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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.

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Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max (L.) Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins. We isolated two high-seed protein (HP) soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes ß-conglycinin (GmCG-1) and HP2 encodes Sucrose Binding Protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits.

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Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

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The advanced model of floral morphogenesis is based largely on data from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but this process is less well understood in the Triticeae. Here, we investigated a sterile barley (Hordeum vulgare) mutant with malformed floral organs (designated mfo1), of which the paleae, lodicules, and stamens in each floret were all converted into lemma-like organs, and the ovary was abnormally shaped. Combining bulked-segregant analysis, whole-genome resequencing, and TILLING approaches, the mfo1 mutant was attributed to loss-of-function mutations in the MADS-box transcription factor gene HvAGL6, a key regulator in the ABCDE floral morphogenesis model. Through transcriptomic analysis between young inflorescences of wild-type and mfo1 plants, 380 genes were identified as differentially expressed, most of which function in DNA binding, protein dimerization, cell differentiation, or meristem determinacy. Regulatory pathway enrichment showed HvAGL6 associates with transcriptional abundance of many MADS-box genes, including the B-class gene HvMADS4. Mutants with deficiency in HvMADS4 exhibited the conversion of stamens into supernumerary pistils, producing multiple ovaries resembling the completely sterile multiple ovaries 3.h (mov3.h) mutant. These findings demonstrate that the regulatory model of floral morphogenesis is conserved across plant species and provides insights into the interactions between HvAGL6 and other MADS-box regulators.

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BACKGROUND: Semi-dwarfing alleles are used widely in cereals to confer improved lodging resistance and assimilate partitioning. The most widely deployed semi-dwarfing alleles in rice and barley encode the gibberellin (GA)-biosynthetic enzyme GA 20-OXIDASE2 (GA20OX2). The hexaploid wheat genome carries three homoeologous copies of GA20OX2, and because of functional redundancy, loss-of-function alleles of a single homoeologue would not be selected in wheat breeding programmes. Instead, approximately 70% of wheat cultivars carry gain-of-function mutations in REDUCED HEIGHT 1 (RHT1) genes that encode negative growth regulators and are degraded in response to GA. Semi-dwarf Rht-B1b or Rht-D1b alleles encode proteins that are insensitive to GA-mediated degradation. However, because RHT1 is expressed ubiquitously these alleles have pleiotropic effects that confer undesirable traits in some environments. RESULTS: We have applied reverse genetics to combine loss-of-function alleles in all three homoeologues of wheat GA20OX2 and its paralogue GA20OX1 and evaluated their performance in three years of field trials. ga20ox1 mutants exhibited a mild height reduction (approximately 3%) suggesting GA20OX1 plays a minor role in stem elongation in wheat. ga20ox2 mutants have reduced GA1 content and are 12-32% shorter than their wild-type segregants, comparable to the effect of the Rht-D1b 'Green Revolution' allele. The ga20ox2 mutants showed no significant negative effects on yield components in the spring wheat variety 'Cadenza'. CONCLUSIONS: Our study demonstrates that chemical mutagenesis can expand genetic variation in polyploid crops to uncover novel alleles despite the difficulty in identifying appropriate mutations for some target genes and the negative effects of background mutations. Field experiments demonstrate that mutations in GA20OX2 reduce height in wheat, but it will be necessary to evaluate the effect of these alleles in different genetic backgrounds and environments to determine their value in wheat breeding as alternative semi-dwarfing alleles.

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Treatment of plants with chemical mutagens results primarily in the production of novel single nucleotide variants. Mutagenesis is a mostly random process and as such plants derived from mutagenesis of different seeds or in vitro material are expected to accumulate different mutations. An important step in the creation of a mutant population for forward or reverse genetics is the choice of treatment conditions (e.g., dosage) such that sufficient mutations accumulate while not adversely affecting propagation of the plant. DNA sequencing provides a quick method to evaluate the effect of different treatment conditions and their effect on the density and spectrum of accumulated mutations. Whole genome sequencing or reduced representation sequencing is carried out followed by mapping to a reference genome and production of a Variant Call Format (VCF) file. We provide here a method for generating a multi-sample VCF from mutagenized plants and describe a new tool to streamline the process of recovering unique induced mutations and determining their possible effect on gene function.

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The innovations and progress in genome editing/new breeding technologies have revolutionized research in the field of functional genomics and crop improvement. This revolution has expanded the horizons of agricultural research, presenting fresh possibilities for creating novel plant varieties equipped with desired traits that can effectively combat the challenges posed by climate change. However, the regulation and social acceptance of genome-edited crops still remain as major barriers. Only a few countries considered the site-directed nuclease 1 (SDN1) approach-based genome-edited plants under less or no regulation. Hence, the present review aims to comprise information on the research work conducted using SDN1 in crops by various genome editing tools. It also elucidates the promising candidate genes that can be used for editing and has listed the studies on non-transgenic crops developed through SDN1 either by Agrobacterium-mediated transformation or by ribo nucleoprotein (RNP) complex. The review also hoards the existing regulatory landscape of genome editing and provides an overview of globally commercialized genome-edited crops. These compilations will enable confidence in researchers and policymakers, across the globe, to recognize the full potential of this technology and reconsider the regulatory aspects associated with genome-edited crops. Furthermore, this compilation serves as a valuable resource for researchers embarking on the development of customized non-transgenic crops through the utilization of SDN1.

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OBJECTIVE: Little research has been done on managing soil health for large-scale, outdoor hemp production, contributing to the possible overuse of black plastic for weed suppression. Our experiment aimed to understand the performance of alternative ground covers including forage crops and hay as well as a less disruptive tilling method called strip-tilling compared to black plastic. RESULTS: Yield and soil health data were taken from three experimental plantings from two different outdoor CBD hemp farms in Vermont, USA. We find that hay may be a competitive alternative to black plastic in terms of producing heavier plants. Our research also found that clover seed and hay are both more cost-effective options than black plastic which may sway some farmers to adopt these alternative ground cover options.

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Since its introduction in 2000, the TILLING strategy has been widely used in plant research to create novel genetic diversity. TILLING is based on chemical or physical mutagenesis followed by the rapid identification of mutations within genes of interest. TILLING mutants may be used for functional analysis of genes and being nontransgenic, they may be directly used in pre-breeding programs. Nevertheless, classical mutagenesis is a random process, giving rise to mutations all over the genome. Therefore TILLING mutants carry background mutations, some of which may affect the phenotype and should be eliminated, which is often time-consuming. Recently, new strategies of targeted genome editing, including CRISPR/Cas9-based methods, have been developed and optimized for many plant species. These methods precisely target only genes of interest and produce very few off-targets. Thus, the question arises: is it the end of TILLING era in plant studies? In this review, we recap the basics of the TILLING strategy, summarize the current status of plant TILLING research and present recent TILLING achievements. Based on these reports, we conclude that TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects.

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Polyphenol oxidase (PPO) activity is a major cause of the undesirable brown color of wheat-based products. Ppo1, a major gene for PPO activity, was cloned based on sequence homology in previous studies; however, its function and regulation mechanism remain unclear. In this study, the function and genetic regulation of Ppo1 were analyzed using RNA interference (RNAi) and Targeting Induced Local Lesions IN Genomes (TILLING) technology, and superior mutants were identified. Compared with the control, the level of Ppo1 transcript in RNAi transgenic lines was drastically decreased by 15.5%-60.9% during grain development, and PPO activity was significantly reduced by 12.9%-20.4%, confirming the role of Ppo1 in PPO activity. Thirty-two Ppo1 mutants were identified in the ethyl methanesulfonate (EMS)-mutagenized population, including eight missense mutations, 16 synonymous mutations, and eight intron mutations. The expression of Ppo1 was reduced significantly by 6.7%-37.1% and 10.1%-54.4% in mutants M092141 (G311S) and M091098 (G299R), respectively, in which PPO activity was decreased by 29.7% and 28.8%, respectively, indicating that mutation sites of two mutants have important effects on PPO1 function. Sequence and structure analysis revealed that the two sites were highly conserved among 74 plant species, where the frequency of glycine was 94.6% and 100%, respectively, and adjacent to the entrance of the hydrophobic pocket of the active site. The M092141 and M091098 mutants can be used as important germplasms to develop wheat cultivars with low grain PPO activity. This study provided important insights into the molecular mechanism of Ppo1 and the genetic improvement of wheat PPO activity.

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The intensification of food production via conventional crop breeding alone is inadequate to cater for global hunger. The development of precise and expeditious high throughput reverse genetics approaches has hugely benefited modern plant breeding programs. Targeting Induced Local Lesions in Genomes (TILLING) is one such reverse genetics approach which employs chemical/physical mutagenesis to create new genetic sources and identifies superior/novel alleles. Owing to technical limitations and sectional applicability of the original TILLING protocol, it has been timely modified. Successions include: EcoTILLING, Double stranded EcoTILLING (DEcoTILLING), Self-EcoTILLING, Individualized TILLING (iTILLING), Deletion-TILLING (De-TILLING), PolyTILLING, and VeggieTILLING. This has widened its application to a variety of crops and needs. They can characterize mutations in coding as well as non-coding regions and can overcome complexities associated with the large genomes. Combining next generation sequencing tools with the existing TILLING protocols has enabled screening of huge germplasm collections and mutant populations for the target genes. In silico TILLING platforms have transformed TILLING into an exciting breeding approach. The present review outlines these multifarious TILLING modifications for precise mutation detection and their application in advance breeding programmes together with relevant case studies. Appropriate use of these protocols will open up new avenues for crop improvement in the twenty first century.

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MAIN CONCLUSION: The present review illustrates a comprehensive overview of the allele mining for genetic improvement in vegetable crops, and allele exploration methods and their utilization in various applications related to pre-breeding of economically important traits in vegetable crops. Vegetable crops have numerous wild descendants, ancestors and terrestrial races that could be exploited to develop high-yielding and climate-resilient varieties resistant/tolerant to biotic and abiotic stresses. To further boost the genetic potential of economic traits, the available genomic tools must be targeted and re-opened for exploitation of novel alleles from genetic stocks by the discovery of beneficial alleles from wild relatives and their introgression to cultivated types. This capability would be useful for giving plant breeders direct access to critical alleles that confer higher production, improve bioactive compounds, increase water and nutrient productivity as well as biotic and abiotic stress resilience. Allele mining is a new sophisticated technique for dissecting naturally occurring allelic variants in candidate genes that influence important traits which could be used for genetic improvement of vegetable crops. Target-induced local lesions in genomes (TILLINGs) is a sensitive mutation detection avenue in functional genomics, particularly wherein genome sequence information is limited or not available. Population exposure to chemical mutagens and the absence of selectivity lead to TILLING and EcoTILLING. EcoTILLING may lead to natural induction of SNPs and InDels. It is anticipated that as TILLING is used for vegetable crops improvement in the near future, indirect benefits will become apparent. Therefore, in this review we have highlighted the up-to-date information on allele mining for genetic enhancement in vegetable crops and methods of allele exploration and their use in pre-breeding for improvement of economic traits.

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Alkalinity is an important environmental factor that affects crop production and will be exacerbated in the current climate change scenario. Thus, the presence of carbonates and high pH in soils negatively impacts nutrient assimilation and photosynthesis and causes oxidative stress. A potential strategy to improve tolerance to alkalinity could be the modification of cation exchanger (CAX) activity, given that these transporters are involved in calcium (Ca2+) signaling under stresses. In this study, we used three Brassica rapa mutants (BraA.cax1a-4, BraA.cax1a-7, and BraA.cax1a-12) from the parental line 'R-o-18' that were generated by Targeting Induced Local Lesions in Genomes (TILLING) and grown under control and alkaline conditions. The objective was to assess the tolerance of these mutants to alkalinity stress. Biomass, nutrient accumulation, oxidative stress, and photosynthesis parameters were analyzed. The results showed that BraA.cax1a-7 mutation was negative for alkalinity tolerance because it reduced plant biomass, increased oxidative stress, partially inhibited antioxidant response, and lowered photosynthesis performance. Conversely, the BraA.cax1a-12 mutation increased plant biomass and Ca2+ accumulation, reduced oxidative stress, and improved antioxidant response and photosynthesis performance. Hence, this study identifies BraA.cax1a-12 as a useful CAX1 mutation to enhance the tolerance of plants grown under alkaline conditions.

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Gene co-expression network analysis is an efficient systems biology approach for the discovery of novel gene functions and trait-associated gene modules. To identify clusters of functionally related genes involved in soybean nodule formation and development, we performed a weighted gene co-expression network analysis. Two nodule-specific modules (NSM-1 and NSM-2, containing 304 and 203 genes, respectively) were identified. The NSM-1 gene promoters were significantly enriched in cis-binding elements for ERF, MYB, and C2H2-type zinc transcription factors, whereas NSM-2 gene promoters were enriched in cis-binding elements for TCP, bZIP, and bHLH transcription factors, suggesting a role of these regulatory factors in the transcriptional activation of nodule co-expressed genes. The co-expressed gene modules included genes with potential novel roles in nodulation, including those involved in xylem development, transmembrane transport, the ethylene signalling pathway, cytoskeleton organization, cytokinesis and regulation of the cell cycle, regulation of meristem initiation and growth, transcriptional regulation, DNA methylation, and histone modifications. Functional analysis of two co-expressed genes using TILLING mutants provided novel insight into the involvement of unsaturated fatty acid biosynthesis and folate metabolism in nodule formation and development. The identified gene co-expression modules provide valuable resources for further functional genomics studies to dissect the genetic basis of nodule formation and development in soybean.

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The opium poppy's ability to produce various alkaloids is both useful and problematic. Breeding of new varieties with varying alkaloid content is therefore an important task. In this paper, the breeding technology of new low morphine poppy genotypes, based on a combination of a TILLING approach and single-molecule real-time NGS sequencing, is presented. Verification of the mutants in the TILLING population was obtained using RT-PCR and HPLC methods. Only three of the single-copy genes of the morphine pathway among the eleven genes were used for the identification of mutant genotypes. Point mutations were obtained only in one gene (CNMT) while an insertion was obtained in the other (SalAT). Only a few expected transition SNPs from G:C to A:T were obtained. In the low morphine mutant genotype, the production of morphine was decreased to 0.1% from 1.4% in the original variety. A comprehensive description of the breeding process, a basic characterization of the main alkaloid content, and a gene expression profile for the main alkaloid-producing genes is provided. Difficulties with the TILLING approach are also described and discussed.

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Micronutrient malnutrition affects more than half of the world population. Reduced bioavailability of microelements in the raw materials is considered one of the main causes of mineral deficiency in populations whose diet is largely based on the consumption of staple crops. In this context, the production of low phytic acid (lpa) cereals is a main goal of the breeding programs, as phytic acid (PA) binds essential mineral cations such as iron (Fe), zinc (Zn), manganese (Mn), potassium (K), calcium (Ca) and magnesium (Mg) precipitating in the form of phytate salts poorly digested by monogastric animals, including humans, due to the lack of phytases in the digestive tract. Since PA limits the bioavailability of microelements, it is widely recognized as an anti-nutritional compound. A Targeting Induced Local Lesions IN Genomes (TILLING) approach has been undertaken to silence the genes encoding the TdABCC13 proteins, known as Multidrug-Resistance associated Proteins 3 (TdMRP3), transporters involved in the accumulation of PA inside the vacuole in durum wheat. The TdMRP3 complete null genotypes showed a significant reduction in the content of PA and were able to accumulate a higher amount of essential micronutrients (Fe, Zn, Mn) compared to the control. The number of spikelets and seeds per spike, traits associated with the agronomic performances, were reduced compared to the control, but the negative effect was in part balanced by the increased grain weight. The TdMRP3 mutant lines showed morphological differences in the root apparatus such as a significant decrease in the number of root tips, root length, volume and surface area and an increase in root average diameter compared to the control plants. These materials represent a promising basis for obtaining new commercial durum wheats with higher nutritional value.

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There is limited information regarding the morphometric relationships of panicle traits in oat (Avena sativa) and their contribution to phenology and growth, physiology, and pathology traits important for yield. To model panicle growth and development and identify genomic regions associated with corresponding traits, 10 diverse spring oat mapping populations (n = 2,993) were evaluated in the field and 9 genotyped via genotyping-by-sequencing. Representative panicles from all progeny individuals, parents, and check lines were scanned, and images were analyzed using manual and automated techniques, resulting in over 60 unique panicle, rachis, and spikelet variables. Spatial modeling and days to heading were used to account for environmental and phenological variances, respectively. Panicle variables were intercorrelated, providing reproducible archetypal and growth models. Notably, adult plant resistance for oat crown rust was most prominent for taller, stiff stalked plants having a more open panicle structure. Within and among family variance for panicle traits reflected the moderate-to-high heritability and mutual genome-wide associations (hotspots) with numerous high-effect loci. Candidate genes and potential breeding applications are discussed. This work adds to the growing genetic resources for oat and provides a unique perspective on the genetic basis of panicle architecture in cereal crops.

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Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

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Transcriptional reprogramming is an essential feature of plant immunity and is governed by transcription factors (TFs) and co-regulatory proteins associated with discrete transcriptional complexes. On the other hand, effector proteins from pathogens have been shown to hijack these vast repertoires of plant TFs. Our current knowledge of host genes' role (including TFs) involved in pathogen colonization is based on research employing model plants such as Arabidopsis and rice with minimal efforts in wheat rust interactions. In this study, we begun the research by identifying wheat genes that benefit rust pathogens during infection and editing those genes to provide wheat with passive resistance to rust. We identified the wheat MYC4 transcription factor (TF) located on chromosome 1B (TaMYC4-1B) as a rust pathogen target. The gene was upregulated only in susceptible lines in the presence of the pathogens. Down-regulation of TaMYC4-1B using barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) in the susceptible cultivar Chinese Spring enhanced its resistance to the stem rust pathogen. Knockout of the TaMYC4-1BL in Cadenza rendered new resistance to races of stem, leaf, and stripe rust pathogens. We developed new germplasm in wheat via modifications of the wheat TaMYC4-1BL transcription factor.