Complete mitochondrial genome of Agropyron cristatum reveals gene transfer and RNA editing events.

BACKGROUND: As an important forage in arid and semi-arid regions, Agropyron cristatum provides livestock with exceptionally high nutritional value. Additionally, A. cristatum exhibits outstanding genetic characteristics to endure drought and disease. Therefore, rich genetic diversity serves as a cornerstone for the improvement of major food crops. The purposes of this study were to systematically describe mitogenome of A.cristatum and preliminarily analyze its internal variations. RESULT: The A. cristatum mitogenome was a single-ring molecular structure of 381,065 bp that comprised 52 genes, including 35 protein-coding, 3 rRNA and 14 tRNA genes. Among these, two pseudoprotein-coding genes and multiple copies of tRNA genes were observed. A total of 320 repetitive sequences was found to cover more than 10% of the mitogenome (105 simple sequences, 185 dispersed and 30 tandem repeats), which led to a large number of fragment rearrangements in the mitogenome of A. cristatum. Leucine was the most frequent amino acid (n = 1087,10.8%) in the protein-coding genes of A. cristatum mitogenome, and the highest usage codon was ATG (initiation codon). The number of A/T changes at the third base of the codon was much higher than that of G/C. Among 23 PCGs, the range of Pi values is from 0.0021 to 0.0539, with an average of 0.013. Additionally, 81 RNA editing sites were predicted, which were considerably fewer than those reported in other plant mitogenomes. Most of the RNA editing site base positions were concentrated at the first and second codon bases, which were C to T transitions. Moreover, we identified 95 sequence fragments (total length of 34, 343 bp) that were transferred from the chloroplast to mitochondria genes, introns, and intergenic regions. The stability of the tRNA genes was maintained during this process. Selection pressure analysis of 23 protein-coding genes shared by 15 Poaceae plants, showed that most genes were subjected to purifying selection during evolution, whereas rps4, cob, mttB, and ccmB underwent positive selection in different plants. Finally, a phylogenetic tree was constructed based on 22 plant mitogenomes, which showed that Agropyron plants have a high degree of independent heritability in Triticeae. CONCLUSION: The findings of this study provide new data for a better understanding of A. cristatum genes, and demonstrate that mitogenomes are suitable for the study of plant classifications, such as those of Agropyron. Moreover, it provides a reference for further exploration of the phylogenetic relationships within Agropyron species, and establishes a theoretical basis for the subsequent development and utilization of A. cristatum plant germplasm resources.

Disentangling sources of gene tree discordance for Hordeum species via target-enriched sequencing assays.

Hordeum is an economically and evolutionarily important genus within the Triticeae tribe of the family Poaceae, and contains 33 widely distributed and diverse species which cytologically represent four subgenomes (H, Xa, Xu and I). These wild species (except Hordeum spontaneum, which is the primary gene pool of barley) are secondary or tertiary gene-pool germplasms for barley and wheat improvement, and uncovering their complicated evolutionary relationships would benefit for future breeding programs. Here, we developed a complexity-reduced pipeline via capturing genome-wide distributed fragments via two novel target-enriched assays (HorCap v1.0 and BarPlex v1.0) in conjugation with high-throughput sequencing of the enrichments. Both assays were tested for genotyping 40 species from three genera (Hordeum, Triticum, and Aegilops) containing 82 samples 67 accessions. Either of both assays worked efficiently in genotyping, while integration of both assays can significantly improve the robustness and resolution of the Hordeum phylogenetic trees. Interestingly, the incomplete lineage sorting (ILS) was inferred for the first time as the major factor causing phylogenetic discordance among the four subgenomes, whereas in New World species (carrying I genome) post-speciation introgression events were revealed. Through revising the evolutionary relationships of the Hordeum species based on an ancestral state reconstruction for the diploids and parental donor inference for the polyploids, our results raised new queries about the Hordeum phylogeny. Moreover, both newly-developed assays are applicable in genotyping and phylogenetic analysis of Hordeum and other Triticeae wild species.

Alien Chromatin from Hordeeae Grasses Enhances the Compatibility of Epichloë Endophyte Symbiosis with the Hexaploid Wheat Triticum aestivum.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat-alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat-Epichloë associations to be created.

A chromosome level genome assembly of Pseudoroegneria Libanotica reveals a key Kcs gene involves in the cuticular wax elongation for drought resistance.

BACKGROUND: The genus Pseudoroegneria (Nevski) Löve (Triticeae, Poaceae), whose genome symbol was designed as "St", accounts for more than 60% of perennial Triticeae species. The diploid species Psudoroegneria libanotica (2n = 14) contains the most ancient St genome, exhibited strong drought resistance, and was morphologically covered by cuticular wax on the aerial part. Therefore, the St-genome sequencing data could provide fundamental information for studies of genome evolution and reveal its mechanisms of cuticular wax and drought resistance. RESULTS: In this study, we reported the chromosome-level genome assembly for the St genome of Pse. libanotica, with a total size of 2.99 Gb. 46,369 protein-coding genes annotated and 71.62% was repeat sequences. Comparative analyses revealed that the genus Pseudoroegneria diverged during the middle and late Miocene. During this period, unique genes, gene family expansion, and contraction in Pse. libanotica were enriched in biotic and abiotic stresses, such as fatty acid biosynthesis which may greatly contribute to its drought adaption. Furthermore, we investigated genes associated with the cuticular wax formation and water deficit and found a new Kcs gene evm.TU.CTG175.54. It plays a critical role in the very long chain fatty acid (VLCFA) elongation from C18 to C26 in Pse. libanotica. The function needs more evidence to be verified. CONCLUSIONS: We sequenced and assembled the St genome in Triticeae and discovered a new KCS gene that plays a role in wax extension to cope with drought. Our study lays a foundation for the genome diversification of Triticeae species and deciphers cuticular wax formation genes involved in drought resistance.

A guide to barley mutants.

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments. RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections. CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.

Kinase fusion proteins: intracellular R-proteins in plant immunity.

Resistance (R) genes in the Triticeae tribe include not only genes encoding the canonical intracellular nucleotide-binding leucine-rich-repeat proteins (NLRs) but also genes encoding kinase fusion proteins (KFPs). Exploring these unconventional KFPs may expand the scope of effector-triggered immunity (ETI) and will have significant implications for crop improvement.

Identification of Whirly transcription factors in Triticeae species and functional analysis of TaWHY1-7D in response to osmotic stress.

Osmotic stress poses a threat to the production and quality of crops. Whirly transcription factors have been investigated to enhance stress tolerance. In this study, a total of 18 Whirly genes were identified from six Triticeae species, which were classified into Whirly1 and Whirly2. The exon-intron structure, conserved motif, chromosomal location, collinearity, and regulatory network of Whirly genes were also analyzed. Real-time PCR results indicated that TaWHY1 genes exhibited higher expression levels in leaf sheaths and leaves during the seedling stage, while TaWHY2 genes were predominantly expressed in roots. Under PEG stress, the expression levels of TaWHY1-7A, TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D were increased, TaWHY1-7D was reduced, and TaWHY1-4A had no significant change. All TaWHY genes were significantly up-regulated in response to NaCl stress treatment. In addition, TaWHY1-7A and TaWHY1-7D mainly enhanced the tolerance to oxidative stress in yeast cells. TaWHY2s mainly improved NaCl stress tolerance and were sensitive to oxidative stress in yeast cells. All TaWHYs slightly improved the yeast tolerance to d-sorbitol stress. The heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis. In conclusion, these results provide the foundation for further functional study of Whirly genes aimed at improving osmotic stress tolerance in wheat.

Volatile metabolomics reveals the characteristics of the unique flavor substances in oats.

Oats is a cereal well known for its high nutritional value and unique flavor. This study investigated the metabolomics data from oats, wheat, and barley using broadly targeted GC-MS metabonomic techniques. A total of 437 volatile organic compounds (VOCs) were identified, of which 414 were shared metabolites, with three metabolites unique to oats. Three hundred and seven differentially accumulated metabolites (DAMs) were screened from all the comparison groups, of which 27 metabolites were shared by oats and barley, and 121 shared by oats and wheat. Terpenoids and esters were the key metabolites determining the differences in flavor. A KEGG analysis indicated that the alpha-linolenic acid and phenylalanine pathways were the most significant metabolic pathways. The 42 DAMs found may be the main substances leading to the flavor differences between the different varieties. Overall, this study reveals the main reasons for the unique flavor of oats through metabolomic evidence.

The Triticeae CBF Gene Cluster-To Frost Resistance and Beyond.

The pivotal role of CBF/DREB1 transcriptional factors in Triticeae crops involved in the abiotic stress response has been highlighted. The CBFs represent an important hub in the ICE-CBF-COR pathway, which is one of the most relevant mechanisms capable of activating the adaptive response to cold and drought in wheat, barley, and rye. Understanding the intricate mechanisms and regulation of the cluster of CBF genes harbored by the homoeologous chromosome group 5 entails significant potential for the genetic improvement of small grain cereals. Triticeae crops seem to share common mechanisms characterized, however, by some peculiar aspects of the response to stress, highlighting a combined landscape of single-nucleotide variants and copy number variation involving CBF members of subgroup IV. Moreover, while chromosome 5 ploidy appears to confer species-specific levels of resistance, an important involvement of the ICE factor might explain the greater tolerance of rye. By unraveling the genetic basis of abiotic stress tolerance, researchers can develop resilient varieties better equipped to withstand extreme environmental conditions. Hence, advancing our knowledge of CBFs and their interactions represents a promising avenue for improving crop resilience and food security.

Genome Analysis of Thinopyrum intermedium and Its Potential Progenitor Species Using Oligo-FISH.

The genome composition of intermediate wheatgrass (IWG) is complex and continues to be a subject of investigation. In this study, molecular cytogenetics were used to investigate the karyotype composition of Th. intermedium and its relative diploid species. St2-80 developed from Pseudowroegneria strigose and pDb12H developed from Dasypyrum breviaristatum were used as probes in fluorescence in situ hybridization (FISH) to classify the chromosomes of Th. intermedium into three groups, expressed as JvsJvsJrJrStSt. A combined multiplex oligonucleotide probe, including pSc119.2-1, (GAA)10, AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6, was used to establish the FISH karyotype of ten accessions of Th. intermedium. Variability among and within the studied accessions of intermediate wheatgrass was observed in their FISH patterns. Results of this study led to the conclusions that Jvs had largely been contributed from Da. breviaristatum, but not the present-day Da. villosum; IWG had only one J genome, Jr, which was related to either Th. elongatum or Th. bessarabicum; and St was contributed from the genus Pseudoroegneria by hybridization with Th. junceiforme or Th. sartorii.

Corrigendum: Triticeae crop genome biology: an endless frontier.

[This corrects the article DOI: 10.3389/fpls.2023.1222681.].

Integrative gene duplication and genome-wide analysis as an approach to facilitate wheat reverse genetics: An example in the TaCIPK family.

INTRODUCTION: Reverse genetic studies conducted in the plant with a complex or polyploidy genome enriched with large gene families (like wheat) often meet challenges in identifying the key candidate genes related to important traits and prioritizing the genes for functional experiments. OBJECTIVE: To overcome the above-mentioned challenges of reverse genetics, this work aims to establish an efficient multi-species strategy for genome-wide gene identification and prioritization of the key candidate genes. METHODS: We established the integrative gene duplication and genome-wide analysis (iGG analysis) as a strategy for pinpointing key candidate genes deserving functional research. The iGG captures the evolution, and the expansion/contraction of large gene families across phylogeny-related species and integrates spatial-temporal expression information for gene function inference. Transgenic approaches were also employed to functional validation. RESULTS: As a proof-of-concept for the iGG analysis, we took the wheat calcineurin B-like protein-interacting protein kinases (CIPKs) family as an example. We identified CIPKs from seven monocot species, established the orthologous relationship of CIPKs between rice and wheat, and characterized Triticeae-specific CIPK duplicates (e.g., CIPK4 and CIPK17). Integrated with our analysis of CBLs and CBL-CIPK interaction, we revealed that divergent expressions of TaCBLs and TaCIPKs could play an important role in keeping the stoichiometric balance of CBL-CIPK. Furthermore, we validated the function of TaCIPK17-A2 in the regulation of drought tolerance by using transgenic approaches. Overexpression of TaCIPK17 enhanced antioxidant capacity and improved drought tolerance in wheat. CONCLUSION: The iGG analysis leverages evolutionary and comparative genomics of crops with large genomes to rapidly highlight the duplicated genes potentially associated with speciation, domestication and/or particular traits that deserve reverse-genetic functional studies. Through the identification of Triticeae-specific TaCIPK17 duplicates and functional validation, we demonstrated the effectiveness of the iGG analysis and provided a new target gene for improving drought tolerance in wheat.

Triticeae crop genome biology: an endless frontier.

Triticeae, the wheatgrass tribe, includes several major cereal crops and their wild relatives. Major crops within the Triticeae are wheat, barley, rye, and oat, which are important for human consumption, animal feed, and rangeland protection. Species within this tribe are known for their large genomes and complex genetic histories. Powered by recent advances in sequencing technology, researchers worldwide have made progress in elucidating the genomes of Triticeae crops. In addition to assemblies of high-quality reference genomes, pan-genome studies have just started to capture the genomic diversities of these species, shedding light on our understanding of the genetic basis of domestication and environmental adaptation of Triticeae crops. In this review, we focus on recent signs of progress in genome sequencing, pan-genome analyses, and resequencing analysis of Triticeae crops. We also propose future research avenues in Triticeae crop genomes, including identifying genome structure variations, the association of genomic regions with desired traits, mining functions of the non-coding area, introgression of high-quality genes from wild Triticeae resources, genome editing, and integration of genomic resources.

HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley.

Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1, and propose causative gene sequences: a C2H2 zinc-finger gene HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels, resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data, we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement.

Genome-wide characterization of the VQ genes in Triticeae and their functionalization driven by polyploidization and gene duplication events in wheat.

Valine-glutamine motif-containing (VQ) proteins are transcriptional cofactors widely involved in plant growth, development, and response to various stresses. Although the VQ family has been genome-wide identified in some species, but the knowledge regarding duplication-driven functionalization of VQ genes among evolutionarily related species is still lacking. Here, 952 VQ genes have been identified from 16 species, emphasizing seven Triticeae species including the bread wheat. Comprehensive phylogenetic and syntenic analyses allow us to establish the orthologous relationship of VQ genes from rice (Oryza sativa) to bread wheat (Triticum aestivum). The evolutionary analysis revealed that whole-genome duplication (WGD) drives the expansion of OsVQs, while TaVQs expansion is associated with a recent burst of gene duplication (RBGD). We also analyzed the motif composition and molecular properties of TaVQ proteins, enriched biological functions, and expression patterns of TaVQs. We demonstrate that WGD-derived TaVQs have become divergent in both protein motif composition and expression pattern, while RBGD-derived TaVQs tend to adopt specific expression patterns, suggesting their functionalization in certain biological processes or in response to specific stresses. Furthermore, some RBGD-derived TaVQs are found to be associated with salt tolerance. Several of the identified salt-related TaVQ proteins were located in the cytoplasm and nucleus and their salt-responsive expression patterns were validated by qPCR analysis. Yeast-based functional experiments confirmed that TaVQ27 may be a new regulator to salt response and regulation. Overall, this study lays the foundation for further functional validation of VQ family members within the Triticeae species.

Form follows function in Triticeae inflorescences.

Grass inflorescences produce grains, which are directly connected to our food. In grass crops, yields are mainly affected by grain number and weight; thus, understanding inflorescence shape is crucially important for cereal crop breeding. In the last two decades, several key genes controlling inflorescence shape have been elucidated, thanks to the availability of rich genetic resources and powerful genomics tools. In this review, we focus on the inflorescence architecture of Triticeae species, including the major cereal crops wheat and barley. We summarize recent advances in our understanding of the genetic basis of spike branching, and spikelet and floret development in the Triticeae. Considering our changing climate and its impacts on cereal crop yields, we also discuss the future orientation of research.

A survey of lineage-specific genes in Triticeae reveals de novo gene evolution from genomic raw material.

Diploid plant genomes typically contain ~35,000 genes, almost all belonging to highly conserved gene families. Only a small fraction are lineage-specific, which are found in only one or few closely related species. Little is known about how genes arise de novo in plant genomes and how often this occurs; however, they are believed to be important for plants diversification and adaptation. We developed a pipeline to identify lineage-specific genes in Triticeae, using newly available genome assemblies of wheat, barley, and rye. Applying a set of stringent criteria, we identified 5942 candidate Triticeae-specific genes (TSGs), of which 2337 were validated as protein-coding genes in wheat. Differential gene expression analyses revealed that stress-induced wheat TSGs are strongly enriched in putative secreted proteins. Some were previously described to be involved in Triticeae non-host resistance and cold response. Additionally, we show that 1079 TSGs have sequence homology to transposable elements (TEs), ~68% of them deriving from regulatory non-coding regions of Gypsy retrotransposons. Most importantly, we demonstrate that these TSGs are enriched in transmembrane domains and are among the most highly expressed wheat genes overall. To summarize, we conclude that de novo gene formation is relatively rare and that Triticeae probably possess ~779 lineage-specific genes per haploid genome. TSGs, which respond to pathogen and environmental stresses, may be interesting candidates for future targeted resistance breeding in Triticeae. Finally, we propose that non-coding regions of TEs might provide important genetic raw material for the functional innovation of TM domains and the evolution of novel secreted proteins.

Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.).

The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.