Community Resource: Large-Scale Proteogenomics to Refine Wheat Genome Annotations.

Triticum aestivum is an important crop whose reference genome (International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v2.1) offers a valuable resource for understanding wheat genetic structure, improving agronomic traits, and developing new cultivars. A key aspect of gene model annotation is protein-level evidence of gene expression obtained from proteomics studies, followed up by proteogenomics to physically map proteins to the genome. In this research, we have retrieved the largest recent wheat proteomics datasets publicly available and applied the Basic Local Alignment Search Tool (tBLASTn) algorithm to map the 861,759 identified unique peptides against IWGSC RefSeq v2.1. Of the 92,719 hits, 83,015 unique peptides aligned along 33,612 High Confidence (HC) genes, thus validating 31.4% of all wheat HC gene models. Furthermore, 6685 unique peptides were mapped against 3702 Low Confidence (LC) gene models, and we argue that these gene models should be considered for HC status. The remaining 2934 orphan peptides can be used for novel gene discovery, as exemplified here on chromosome 4D. We demonstrated that tBLASTn could not map peptides exhibiting mid-sequence frame shift. We supply all our proteogenomics results, Galaxy workflow and Python code, as well as Browser Extensible Data (BED) files as a resource for the wheat community via the Apollo Jbrowse, and GitHub repositories. Our workflow could be applied to other proteomics datasets to expand this resource with proteins and peptides from biotically and abiotically stressed samples. This would help tease out wheat gene expression under various environmental conditions, both spatially and temporally.

Bioaccessibility of Phenolic Compounds, Resistant Starch, and Dietary Fibers from Australian Green Banana during In Vitro Digestion and Colonic Fermentation.

Green bananas contain a substantial amount of resistant starch (RS), dietary fiber (DF), and phytochemicals, which exhibit potent antioxidant capabilities, primarily attributable to the abundance of polyphenols. The objective of this study was to assess the variations in the contents and bioaccessibility of RS, DF, and phenolic compounds in three types of Australian green bananas (Cavendish "Musa acuminata", Ladyfinger "Musa paradisiaca L.", and Ducasse "Musa balbisiana"), along with their antioxidant capacities, and the production of short-chain fatty acids (SCFAs) following in vitro simulated gastrointestinal digestion and colonic fermentation. The studied cultivars exhibited significant levels of RS, with Ladyfinger showing the greatest (49%). However, Ducasse bananas had the greatest DF concentration (38.73%). Greater TPC levels for Ladyfinger (2.32 mg GAE/g), as well as TFC and TTC (0.06 mg QE/g and 3.2 mg CE/g, respectively) in Cavendish, together with strong antioxidant capacities (DPPH, 0.89 mg TE/g in Cavendish), have been detected after both intestinal phase and colonic fermentation at 12 and 24 h. The bioaccessibility of most phenolic compounds from bananas was high after gastric and small intestinal digestion. Nevertheless, a significant proportion of kaempferol (31% in Cavendish) remained detectable in the residue after colonic fermentation. The greatest production of SCFAs in all banana cultivars was observed after 24 h of fermentation, except valeric acid, which exhibited the greatest output after 12 h of fermentation. In conclusion, the consumption of whole green bananas may have an advantageous effect on bowel health and offer antioxidant characteristics.

Large flour aggregates containing ordered B + V starch crystals significantly improved the digestion resistance of starch in pretreated multigrain flour.

The starch digestibility of flour is influenced by both physicochemical treatment and flour particle size, but the interactive effect of these two factors is still unclear. In this study, the effect of pullulanase debranching, combined with heat-moisture treatment (P-HMT), on starch digestibility of multi-grain flours (including oat, buckwheat and wheat) differing in particle size was investigated. The results showed that the larger-size flour always resulted in a higher resistant starch (RS) content either in natural or treated multi-grain flour (NMF or PHF). P-HMT doubled the RS content in NMFs and the large-size PHF yielded the highest RS content (78.43 %). In NMFs, the cell wall integrity and flour particle size were positively related to starch anti-digestibility. P-HMT caused the destruction of cell walls and starch granules, as well as the formation of rigid flour aggregates with B + V starch crystallite. The largest flour aggregates with the most ordered B + V starch were found in large-size PHF, which contributed to its highest RS yield, while the medium- and small-size PHFs with smaller aggregates were sensitive to P-HMT, resulting in the lower ordered starch but stronger interactions between starch and free lipid or monomeric proteins, eventually leading to their lower RS but higher SDS yield.

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Wheat Water-Soluble Carbohydrate Remobilisation under Water Deficit by 1-FEH w3.

Fructan 1-exohydrolase (1-FEH) is one of the major enzymes in water-soluble carbohydrate (WSC) remobilisation for grains in wheat. We investigated the functional role of 1-FEH w1, w2, and w3 isoforms in WSC remobilisation under post-anthesis water deficit using mutation lines derived from the Australian wheat variety Chara. F1 seeds, developed by backcrossing the 1-FEH w1, w2, and w3 mutation lines with Chara, were genotyped using the Infinium 90K SNP iSelect platform to characterise the mutated region. Putative deletions were identified in FEH mutation lines encompassing the FEH genomic regions. Mapping analysis demonstrated that mutations affected significantly longer regions than the target FEH gene regions. Functional roles of the non-target genes were carried out utilising bioinformatics and confirmed that the non-target genes were unlikely to confound the effects considered to be due to the influence of 1-FEH gene functions. Glasshouse experiments revealed that the 1-FEH w3 mutation line had a slower degradation and remobilisation of fructans than the 1-FEH w2 and w1 mutation lines and Chara, which reduced grain filling and grain yield. Thus, 1-FEH w3 plays a vital role in reducing yield loss under drought. This insight into the distinct role of the 1-FEH isoforms provides new gene targets for water-deficit-tolerant wheat breeding.

Centromere repositioning and shifts in wheat evolution.

The centromere is the region of a chromosome that directs its separation and plays an important role in cell division and reproduction of organisms. Elucidating the dynamics of centromeres is an alternative strategy for exploring the evolution of wheat. Here, we comprehensively analyzed centromeres from the de novo-assembled common wheat cultivar Aikang58 (AK58), Chinese Spring (CS), and all sequenced diploid and tetraploid ancestors by chromatin immunoprecipitation sequencing, whole-genome bisulfite sequencing, RNA sequencing, assay for transposase-accessible chromatin using sequencing, and comparative genomics. We found that centromere-associated sequences were concentrated during tetraploidization and hexaploidization. Centromeric repeats of wheat (CRWs) have undergone expansion during wheat evolution, with strong interweaving between the A and B subgenomes post tetraploidization. We found that CENH3 prefers to bind with younger CRWs, as directly supported by immunocolocalization on two chromosomes (1A and 2A) of wild emmer wheat with dicentromeric regions, only one of which bound with CENH3. In a comparison of AK58 with CS, obvious centromere repositioning was detected on chromosomes 1B, 3D, and 4D. The active centromeres showed a unique combination of lower CG but higher CHH and CHG methylation levels. We also found that centromeric chromatin was more open than pericentromeric chromatin, with higher levels of gene expression but lower gene density. Frequent introgression between tetraploid and hexaploid wheat also had a strong influence on centromere position on the same chromosome. This study also showed that active wheat centromeres were genetically and epigenetically determined.

Effects of Composition and Strength of Wheat Gluten on Starch Structure, Digestion Properties and the Underlying Mechanism.

To understand the effect of gluten on starch digestion characteristics, the structural characteristics of protein, starch, and starch digestion attributes were explored by using flours of four wheat near-isogenic lines. Protein and starch fractions from the four flours were used to form so-called recombinant flours where glutenin and gliadin protein fractions, in different ratios, were combined with starch and heated in a water slurry at 80 °C for 5 min. We found that starch digestibility of the recombinant flours could be reproducibly modified by altering the long- and short-range molecular order of starch through varying the attributes of the gluten protein by virtue of the gluten strength as well as the proportions of glutenin and gliadins. The gluten composition changes of strong-gluten flour did not improve the starch digestion resistibility, however, for the moderate- and weak-gluten flours, the proportional increase of glutenin improved the resistance of starch to digestion through the increased long- and short-range molecular order of starch. The resistance of starch to digestion could also be enhanced with increasing gliadin, and was associated with the modified short-range molecular order of starch. We propose that flour mixtures can be optimized for specified product quality by manipulating the amounts of both gliadin and glutenin.

Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Macromolecular networks interactions in wheat flour dough matrices during sequential thermal-mechanical treatment.

Differences in Mixolab measurements of dough processing were examined using, as a base, flour from pure breeding, isogenic, wheat lines carrying either the high molecular weight glutenin subunits 5 + 10 or 2 + 12. Before dough pasting, subunits 5 + 10 tend to form a stable gluten network relying mainly on disulfide bonds and hydrogen bonds, but 2 + 12 flour was prone to generating fragile protein aggregates dominated by disulfide bonds and hydrophobicity. During dough pasting, a broader protein network rich in un-extractable polymeric proteins, disulfide bonds and ß-sheets was formed in the dough with subunits 5 + 10, thus resulting in an extensive and compact protein-starch complex which was characterized by high thermal stability and low starch gelatinization, while in the dough of the 2 + 12 line, a porous protein-starch gel with fragmented protein aggregates was controlled by the combination of disulfide bonds, hydrophobicity and hydrogen bonds that facilitated the formation of antiparallel ß-sheets.

Optical maps refine the bread wheat Triticum aestivum cv. Chinese Spring genome assembly.

Until recently, achieving a reference-quality genome sequence for bread wheat was long thought beyond the limits of genome sequencing and assembly technology, primarily due to the large genome size and > 80% repetitive sequence content. The release of the chromosome scale 14.5-Gb IWGSC RefSeq v1.0 genome sequence of bread wheat cv. Chinese Spring (CS) was, therefore, a milestone. Here, we used a direct label and stain (DLS) optical map of the CS genome together with a prior nick, label, repair and stain (NLRS) optical map, and sequence contigs assembled with Pacific Biosciences long reads, to refine the v1.0 assembly. Inconsistencies between the sequence and maps were reconciled and gaps were closed. Gap filling and anchoring of 279 unplaced scaffolds increased the total length of pseudomolecules by 168 Mb (excluding Ns). Positions and orientations were corrected for 233 and 354 scaffolds, respectively, representing 10% of the genome sequence. The accuracy of the remaining 90% of the assembly was validated. As a result of the increased contiguity, the numbers of transposable elements (TEs) and intact TEs have increased in IWGSC RefSeq v2.1 compared with v1.0. In total, 98% of the gene models identified in v1.0 were mapped onto this new assembly through development of a dedicated approach implemented in the MAGAAT pipeline. The numbers of high-confidence genes on pseudomolecules have increased from 105 319 to 105 534. The reconciled assembly enhances the utility of the sequence for genetic mapping, comparative genomics, gene annotation and isolation, and more general studies on the biology of wheat.

Integrating Wheat Nucleolus Structure and Function: Variation in the Wheat Ribosomal RNA and Protein Genes.

We review the coordinated production and integration of the RNA (ribosomal RNA, rRNA) and protein (ribosomal protein, RP) components of wheat cytoplasmic ribosomes in response to changes in genetic constitution, biotic and abiotic stresses. The components examined are highly conserved and identified with reference to model systems such as human, Arabidopsis, and rice, but have sufficient levels of differences in their DNA and amino acid sequences to form fingerprints or gene haplotypes that provide new markers to associate with phenotype variation. Specifically, it is argued that populations of ribosomes within a cell can comprise distinct complements of rRNA and RPs to form units with unique functionalities. The unique functionalities of ribosome populations within a cell can become central in situations of stress where they may preferentially translate mRNAs coding for proteins better suited to contributing to survival of the cell. In model systems where this concept has been developed, the engagement of initiation factors and elongation factors to account for variation in the translation machinery of the cell in response to stresses provided the precedents. The polyploid nature of wheat adds extra variation at each step of the synthesis and assembly of the rRNAs and RPs which can, as a result, potentially enhance its response to changing environments and disease threats.

Origin and adaptation to high altitude of Tibetan semi-wild wheat.

Tibetan wheat is grown under environmental constraints at high-altitude conditions, but its underlying adaptation mechanism remains unknown. Here, we present a draft genome sequence of a Tibetan semi-wild wheat (Triticum aestivum ssp. tibetanum Shao) accession Zang1817 and re-sequence 245 wheat accessions, including world-wide wheat landraces, cultivars as well as Tibetan landraces. We demonstrate that high-altitude environments can trigger extensive reshaping of wheat genomes, and also uncover that Tibetan wheat accessions accumulate high-altitude adapted haplotypes of related genes in response to harsh environmental constraints. Moreover, we find that Tibetan semi-wild wheat is a feral form of Tibetan landrace, and identify two associated loci, including a 0.8-Mb deletion region containing Brt1/2 homologs and a genomic region with TaQ-5A gene, responsible for rachis brittleness during the de-domestication episode. Our study provides confident evidence to support the hypothesis that Tibetan semi-wild wheat is de-domesticated from local landraces, in response to high-altitude extremes.

Resequencing of 145 Landmark Cultivars Reveals Asymmetric Sub-genome Selection and Strong Founder Genotype Effects on Wheat Breeding in China.

Controlled pedigrees and the multi-decade timescale of national crop plant breeding programs offer a unique experimental context for examining how selection affects plant genomes. More than 3000 wheat cultivars have been registered, released, and documented since 1949 in China. In this study, a set of 145 elite cultivars selected from historical points of wheat breeding in China were re-sequenced. A total of 43.75 Tb of sequence data were generated with an average read depth of 17.94× for each cultivar, and more than 60.92 million SNPs and 2.54 million InDels were captured, based on the Chinese Spring RefSeq genome v1.0. Seventy years of breeder-driven selection led to dramatic changes in grain yield and related phenotypes, with distinct genomic regions and phenotypes targeted by different breeders across the decades. There are very clear instances illustrating how introduced Italian and other foreign germplasm was integrated into Chinese wheat programs and reshaped the genomic landscape of local modern cultivars. Importantly, the resequencing data also highlighted significant asymmetric breeding selection among the three sub-genomes: this was evident in both the collinear blocks for homeologous chromosomes and among sets of three homeologous genes. Accumulation of more newly assembled genes in newer cultivars implied the potential value of these genes in breeding. Conserved and extended sharing of linkage disequilibrium (LD) blocks was highlighted among pedigree-related cultivars, in which fewer haplotype differences were detected. Fixation or replacement of haplotypes from founder genotypes after generations of breeding was related to their breeding value. Based on the haplotype frequency changes in LD blocks of pedigree-related cultivars, we propose a strategy for evaluating the breeding value of any given line on the basis of the accumulation (pyramiding) of beneficial haplotypes. Collectively, our study demonstrates the influence of "founder genotypes" on the output of breeding efforts over many decades and also suggests that founder genotype perspectives are in fact more dynamic when applied in the context of modern genomics-informed breeding.

Proteomic profiling of developing wheat heads under water-stress.

A replicated iTRAQ (isobaric tags for relative and absolute quantification) study on developing wheat heads from two doubled haploid (DH) lines identified from a cross between cv Westonia x cv Kauz characterized the proteome changes influenced by reproductive stage water-stress. All lines were exposed to 10 days of water-stress from early booting (Zadok 40), with sample sets taken from five head developmental stages. Two sample groups (water-stressed and control) account for 120 samples that required 18 eight-plex iTRAQ runs. Based on the IWGSC RefSeq v1 wheat assembly, among the 4592 identified proteins, a total of 132 proteins showed a significant response to water-stress, including the down-regulation of a mitochondrial Rho GTPase, a regulator of intercellular fundamental biological processes (7.5 fold) and cell division protein FtsZ at anthesis (6.0 fold). Up-regulated proteins included inosine-5'-monophosphate dehydrogenase (3.83 fold) and glycerophosphodiester phosphodiesterase (4.05 fold). The Pre-FHE and FHE stages (full head emerged) of head development were differentiated by 391 proteins and 270 proteins differentiated the FHE and Post-FHE stages. Water-stress during meiosis affected seed setting with 27% and 6% reduction in the progeny DH105 and DH299 respectively. Among the 77 proteins that differentiated between the two DH lines, 7 proteins were significantly influenced by water-stress and correlated with the seed set phenotype response of the DH lines to water-stress (e.g. the up-regulation of a subtilisin-like protease in DH 299 relative to DH 105). This study provided unique insights into the biological changes in developing wheat head that occur during water-stress.

Status and Potential of Single-Cell Transcriptomics for Understanding Plant Development and Functional Biology.

The advent of modern "omics" technologies (genomics, transcriptomics, proteomics, and metabolomics) are attributed to innovative breakthroughs in genome sequencing, bioinformatics, and analytic tools. An organism's biological structure and function is the result of the concerted action of single cells in different tissues. Single cell genomics has emerged as a ground-breaking technology that has greatly enhanced our understanding of the complexity of gene expression at a microscopic resolution and holds the potential to revolutionize the way we characterize complex cell assemblies and study their spatial organization, dynamics, clonal distribution, pathways, function, and networking. Mammalian systems have benefitted immensely from these approaches to dissect complex systems such as cancer, immunological disorders, epigenetic controls of diseases, and understanding of developmental biology. However, the applications of single-cell omics in plant research are just starting. The potential to decipher the fundamentals of developmental and functional biology of large and complex plant species at the single-cell resolution are now becoming important drivers of research. In this review, we present the status, challenges and potential of one important and most commonly used single-cell omics technique in plants, namely single cell transcriptomics. © 2020 International Society for Advancement of Cytometry.

The Battle to Sequence the Bread Wheat Genome: A Tale of the Three Kingdoms.

In the year 2018, the world witnessed the finale of the race to sequence the genome of the world's most widely grown crop, the common wheat. Wheat has been known to bear a notoriously large and complicated genome of a polyploidy nature. A decade competition to sequence the wheat genome initiated with a single consortium of multiple countries, taking a conventional strategy similar to that for sequencing Arabidopsis and rice, became ferocious over time as both sequencing technologies and genome assembling methodologies advanced. At different stages, multiple versions of genome sequences of the same variety (e.g., Chinese Spring) were produced by several groups with their special strategies. Finally, 16 years after the rice genome was finished and 9 years after that of maize, the wheat research community now possesses its own reference genome. Armed with these genomics tools, wheat will reestablish itself as a model for polyploid plants in studying the mechanisms of polyploidy evolution, domestication, genetic and epigenetic regulation of homoeolog expression, as well as defining its genetic diversity and breeding on the genome level. The enhanced resolution of the wheat genome should also help accelerate development of wheat cultivars that are more tolerant to biotic and/or abiotic stresses with better quality and higher yield.

Signatures for torque variation in wheat dough structure are affected by enzymatic treatments and heating.

Molecular interactions in dough are poorly defined but affect final product usage. By monitoring changes in torque as dough is formed, we identified 80-85 °C as a gateway stage determining dough collapse during the mixing/heating process. We propose that this phenomenon is a diagnostic signature linked to integral features of dough complexes formed by some wheat varieties but not others. We found the dough at 80-85 °C was stabilized by increasing the starting bowl temperature (before a standard linear increase in temperature) of the mixing process and demonstrated the significance of specific macromolecular interactions that are formed early in the mixing process. Enzymes including papain, alpha-amylase, glucose oxidase and phytase stabilized dough structure to facilitate transition through the gateway temperatures between 80 and 85 °C. Our results show that if the dough initially formed a protein-starch complex that was too large, instability and collapse of the structure can occur later.

Solubility variation of wheat dough proteins: A practical way to track protein behaviors in dough processing.

To understand wheat dough protein behavior under dual mixing and thermal treatment, solubility of Mixolab-dough proteins were investigated using nine extraction buffers of different dissociation capacities. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) demonstrated that overall changes of protein fractions and dynamic responses of specific proteins during dough processing were well reflected by their solubility variations. After starch pasting, the abundance of 0.5 M NaCl extractable proteins were decreased except for six protein groups including α-amylase inhibitors and superoxide dismutase (SOD). The solubility loss of glutenin proteins at C3 (32 min; 80 â„ƒ) was mainly ascribed to the un-extractable HMW-GSs, LMW-GSs, globulin and triticin, while the extract yield of α-, ß-, γ-gliadins and avenin-like proteins (ALPs) increased after starch pasting. Differential responses of dough proteins to extraction systems provides the basis for further exploring wheat protein dynamics in processing.

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat.

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication-related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.