DMC1 stabilizes crossovers at high and low temperatures during wheat meiosis.

Effective chromosome synapsis and crossover formation during meiosis are essential for fertility, especially in grain crops such as wheat. These processes function most efficiently in wheat at temperatures between 17-23 °C, although the genetic mechanisms for such temperature dependence are unknown. In a previously identified mutant of the hexaploid wheat reference variety 'Chinese Spring' lacking the long arm of chromosome 5D, exposure to low temperatures during meiosis resulted in asynapsis and crossover failure. In a second mutant (ttmei1), containing a 4 Mb deletion in chromosome 5DL, exposure to 13 °C led to similarly high levels of asynapsis and univalence. Moreover, exposure to 30 °C led to a significant, but less extreme effect on crossovers. Previously, we proposed that, of 41 genes deleted in this 4 Mb region, the major meiotic gene TaDMC1-D1 was the most likely candidate for preservation of synapsis and crossovers at low (and possibly high) temperatures. In the current study, using RNA-guided Cas9, we developed a new Chinese Spring CRISPR mutant, containing a 39 bp deletion in the 5D copy of DMC1, representing the first reported CRISPR-Cas9 targeted mutagenesis in Chinese Spring, and the first CRISPR mutant for DMC1 in wheat. In controlled environment experiments, wild-type Chinese Spring, CRISPR dmc1-D1 and backcrossed ttmei1 mutants were exposed to either high or low temperatures during the temperature-sensitive period from premeiotic interphase to early meiosis I. After 6-7 days at 13 °C, crossovers decreased by over 95% in the dmc1-D1 mutants, when compared with wild-type plants grown under the same conditions. After 24 hours at 30 °C, dmc1-D1 mutants exhibited a reduced number of crossovers and increased univalence, although these differences were less marked than at 13 °C. Similar results were obtained for ttmei1 mutants, although their scores were more variable, possibly reflecting higher levels of background mutation. These experiments confirm our previous hypothesis that DMC1-D1 is responsible for preservation of normal crossover formation at low and, to a certain extent, high temperatures. Given that reductions in crossovers have significant effects on grain yield, these results have important implications for wheat breeding, particularly in the face of climate change.

Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology.

The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

ZIP4 is required for normal progression of synapsis and for over 95% of crossovers in wheat meiosis.

Tetraploid (AABB) and hexaploid (AABBDD) wheat have multiple sets of similar chromosomes, with successful meiosis and preservation of fertility relying on synapsis and crossover (CO) formation only taking place between homologous chromosomes. In hexaploid wheat, the major meiotic gene TaZIP4-B2 (Ph1) on chromosome 5B, promotes CO formation between homologous chromosomes, whilst suppressing COs between homeologous (related) chromosomes. In other species, ZIP4 mutations eliminate approximately 85% of COs, consistent with loss of the class I CO pathway. Tetraploid wheat has three ZIP4 copies: TtZIP4-A1 on chromosome 3A, TtZIP4-B1 on 3B and TtZIP4-B2 on 5B. Here, we have developed single, double and triple zip4 TILLING mutants and a CRISPR Ttzip4-B2 mutant, to determine the effect of ZIP4 genes on synapsis and CO formation in the tetraploid wheat cultivar 'Kronos'. We show that disruption of two ZIP4 gene copies in Ttzip4-A1B1 double mutants, results in a 76-78% reduction in COs when compared to wild-type plants. Moreover, when all three copies are disrupted in Ttzip4-A1B1B2 triple mutants, COs are reduced by over 95%, suggesting that the TtZIP4-B2 copy may also affect class II COs. If this is the case, the class I and class II CO pathways may be interlinked in wheat. When ZIP4 duplicated and diverged from chromosome 3B on wheat polyploidization, the new 5B copy, TaZIP4-B2, could have acquired an additional function to stabilize both CO pathways. In tetraploid plants deficient in all three ZIP4 copies, synapsis is delayed and does not complete, consistent with our previous studies in hexaploid wheat, when a similar delay in synapsis was observed in a 59.3 Mb deletion mutant, ph1b, encompassing the TaZIP4-B2 gene on chromosome 5B. These findings confirm the requirement of ZIP4-B2 for efficient synapsis, and suggest that TtZIP4 genes have a stronger effect on synapsis than previously described in Arabidopsis and rice. Thus, ZIP4-B2 in wheat accounts for the two major phenotypes reported for Ph1, promotion of homologous synapsis and suppression of homeologous COs.

The wheat stem rust resistance gene Sr43 encodes an unusual protein kinase.

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62.

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement.

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.

Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

CRISPR-Cas9 Based Genome Editing in Wheat.

The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.

An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat.

Wheat, though a key crop plant with considerable influence on world food security, has nonetheless trailed behind other major cereals in the advancement of gene transformation technology for its improvement. New breeding technologies such as genome editing allow precise DNA manipulation, but their potential is limited by low regeneration efficiencies in tissue culture and the lack of transformable genotypes. We developed, in the hexaploid spring wheat cultivar "Fielder," a robust, reproducible Agrobacterium tumefaciens-mediated transformation system with transformation efficiencies of up to 33%. The system requires immature embryos as starting material and includes a centrifugation pretreatment before the inoculation with Agrobacterium. This high-throughput, highly efficient, and repeatable transformation system has been used effectively to introduce genes of interest for overexpression, RNA interference, and CRISPR-Cas-based genome editing. With slight modifications reported here, the standard protocol can be applied to the hexaploid wheat "Cadenza" and the tetraploid durum wheat "Kronos" with efficiencies of up to 4% and 10%, respectively. The system has also been employed to assess the developmental gene fusion GRF-GIF with outstanding results. In our hands, this technology combined with our transformation system improved transformation efficiency to 77.5% in Fielder. This combination should help alleviate the genotype dependence of wheat transformation, allowing new genome-editing tools to be used directly in more elite wheat varieties. © 2021 The Authors. Basic Protocol 1: Growing of donor plants Basic Protocol 2: Transformation of Agrobacterium with vector by electroporation Basic Protocol 3: Starting material collection, sterilization, and embryo inoculation Basic Protocol 4: Selection, regeneration, rooting, and acclimatization of transformants.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

A roadmap for gene functional characterisation in crops with large genomes: Lessons from polyploid wheat.

Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite their importance, a lack of genomic information and resources has hindered the functional characterisation of genes in major crops. The recent release of high-quality reference sequences for these crops underpins a suite of genetic and genomic resources that support basic research and breeding. For wheat, these include gene model annotations, expression atlases and gene networks that provide information about putative function. Sequenced mutant populations, improved transformation protocols and structured natural populations provide rapid methods to study gene function directly. We highlight a case study exemplifying how to integrate these resources. This review provides a helpful guide for plant scientists, especially those expanding into crop research, to capitalise on the discoveries made in Arabidopsis and other plants. This will accelerate the improvement of crops of vital importance for food and nutrition security.

Correction to: An efficient and reproducible Agrobacterium- mediated transformation method for hexaploid wheat (Triticum aestivum L.).

[This corrects the article DOI: 10.1186/s13007-019-0503-z.].

An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.).

BACKGROUND: Despite wheat being a worldwide staple, it is still considered the most difficult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and effective wheat transformation system is still greatly needed. RESULTS: We have developed and optimised a reproducible Agrobacterium-mediated transformation system for the spring wheat cv 'Fielder' that yields transformation efficiencies of up to 25%. We report on some of the important factors that influence transformation efficiencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for independent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efficiencies might be even higher than those reported. CONCLUSION: We reported here a high-throughput, highly efficient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR-Cas9 based genome editing.

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research.

'Speed breeding' (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants' daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber-based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.

Agrobacterium-mediated transformation systems of Primula vulgaris.

BACKGROUND: Genetic transformation is a valuable tool and an important procedure in plant functional genomics contributing to gene discovery, allowing powerful insights into gene function and genetically controlled characteristics. Primulaceae species provide one of the best-known examples of heteromorphic flower development, a breeding system which has attracted considerable attention, including that of Charles Darwin. Molecular approaches, including plant transformation give the best opportunity to define and understand the role of genes involved in floral heteromorphy in the common primrose, Primula vulgaris, along with other Primula species. RESULTS: Two transformation systems have been developed in P. vulgaris. The first system, Agrobacterium-mediated vacuum infiltration of seedlings, enables the rapid testing of transgenes, transiently in planta. GUS expression was observed in the cotyledons, true leaves, and roots of Primula seedlings. The second system is based on Agrobacterium tumefaciens infection of pedicel explants with an average transformation efficiency of 4.6%. This transformation system, based on regeneration and selection of transformants within in vitro culture, demonstrates stable transgene integration and transmission to the next generation. CONCLUSION: The two transformation systems reported here will aid fundamental research into important traits in Primula. Although, stable integration of transgenes is the ultimate goal for such analyses, transient gene expression via Agrobacterium-mediated DNA transfer, offers a simple and fast method to analyse transgene functions. The second system describes, for the first time, stable Agrobacterium-mediated transformation of Primula vulgaris, which will be key to characterising the genes responsible for the control of floral heteromorphy.

Magnesium Increases Homoeologous Crossover Frequency During Meiosis in ZIP4 (Ph1 Gene) Mutant Wheat-Wild Relative Hybrids.

Wild relatives provide an important source of useful traits in wheat breeding. Wheat and wild relative hybrids have been widely used in breeding programs to introduce such traits into wheat. However, successful introgression is limited by the low frequency of homoeologous crossover (CO) between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70 Mb deletion on chromosome 5B (ph1b) and wild relatives, have been exploited to increase the level of homoeologous CO, allowing chromosome exchange between their chromosomes. In ph1b-rye hybrids, CO number increases from a mean of 1 CO to 7 COs per cell. CO number can be further increased up to a mean of 12 COs per cell in these ph1b hybrids by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic crossover gene ZIP4 on chromosome 5B (TaZIP4-B2) within the 70 Mb deletion, was responsible for the restriction of homoeologous COs in wheat-wild relative hybrids, confirming the ph1b phenotype as a complete Tazip4-B2 deletion mutant (Tazip4-B2 ph1b). In this study, we have identified the particular Hoagland solution constituent responsible for the increased chiasma frequency in Tazip4-B2 ph1b mutant-rye hybrids and extended the analysis to Tazip4-B2 TILLING and CRISPR mutant-Ae variabilis hybrids. Chiasma frequency at meiotic metaphase I, in the absence of each Hoagland solution macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O) was analyzed. A significant decrease in homoeologous CO frequency was observed when the Mg2+ ion was absent. A significant increase of homoeologous CO frequency was observed in all analyzed hybrids, when plants were irrigated with a 1 mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.