Redirecting barley breeding for grass production through genome editing of Photoperiod-H1

Abstract

Genome editing enables precise modification to harness an elite grain-producing barley (Hordeum vulgare L.) cultivar for grass production.

Figure 1.

Creation of the ppd-H1 mutants by in planta particle bombardment. A) Schematic representation of the Ppd-H1 gene with gRNA design. The white and gray boxes signify untranslated regions and exons, respectively. Red characters within the gRNA sequences denote the protospacer adjacent motif (PAM), while underlines highlight the restriction enzyme sites intended for CAPS assays. B) Noted genome-edited mutations in each CAPS-positive E₀ plant. Mutations introduced by genome editing are highlighted. C) Genotypic analysis of E₁ offspring derived from E₀ NH7 and E₀ NH3 plants. “−” and “+” denote digestion with and without restriction enzymes, respectively. D) Detailed sequence evaluation of genome-edited E₁ homozygous plants, specifically NH7-6 and NH3-2. E) Overview of the genome editing experiments.

Figure 2.

Phenotype of the ppd-H1 mutants and expression analyses of flowering genes. A and B) Examination of the heading date in wild-type plants vis-à-vis the ppd-H1 mutants. Both wild-type and ppd-H1 mutant plants were cultivated under LD conditions (16 h of light followed by 8 h of darkness). Center lines in boxplots show the medians; box limits indicate the 25th and 75th percentiles; whiskers are the minimum and maximum of all data (n = 6). Distinct letters indicate significant differences as determined by Tukey's test (P < 0.01). The scale bar represents 20 cm. DAG, days after germination. C) Grass production assessment of ppd-H1 mutants. The aboveground segments of plants, taken just prior to inflorescence evolution, underwent drying at 85 °C over a 7-d period before weighing. Center lines in boxplots show the medians; box limits indicate the 25th and 75th percentiles; whiskers are the minimum and maximum of all data (n = 6). Distinct letters indicate significant differences as determined by Tukey's test (P < 0.01). D) Circadian patterns of flowering gene expressions in ppd-H1 mutants. Total RNA was procured from mature leaves of 14-d-old plants grown in LD conditions (16 h light/8 h dark intervals). The graph’s shaded area marks the dark phase. The provided data are averaged with the Se based on 4 replications (n = 4). An asterisk indicates a time point at which both ppd-H1 mutants demonstrated significant differences compared with the wild type, as determined by Tukey's test (P < 0.05). A detailed summary of the statistical analysis is presented in Supplementary Table S2. Specific primers employed for the RT-qPCR can be found in Supplementary Table S1. E)HvFT1 gene expression observed during the vegetative growth phase. The total RNA was isolated from mature leaves at sequential time points: 7, 14, and 21 DAG. Data delineates the mean ± Se with 4 replicates (n = 4). Distinct letters signify remarkable differences based on Tukey's test (P < 0.01).