Wheat Long Noncoding RNAs from Organelle and Nuclear Genomes Carry Conserved microRNA Precursors Which May Together Comprise Intricate Networks in Insect Responses.

Long noncoding RNAs (lncRNAs) are a diverse class of noncoding RNAs that are typically longer than 200 nucleotides but lack coding potentials. Advances in deep sequencing technologies enabled a better exploration of this type of noncoding transcripts. The poor sequence conservation, however, complicates the identification and annotation of lncRNAs at a large scale. Wheat is among the leading food staples worldwide whose production is threatened by both biotic and abiotic stressors. Here, we identified putative lncRNAs from durum wheat varieties that differ in stem solidness, a major source of defense against wheat stem sawfly, a devastating insect pest. We also analyzed and annotated lncRNAs from two bread wheat varieties, resistant and susceptible to another destructive pest, orange wheat blossom midge, with and without infestation. Several putative lncRNAs contained potential precursor sequences and/or target regions for microRNAs, another type of regulatory noncoding RNAs, which may indicate functional networks. Interestingly, in contrast to lncRNAs themselves, microRNAs with potential precursors within the lncRNA sequences appeared to be highly conserved at the sequence and family levels. We also observed a few putative lncRNAs that have perfect to near-perfect matches to organellar genomes, supporting the recent observations that organellar genomes may contribute to the noncoding transcript pool of the cell.

The complete genome sequence of elite bread wheat cultivar, "Sonmez".

High-yielding crop varieties will become critical in meeting the future food demand in the face of worsening weather extremes and threatening biotic stressors. The bread wheat cultivar Sonmez-2001 is a registered variety that is notable for its performance under low-irrigation conditions, which further improves upon irrigation. Additionally, Sonmez-2001 is resilient against certain biotic stressors, particularly soil-borne pathogens. Here, we provide a reference-guided whole genome sequence of Sonmez-2001, assembled into 21 chromosomes of the A, B and D genomes and totaling 13.3 gigabase-pairs in length. Additionally, a de novo assembly of an additional 1.05 gigabase-pairs was generated that represents either Sonmez-specific sequences or sequences that considerably diverged between Sonmez and Chinese Spring. Within this de novo assembly, we identified 35 gene models, of which 11 were high-confidence, that may contribute to the favorable traits of this high-performing variety. We identified up to 24 million sequence variants, of which up to 2.4% reside in coding sequences, that can be used to develop molecular markers that should be of immediate use to the cereal community.

Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes.

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Multiple Variant Calling Pipelines in Wheat Whole Exome Sequencing.

The highly challenging hexaploid wheat (Triticum aestivum) genome is becoming ever more accessible due to the continued development of multiple reference genomes, a factor which aids in the plight to better understand variation in important traits. Although the process of variant calling is relatively straightforward, selection of the best combination of the computational tools for read alignment and variant calling stages of the analysis and efficient filtering of the false variant calls are not always easy tasks. Previous studies have analyzed the impact of methods on the quality metrics in diploid organisms. Given that variant identification in wheat largely relies on accurate mining of exome data, there is a critical need to better understand how different methods affect the analysis of whole exome sequencing (WES) data in polyploid species. This study aims to address this by performing whole exome sequencing of 48 wheat cultivars and assessing the performance of various variant calling pipelines at their suggested settings. The results show that all the pipelines require filtering to eliminate false-positive calls. The high consensus among the reference SNPs called by the best-performing pipelines suggests that filtering provides accurate and reproducible results. This study also provides detailed comparisons for high sensitivity and precision at individual and population levels for the raw and filtered SNP calls.

OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security.

The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.

Pan-Genome miRNomics in Brachypodium.

Pan-genomes are efficient tools for the identification of conserved and varying genomic sequences within lineages of a species. Investigating genetic variations might lead to the discovery of genes present in a subset of lineages, which might contribute into beneficial agronomic traits such as stress resistance or yield. The content of varying genomic regions in the pan-genome could include protein-coding genes as well as microRNA(miRNAs), small non-coding RNAs playing key roles in the regulation of gene expression. In this study, we performed in silico miRNA identification from the genomic sequences of 54 lineages of Brachypodium distachyon, aiming to explore varying miRNA contents and their functional interactions. A total of 115 miRNA families were identified in 54 lineages, 56 of which were found to be present in all lineages. The miRNA families were classified based on their conservation among lineages and potential mRNA targets were identified. Obtaining information about regulatory mechanisms stemming from these miRNAs offers strong potential to provide a better insight into the complex traits that were potentially present in some lineages. Future work could lead us to introduce these traits to different lineages or other economically important plant species in order to promote their survival in different environmental conditions.

We're in this Together: Sensation of the Host Cell Environment by Endosymbiotic Bacteria.

Bacteria inhabit diverse environments, including the inside of eukaryotic cells. While a bacterial invader may initially act as a parasite or pathogen, a subsequent mutualistic relationship can emerge in which the endosymbiotic bacteria and their host share metabolites. While the environment of the host cell provides improved stability when compared to an extracellular environment, the endosymbiont population must still cope with changing conditions, including variable nutrient concentrations, the host cell cycle, host developmental programs, and host genetic variation. Furthermore, the eukaryotic host can deploy mechanisms actively preventing a bacterial return to a pathogenic state. Many endosymbionts are likely to use two-component systems (TCSs) to sense their surroundings, and expanded genomic studies of endosymbionts should reveal how TCSs may promote bacterial integration with a host cell. We suggest that studying TCS maintenance or loss may be informative about the evolutionary pathway taken toward endosymbiosis, or even toward endosymbiont-to-organelle conversion.

An Unusual Amino Acid Substitution Within Hummingbird Cytochrome c Oxidase Alters a Key Proton-Conducting Channel.

Hummingbirds in flight exhibit the highest mass-specific metabolic rate of all vertebrates. The bioenergetic requirements associated with sustained hovering flight raise the possibility of unique amino acid substitutions that would enhance aerobic metabolism. Here, we have identified a non-conservative substitution within the mitochondria-encoded cytochrome c oxidase subunit I (COI) that is fixed within hummingbirds, but not among other vertebrates. This unusual change is also rare among metazoans, but can be identified in several clades with diverse life histories. We performed atomistic molecular dynamics simulations using bovine and hummingbird COI models, thereby bypassing experimental limitations imposed by the inability to modify mtDNA in a site-specific manner. Intriguingly, our findings suggest that COI amino acid position 153 (bovine numbering convention) provides control over the hydration and activity of a key proton channel in COX. We discuss potential phenotypic outcomes linked to this alteration encoded by hummingbird mitochondrial genomes.

Chromosome-based survey sequencing reveals the genome organization of wild wheat progenitor Triticum dicoccoides.

Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome-based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein-coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small-scale evolutionary rearrangements, in addition to providing evidence for the 4AL-5AL-7BS translocation in wild emmer wheat. Chromosome-based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

A large-scale chromosome-specific SNP discovery guideline.

Single-nucleotide polymorphisms (SNPs) are the most prevalent type of variation in genomes that are increasingly being used as molecular markers in diversity analyses, mapping and cloning of genes, and germplasm characterization. However, only a few studies reported large-scale SNP discovery in Aegilops tauschii, restricting their potential use as markers for the low-polymorphic D genome. Here, we report 68,592 SNPs found on the gene-related sequences of the 5D chromosome of Ae. tauschii genotype MvGB589 using genomic and transcriptomic sequences from seven Ae. tauschii accessions, including AL8/78, the only genotype for which a draft genome sequence is available at present. We also suggest a workflow to compare SNP positions in homologous regions on the 5D chromosome of Triticum aestivum, bread wheat, to mark single nucleotide variations between these closely related species. Overall, the identified SNPs define a density of 4.49 SNPs per kilobyte, among the highest reported for the genic regions of Ae. tauschii so far. To our knowledge, this study also presents the first chromosome-specific SNP catalog in Ae. tauschii that should facilitate the association of these SNPs with morphological traits on chromosome 5D to be ultimately targeted for wheat improvement.

Root precursors of microRNAs in wild emmer and modern wheats show major differences in response to drought stress.

MicroRNAs, small regulatory molecules with significant impacts on the transcriptional network of all living organisms, have been the focus of several studies conducted mostly on modern wheat cultivars. In this study, we investigated miRNA repertoires of modern durum wheat and its wild relatives, with differing degrees of drought tolerance, to identify miRNA candidates and their targets involved in drought stress response. Root transcriptomes of Triticum turgidum ssp. durum variety Kiziltan and two Triticum turgidum ssp. dicoccoides genotypes TR39477 and TTD-22 under control and drought conditions were assembled from individual RNA-Seq reads and used for in silico identification of miRNAs. A total of 66 miRNAs were identified from all species, across all conditions, of which 46 and 38 of the miRNAs identified from modern durum wheat and wild genotypes, respectively, had not been previously reported. Genotype- and/or stress-specific miRNAs provide insights into our understanding of the complex drought response. Particularly, miR1435, miR5024, and miR7714, identified only from drought-stress roots of drought-tolerant genotype TR39477, can be candidates for future studies to explore and exploit the drought response to develop tolerant varieties.

Molecular organization and comparative analysis of chromosome 5B of the wild wheat ancestor Triticum dicoccoides.

Wild emmer wheat, Triticum turgidum ssp. dicoccoides is the wild relative of Triticum turgidum, the progenitor of durum and bread wheat, and maintains a rich allelic diversity among its wild populations. The lack of adequate genetic and genomic resources, however, restricts its exploitation in wheat improvement. Here, we report next-generation sequencing of the flow-sorted chromosome 5B of T. dicoccoides to shed light into its genome structure, function and organization by exploring the repetitive elements, protein-encoding genes and putative microRNA and tRNA coding sequences. Comparative analyses with its counterparts in modern and wild wheats suggest clues into the B-genome evolution. Syntenic relationships of chromosome 5B with the model grasses can facilitate further efforts for fine-mapping of traits of interest. Mapping of 5B sequences onto the root transcriptomes of two additional T. dicoccoides genotypes, with contrasting drought tolerances, revealed several thousands of single nucleotide polymorphisms, of which 584 shared polymorphisms on 228 transcripts were specific to the drought-tolerant genotype. To our knowledge, this study presents the largest genomics resource currently available for T. dicoccoides, which, we believe, will encourage the exploitation of its genetic and genomic potential for wheat improvement to meet the increasing demand to feed the world.

The physical map of wheat chromosome 5DS revealed gene duplications and small rearrangements.

BACKGROUND: The substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution. RESULTS: The physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins. CONCLUSIONS: Here, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.

Stress responsive miRNAs and isomiRs in cereals.

Abiotic and biotic stress conditions are vital determinants in the production of cereals, the major caloric source in human nutrition. Small RNAs, miRNAs and isomiRs are central to post-transcriptional regulation of gene expression in a variety of cellular processes including development and stress responses. Several miRNAs have been identified using new technologies and have roles in stress responses in plants, including cereals. The overall knowledge about the cereal miRNA repertoire, as well as an understanding of complex miRNA mediated mechanisms of target regulation in response to stress conditions, is far from complete. Ongoing efforts that add to our understanding of complex miRNA machinery have implications in plant response to stress conditions. Additionally, sequence variants of miRNAs (isomiRNAs or isomiRs), regulation of their expression through dissection of upstream regulatory elements, the role of Processing-bodies (P-bodies) in miRNA exerted gene regulation and yet unveiled organellar plant miRNAs are newly emerging topics, which will contribute to the elucidation of the miRNA machinery and its role in cereal tolerance against abiotic and biotic stresses.

Next-generation sequencing of flow-sorted wheat chromosome 5D reveals lineage-specific translocations and widespread gene duplications.

BACKGROUND: The ~17 Gb hexaploid bread wheat genome is a high priority and a major technical challenge for genomic studies. In particular, the D sub-genome is relatively lacking in genetic diversity, making it both difficult to map genetically, and a target for introgression of agriculturally useful traits. Elucidating its sequence and structure will therefore facilitate wheat breeding and crop improvement. RESULTS: We generated shotgun sequences from each arm of flow-sorted Triticum aestivum chromosome 5D using 454 FLX Titanium technology, giving 1.34× and 1.61× coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. By a combination of sequence similarity and assembly-based methods, ~74% of the sequence reads were classified as repetitive elements, and coding sequence models of 1314 (5DS) and 2975 (5DL) genes were generated. The order of conserved genes in syntenic regions of previously sequenced grass genomes were integrated with physical and genetic map positions of 518 wheat markers to establish a virtual gene order for chromosome 5D. CONCLUSIONS: The virtual gene order revealed a large-scale chromosomal rearrangement in the peri-centromeric region of 5DL, and a concentration of non-syntenic genes in the telomeric region of 5DS. Although our data support the large-scale conservation of Triticeae chromosome structure, they also suggest that some regions are evolving rapidly through frequent gene duplications and translocations. SEQUENCE ACCESSIONS: EBI European Nucleotide Archive, Study no. ERP002330.

Genomics approaches for crop improvement against abiotic stress.

As sessile organisms, plants are inevitably exposed to one or a combination of stress factors every now and then throughout their growth and development. Stress responses vary considerably even in the same plant species; stress-susceptible genotypes are at one extreme, and stress-tolerant ones are at the other. Elucidation of the stress responses of crop plants is of extreme relevance, considering the central role of crops in food and biofuel production. Crop improvement has been a traditional issue to increase yields and enhance stress tolerance; however, crop improvement against abiotic stresses has been particularly compelling, given the complex nature of these stresses. As traditional strategies for crop improvement approach their limits, the era of genomics research has arisen with new and promising perspectives in breeding improved varieties against abiotic stresses.

Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A.

BACKGROUND: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC-based or 583 LTC-based contigs. CONCLUSIONS/SIGNIFICANCE: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A.

Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI-MS/MS.

To elucidate differentially expressed proteins and to further understand post-translational modifications of transcripts, full leaf proteome profiles of two wild emmer (Triticum turgidum ssp. dicoccoides TR39477 and TTD22) and one modern durum wheat (Triticum turgidum ssp. durum cv. Kiziltan) genotypes were compared upon 9-day drought stress using two-dimensional gel electrophoresis and nano-scale liquid chromatographic electrospray ionization tandem mass spectrometry methods. The three genotypes compared exhibit distinctive physiological responses to drought as previously shown by our group. Results demonstrated that many of the proteins were common in both wild emmer and modern wheat proteomes; of which, 75 were detected as differentially expressed proteins. Several proteins identified in all proteomes exhibited drought regulated patterns of expression. A number of proteins were observed with higher expression levels in response to drought in wild genotypes compared to their modern relative. Eleven protein spots with low peptide matches were identified as candidate unique drought responsive proteins. Of the differentially expressed proteins, four were selected and further analyzed by quantitative real-time PCR at the transcriptome level to compare with the proteomic data. The present study provides protein level differences in response to drought in modern and wild genotypes of wheat that may account for the differences of the overall responses of these genotypes to drought. Such comparative proteomics analyses may aid in the better understanding of complex drought response and may suggest candidate genes for molecular breeding studies to improve tolerance against drought stress and, thus, to enhance yields.

Subgenomic analysis of microRNAs in polyploid wheat.

In this study, a survey of miRNAs using the next-generation sequencing data was performed at subgenomic level. After analyzing shotgun sequences from chromosome 4A of bread wheat (Triticum aestivum L.), a total of 68 different miRNAs were predicted in silico, of which 37 were identified in wheat for the first time. The long arm of the chromosome was found to harbor a higher variety (51) and representation (3,928) of miRNAs compared with the short arm (49; 2,226). Out of the 68 miRNAs, 32 were detected to be common to both arms, revealing the presence of separate miRNA clusters in the two chromosome arms. The differences in degree of representation of the different miRNAs were found to be highly variable, ranging 592-fold, which may have an effect on target regulation. Targets were retrieved for 62 (out of 68) of wheat-specific, newly identified miRNAs indicated that fundamental aspects of plant morphology such as height and flowering were predicted to be affected. In silico expression blast analysis indicated 24 (out of 68) were found to give hits to expressed sequences. This is the first report of species- and chromosome-specific miRNAs.

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(®) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.