Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors

The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the SiFMBP, SiDof4, SiBADH2, SiGBSS1, and SiIPK1 genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an Agrobacterium-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T0 generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the SiALS and SiACC genes of foxtail millet. By utilizing CBE to target the SiALS gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.

Downregulation of PyHRG1, encoding a novel secretory protein in the red alga Pyropia yezoensis, enhances heat tolerance

An increase in seawater temperature owing to global warming is expected to substantially limit the growth of marine algae, including Pyropia yezoensis, a commercially valuable red alga. To improve our knowledge of the genes involved in the acquisition of heat tolerance in P. yezoensis, transcriptomes sequences were obtained from both the wild-type SG104 P. yezoensis and heat-tolerant mutant Gy500. We selected 1,251 differentially expressed genes that were up- or downregulated in response to the heat stress condition and in the heat-tolerant mutant Gy500, based on fragment per million reads expression values. Among them, PyHRG1 was downregulated under heat stress in SG104 and expressed at a low level in Gy500. PyHRG1 encodes a secretory protein of 26.5 kDa. PyHRG1 shows no significant sequence homology with any known genes deposited in public databases to date. However, PyHRG1 homologs were found in other red algae, including other Pyropia species. When PyHRG1 was introduced into the single-cell green alga Chlamydomonas reinhardtii, transformed cells overexpressing PyHRG1 showed severely retarded growth. These results demonstrate that PyHRG1 encodes a novel red algae-specific protein and plays a role in heat tolerance in algae. The transcriptome sequences obtained in this study, which include PyHRG1, will facilitate future studies to understand the molecular mechanisms involved in heat tolerance in red algae.

Field performance of heat tolerant mutant rice lines generated from Oryza sativa and Oryza glaberrima

This study evaluated mutant lines developed from two cultivated species of upland rice, Oryza sativa, and Oryza glaberrima, in field experiments conducted during the hot and dry seasons of 2014/2015 in Morogoro, Tanzania. The growth yield and yield components of 34 and 14 Gamma induced mutant upland rice lines developed from O. sativa (Kihogo red) and O. glaberrima were evaluated, respectively. The mutant lines were selected based on variable expression of heat shock protein genes (HSPs) in previously conducted heat tolerance studies. The minimum and maximum temperatures and rainfall during the field performance experiment were measured between 20 oC and 35 oC, and 32.7 mm and 155.5 mm, respectively. The data for 12 yield and yield component parameters such as days to early and 50% flowering, days to physical maturity, plant height, number of tillers, number of panicles, spikelets, filled grains, unfilled grains and 1,000 grain weights were collected and analysed using ANOVA and Principal Component Analysis. Significant differences (P≤0.05) were obtained among the mutant lines in terms of grain yield, spikelet sterility and other variables, which were further used as criteria for selection of heat and drought tolerant rice lines. Eight heat and drought tolerant mutant rice lines with high yields (over 3.5 ton/ha) and low spikelet sterility were selected for further advancement in breeding programmes

National repository of EMS induced mutants of an upland rice cultivar Nagina 22: progress update on characterization and utilization.

The Indian initiative for creating mutant resources in rice has generated 87,000 mutants in the background of a popular drought- and heat-tolerant upland cultivar, Nagina 22 (N22), through EMS mutagenesis. So far, 541 macro-mutants from this resource have been identified, maintained in the mutant garden and characterized in detail based on 44 descriptors pertaining to distinctness, uniformity and stability (DUS) of rice and other agronomic parameters. The similarity index of the mutants was more than 0.6 for nearly 90% of the mutants with respect to DUS descriptors, further establishing the validity of the mutants. The available high-quality sequence resource of N22 has been improved by reducing the gaps by 0.02% in the coding sequence (CDS) region. This was made possible using the newly synthesized whole-genome data of N22 which helped to remove 9006 'Ns' and replace 12,746 existing nucleotides with the accurate ones. These sequence and morphological details have been updated in the mutant database 'EMSgardeN22'. Further, 1058 mutants have been identified for low-P tolerance, tolerance to sheath blight, blast, drought, heat, higher photosynthetic efficiency and agronomic and root traits from this resource. A novel herbicide-tolerant (imazethapyr) mutant earlier identified and characterized from this resource is now being used in introgressing the herbicide-tolerant trait in eight major rice varieties in India. Further, robust and simpler screening systems have been tested for studying low-P tolerance of the mutants. A grain-size mutant, heat-tolerant mutant, drought-tolerant mutant, stay-green mutant and low-P tolerant and water-use efficient high-root-volume mutants have been characterized at morphological and molecular levels. A brief account of all these mutants, the entire mutant resource and the elaborate trait-based screenings is presented in this chapter.