Targeted genome modifications in cereal crops

The recent advent of customizable endonucleases has led to remarkable advances in genetic engineering, as these molecular scissors allow for the targeted introduction of mutations or even precisely predefined genetic modifications into virtually any genomic target site of choice. Thanks to its unprecedented precision, efficiency, and functional versatility, this technology, commonly referred to as genome editing, has become an effective force not only in basic research devoted to the elucidation of gene function, but also for knowledgebased improvement of crop traits. Among the different platforms currently available for site-directed genome modifications, RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases have proven to be the most powerful. This review provides an application-oriented overview of the development of customizable endonucleases, current approaches to cereal crop breeding, and future opportunities in this field

Design in Type-I, Run in Type-III: Fast and Scalable Bilinear-Type Conversion using Integer Programming

Bilinear-type conversion is to convert cryptographic schemes designed over symmetric groups instantiated with imperilled curves into ones that run over more secure and efficient asymmetric groups. In this paper we introduce a novel type conversion method called {\em IPConv} using 0-1 Integer Programming. Instantiated with a widely available IP solver, it instantly converts existing intricate schemes, and can process large-scale schemes that involves more than a thousand variables and hundreds of pairings. Such a quick and scalable method allows a new approach in designing cryptographic schemes over asymmetric bilinear groups. Namely, designers work without taking much care about asymmetry of computation but the converted scheme runs well in the asymmetric setting. We demonstrate the usefulness of conversion-aided design by presenting somewhat counter-intuitive examples where converted DLIN-based Groth-Sahai proofs are more compact than manually built SXDH-based proofs

Protocol for Genome Editing to Produce Multiple Mutants in Wheat

Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019)

Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar ‘Fielder’

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar ‘Fielder’, an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies

Tamyb10-D1 restores red grain color and increases grain dormancy via suppressing expression of TaLTP2.128, non-specific lipid transfer protein in wheat

Grain dormancy of wheat is closely associated with grain color: red-grained lines show higher dormancy than white-grained lines. The production of red pigments is regulated by R-1, Tamyb10 gene. However, the relation between grain color and dormancy remains unknown. For this study, we generated transgenic lines which were introduced a DNA fragment containing Tamyb10-D1 gene and its a 2 kb promoter including the 5′ untranslated region into white-grained wheat. Transgenic lines showed red-grained and higher dormant traits. Contents of plant hormones and gene expression of embryos at 30 days after pollination were examined in a wild type and a transgenic line. No differences were observed in the contents of plant hormones, but several genes are differentially expressed between these lines. One differentially expressed gene, TaLTP2.128, is a member of non-specific lipid transfer proteins. It was expressed higher in white grains than in red grains. A putative amino acid sequence showed similarity to that of OsHyPRP5, which is identified as QTL controlling low-temperature germinability in rice. Expression of TaLTP2.128 was increased by grain imbibition. The increasing levels were higher not only in other white-grained lines, but also in non-dormant red-grained lines. TaLTP2.128 was expressed at a quite early stage of germination. These study findings indicate that Tamyb10 regulates dormancy release by the modification of TaLTP2.128 acting as trigger of germination

Root Cortex Provides a Venue for Gas-Space Formation and Is Essential for Plant Adaptation to Waterlogging

Lysigenous aerenchyma, which develops by death and subsequent lysis of the cortical cells in roots, is essential for internal long-distance oxygen transport from shoot base to root tips of plants in waterlogged soil. Although many studies focus on the amounts of aerenchyma in roots, significance of the size of the root cortex in which aerenchyma forms has received less research attention. In the present study, we evaluated the cross-sectional area of each root tissue in adventitious roots of upland crops, wheat (Triticum aestivum) and maize (Zea mays ssp. mays), and the wetland crop, rice (Oryza sativa) under aerated or stagnant deoxygenated conditions; the latter can mimic the changes in gas composition in waterlogged soils. Our analyses revealed that the areas of whole root and cortex of the three species increased under stagnant conditions. In rice roots, cortex to stele ratio (CSR) and aerenchyma to cortex ratio (ACR), which is associated with the areas of gas spaces, were much higher than those in wheat and maize roots, suggesting that these anatomical features are essential for a high capacity for oxygen transport along roots. To test this hypothesis, rates of radial oxygen loss (ROL), which is the diffusive flux of oxygen from within a root to the external medium, from thick and thin adventitious roots of rice were measured using a cylindrical (root-sleeving) oxygen electrode, for plants with shoots in air and roots in an oxygen-free medium. As expected, the rate of ROL from thick roots, which have larger cortex and aerenchyma areas, was higher than that of thin roots. The rate of ROL was highest at the apical part of rice roots, where aerenchyma was hardly detected, but at which cuboidal cell arrangement in the cortex provides tissue porosity. We conclude that high CSR in combination with large root diameter is a feature which promotes oxygen transport from shoot base to root tips of plants. Moreover, we propose that CSR should be a useful quantitative index for the evaluation and improvement of root traits contributing to tolerance of crops to soil waterlogging

Development of a high-resolution two-dimensional detector-based dose verification system for tumor-tracking irradiation in the CyberKnife system

We aim to evaluate the basic characteristics of SRS MapCHECK (SRSMC) for CyberKnife (CK) and establish a dose verification system using SRSMC for the tumor-tracking irradiation for CK. The field size and angular dependence of SRSMC were evaluated for basic characterization. The output factors (OPFs) and absolute doses measured by SRSMC were compared with those measured using microDiamond and microchamber detectors and those calculated by the treatment planning system (TPS). The angular dependence was evaluated by comparing the SRSMC with a microchamber. The tumor-tracking dose verification system consists of SRSMC and a moving platform. The doses measured using SRSMC were compared with the doses measured using a microchamber and radiochromic film. The OPFs and absolute doses of SRSMC were within ±3.0% error for almost all field sizes, and the angular dependence was within ±2.0% for all incidence angles. The absolute dose errors between SRSMC and TPS tended to increase when the field size was smaller than 10 mm. The absolute doses of the tumor-tracking irradiation measured using SRSMC and those measured using a microchamber agreed within 1.0%, and the gamma pass rates of SRSMC in comparison with those of the radiochromic film were greater than 95%. The basic characteristics of SRSMC for CK presented acceptable results for clinical use. The results of the tumor-tracking dose verification system realized using SRSMC were equivalent to those of conventional methods, and this system is expected to contribute toward improving the efficiency of quality control in many facilities

Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs

Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3⁺–basal cells (Tbx3⁺-BCs) and their neighboring cells where Adam8–extracellular signal–regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3⁺-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3⁺-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3⁺-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin

Genome-Edited Triple-Recessive Mutation AltersSeed Dormancy in Wheat

1Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley

Remarks on mix-network based on permutation networks

Abstract. This paper addresses the security and efficiency issues of the Mix-net based on permutation networks introduced in [1]. We first show that the original construction results in a Mix-net that yields biased permutation, so it gives some advantage to adversaries. A simple repair is provided. We then observe that one of the original schemes can be improved so that the servers and verifier enjoy more efficient computation and communication.