Fig 3 - available from: Plant Methods
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Locations of HvMet1A variants. Conserved domains are shown in grey, first the BAH domain (Bromo Adjacent Homology domain), followed by the Cytosine-C5 specific DNA methyltransferase domain. Identified variants are shown by arrows, red arrows highlight nonsynonymous variants, orange synonymous and green intron variants. A detailed summary of the variants is given in Additional file 7: Table S6. The size bar represents 1 kb (Figure was generated using http://wormw eb.org/exoni ntron )
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Background:
We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene f...
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... S6). As the original plants came from a bulk harvest of the previous generation, we could not assume categorically that all individuals were unique. A C2618T variant was identified in five plants, and three variants C2614T, G3977A and C4295T occurred in two plants. Thus, from the original 30 mutations, 23 independent variants were retained ( Fig. 3) and classified as three intron mutations, five synonymous mutations and 15 nonsynonymous mutations. Most of the exon mutations were in the targeted 'conserveddomains' of HvMET1A. For the nonsynonymous mutations we calculated the PROVEAN score, the smaller the value the higher the confidence that this mutation might be deleterious for ...
Citations
... The overall density of mutations was high (one mutation per 405 kb), what was slightly higher than the average mutation density in the HorTILLUS population estimated from TILLING analysis of 32 different genes, 1/477 kb (Szurman-Zubrzycka et al. 2018). The latest research in other barley TILLING populations showed an even higher mutation density of 1/154 kb or 1/256 kb, but with the use of whole exome capture sequencing and whole genome sequencing, respectively (Schreiber et al. 2019;Jiang et al. 2022). ...
Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar ‘Sebastian’. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the wild-type (WT). To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar ‘Sebastian’ under both optimal watering conditions and 10 days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and WT plants under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis -regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 ( expansin 8 ), HvXTH ( xyloglucan endotransglucosylase/hydrolase ), and HvPAE9 ( pectin acetylesterase 9 ), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size and reopening.
... However, these results were not replicated in other species such as Arabidopsis (Garcia-Molina & Leister, 2020) and potato (Solanum tuberosum; Lehretz et al., 2022), indicating that the complexity of light environmental conditions requires tailored and species-specific optimization of the response. BEST-CROP will attempt to ameliorate NPQ kinetics by exploiting barley natural genetic variability collected at the Cranachan database (https://barley.hutton.ac.uk/), as well as barley mutant populations available at the James Hutton Institute (Caldwell et al., 2004;Schreiber et al., 2019). ...
There is a need for ground‐breaking technologies to boost crop yield, both grains and biomass, and their processing into economically competitive materials. Novel cereals with enhanced photosynthesis and assimilation of greenhouse gasses, such as carbon dioxide and ozone, and tailored straw suitable for industrial manufacturing, open a new perspective for the circular economy. Here we describe the vision, strategies, and objectives of BEST‐CROP, a Horizon‐Europe and United Kingdom Research and Innovation (UKRI) funded project that relies on an alliance of academic plant scientists teaming up with plant breeding companies and straw processing companies to use the major advances in photosynthetic knowledge to improve barley biomass and to exploit the variability of barley straw quality and composition. We adopt the most promising strategies to improve the photosynthetic properties and ozone assimilation capacity of barley: (i) tuning leaf chlorophyll content and modifying canopy architecture; (ii) increasing the kinetics of photosynthetic responses to changes in irradiance; (iii) introducing photorespiration bypasses; (iv) modulating stomatal opening, thus increasing the rate of carbon dioxide fixation and ozone assimilation. We expect that by improving our targeted traits we will achieve increases in aboveground total biomass production without modification of the harvest index, with added benefits in sustainability via better resource‐use efficiency of water and nitrogen. In parallel, the resulting barley straw is tailored to: (i) increase straw protein content to make it suitable for the development of alternative biolubricants and feed sources; (ii) control cellulose/lignin contents and lignin properties to develop straw‐based construction panels and polymer composites. Overall, by exploiting natural‐ and induced‐genetic variability as well as gene editing and transgenic engineering, BEST‐CROP will lead to multi‐purpose next generation barley cultivars supporting sustainable agriculture and capable of straw‐based applications.
... Up to now, several barley TILLING populations have been developed, including the cultivar Golden Promise population as the reference genotype for barley transformation, with 3072 M 2 mutants [11]. Malting cultivar Barke was used to generate a TILLING resource comprising 10,279 M 2 mutants [12], and leaf material and seeds from approximately 20,000 M 2 plants in cultivar Optic were individually harvested to develop its mutant population [10]. ...
Mutagenesis is an important tool in crop improvement and free of the regulatory restrictions imposed on genetically modified organisms. Barley (Hordeum vulgare L.) is a diploid species with a genome smaller than those of other members of the Triticeae crops, making it an attractive model for genetic studies in Triticeae crops. In this study, we report an ethyl methane sulfonate (EMS)-mutagenized population in the Chinese barley landrace TX9425, which is tolerant to both abiotic and biotic stress. A TILLING (Targeting Induced Locus Lesion in Genomes) population consisting of 2000 M2 lines was also constructed based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment. The mutant phenotypes of the M2 and M3 generations were scored and revealed the presence of a wide spectrum of morphological diversity. The population was evaluated by screening for induced mutations in five genes of interest. A detailed analysis was performed for the HvGLR3.5 gene and three mutations were identified by screening in 2000 M2 lines. Two of three mutations displayed tuft and yellow striped leaves compared to the wild type. Altogether, our study shows the efficiency of screening and the great potential of the new TILLING population for genetic studies in the barley crop model system.
... Higher proportions of transposable elements in larger genomes may cause stronger genetic and developmental p.4/43 defects in met1 mutants through their remobilisation (Mirouze & Vitte, 2014). This hypothesis is supported by the lack of null mutants in MET1 orthologs identified in crop species with large genomes including in a barley TILLING population (genome size of ~5Gb) (Schreiber et al., 2019) and in maize TILLING and transposon populations (genome size of ~2.4 Gb) . ...
... Similarly, we were unable to recover any null met1-1 mutants in tetraploid wheat, in which both the A and B homoeologs are interrupted by a premature termination codon mutation (Supplementary Table 3). This indicates that complete loss of MET1-1 is lethal in wheat, consistent with other monocotyledonous crop species with large genomes such as maize and barley Schreiber et al., 2019). ...
DNA methylation is conserved across biological kingdoms, playing important roles in gene expression, transposable element silencing and genome stability. Altering DNA methylation could generate additional phenotypic variation for crop breeding, however the lethality of epigenetic mutants in crop species has hindered its investigation. Here, we exploit partial redundancy between homoeologs in polyploid wheat to generate viable mutants in the DNA methyltransferase 1-1 (MET1-1) gene with altered methylation profiles. In both Triticum turgidum (tetraploid wheat) and Triticum aestivum (hexaploid wheat) we identified clear segregation distortions of higher-order mutants (5/6 and 6/6 mutant met1-1 copies in hexaploid and 3/4 and 4/4 copies in tetraploid) when genotyping segregating seeds and seedlings, which we attribute to reduced transmission of null mutant gametes. We found that the reduced transmission occurred from both the maternal and paternal gametes, however, we did not detect any deleterious effects on pollen development. The loss of four or more functional copies of MET1-1 results in decreased CG methylation in hexaploid wheat. Changes to gene expression increase stepwise with the number of mutant alleles suggesting a dosage dependent effect. Finally, we identify heritable changes to flowering and awn phenotypes which segregate independently of MET1-1. Together our results demonstrate that polyploidy can be leveraged to generate quantitative changes to CG methylation without the lethal consequences observed in other crops, opening the potential to exploit novel epialleles in plant breeding.
... This crop is increasingly utilised in genetic studies due to its diploid nature, low chromosome number, ease of cross-breeding and cultivation in different climatic conditions. In recent years, considerable progress has been made in generating novel genetic resources in barley, including a high-quality genome, gene knockout collections for reverse genetics, marker rich genetic diversity panels and synthetic recombinant populations for association genetics (Nice et al., 2016;Mascher et al., 2017;Schreiber et al., 2019). ...
Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources.
Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short‐root phenotype compared with wild‐type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array‐based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles.
Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a.
We conclude that barley PIN1 genes have sub‐functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.
... This crop is increasingly utilised in genetic studies due to its diploid nature, low chromosome number, ease of cross-breeding and cultivation in different climatic conditions. In recent years, considerable progress has been made in generating novel genetic resources in barley, including a high-quality genome, gene knockout collections for reverse genetics, marker rich genetic diversity panels and synthetic recombinant populations for association genetics (Nice et al., 2016;Mascher et al., 2017;Schreiber et al., 2019). ...
Root angle is a critical factor in optimising the acquisition of essential resources from different soil depths. The regulation of root angle relies on the auxin-mediated root gravitropism machinery. While the influence of ethylene on auxin levels is known, its specific role in governing root gravitropism and angle remains uncertain, particularly when Arabidopsis (Arabidopsis thaliana) core ethylene signaling mutants show no gravitropic defects. Our research, focusing on rice (Oryza sativa L.) and maize (Zea mays), clearly reveals the involvement of ethylene in root angle regulation in cereal crops through the modulation of auxin biosynthesis and the root gravitropism machinery. We elucidated the molecular components by which ethylene exerts its regulatory effect on auxin biosynthesis to control root gravitropism machinery. The ethylene-insensitive mutants ethylene insensitive2 (osein2) and ethylene insensitive like1 (oseil1), exhibited substantially shallower crown root angle compared to the wild type. Gravitropism assays revealed reduced root gravitropic response in these mutants. Hormone profiling analysis confirmed decreased auxin levels in the root tips of the osein2 mutant, and exogenous auxin (NAA) application rescued root gravitropism in both ethylene-insensitive mutants. Additionally, the auxin-biosynthetic mutant mao hu zi10 (mhz10)/tryptophan aminotransferase2 (ostar2) showed impaired gravitropic response and shallow crown root angle phenotypes. Similarly, maize ethylene-insensitive mutants (zmein2) exhibited defective gravitropism and root angle phenotypes. In conclusion, our study highlights that ethylene controls the auxin-dependent root gravitropism machinery to regulate root angle in rice and maize, revealing a functional divergence in ethylene signaling between Arabidopsis and cereal crops. These findings contribute to a better understanding of root angle regulation and have implications for improving resource acquisition in agricultural systems.
... Commonly used chemical mutagens include sodium azide (NaN 3 ), N-methyl-N-nitrosourea (MNU), and ethyl methanesulfonate (EMS), each of which induces nucleotide transitions (predominantly G-A, or C-T). Several chemically induced mutant populations have been developed in major crops and other economically important plants (Abe et al. 2012;Henry et al. 2014;Jiao et al. 2016;Gupta et al. 2017;Li et al. 2017;Schreiber et al. 2019;Gao et al. 2020;Sashidhar et al. 2020;Nie et al. 2021). Mutants in an elite parental cultivar/variety background with desirable performance for a specific trait (e.g., salt tolerance) can readily be used to develop new varieties via genetic improvement (Takagi et al. 2015). ...
Induced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley ( Hordeum vulgare ) cultivar ‘Hua30’ and landrace ‘HTX’. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M 1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).
... A c c e p t e d M a n u s c r i p t Diversity in floral structure can be obtained through specific targeting of regulatory genes (e.g. CRISPR/Cas9; Selva et al., 2021;Li et al., 2021), through the exploitation of natural genetic variation (e.g. in barley;Caldwell et al., 2004;Talamè et al., 2008;Druka et al., 2011;Szarejko et al., 2017;Szurman-Zubrzycka et al., 2018;Schreiber et al., 2019;and wheat;Krasileva et al., 2017), or through the induction of mutations by chemical mutagenesis (Knudsen et al., 2021). In terms of the latter, historical forward genetic screens have uncovered an array of floral phenotypes. ...
Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1 and propose causative gene sequences: a C2H2 zinc-finger HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement.
... For instance, inbreeding may cause plants to be more susceptible to natural enemies [84,85]. Inbreeding can reduce the expression of genes whose products contribute to the formation of defence compounds [86,87], phytohormones [88] and metabolites [86,89] that are critical for defence signalling [90]. These changes in inbred plants may contribute to increased nutritional losses, leading to inbreeding depression in the presence of herbivores [89]. ...
Inbreeding is the crossing of closely related individuals in nature or a plantation or self-pollinating plants, which produces plants with high homozygosity. This process can reduce genetic diversity in the offspring and decrease heterozygosity, whereas inbred depression (ID) can often reduce viability. Inbred depression is common in plants and animals and has played a significant role in evolution. In the review, we aim to show that inbreeding can, through the action of epigenetic mechanisms, affect gene expression, resulting in changes in the metabolism and phenotype of organisms. This is particularly important in plant breeding because epigenetic profiles can be linked to the deterioration or improvement of agriculturally important characteristics.
... The EMS an alkylating mutagenic agent is especially efficient because it produces adducts with DNA bases, leading to damage the nucleotide complementation, hence modifying the base pairs. It causes G/C-to-A/T transitions that give a higher rate of random mutations all over the genome (Schreiber et al., 2019). Endonuclease cleave successfully with several missenses in the unknown sequence of DNA heteroduplex and duplex to that of known sequence unveils the polymorphism at different positions. ...
... TILLING is a nontransgenic approach and provides an alternative safer platform to overcome the limitations faced by transgenic and genome editing technologies. Because in TILLING, there is no introduction of any foreign DNA into the host cell which make, it more preferable and address all the regulatory, societal, and consumer concerns for their safe use (Schreiber et al., 2019;Sestili et al., 2019). Also due to is greater effectiveness in mutation screening, TILLING has attained more acceptability in public since, the crops designed by employing this method are released from the strict biosafety regulations enforced by GM regulatory bodies (Sestili et al., 2010). ...
... The shelf life of tomato fruits was increased by disrupting the ETHYLENE RESPONSE1 (SlETR1) gene via TILLING technique (Schreiber et al., 2019). TILLING has also been applied to improve the legumes such as chickpea (Amri-Tiliouine et al., 2018), mungbean (Varadaraju et al., 2020), sorghum (Xin et al., 2008), and barley (Schreiber et al., 2019) (Table 10.1). ...
This comprehensive three-volume set book, Biotechnologies and
Genetics in Plant Mutation Breeding, aims to help combat the
challenge of providing enough food for the world by use of the advanced
process of genetics to improve crop production, in both quantity and quality.
Volume 2: Mutagenesis and Crop Improvement first deals with
mutagenesis, cytotoxicity, and crop improvement. It discusses the processes,
mutagenic effectiveness, and efficiency and mechanisms of mutagenesis and
covers the principles, applications, and scope of mutagenesis as well. Several
chapters focus on mutation-induced cytological aberrations and cytotoxicity.
There is also emphasis on improvement of agronomic characteristics by
manipulating the genotype of plant species, resulting in increase in
productivity.
