Biology subjects

Harwood, W.

Publications and source records attributed to Harwood, W..

9 recordsLinked to original sources

bioRxiv2026

The demonstration of a single origin for nodule evolution, with nodule engineering in a non-nodulating species

The nitrogen-fixing root-nodule symbiosis provides a sustainable source of nitrogen for plants within the Nitrogen (N)-fixing clade (NFC). A debate has raged over whether nodulation evolved once, with many losses or multiple times following a predisposition event. Here we demonstrate that nodule-organogenesis is fully conserved between an actinorhizal nodulator Datisca glomerata and the legume Medicago truncatula, showing entirely conserved programmes for Nodule INception (NIN)-controlled development leading to nodule emergence. Convergent losses of N-fixation within the NFC is associated with loss of NIN and we show the engineering of nodule-like development into strawberry, a non-nodulating member of the NFC, through the repair of NIN-functionality. Similar NIN-engineering resulted in altered-root developmental responses in barley. Our work is consistent with the single-gain hypothesis, where repair of NIN can recapitulate nodules in species within the NFC, demonstrating that understanding the ancestral state of nodulation facilitates its engineering.

plant biology↗

bioRxiv2022

Highly efficient genome editing in barley using novel LbCas12a variants and impact of sgRNA architecture

We report the first successful, high efficiency use of LbCas12a in barley and describe the development and application of two novel Cas12a variants. In total we compared five coding sequence (CDS) variants including the two novel ones and two guide architectures over 5 different target genes using twenty different guides. We found large differences in editing efficiencies between the different CDS versions (0-87%) and guide architectures (0-70%) and show our two novel CDS versions massively outperform the others on test in this species. We show heritability of mutations generated. Our findings highlight the importance of optimising CRISPR systems for individual species and are likely to aid the use of LbCas12a in other monocot species.

genomics↗

bioRxiv2021

In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

plant biology↗

bioRxiv2021

Evolution of the bread wheat D-subgenome and enriching it with diversity from Aegilops tauschii

Aegilops tauschii, the diploid wild progenitor of the D-subgenome of bread wheat, constitutes a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. To better define and understand this diversity, we sequenced 242 Ae. tauschii accessions and compared them to the wheat D-subgenome. We characterized a rare, geographically-restricted lineage of Ae. tauschii and discovered that it contributed to the wheat D-subgenome, thereby elucidating the origin of bread wheat from at least two independent hybridizations. We then used k-mer-based association mapping to identify 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 synthetic hexaploids incorporating diverse Ae. tauschii genomes. This pipeline permits rapid trait discovery in the diploid ancestor through to functional genetic validation in a hexaploid background amenable to breeding.

plant biology↗

bioRxiv2020

CRISPR-Cas9-mediated editing of myb28 impairs glucoraphanin accumulation of Brassica oleracea in the field

We sought to quantify the role of MYB28 in the regulation of aliphatic glucosinolate biosynthesis and associated sulphur metabolism in field-grown B. oleracea with the use of CRISPR-Cas9-mediated gene editing technology. We describe the first characterised myb28 knockout mutant in B. oleracea, and the first UK field trial of CRISPR-Cas9-mediated gene edited plants under the European Court of Justice interpretation of the 2001/18 EU GMO directive. We report that knocking-out myb28 results in downregulation of aliphatic glucosinolate biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants. There were no significant changes to the accumulation of sulphate, S-methyl cysteine sulfoxide and indole glucosinolate in leaf and floret tissues.

plant biology↗

bioRxiv2019

TRA1: a locus responsible for controlling Agrobacterium-mediated transformability in barley.

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally large embryos were more or less amenable to transformation and discovered that mutant M1460 had a transformation efficiencies similar to that of Golden Promise. The large-embryo phenotype of M1460 is due to mutation at the LYS3 locus. There are three other barley lines with independent mutations at the same LYS3 locus, and one of these, Riso1508 has an identical missense mutation to that in M1460. However, none of the lys3 mutants except M1460 were transformable showing that the locus responsible for transformation efficiency, TRA1, was not LYS3 but another locus unique to M1460. To identify TRA1, we generated a mapping population by crossing M1460 to the cultivar Optic, which is recalcitrant to transformation. After four rounds of backcrossing to Optic, plants were genotyped and their progeny were tested for transformability. Some of the progeny lines were transformable at high efficiencies similar to those seen for the parent M1460 and some were not transformable, like Optic. A region on chromosome 2H inherited from M1460 is present in transformable lines only. We propose that one of the 225 genes in this region is TRA1.

plant biology↗

bioRxiv2018

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

1.To meet the challenge of feeding a growing population, breeders and scientists are continuously looking for ways to increase genetic gain in crop breeding. One way this can be achieved is through \"speed breeding\" (SB), which shortens the breeding cycle and accelerates research studies through rapid generation advancement. The SB method can be carried out in a number of ways, one of which involves extending the duration of a plants daily exposure to light (photoperiod) combined with early seed harvest in order to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. Here we present glasshouse and growth chamber-based SB protocols with supporting data from experimentation with several crop species. These protocols describe the growing conditions, including soil media composition, lighting, temperature and spacing, which promote rapid growth of spring and winter bread wheat, durum wheat, barley, oat, various members of the Brassica family, chickpea, pea, grasspea, quinoa and the model grass 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) purposes. Conversely, instructions on how to perform SB on a small-scale by creating a benchtop SB growth cabinet that enables optimization of parameters at a low cost are provided. We also outline the procedure for harvesting and germinating premature wheat, barley and pea seed to reduce generation time. Finally, we provide troubleshooting suggestions to avoid potential pitfalls.

plant biology↗

bioRxiv2018

Magnesium increases homoeologous crossover frequency in ZIP4 (Ph1) 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 70Mb 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 70Mb 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{middle dot}4H2O or Mg SO4{middle dot}7H2O) was analysed. 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 analysed hybrids, when plants were irrigated with a 1mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.

cell biology↗

bioRxiv2017

Speed breeding: a powerful tool to accelerate crop research and breeding

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand [1]. This slow improvement rate is attributed partly to the long generation times of crop plants. Here we present a method called speed breeding, which greatly shortens generation time and accelerates breeding and research programs. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum), and pea (Pisum sativum) and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully-enclosed controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies, and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent and potential for adaptation to larger-scale crop improvement programs. Cost-saving through LED supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing, and genomic selection, accelerating the rate of crop improvement.

plant biology↗