miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana.

Many plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro-RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared with Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.

Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins.

Nuclear male-sterile mutants with non-conditional, recessive and strictly monogenic inheritance are useful for both hybrid and conventional breeding systems, and have long been a research focus for many crops. In allohexaploid wheat, however, genic redundancy results in rarity of such mutants, with the ethyl methanesulfonate-induced mutant ms5 among the few reported to date. Here, we identify TaMs5 as a glycosylphosphatidylinositol-anchored lipid transfer protein required for normal pollen exine development, and by transgenic complementation demonstrate that TaMs5-A restores fertility to ms5. We show ms5 locates to a centromere-proximal interval and has a sterility inheritance pattern modulated by TaMs5-D but not TaMs5-B. We describe two allelic forms of TaMs5-D, one of which is non-functional and confers mono-factorial inheritance of sterility. The second form is functional but shows incomplete dominance. Consistent with reduced functionality, transcript abundance in developing anthers was found to be lower for TaMs5-D than TaMs5-A. At the 3B homoeolocus, we found only non-functional alleles among 178 diverse hexaploid and tetraploid wheats that include landraces and Triticum dicoccoides. Apparent ubiquity of non-functional TaMs5-B alleles suggests loss-of-function arose early in wheat evolution and, therefore, at most knockout of two homoeoloci is required for sterility. This work provides genetic information, resources and tools required for successful implementation of ms5 sterility in breeding systems for bread and durum wheats.

CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of male-sterile hexaploid wheat lines for use in hybrid seed production.

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male-sterile lines were potentially non-transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.

Effects of Rht-B1 and Ppd-D1 loci on pollinator traits in wheat.

KEY MESSAGE: Elite wheat pollinators are critical for successful hybrid breeding. We identified Rht-B1 and Ppd-D1 loci affecting multiple pollinator traits and therefore represent major targets for improving hybrid seed production. Hybrid breeding has a great potential to significantly boost wheat yields. Ideal male pollinators would be taller in stature, contain many spikelets well-spaced along the spike and exhibit high extrusion of large anthers. Most importantly, flowering time would match with that of the female parent. Available genetic resources for developing an elite wheat pollinator are limited, and the genetic basis for many of these traits is largely unknown. Here, we report on the genetic analysis of pollinator traits using biparental mapping populations. We identified two anther extrusion QTLs of medium effect, one on chromosome 1BL and the other on 4BS coinciding with the semi-dwarfing Rht-B1 locus. The effect of Rht-B1 alleles on anther extrusion is genotype dependent, while tall plant Rht-B1a allele is consistently associated with large anthers. Multiple QTLs were identified at the Ppd-D1 locus for anther length, spikelet number and spike length, with the photoperiod-sensitive Ppd-D1b allele associated with favourable pollinator traits in the populations studied. We also demonstrated that homeoloci, Rht-D1 and Ppd-B1, influence anther length among other traits. These results suggest that combinations of Rht-B1 and Ppd-D1 alleles control multiple pollinator traits and should be major targets of hybrid wheat breeding programs.

Wheat TaMs1 is a glycosylphosphatidylinositol-anchored lipid transfer protein necessary for pollen development.

BACKGROUND: In flowering plants, lipid biosynthesis and transport within anthers is essential for male reproductive success. TaMs1, a dominant wheat fertility gene located on chromosome 4BS, has been previously fine mapped and identified to encode a glycosylphosphatidylinositol (GPI)-anchored non-specific lipid transfer protein (nsLTP). Although this gene is critical for pollen exine development, details of its function remains poorly understood. RESULTS: In this study, we report that TaMs1 is only expressed from the B sub-genome, with highest transcript abundance detected in anthers containing microspores undergoing pre-meiosis through to meiosis. ß-glucuronidase transcriptional fusions further revealed that TaMs1 is expressed throughout all anther cell-types. TaMs1 was identified to be expressed at an earlier stage of anther development relative to genes reported to be necessary for sporopollenin precursor biosynthesis. In anthers missing a functional TaMs1 (ms1c deletion mutant), these same genes were not observed to be mis-regulated, indicating an independent function for TaMs1 in pollen development. Exogenous hormone treatments on GUS reporter lines suggest that TaMs1 expression is increased by both indole-3-acetic acid (IAA) and abscisic acid (ABA). Translational fusion constructs showed that TaMs1 is targeted to the plasma membrane. CONCLUSIONS: In summary, TaMs1 is a wheat fertility gene, expressed early in anther development and encodes a GPI-LTP targeted to the plasma membrane. The work presented provides a new insight into the process of wheat pollen development.

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection.

Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Genetic mapping and marker development for resistance of wheat against the root lesion nematode Pratylenchus neglectus.

BACKGROUND: The Rlnn1 locus, which resides on chromosome 7A of bread wheat (Triticum aestivum L.) confers moderate resistance against the root lesion nematode Pratylenchus neglectus. Prior to this research, the exact linkage relationships of Rlnn1 with other loci on chromosome 7A were not clear and there were no simple codominant markers available for selection of Rlnn1 in wheat breeding. The objectives of the research reported here were to (1) develop an improved genetic map of the Rlnn1 region of chromosome 7A and (2) develop molecular markers that could be used in marker-assisted selection to improve resistance of wheat against P. neglectus. RESULTS: A large-effect quantitative trait locus (QTL) for resistance against P. neglectus was genetically mapped using a population of Excalibur/Kukri doubled haploid lines. This QTL coincides in position with the rust resistance gene(s) Lr20/Sr15, the phytoene synthase gene Psy-A1 and 10 molecular markers, including five new markers designed using wheat-rice comparative genomics and wheat expressed sequence tags. Two of the new markers are suitable for use as molecular diagnostic tools to distinguish plants that carry Rlnn1 and Lr20/Sr15 from those that do not carry these resistance genes. CONCLUSIONS: The genomic location of Rlnn1 was confirmed to be in the terminal region of the long arm of chromosome 7A. Molecular markers were developed that provide simple alternatives to costly phenotypic assessment of resistance against P. neglectus in wheat breeding. In Excalibur, genetic recombination seems to be completely suppressed in the Rlnn1 region.

Expression and functional analysis of TaASY1 during meiosis of bread wheat (Triticum aestivum).

BACKGROUND: Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. RESULTS: The full length wheat cDNA and genomic clone, TaASY1, has been isolated, sequenced and characterised. TaASY1 is located on chromosome Group 5 and the open reading frame displays significant nucleotide sequence identity to OsPAIR2 (84%) and AtASY1 (63%). Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occur. Immunolocalisation using transmission electron microscopy showed TaASY1 interacts with chromatin that is associated with both axial elements before SC formation as well as lateral elements of formed SCs. CONCLUSION: TaASY1 is a homologue of ScHOP1, AtASY1 and OsPAIR2 and is the first gene to be isolated from bread wheat that is involved in pairing and synapsis of homologous chromosomes.

Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding.

The current rate of yield gain in crops is insufficient to meet the predicted demands. Capturing the yield boost from heterosis is one of the few technologies that offers rapid gain. Hybrids are widely used for cereals, maize and rice, but it has been a challenge to develop a viable hybrid system for bread wheat due to the wheat genome complexity, which is both large and hexaploid. Wheat is our most widely grown crop providing 20% of the calories for humans. Here, we describe the identification of Ms1, a gene proposed for use in large-scale, low-cost production of male-sterile (ms) female lines necessary for hybrid wheat seed production. We show that Ms1 completely restores fertility to ms1d, and encodes a glycosylphosphatidylinositol-anchored lipid transfer protein, necessary for pollen exine development. This represents a key step towards developing a robust hybridization platform in wheat.Heterosis can rapidly boost yield in crop species but development of hybrid-breeding systems for bread wheat remains a challenge. Here, Tucker et al. describe the molecular identification of the wheat Ms1 gene and discuss its potential for large-scale hybrid seed production in wheat.

Genotyping by high-resolution melting analysis.

High-resolution melting (HRM) analysis is a simple, closed tube, post-PCR method used to identify genetic variation. The method is highly sensitive and can discriminate DNA sequence variants based on length (such as insertions or deletions), composition (such as single nucleotide polymorphisms, i.e., SNP) or strand complementarity (such as heterozygous or homozygous material). The technique involves PCR amplification of a target sequence in the presence of a fluorescent double-stranded DNA (dsDNA) binding dye, melting of the fluorescent amplicons, and subsequent interpretation of melt curve profiles. Here, we describe general considerations for assay design, PCR amplification, and HRM analysis.

A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem.

A long-standing question in plants and animals is how spatial patterns are maintained within stem cell niches despite ongoing cell divisions. Here we address how, during shoot meristem formation in Arabidopsis thaliana, the three apical cell layers acquire stem cell identity. Using a sensitized mutant screen, we identified miR394 as a mobile signal produced by the surface cell layer (the protoderm) that confers stem cell competence to the distal meristem by repressing the F box protein LEAF CURLING RESPONSIVENESS. This repression is required to potentiate signaling from underneath the stem cells by the transcription factor WUSCHEL, maintaining stem cell pluripotency. The interaction of two opposing signaling centers provides a mechanistic framework of how stem cells are localized at the tip of the meristem. Although the constituent cells change, the surface layer provides a stable point of reference in the self-organizing meristem.

Genetic analysis of abiotic stress tolerance in crops.

Abiotic stress tolerance is complex, but as phenotyping technologies improve, components that contribute to abiotic stress tolerance can be quantified with increasing ease. In parallel with these phenomics advances, genetic approaches with more complex genomes are becoming increasingly tractable as genomic information in non-model crops increases and even whole crop genomes can be re-sequenced. Thus, genetic approaches to elucidating the molecular basis to abiotic stress tolerance in crops are becoming more easily achievable.

Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo.

Stem cells are maintained in an undifferentiated state by signals from their microenvironment, the stem cell niche. Despite its central role for organogenesis throughout the plant's life, little is known about how niche development is regulated in the Arabidopsis embryo. Here we show that, in the absence of functional ZWILLE (ZLL), which is a member of the ARGONAUTE (AGO) family, stem cell-specific expression of the signal peptide gene CLAVATA3 (CLV3) is not maintained despite increased levels of the homeodomain transcription factor WUSCHEL (WUS), which is expressed in the organising centre (OC) of the niche and normally promotes stem cell identity. Tissue-specific expression indicates that ZLL acts to maintain the stem cells from the neighbouring vascular primordium, providing direct evidence for a non-cell-autonomous mechanism. Furthermore, mutant and marker gene analyses suggest that during shoot meristem formation, ZLL functions in a similar manner but in a sequential order with its close homologue AGO1, which mediates RNA interference. Thus, WUS-dependent OC signalling to the stem cells is promoted by AGO1 and subsequently maintained by a provascular ZLL-dependent signalling pathway.

WUSCHEL regulates cell differentiation during anther development.

During anther development a series of cell specification events establishes the male gametophyte and the surrounding sporophytic structure. Here we show that the homeobox gene WUSCHEL, originally identified as a central regulator of stem cell maintenance, plays an important role in cell type specification during male organogenesis. WUS expression is initiated very early during anther development in the precursor cells of the stomium and terminates just before the stomium cells enter terminal differentiation. At this stage the stomium cells and the neighboring septum cells that separate the pollen sacs undergo typical cell wall thickening and degenerate which leads to rupture of the anther and pollen release. In wus mutants, neither stomium cells nor septum cells differentiate or undergo cell death and degenerate. As a consequence, the anther stays intact and pollen is not released. CLAVATA3 which is activated by WUS in stem cell maintenance, is not activated in anthers indicating a novel pathway regulated by WUS. Comparing WUS function in stem cell maintenance and sexual organ development suggests that WUS expressing cells represent a conserved signaling module that regulates behavior and communication of undifferentiated cells.