Population‐dependent reproducible deviation from natural bread wheat genome in synthetic hexaploid wheat

Sequence elimination is one of the main mechanisms that increases the divergence among homoeologous chromosomes after allopolyploidization to enhance the stability of recently established lineages, but it can cause a loss of some economically important genes. Synthetic hexaploid wheat (SHW) is an important source of genetic variation to the natural hexaploid wheat (NHW) genepool that has low genetic diversity. Here, we investigated the change between SHW and NHW genomes by utilizing a large germplasm set of primary synthetics and synthetic derivatives. Reproducible segment elimination (RSE) was declared if a large chromosomal chunk (>5 cM) produced no aligned reads in more than five SHWs. RSE in five genomic regions was the major source of variation between SHW and NHW. One RSE eliminated almost the complete short arm of chromosome 1B, which contains major genes for flour quality, disease resistance and different enzymes. The occurrence of RSE was highly dependent on the choice of diploid and tetraploid parental lines, their ancestral subpopulation and admixture, e.g. SHWs derived from Triticum dicoccon or from one of two Aegilops tauschii subpopulations were almost free of RSE, while highly admixed parents had higher RSE rates. The rate of RSE in synthetic derivatives was almost double that in primary synthetics. Genome‐wide association analysis detected four loci with minor effects on the occurrence of RSE, indicating that both parental lines and genetic factors were affecting the occurrence of RSE. Therefore, pre‐pre‐breeding strategies should be applied before introducing SHW into pre‐breeding programs to ensure genomic stability and avoid undesirable gene loss.     

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole‐genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co‐cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low‐density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info .     

Identification and molecular characterization of the nicotianamine synthase gene family in bread wheat

Nicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.     

High‐density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high‐density and high‐throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.     

Resistance evaluation of wheat germplasm containing Dn4 or Dny against Russian wheat aphid biotype RWASA3

Host plant resistance can effectively manage Russian wheat aphid (Diuraphis noxia) Kurdjumov (Homoptera: Aphididae) in areas where it is an economically important pest of wheat. However, biotypes of D. noxia virulent on wheat containing resistance gene Dn4 have been reported in both the United States and South Africa. Thirty wheat genotypes, including susceptible Yuma, resistant CItr2401, as well as 25 genotypes containing Dn4 and three genotypes containing Dny were planted under greenhouse conditions in Bethlehem, South Africa, and screened with D. noxia biotype RWASA3. RWASA3 caused susceptible damage symptoms in MTRWA92‐145, Ankor, Halt, Bond CL, 18FAWWON‐SA 262, Prowers99, 18FAWWON‐SA 264, Hatcher, Yumar, Corwa and Thunder CL all reported to contain the Dn4 resistance gene. Genotypes PI586956, Stanton and 18FAWWON‐SA 257, containing the Dny‐resistance gene were susceptible to RWASA3. Similarly, coinciding development of virulence to resistance genes Dn4 and Dny was reported in the United States. However, in this study, 13 Dn4‐containing genotypes showed moderate resistance when screened with RWASA3 alluding to a more complex biotype‐gene‐interaction. These findings could indicate that Dn4 and Dny may be related and possibly share a similar or common resistance factor. Further studies will be aimed at explaining these results investigating the possibility of an allelic cluster or series for Dn4, possibly including Dny.     

CropSNPdb: a database of SNP array data for Brassica crops and hexaploid bread wheat

Advances in sequencing technology have led to a rapid rise in the genomic data available for plants, driving new insights into the evolution, domestication and improvement of crops. Single nucleotide polymorphisms (SNPs) are a major component of crop genomic diversity, and are invaluable as genetic markers in research and breeding programs. High‐throughput SNP arrays, or ‘SNP chips’, can generate reproducible sets of informative SNP markers and have been broadly adopted. Although there are many public repositories for sequencing data, which are routinely uploaded, there are no formal repositories for crop SNP array data. To make SNP array data more easily accessible, we have developed CropSNPdb ( http://snpdb.appliedbioinformatics.com.au ), a database for SNP array data produced by the Illumina Infinium? hexaploid bread wheat (Triticum aestivum) 90K and Brassica 60K arrays. We currently host SNPs from datasets covering 526 Brassica lines and 309 bread wheat lines, and provide search, download and upload utilities for users. CropSNPdb provides a useful repository for these data, which can be applied for a range of genomics and molecular crop‐breeding activities.     

ADP‐glucose pyrophosphorylase genes,associated with kernel weight,underwent selection during wheat domestication and breeding

ADP‐glucose pyrophosphorylase, comprising two small subunits and two large subunits, is considered a key enzyme in the endosperm starch synthesis pathway in wheat (Triticum aestivum L.). Two genes, TaAGP‐S1‐7A and TaAGP‐L‐1B, were investigated in this study. Haplotypes of these genes were associated with thousand kernel weight (TKW) in different populations. Mean TKWs of favoured haplotypes were significantly higher than those of nonfavoured ones. Two molecular markers developed to distinguish these haplotypes could be used in molecular breeding. Frequencies of favoured haplotypes were dramatically increased in cultivars released in China after the 1940s. These favoured haplotypes were also positively selected in six major wheat production regions globally. Selection of AGP‐S1 and AGP‐L‐1B in wheat mainly occurred during and after hexaploidization. Strong additive effects of the favoured haplotypes of with other genes for starch synthesis were also detected in different populations.     

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS‐containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8‐mediated powdery mildew resistance in lines containing Pm8 in a transient single‐cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post‐translational mechanism is involved in suppression of Pm8. Co‐expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co‐immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding.     

The wheat TaGBF1 gene is involved in the blue‐light response and salt tolerance

Light and abiotic stress both strongly modulate plant growth and development. However, the effect of light‐responsive factors on growth and abiotic stress responses in wheat (Triticum aestivum) is unknown. G–box binding factors (GBFs) are blue light‐specific components, but their function in abiotic stress responses has not been studied. Here we identified a wheat GBF1 gene that mediated both the blue light‐ and abiotic stress‐responsive signaling pathways. TaGBF1 was inducible by blue light, salt and exposure to abscisic acid (ABA). TaGBF1 interacted with a G–box light‐responsive element in vitro and promoted a blue‐light response in wheat and Aradidopsis thaliana. Both TaGBF1 over‐expression in wheat and its heterologous expression in A. thaliana heighten sensitivity to salinity and ABA, but its knockdown in wheat conferred resistance to high salinity and ABA. The expression of AtABI5, a key component of the ABA signaling pathway in A. thaliana, and its homolog Wabi5 in wheat was increased by transgenic expression of TaGBF1. The hypersensitivity to salt and ABA caused by TaGBF1 was not observed in the abi5 mutant background, showing that ABI5 is the mediator in TaGBF1‐induced abiotic stress responses. However, the hypersensitivity to salt conferred by TaGBF1 is not dependent on light. This suggests that TaGBF1 is a common component of blue light‐ and abiotic stress‐responsive signaling pathways.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Self-assemblies of Rab- and Arf-family small GTPases on lipid bilayers in membrane tethering

Small GTPases of the Ras superfamily, which include Ras-, Rho-, Rab-, Arf-, and Ran-family isoforms, are generally known to function as a nucleotide-dependent molecular switch in eukaryotic cells. In the GTP-loaded forms, they selectively recruit their cognate interacting proteins or protein complexes, termed “effectors,” to the cytoplasmic face of subcellular membrane compartments, thereby switching on the downstream effector functions, which are vital for fundamental cellular events, such as cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. Nevertheless, in addition to acting as the classic nucleotide-dependent switches for the effectors, recent studies have uncovered that small GTPases themselves can be self-assembled specifically into homo-dimers or higher-order oligomers on membranes, and these assembly processes are likely responsible for their physiological functions. This Review focuses particularly on the self-assembly processes of Rab- and Arf-family isoforms during membrane tethering, the most critical step to ensure the fidelity of membrane trafficking. A summary of the current experimental evidence for self-assemblies of Rab and Arf small GTPases on lipid bilayers in chemically defined reconstitution system is provided     

TaDA1, a conserved negative regulator of kernel size,has an additive effect with TaGW2 in common wheat (Triticum aestivum L.)

Kernel size is an important trait determining cereal yields. In this study, we cloned and characterized TaDA1, a conserved negative regulator of kernel size in wheat (Triticum aestivum). The overexpression of TaDA1 decreased the size and weight of wheat kernels, while its down‐regulation using RNA interference (RNAi) had the opposite effect. Three TaDA1‐A haplotypes were identified in Chinese wheat core collections, and a haplotype association analysis showed that TaDA1‐A‐HapI was significantly correlated with the production of larger kernels and higher kernel weights in modern Chinese cultivars. The haplotype effect resulted from a difference in TaDA1‐A expression levels between genotypes, with TaDA1‐A‐HapI resulting in lower TaDA1‐A expression levels. This favourable haplotype was found having been positively selected during wheat breeding over the last century. Furthermore, we demonstrated that TaDA1‐A physically interacts with TaGW2‐B. The additive effects of TaDA1‐A and TaGW2‐B on kernel weight were confirmed not only by the phenotypic enhancement arising from the simultaneous down‐regulation of TaDA1 and TaGW2 expression, but also by the combinational haplotype effects estimated from multi‐environment field data from 348 wheat cultivars. A comparative proteome analysis of developing transgenic and wild‐type grains indicated that TaDA1 and TaGW2 are involved in partially overlapping but relatively independent protein regulatory networks. Thus, we have identified an important gene controlling kernel size in wheat and determined its interaction with other genes regulating kernel weight, which could have beneficial applications in wheat breeding.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

APETALA 2‐like genes AP2L2 and Q specify lemma identity and axillary floral meristem development in wheat

The spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss‐of‐function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma‐like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2‐like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.     

Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.