is not affected by cAMP, the catalytic product of the AC, but, interestingly, causes cytosolic cAMP accumulation. These findings are consistent with a role for AtKUP5 as K+ flux sensor, where the flux-dependent cAMP increases modulate downstream components essential for K+ homeostasis, such as cyclic nucleotide gated channels.

The Genome Sequence of the Wild Tomato Solanum pimpinellifolium Provides Insights Into Salinity Tolerance.

Solanum pimpinellifolium, a wild relative of cultivated tomato, offers a wealth of breeding potential for desirable traits such as tolerance to abiotic and biotic stresses. Here, we report the genome assembly and annotation of S. pimpinellifolium 'LA0480.' Moreover, we present phenotypic data from one field experiment that demonstrate a greater salinity tolerance for fruit- and yield-related traits in S. pimpinellifolium compared with cultivated tomato. The 'LA0480' genome assembly size (811 Mb) and the number of annotated genes (25,970) are within the range observed for other sequenced tomato species. We developed and utilized the Dragon Eukaryotic Analyses Platform (DEAP) to functionally annotate the 'LA0480' protein-coding genes. Additionally, we used DEAP to compare protein function between S. pimpinellifolium and cultivated tomato. Our data suggest enrichment in genes involved in biotic and abiotic stress responses. To understand the genomic basis for these differences in S. pimpinellifolium and S. lycopersicum, we analyzed 15 genes that have previously been shown to mediate salinity tolerance in plants. We show that S. pimpinellifolium has a higher copy number of the inositol-3-phosphate synthase and phosphatase genes, which are both key enzymes in the production of inositol and its derivatives. Moreover, our analysis indicates that changes occurring in the inositol phosphate pathway may contribute to the observed higher salinity tolerance in 'LA0480.' Altogether, our work provides essential resources to understand and unlock the genetic and breeding potential of S. pimpinellifolium, and to discover the genomic basis underlying its environmental robustness.

Identification of Putative Transmembrane Proteins Involved in Salinity Tolerance in Chenopodium quinoa by Integrating Physiological Data, RNAseq, and SNP Analyses.

Chenopodium quinoa (quinoa) is an emerging crop that produces nutritious grains with the potential to contribute to global food security. Quinoa can also grow on marginal lands, such as soils affected by high salinity. To identify candidate salt tolerance genes in the recently sequenced quinoa genome, we used a multifaceted approach integrating RNAseq analyses with comparative genomics and topology prediction. We identified 219 candidate genes by selecting those that were differentially expressed in response to salinity, were specific to or overrepresented in quinoa relative to other Amaranthaceae species, and had more than one predicted transmembrane domain. To determine whether these genes might underlie variation in salinity tolerance in quinoa and its close relatives, we compared the response to salinity stress in a panel of 21 Chenopodium accessions (14 C. quinoa, 5 C. berlandieri, and 2 C. hircinum). We found large variation in salinity tolerance, with one C. hircinum displaying the highest salinity tolerance. Using genome re-sequencing data from these accessions, we investigated single nucleotide polymorphisms and copy number variation (CNV) in the 219 candidate genes in accessions of contrasting salinity tolerance, and identified 15 genes that could contribute to the differences in salinity tolerance of these Chenopodium accessions.