Healthy values and de novo domestication of sand rice (Agriophyllum squarrosum), a comparative view against Chenopodium quinoa.

Sand rice (Agriophyllum squarrosum) is prized for its well-balanced nutritional properties, broad adaptability in Central Asia and highly therapeutic potentials. It has been considered as a potential climate-resilient crop. Its seed has comparable metabolite profile with Chenopodium quinoa and is rich in proteins, essential amino acids, minerals, polyunsaturated fatty acids, and phenolics, but low in carbohydrates. Phenolics like protocatechuic acid and quercetins have been characterized with biological functions on regulation of lipid and glucose metabolism in addition to anti-inflammatory and antioxidant activities. Sand rice is thus an important source for developing functional and nutraceutical products. Though historical consumption has been over 1300 years, sand rice has undergone few agronomic improvements until recently. Breeding by individual selection has been performed and yield of the best genotype can reach up to 1295.5 kg/ha. Furthermore, chemical mutagenesis has been used to modify the undesirable traits and a case study of a dwarf line (dwarf1), which showed the Green Revolution-like phenotypes, is presented. Utilization of both breeding methodologies will accelerate its domestication process. As a novel crop, sand rice research is rather limited compared with quinoa. More scientific input is urgently required if the nutritional and commercial potentials are to be fully realized.Supplemental data for this article is available online at https://doi.org/10.1080/10408398.2021.1999202 .

Precipitation and local environment shape the geographic variation of seed size across natural populations of sand rice (Agriophyllum squarrosum).

Sand rice (Agriophyllum squarrosum) is widely distributed on dunes in the Asian interior arid zone, and its large intraspecific trait variation makes it a very good model for investigating the ecological processes underlying its adaptation to the desert environment. In this study, seed size variation across 68 natural populations was used to establish geographic patterns and to quantify the effects of the climate, soil, and collection-year weather variables. The length of the seed major axis and thousand seed weight (TSW) both showed significant longitudinal patterns. Long-term climate variables accounted for most of the explained variances for seed major axis (57.20%) and TSW (91.54%). Specifically, annual precipitation and minimum monthly precipitation had the most significantly positive and negative effects, indicating that longitudinal clines are driven by a precipitation gradient across the species' distribution range. A substantial unique effect of soil variables (27.27%) was found for seed major axis variation, but only 3.64% of TSW variation was explained by soil variables. Two extreme groups were selected to evaluate the genetic and plastic effects on seed size in a common garden experiment. Large-seeded individuals were more competitive in semi-arid regions, and had stronger adaptive plasticity as well as better performance in early seedling establishment, and hence they have potential for use in future domestication projects.

Genomic Variation Underlying the Breeding Selection of Quinoa Varieties Longli-4 and CA3-1 in China.

Quinoa (Chenopodium quinoa) is a well-known climate-resilient crop and has been introduced into multiple marginal lands across the world, including China, to improve food security and/or balanced nutrient supplies. Conventional breeding has been widely applied in the selection and breeding of quinoa varieties in China since 1980s; however, few studies have been implemented on the genetic variances among different varieties developed by diversity breeding objectives. In this study, the phenotypic and genetic differences between two varieties (Longli-4 and CA3-1) from China were systematically analyzed. A total of 407,651 and 2,731,411 single nucleotide polymorphisms (SNPs) and 212,724 and 587,935 small insertion and deletion (INDELs) were detected for Longli-4 and CA3-1, respectively, when compared with the reference genome of PI614886. The SNPs/INDELs were unevenly distributed across each chromosome for both varieties. There were 143,996 SNPs and 83,410 INDELs shared between Longli-4 and CA3-1, accounting for 4% of the total variances. The variation was then screened based on the SNP effects. There were 818 and 73 genes with the variety-specific non-synonymous and stop-gain variation in Longli-4, whereas there were 13,701 and 733 genes in CA3-1. Specifically, 3501 genes with the non-synonymous variation and 74 genes with the stop-gain variation were found in both Longli-4 and CA3-1. These results suggest that convergent selection occurred during the different breeding processes. A set of candidate genes related to agronomic traits and domestication were further selected to detect the genetic divergence in detail in the two varieties. Only one domestication gene was identified having Longli-4-specific stop-gain variation. Twelve candidate genes related to betalain (1), flowering (4), seed size (2), domestication (1), and saponin (4) were identified having CA3-1-specific stop-gain variation. Interestingly, one seed size gene homologous of CKX1 (cytokinin oxidase/dehydrogenase 1) had the stop-gain variation in both varieties. This research will therefore provide guidance for the molecular-assisted breeding in quinoa.

Transcriptomic analysis of a psammophyte food crop, sand rice (Agriophyllum squarrosum) and identification of candidate genes essential for sand dune adaptation.

BACKGROUND: Sand rice (Agriophyllum squarrosum) is an annual desert plant adapted to mobile sand dunes in arid and semi-arid regions of Central Asia. The sand rice seeds have excellent nutrition value and have been historically consumed by local populations in the desert regions of northwest China. Sand rice is a potential food crop resilient to ongoing climate change; however, partly due to the scarcity of genetic information, this species has undergone only little agronomic modifications through classical breeding during recent years. RESULTS: We generated a deep transcriptomic sequencing of sand rice, which uncovers 67,741 unigenes. Phylogenetic analysis based on 221 single-copy genes showed close relationship between sand rice and the recently domesticated crop sugar beet. Transcriptomic comparisons also showed a high level of global sequence conservation between these two species. Conservation of sand rice and sugar beet orthologs assigned to response to salt stress gene ontology term suggests that sand rice is also a potential salt tolerant plant. Furthermore, sand rice is far more tolerant to high temperature. A set of genes likely relevant for resistance to heat stress, was functionally annotated according to expression levels, sequence annotation, and comparisons corresponding transcriptome profiling results in Arabidopsis. CONCLUSIONS: The present work provides abundant genomic information for functional dissection of the important traits in sand rice. Future screening the genetic variation among different ecotypes and constructing a draft genome sequence will further facilitate agronomic trait improvement and final domestication of sand rice.

Genomic variation induced by a low concentration of ethyl methanesulfonate (EMS) in quinoa 'Longli-4' variety.

Quinoa (Chenopodium quinoa, 2n = 4x = 36), a super pseudocereal crop, has been introduced into China nearly 60 years. Many excellent varieties have been developed through massive selection; however, few are developed through mutagenesis breeding. In this study, the 'Longli-4' variety, locally cultivated in Gansu province, Northwest China, was selected for experimentation. The grains of 'Longli-4' were treated with ethyl methanesulfonate (EMS) at a concentration of 0.8% for 8 h. Nine plants from independent M2 families were randomly selected to investigate the mutagenesis effect of EMS on the quinoa genome. The results indicated that the single nucleotide polymorphisms (SNPs) induced by EMS were unevenly distributed across all 18 chromosomes, with an average mutation frequency of 91.2 SNPs/Mb, ranging from 4.5 to 203.5 SNPs/Mb. A significant positive correlation between the number of SNPs and chromosome length was identified through linear model analysis. Transitions from G/C to A/T were the most predominated in all variant categories, accounting for 34.4-67.2% of the mutations, and SNPs were significantly enriched in intergenic regions, representing 69.2-75.1% of the total mutations. This study provides empirical support for the application of low concentration EMS treatment in quinoa breeding.

MAIGO2 is involved in abscisic acid-mediated response to abiotic stresses and Golgi-to-ER retrograde transport.

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50-interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2-like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)-stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi-to-ER retrograde trafficking. Therefore, a new role for ER-Golgi membrane trafficking in abiotic-stress and ER-stress responses is discovered.

An Alternative Class of Targets for microRNAs Containing CG Dinucleotide.

MicroRNAs (miRNAs) are endogenous ~23 nt RNAs which regulate message RNA (mRNA) targets mainly through perfect pairing with their seed region (positions 2-7). Several instances of UTR sequence with an additional nucleotide that might form a bulge within the pairing region, can also be recognized by miRNA as their target (bugle-target). But the prevalence of such imperfect base pairings in human and their roles in the evolution are incompletely understood. We found that human miRNAs with the CG dinucleotides (CG dimer) in their seed region have a significant low mutation rate than their putative binding sites in mRNA targets. Interspecific comparation shows that these miRNAs had very few conservative targets with the perfect seed-pairing, while potentially having a subclass of bulge-targets. Compared with the canonical target (perfect seed-pairing), these bulge-targets had a lower negative correlation with the miRNA expression, and either were down-regulated in the miRNA overexpression experiment or up-regulated in the miRNA knock-down experiment. Our results show that the bulge-targets are widespread in the miRNAs with CG dinucleotide within their seed regions, which could in part explain the rare conserved targets of these miRNAs based on seed rule. Incorporating these bulge-targets, together with conservation information, could more accurately predict the entire targets of these miRNAs.

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana.

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.

Transcriptional responses to phosphate starvation in Brachypodium distachyon roots.

Brachypodium distachyon is a model plant that has recently emerged in grass research. Although the growth and photochemical efficiency of this species respond strongly to phosphate (Pi) availability, its Pi starvation response mechanism, which controls the Pi homeostasis, remains largely unknown. This study presents the transcriptomic response profiles of Pi-deficient roots at growth stages during which the plants were starved but obvious growth defects were absent. The results identify several phosphate transporters (i.e., PHO1), purple acid phosphatases, and SYG1/PHO81/XPR1 (SPX) domain-containing proteins out of a total of 1740 differentially expressed genes (DEGs). In particular, the transcription factor ethylene response factor (ERF), basic helix-loop-helix (bHLH), and WRKY family genes were the three most abundant DEG groups and the latter was significantly enriched. Comparative transcriptome analysis of Brachypodium versus Arabidopsis and rice revealed the presence of several common components in response to Pi fluctuations. Most significantly, jasmonic acid (JA) signaling-related genes were overrepresented in gene ontology (GO) enrichment tests. The presence of a possible link between low Pi response, inositol polyphosphates, and JA signaling is therefore discussed.

Synteny-based mapping of causal point mutations relevant to sand rice (Agriophyllum squarrosum) trichomeless1 mutant by RNA-sequencing.

Sand rice (Agriophyllum squarrosum), a diploid Amaranthaceae species within the order Caryophyllalles, has potential as a crop in response to concerns about ongoing climate change and future food security. Modifying the weedy traits, such as dense trichomes, is important for the domestication of sand rice. In this study, an ethyl methanesulfonate (EMS) induced Agriophyllum squarrosum trichomeless mutant (astcl1) was isolated and genetic analysis revealed that this mutant was controlled by a single recessive locus. RNA sequencing was employed to analyze sequence variations between the mutant and wild-type individuals based on allele frequencies. Synteny-based mapping against two closely related and sequenced species finally delimited the causal mutations into an approximately 18.97 mega base pair (Mb) interval on the top portion of the chromosome 6 (Bv6) of sugar beet (Beta vulgaris) and two intervals (5.56 and 2.69 Mb) on the chromosomes 14 (CqA14) and 6 (CqB06) of quinoa (Chenopodium quinoa). These two quinoa intervals were located in the orthologous chromosomal regions against sugar beet Bv6. Differential expression analysis revealed that the astcl1 mutation only affects the expression of a small subset of genes. Most significantly, 17 lipid transport and metabolism related genes, such as GDSL esterases and very-long-chain 3-oxoacyl-CoA reductase 1, and two R2R3 MYB genes, MYB39 and RAX3, were down-regulated in astcl1 mutants. These results imply that the Astcl1 protein coordinately regulates trichome initiation and cuticle biosynthesis in sand rice.

A comparative transcriptomic analysis reveals the core genetic components of salt and osmotic stress responses in Braya humilis.

Braya humilis is a member of the Euclidieae tribe within the family Brassicaceae. This species exhibits a broad range of adaptations to different climatic zones and latitudes as it has a distribution that ranges from northern Asia to the arctic-alpine regions of northern North America. In China, B. humilis is mainly found on the Qinghai-Tibetan Plateau (QTP) and in adjacent arid regions. In this study, we sequenced a sample from an arid region adjacent to the QTP using the Illumina platform generating a total of 46,485 highly accurate unigenes, of which 78.41% were annotated by BLASTing versus public protein databases. The B. humilis transcriptome is characterized by a high level of sequence conservation compared with its close relative, Arabidopsis thaliana. We also used reciprocal blast to identify shared orthologous genes between B. humilis and four other sequenced Brassicaceae species (i.e. A. thaliana, A. lyrata, Capsella rubella, and Thellungiella parvula). To enable precise characterization of orthologous genes, the early-diverging basal angiosperm Amborella trichopoda was also included. A total of 6,689 orthologous genes were identified before stricter criteria for the determination of e-values, amino acid hit lengths, and identity values was applied to further reduce this list. This led to a final list of 381 core orthologous genes for B. humilis; 39 out of these genes are involved in salt and osmotic stress responses and estimations of nonsynonymous/synonymous substitution ratios for this species and A. thaliana orthologs show that these genes are under purifying selection in B. humilis. Expression of six genes was detected in B. humilis seedlings under salt and osmotic stress treatments. Comparable expression patterns to their counterparts in Arabidopsis suggest that these orthologous genes are both sequence and functional conservation. The results of this study demonstrate that the environmental adaptations of B. humilis are mainly the results of preexisting genetic components. Future work will be required to characterize the expression patterns of these orthologous genes in natural populations and will provide further insights into the adaptive mechanisms underlying the wide range of B. humilis adaptations.

SNP Discovery and Genetic Variation of Candidate Genes Relevant to Heat Tolerance and Agronomic Traits in Natural Populations of Sand Rice (Agriophyllum squarrosum).

The extreme stress tolerance and high nutritional value of sand rice (Agriophyllum squarrosum) make it attractive for use as an alternative crop in response to concerns about ongoing climate change and future food security. However, a lack of genetic information hinders understanding of the mechanisms underpinning the morphological and physiological adaptations of sand rice. In the present study, we sequenced and analyzed the transcriptomes of two individuals representing semi-arid [Naiman (NM)] and arid [Shapotou (SPT)] sand rice genotypes. A total of 105,868 pairwise single nucleotide polymorphisms (SNPs) distributed in 24,712 Unigenes were identified among SPT and NM samples; the average SNP frequency was 0.3% (one SNP per 333 base pair). Characterization of gene annotation demonstrated that variations in genes involved in DNA recombination were associated with the survival of the NM population in the semi-arid environment. A set of genes predicted to be relevant to heat stress response and agronomic traits was functionally annotated using the accumulated knowledge from Arabidopsis and several crop plants, including rice, barley, maize, and sorghum. Four candidate genes related to heat tolerance (heat-shock transcription factor, HsfA1d), seed size (DA1-Related, DAR1), and flowering (early flowering 3, ELF3 and late elongated hypocotyl, LHY) were subjected to analysis of the genetic diversity in 10 natural populations, representing the core germplasm resource across the area of sand rice distribution in China. Only one SNP was detected in each of HsfA1d and DAR1, among 60 genotypes, with two in ELF3 and four in LHY. Nucleotide diversity ranged from 0.00032 to 0.00118. Haplotype analysis indicated that the NM population carried a specific allele for all four genes, suggesting that divergence has occurred between NM and other populations. These four genes could be further analyzed to determine whether they are associated with phenotype variation and identify alleles favorable for sand rice breeding.

A GDSL-motif esterase/acyltransferase/lipase is responsible for leaf water retention in barley.

The hydrophobic cuticle covers the surface of the most aerial organs of land plants. The barley mutant eceriferum-zv (cer-zv), which is hypersensitive to drought, is unable to accumulate a sufficient quantity of cutin in its leaf cuticle. The mutated locus has been mapped to a 0.02 cM segment in the pericentromeric region of chromosome 4H. As a map-based cloning approach to isolate the gene was therefore considered unlikely to be feasible, a comparison was instead made between the transcriptomes of the mutant and the wild type. In conjunction with extant genomic information, on the basis of predicted functionality, only two genes were considered likely to encode a product associated with cutin formation. When eight independent cer-zv mutant alleles were resequenced with respect to the two candidate genes, it was confirmed that the gene underlying the mutation in each allele encodes a Gly-Asp-Ser-Leu (GDSL)-motif esterase/acyltransferase/lipase. The gene was transcribed in the epidermis, and its product was exclusively deposited in cell wall at the boundary of the cuticle in the leaf elongation zone, coinciding with the major site of cutin deposition. CER-ZV is speculated to function in the deposition of cutin polymer. Its homologs were found in green algae, moss, and euphyllophytes, indicating that it is highly conserved in plant kingdom.

Different Sets of Post-Embryonic Development Genes Are Conserved or Lost in Two Caryophyllales Species (Reaumuria soongorica and Agriophyllum squarrosum).

Reaumuria soongorica and sand rice (Agriophyllum squarrosum) belong to the clade of Caryophyllales and are widely distributed in the desert regions of north China. Both plants have evolved many specific traits and adaptation strategies to cope with recurring environmental threats. However, the genetic basis that underpins their unique traits and adaptation remains unknown. In this study, the transcriptome data of R. soongorica and sand rice were compared with three other species with previously sequenced genomes (Arabidopsis thaliana, Oryza sativa, and Beta vulgaris). Four different gene sets were identified, namely, the genes conserved in both species, those lost in both species, those conserved in R. soongorica only, and those conserved in sand rice only. Gene ontology showed that post-embryonic development genes (PEDGs) were enriched in all gene sets, and different sets of PEDGs were conserved or lost in both the R. soongorica and sand rice genomes. Expression profiles of Arabidopsis orthologs further provided some clues to the function of the species-specific conserved PEDGs. Such orthologs included LEAFY PETIOLE, which could be a candidate gene involved in the development of branch priority in sand rice.

Transcriptomic analysis of a tertiary relict plant, extreme xerophyte Reaumuria soongorica to identify genes related to drought adaptation.

BACKGROUND: Reaumuria soongorica is an extreme xerophyte shrub widely distributed in the desert regions including sand dune, Gobi and marginal loess of central Asia which plays a crucial role to sustain and restore fragile desert ecosystems. However, due to the lacking of the genomic sequences, studies on R. soongorica had mainly limited in physiological responses to drought stress. Here, a deep transcriptomic sequencing of R. soongorica will facilitate molecular functional studies and pave the path to understand drought adaptation for a desert plant. METHODOLOGY/PRINCIPAL FINDINGS: A total of 53,193,660 clean paired-end reads was generated from the Illumina HiSeq™ 2000 platform. By assembly with Trinity, we got 173,700 contigs and 77,647 unigenes with mean length of 677 bp and N50 of 1109 bp. Over 55% (43,054) unigenes were successfully annotated based on sequence similarity against public databases as well as Rfam and Pfam database. Local BLAST and Kyoto Encyclopedia of Genes and Genomes (KEGG) maps were used to further exhausting seek for candidate genes related to drought adaptation and a set of 123 putative candidate genes were identified. Moreover, all the C4 photosynthesis genes existed and were active in R. soongorica, which has been regarded as a typical C3 plant. CONCLUSION/SIGNIFICANCE: The assembled unigenes in present work provide abundant genomic information for the functional assignments in an extreme xerophyte R. soongorica, and will help us exploit the genetic basis of how desert plants adapt to drought environment in the near future.