A route to de novo domestication of wild allotetraploid rice

Reap the crop wild relatives for breeding future crops.

Reap the crop wild relatives for breeding future crops

Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L–1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L–1 1-naphthylacetic acid (NAA), 2.0 mg⋅L–1 6-benzylaminopurine (BAP), and 3.0 mg⋅L–1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L–1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo.

https://www.frontiersin.org/articles/10.3389/fpls.2022.822022/pdf

An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis)

https://www.researchsquare.com/article/rs-513976/v2.pdf?c=1620850052000

Rice breeding in the new era: comparison of useful agronomic traits

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

/pdf/agrobacterium-mediated-genetic-transformation-of-wild-oryza-318gk7sksv.pdf

Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos.

OryzaGenome (
 http://viewer.shigen.info/oryzagenome21detail/index.xhtml
 ), a feature within Oryzabase (
 https://shigen.nig.ac.jp/rice/oryzabase/
 ), is a genomic database for wild Oryza species that provides comparative and evolutionary genomics approaches for the rice research community. Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza species (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula, O. punctata, O. minuta, O. officinalis, O. rhizomatis, O. eichingeri, O. latifolia, O. alta, O. grandiglumis, O. australiensis, O. brachyantha, O. granulata, O. meyeriana, O. ridleyi, and O. longiglumis). These 19 wild species belong to 9 genome types (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ), representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region (e.g., are missense or nonsense mutations). Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication. OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.

/pdf/oryzagenome2-1-database-of-diverse-genotypes-in-wild-oryza-18ms1bhgqf.pdf

OryzaGenome2.1: Database of Diverse Genotypes in Wild Oryza Species.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.

/pdf/specification-of-basal-region-identity-after-asymmetric-s887xpj2e0.pdf

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice

Small RNAs are major components of gene regulatory pathways conserved among eukaryotes. In basic and applied sciences, RNA interference (RNAi) and artificial microRNAs (amiRNAs) are often used to modulate gene expression. The molecular mechanisms of RNAi are mainly studied in nematode or insect cells as models. Functional analyses of endogenous small RNAs, including studies of rice as a model, have greatly contributed to our understanding of plant biology. In plants, small RNA-based gene regulation has unique characteristics not found in animals, and many small RNAs regulate biological phenomena specific to plants. Recently, small RNA profiling using next-generation sequencers became possible, and various small RNA species were identified in plants including rice; their functional analyses are underway. This chapter summarizes the components of small RNA pathways, the molecular species of small RNAs, and the unique function of small RNAs in rice. It also considers the functions of small RNAs in relation to agriculturally important traits.

Toshiya Suzuki

Papers

Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos.

OryzaGenome2.1: Database of Diverse Genotypes in Wild Oryza Species.

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice

Small RNAs in Rice: Molecular Species and Their Functions