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Introduction
Ryan is a plant molecular biologist who's research interests are in floral developmental biology and the underlying molecular mechanisms behind plant responses to abiotic stress. He has previously worked on projects ranging from meiosis to extracellular peptide-based signalling in Arabidopsis and cereals. Ryan completed his PhD at the Co-operative Research Centre for Molecular Plant Breeding at the University of Adelaide, after which he worked as a Molecular Biologist for Bayer Cropscience GmbH (Frankfurt-am-Main, Germany) before a postdoctoral period within the Flemish Interuniversity Institute for Biotechnology (Ghent University, Belgium). He is now leading the hybrid wheat program at the Australian Centre of Plant Functional Genomics (Adelaide, South Australia).
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University of Adelaide
School of Agriculture, Food and Wine
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Senior Researcher
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University of Adelaide
Egyptian Atomic Energy Authority
Bangabandhu Sheikh Mujibur Rahman Science & Technology University
John Innes Centre
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Bayerische Landesanstalt für Landwirtschaft
Ghent University
University College Dublin
KWS Group
The University of Sydney
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New Breeding Technique (NBT) for hybrid wheat seed production
Develop a proprietary wheat fertility control system
Research
Research Items (42)
Global food security demands the development and delivery of new technologies to increase and secure cereal production on finite arable land without increasing water and fertilizer use. There are several options for boosting wheat yields, but most offer only small yield increases. Wheat is an inbred plant, and hybrids hold the potential to deliver a major lift in yield and will open a wide range of new breeding opportunities. A series of technological advances are needed as a base for hybrid wheat programmes. These start with major changes in floral development and architecture to separate the sexes and force outcrossing. Male sterility provides the best method to block self-fertilization, and modifying the flower structure will enhance pollen access. The recent explosion in genomic resources and technologies provides new opportunities to overcome these limitations. This review outlines the problems with existing hybrid wheat breeding systems and explores molecular-based technologies that could improve the hybrid production system to reduce hybrid seed production costs, a prerequisite for a commercial hybrid wheat system.
Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport.
The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.
The Clavata3 (CLV3)/endosperm surrounding region (CLE) signaling peptides are encoded in large plant gene families. CLV3 and the other A-type CLE peptides promote cell differentiation in root and shoot apical meristems, whereas the B-type peptides (CLE41–CLE44) do not. Instead, CLE41 inhibits the differentiation of Zinnia elegans tracheary elements. To test whether CLE genes might code for antagonistic or synergistic functions, peptides from both types were combined through overexpression within or application onto Arabidopsis thaliana seedlings. The CLE41 peptide (CLE41p) promoted proliferation of vascular cells, although delaying differentiation into phloem and xylem cell lineages. Application of CLE41p or overexpression of CLE41 did not suppress the terminal differentiation of the root and shoot apices triggered by A-type CLE peptides. However, in combination, A-type peptides enhanced all of the phenotypes associated with CLE41 gain-of-function, leading to massive proliferation of vascular cells. This proliferation relied on auxin signaling because it was enhanced by exogenous application of a synthetic auxin, decreased by an auxin polar transport inhibitor, and abolished by a mutation in the Monopteros auxin response factor. These findings highlight that vascular patterning is a process controlled in time and space by different CLE peptides in conjunction with hormonal signaling. • cambium • hypocotyl • RAM • SAM • TDIF
The transition to the reproductive phase, inflorescence formation and flower development are crucial elements that ensure maximum reproductive success in a plant’s life cycle. To understand the regulatory mechanisms underlying correct flower development in barley (Hordeum vulgare) we characterised the multiovary 5 (mov5.o) mutant. This mutant develops abnormal flowers that exhibit mosaic floral organs typified by multiple carpels at the total or partial expense of stamens. Genetic mapping positioned mov5 on the long arm of chromosome 2H, incorporating a region that encodes HvLFY, the barley orthologue of LEAFY from Arabidopsis. Sequencing revealed that in mov5.o plants, HvLFY contains a single amino acid substitution in a highly conserved proline residue. CRISPR‐mediated knockout of HvLFY replicated the mov5.o phenotype, suggesting that HvLFYmov5 represents a loss of function allele. In heterologous assays, the HvLFYmov5 polymorphism influenced protein‐protein interactions and affinity for a putative binding site in the promoter of HvMADS58, a C‐class MADS‐box gene. Moreover, molecular analysis indicated that HvLFY interacts with HvUFO and regulates the expression of floral homeotic genes including HvMADS2, HvMADS4 and HvMADS16. Other distinct changes in expression differ from those reported in the rice LFY mutants apo2/rfl, suggesting LFY function in the grasses is modulated in a species‐specific manner. This pathway provides a key entry point for the study of LFY function and multiple ovary formation in barley, as well as cereal species in general.
Meiotic recombination is a critical process for plant breeding, as it creates novel allele combinations that can be exploited for crop improvement. In wheat, a complex allohexaploid that has a diploid-like behaviour, meiotic recombination between homoeologous or alien chromosomes is suppressed through the action of several loci. Here, we report positional cloning of Pairing homoeologous 2 (Ph2) and functional validation of the wheat DNA mismatch repair protein MSH7-3D as a key inhibitor of homoeologous recombination, thus solving a half-century-old question. Similar to ph2 mutant phenotype, we show that mutating MSH7-3D induces a substantial increase in homoeologous recombination (up to 5.5 fold) in wheat-wild relative hybrids, which is also associated with a reduction in homologous recombination. These data reveal a role for MSH7-3D in meiotic stabilisation of allopolyploidy and provides an opportunity to improve wheat’s genetic diversity through alien gene introgression, a major bottleneck facing crop improvement. In the allohexaploid genome of wheat, meiotic recombination between homoeologues is suppressed through the action of several loci. Here, the authors report the cloning of the long sought-after gene Ph2 and show its function in reduction of homoeologous recombination.
Hybrid breeding in wheat has the potential to boost yields. An efficient hybrid seed production system requires elite pollinators; however, such germplasm is limited among modern cultivars. Piko, a winter wheat (Triticum aestivum L.) cultivar, has been identified as a superior pollinator and has been used in Europe. Piko has favourable pollinator traits for anther extrusion, anther length, pollen mass and hybrid seed set. However, the genetic factors responsible for Piko's favourable traits are largely unknown. Here, we report on the genetic analysis of a Piko-derived F2 mapping population. We confirmed that Piko's Rht-D1a allele for tall stature is associated with large anthers and high anther extrusion. However, Rht-D1 was not found to be associated with anther filament length, confirmed by near isogenic lines. Piko's photoperiod sensitive Ppd-B1b allele shows an association with increased spike length, more spikelets and spike architecture traits, while the insensitive Ppd-B1a allele is linked with high anther extrusion and larger anthers. We identified an anther extrusion quantitative trait locus (QTL) on chromosome 6A that showed significantly biased transmission of the favourable Piko allele amongst F2 progenies. The Piko allele is completely absent in the distal 6AS region and the central 6A region revealed a significantly lower ratio (<8%) of F2 with homozygous Piko alleles. Our study provided further evidence for the effects of Rht-D1 and Ppd-B1 loci on multiple pollinator traits and a novel anther extrusion QTL that exhibits segregation distortion.
Ferrochelatase (FC) is the terminal enzyme of heme biosynthesis. In photosynthetic organisms studied so far, there is evidence for two FC isoforms, which are encoded by two genes (FC1 and FC2). Previous studies suggest that these two genes are required for the production of two physiologically distinct heme pools with only FC2-derived heme involved in photosynthesis. We characterised two FCs in barley (Hordeum vulgare L.). The two HvFC isoforms share a common catalytic domain, but HvFC2 additionally contains a C-terminal chlorophyll a/b binding (CAB) domain. Both HvFCs are highly expressed in photosynthetic tissues, with HvFC1 transcripts also being abundant in non-photosynthetic tissues. To determine whether these isoforms differentially affect photosynthesis, transgenic barley ectopically overexpressing HvFC1 and HvFC2 were generated and evaluated for photosynthetic performance. In each case, transgenics exhibited improved photosynthetic rate (A sat), stomatal conductance (g s) and carboxylation efficiency (CE), showing that both FC1 and FC2 play important roles in photosynthesis. Our finding that modified FC expression can improve photosynthesis up to~13% under controlled growth conditions now requires further research to determine if this can be translated to improved yield performance under field conditions.
We investigated the roles of two Ferrochelatases (FCs), which encode the terminal enzyme for heme biosynthesis, in drought and oxidative stress tolerance in model cereal plant barley (Hordeum vulgare). Three independent transgenic lines ectopically overexpressing either barley FC1 or FC2 were selected and evaluated under well-watered, drought, and oxidative stress conditions. Both HvFC1 and HvFC2 overexpressing transgenics showed delayed wilting and maintained higher photosynthetic performance relative to controls, after exposure to soil dehydration. In each case, HvFC overexpression significantly upregulated the nuclear genes associated with detoxification of reactive oxygen species (ROS) upon drought stress. Overexpression of HvFCs, also suppressed photo-oxidative damage induced by the deregulated tetrapyrrole biosynthesis mutant tigrinad12. Previous studies suggest that only FC1 is implicated in stress defense responses, however, our study demonstrated that both FC1 and FC2 affect drought stress tolerance. As FC-derived free heme was proposed as a chloroplast-to-nuclear signal, heme could act as an important signal, stimulating drought responsive nuclear gene expression. This study also highlighted tetrapyrrole biosynthetic enzymes as potential targets for engineering improved crop performance, both in well-watered and water-limited environments.
Hybrid breeding in wheat (Triticum aestivum L.) has the potential to deliver major yield increases. This is a requisite to guarantee food security for increasing population demands and to counterbalance the effects of extreme environmental conditions. Successful hybrid breeding in wheat relies on forced outcrossing while preventing self-pollination. To achieve this, research has been directed towards identifying and improving fertility control systems. To maximise cross-pollination and seed set, however, fertility control systems need to be complemented by breeding phenotypically distinct male and female lines. This review summarises existing and novel male sterility systems for wheat hybridisation. We also consider the genetic resources that can be used to alter wheat's floral development and spike morphology, with a focus on the genetic variation already available. Exploiting these resources can lead to enhanced outcrossing, a key requirement in the progress towards hybrid wheat breeding.
Background: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. Results: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. Conclusions: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.
Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only barley, wheat and rye accumulate delphinidin‐derived (blue) anthocyanins in the aleurone layer of grains, but not rice, maize and sorghum. The underlying genetic basis for this natural occurence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleruone to a ~ 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue‐coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co‐evolved together. We also identified a Pooideae‐specific flavonoid 3’,5’‐hydroxylase (F3’5’H) lineage, termed here Mo_F35H2 which has higher amino acid similarity with eudicot F3’5’Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 which underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, by inquiring the barley germplasm there’s evidence that HvMYB4H and HvMYC4H have underwent human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.
Bread wheat (Triticum aestivum L.) is one of the major crops for human nutrition and an important one for food security. However, wheat yields are highly dependent upon environmental conditions and are affected by various types of abiotic stresses. One strategy for improving wheat yield stability across environments is to harness hybrid vigour. Estimates of yield improvements associated with hybrid vigour in wheat range from 5 to over 20%, which needs to be further enhanced to meet the future global demand. This yield advantage comes with improved yield stability under both biotic and abiotic stress conditions. This chapter focuses on the current status of hybrid wheat breeding, including hybrid seed production systems, hybrid performance under abiotic stresses and prediction of hybrid performance.
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. Abstract 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.
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 EMS‐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 monofactorial 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. This article is protected by copyright. All rights reserved.
Mutation discovery is often key to the identification of genes responsible for major phenotypic traits. In the context of bulked segregant analysis, common reference-based computational approaches are not always suitable as they rely on a genome assembly which may be incomplete or highly divergent from the studied accession. Reference-free methods based on short sequences of length k ( k -mers), such as NIKS, exploit redundancy of information across pools of recombinant genomes. Building on concepts from NIKS we introduce LNISKS, a mutation discovery method which is suited for large and repetitive crop genomes. In our experiments, it rapidly and with high confidence, identified mutations from over 700 Gbp of bread wheat genomic sequence data. LNISKS is publicly available at https://github.com/rsuchecki/LNISKS.
The development and adoption of hybrid seed technology has 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 hybridisation platform utilises 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 hybridisation 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. This article is protected by copyright. All rights reserved.
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. β-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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1557-1) contains supplementary material, which is available to authorized users.
Abstract Non-specific Lipid Transfer Proteins (nsLTPs) are involved in numerous biological processes. To date, only a fraction of wheat (Triticum aestivum L.) nsLTPs (TaLTPs) have been identified, and even fewer have been functionally analysed. In this study, the identification, classification, phylogenetic reconstruction, chromosome distribution, functional annotation and expression profiles of TaLTPs were analysed. 461 putative TaLTPs were identified from the wheat genome and classified into five types (1, 2, C, D and G). Phylogenetic analysis of the TaLTPs along with nsLTPs from Arabidopsis thaliana and rice, showed that all five types were shared across species, however, some type 2 TaLTPs formed wheat-specific clades. Gene duplication analysis indicated that tandem duplications contributed to the expansion of this gene family in wheat. Analysis of RNA sequencing data showed that TaLTPs were expressed in most tissues and stages of wheat development. Further, we refined the expression profile of anther-enriched expressed genes, and identified potential cis-elements regulating their expression specificity. This analysis provides a valuable resource towards elucidating the function of TaLTP family members during wheat development, aids our understanding of the evolution and expansion of the TaLTP gene family and, additionally, provides new information for developing wheat male-sterile lines with application to hybrid breeding.
Bread wheat is strongly autogamous; however, an opportunity for outcrossing occurs when self-pollination fails and florets open. The first phase of floret opening at anthesis is short and induced by lodicule turgidity. Some wheat florets re-open post-anthesis for several days, known as the ‘second opening’, for which the underlying mechanisms are largely unknown. We performed detailed physiological, anatomical, and histological investigations to understand the biological basis of the flower opening process. Wheat florets were observed open when the ovary was unfertilized. Unfertilized ovaries significantly increased in radial size post-anthesis, pushing the lemma and palea apart to open the florets. The absence of fertile pollen was not directly linked to this, but anther filament elongation coincided with initiation of ovary swelling. The pericarp of unfertilized ovaries did not undergo degeneration as normally seen in developing grains, instead pericarp cells remained intact and enlarged, leading to increased ovary radial size. This is a novel role for the ovary pericarp in wheat flower opening, and the knowledge is useful for facilitating cross-pollination in hybrid breeding. Ovary swelling may represent a survival mechanism in autogamous cereals such as wheat and barley, ensuring seed set in the absence of self-fertilization and increasing genetic diversity through cross-pollination.
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.
Drought is one of the major abiotic stresses reducing crop yield. Since the discovery of plant microRNAs (miRNAs), considerable progress has been made in clarifying their role in plant responses to abiotic stresses, including drought. miR827 was previously reported to confer drought tolerance in transgenic Arabidopsis. We examined barley (Hordeum vulgare L. ‘Golden Promise’) plants over-expressing miR827 for plant performance under drought. Transgenic plants constitutively expressing CaMV-35S::Ath-miR827 and drought-inducible Zm-Rab17::Hv-miR827 were phenotyped by non-destructive imaging for growth and whole plant water use efficiency (WUEwp). We observed that the growth, WUEwp, time to anthesis and grain weight of transgenic barley plants expressing CaMV-35S::Ath-miR827 were negatively affected in both well-watered and drought-treated growing conditions compared with the wild-type plants. In contrast, transgenic plants over-expressing Zm-Rab17::Hv-miR827 showed improved WUEwp with no growth or reproductive timing change compared with the wild-type plants. The recovery of Zm-Rab17::Hv-miR827 over-expressing plants also improved following severe drought stress. Our results suggest that Hv-miR827 has the potential to improve the performance of barley under drought and that the choice of promoter to control the timing and specificity of miRNA expression is critical.
Tetrapyrroles such as chlorophyll and heme play a vital role in primary plant metabolic processes such as photosynthesis and respiration. Over the past decades, extensive genetic and molecular analyses have provided valuable insights into the complex regulatory network of the tetrapyrrole biosynthesis. However, tetrapyrroles are also implicated in abiotic stress tolerance, although the mechanisms are largely unknown. With recent reports demonstrating that modified tetrapyrrole biosynthesis in plants confers wilting avoidance, a component physiological trait to drought tolerance, it is now timely that this pathway be reviewed in the context of drought stress signalling. In this review, the significance of tetrapyrrole biosynthesis under drought stress is addressed, with particular emphasis on the inter-relationships with major stress signalling cascades driven by reactive oxygen species (ROS) and organellar retrograde signalling. We propose that unlike the chlorophyll branch, the heme branch of the pathway plays a key role in mediating intracellular drought stress signalling and stimulating ROS detoxification under drought stress. Determining how the tetrapyrrole biosynthetic pathway is involved in stress signalling provides an opportunity to identify gene targets for engineering drought-tolerant crops. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
The recent advent of molecular technologies has changed the way plant breeders identify and select their germplasm as genetic variation can now be assessed at the DNA level. Crop Breeding: Methods and Protocols presents detailed guidelines and tutorials that suit different needs and capacity from small laboratories analyzing molecular markers on a one-by-one basis to the increasingly popular high-throughput protocols for high capacity laboratories. Topics covered include breeding strategy for the selection of an ideal variety or genetic ideotype, protocols for breeders using molecular markers in selection programs and for laboratories providing molecular services to breeding programs, statistical programs and software to aid implementation of molecular data into breeding programs, and methodologies that facilitate the generation of genetic diversity and its characterization. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
In recent years, the interest in new technologies for wheat improvement has increased greatly. As methods for conventional and molecular breeding have advanced, more efficient procedures for phenotyping have become a bottleneck. At the Australian Centre for Plant Functional Genetics (ACPFG), a centre for the improvement of wheat and barley's tolerance to environmental stresses, we developed a phenotyping platform for transgenic wheat and barley. After the initial selection of single/low copy number plants in the T0 generation, the T1 and T2 generation are screened under competitive growth conditions in large plastic bins. At present, the setup consists of 32 bins which can be used two times a year. All bins are fully equipped with irrigation system and sensors for soil water tension, soil temperature, air temperature, and air humidity, recording data at the time step of 5 minutes. The T1 generation is preferably grown in the summer season (January to June) whereas the T2 generation is evaluated in the main growing season (July to December). The first screening of T1 plants serves to remove off-types (for growth duration, plant height, and other growth anomalies) and to select homozygous plants. In the next step, T2 plants are phenotyped under different soil water and nitrogen regimes in a replicated experimental design in comparison with the wild type and null controls (drought and nitrogen response traits). Plant traits recorded in stage 2 for each individual plant were: tiller number, spike number, empty spikes, plant height, number of seeds, seed weight, and above ground biomass. The soil moisture tension curves of the drought treatments did show a gradual water stress increase over several days to weeks, closely mimicking field conditions. The observed average grain yields per plant were realistic for both well watered and droughted conditions, but seemed still to be too high under the low fertility regime. However, interpreting grain yield data from this setup remained difficult mainly because of the relatively high variability between replications. Further trials are needed to show if this variability can be reduced. We concluded that the new high throughput platform for the testing of transgenic wheat growing in a field-like situation but under controlled conditions was functioning. First season results showed that screening conditions for drought and nutrient use efficiency were as planned and that the platform served its objective. Improvements of the platform to achieve more homogenous results are outlined and the establishment of a digital imaging and analysis system is ongoing. These changes are intended to further increase the diagnostic value of the platform.
Climate change is predicted to result in disruption of many farming systems. The Food and Agriculture Organization (FAO) predicts a 15-20% fall in global agricultural production by 2080. Consequently, adaptation of major crop species to climate change will be the biggest challenge for plant breeders this century. Biotechnology will be important when adapting crops to better tolerate changing stresses. It includes using advanced genetic mapping technologies, like molecular markers, in the breeding and development of new varieties. Molecular markers are used to provide greater focus, accuracy and speed in crop breeding programmes with further advances coming. Genetic modification (GM) techniques are providing access to a diversity of genes, used to develop plant varieties more tolerant to the negative impacts of climate change.
A multigene family expressed during early floral development was identified on the short arm of wheat chromosome 3D in the region of the Ph2 locus, a locus controlling homoeologous chromosome pairing in allohexaploid wheat. Physical, genetic and molecular characterisation of the Wheat Meiosis 1 (WM1) gene family identified seven members that localised within a region of 173-kb. WM1 gene family members were sequenced and they encode mainly type Ia plasma membrane-anchored leucine rich repeat-like receptor proteins. In situ expression profiling suggests the gene family is predominantly expressed in floral tissue. In addition to the WM1 gene family, a number of other genes, gene fragments and pseudogenes were identified. It has been predicted that there is approximately one gene every 19-kb and that this region of the wheat genome contains 23 repetitive elements including BARE-1 and Wis2-1 like sequences. Nearly 50% of the repetitive elements identified were similar to known transposons from the CACTA superfamily. Ty1-copia, Ty3-gypsy and Athila LTR retroelements were also prevalent within the region. The WM1 gene cluster is present on 3DS and on barley 3HS but missing from the A and B genomes of hexaploid wheat. This suggests either recent generation of the cluster or specific deletion of the cluster during wheat polyploidisation. The evolutionary significance of the cluster, its possible roles in disease response or floral and early meiotic development and its location at or near the Ph2 locus are discussed.
Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.
Wheat Meiosis 5 (WMS), isolated from an early meiosis anther cDNA library of wheat by cDNA subtraction encodes a novel glycine-serine-proline-alanine-rich protein. The corresponding homologous genes are located on the short arms of chromosomes 3A, 3B and 3D of allohexaploid wheat (Triticum aestivum L.). The copy on 3DS is located within the region deleted in the wheat mutant ph2a that displays increased homoeologous chromosome pairing in crosses with alien species. While WM5 is expressed primarily in young flower buds during early meiosis it is also expressed in shoot meristems, thus indicating functional roles in both meiosis and meristem development. Overall, the WM5 amino acid sequence shares no significant similarity with other known proteins in the NCBI database. However, the carboxyl-terminal region does have similarity with the Arabidopsis PDF1 (Protodermal Factor 1) protein. Comparing WM5 and PDF1 reveals that the two proteins share 33% identity and have similar hydropathy plots and predicted secondary structures. In situ immuno-staining locates the protein to the nuclei of pollen mother cells undergoing meiosis and the epidermal layer of the shoot and flower meristem, including the cell wall and cuticle. We propose that the WM5 protein has a role in shoot and flower development within this economically important cereal crop.
Colinearity in gene content and order between rice and closely related grass species has emerged as a powerful tool for gene identification. Using a comparative genetics approach, we have identified the rice genomic region syntenous to the region deleted in the wheat chromosome pairing mutant ph2a, with a view to identifying genes at the Ph2 locus that control meiotic processes. Utilising markers known to reside within the region deleted in ph2a, and data from wheat, barley and rice genetic maps, markers delimiting the region deleted on wheat chromosome 3DS in the ph2a mutant were used to locate the syntenous region on the short arm of rice chromosome 1. A contig of rice genomic sequence was identified from publicly available sequence information and used in blast searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Southern analysis using a subset of identified wheat ESTs confirmed a syntenous relationship between the rice and wheat genomic regions and defined precisely the extent of the deleted segment in the ph2a mutant. A 6.58-Mb rice contig generated from 60 overlapping rice chromosome 1 P1 artificial chromosome (PAC) clones spanning the syntenous rice region has enabled identification of 218 wheat ESTs putatively located in the region deleted in ph2a. What seems to be a terminal deletion on chromosome 3DS is estimated to be 80 Mb in length. Putative candidate genes that may contribute to the altered meiotic phenotype of ph2a are discussed.
DNA mismatch repair is an essential system for maintaining genetic stability in bacteria and higher eukaryotes. Based on the conserved regions of the bacterial MutS gene and its homologues in yeast and human, a wheat cDNA homologue of MSH6, designated TaMSH7, was isolated by RT-PCR. The deduced amino acid sequence of TaMSH7 shows conserved domains characteristic of other MSH6 genes, with highest similarity to maize MSH7 and Arabidopsis MSH7. TaMSH7 is expressed in meristem tissue associated with a high level of mitotic and meiotic activity, with maximum expression in the reproductive organs of young flower spikes. TaMSH7 is located on the short arms of chromosomes 3A, 3B, and 3D and has been mapped within barley chromosome 3HS. The copy on 3DS is located within the region deleted in the wheat mutant ph2a, which shows altered recombination frequency in the interspecific hybrids. The relationship between the ph2a mutant and TaMSH7 gene function is discussed.
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