The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and “transgene clean” targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.

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Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.

Grain protein content (GPC) affects rice nutrition quality. Here, we identify two stable quantitative trait loci (QTLs), qGPC-1 and qGPC-10 , controlling GPC in a mapping population derived from indica and japonica cultivars crossing. Map-based cloning reveals that OsGluA2 , encoding a glutelin type-A2 precursor, is the candidate gene underlying qGPC-10 . It functions as a positive regulator of GPC and has a pleiotropic effect on rice grain quality. One SNP located in OsGluA2 promoter region is associated with its transcript expression level and GPC diversity. Polymorphisms of this nucleotide can divide all haplotypes into low ( OsGluA2 LET ) and high ( OsGluA2 HET ) expression types. Population genetic and evolutionary analyses reveal that OsGluA2 LET , mainly present in japonica accessions, originates from wild rice. However, OsGluA2 HET , the dominant type in indica , is acquired through mutation of OsGluA2 LET . Our results shed light on the understanding of natural variations of GPC between indica and japonica subspecies.

The genus Allium is one of the largest monocotyledonous genera, containing over 850 species, and most of these species are found in temperate climates of the Northern Hemisphere. Furthermore, as a large number of new Allium species continue to be identified, phylogenetic classification based on morphological characteristics and a few genetic markers will gradually exhibit extremely low discriminatory power. In this study, we present the use of complete chloroplast genome sequences in genome-scale phylogenetic studies of Allium . We sequenced and assembled four Allium chloroplast genomes and retrieved five published chloroplast genomes from GenBank. All nine chloroplast genomes were used for genomic comparison and phylogenetic inference. The chloroplast genomes, ranging from 152,387 bp to 154,482 bp in length, exhibited conservation of genomic structure, and gene organization and order. Subsequently, we observed the expansion of IRs from the basal monocot Acorus americanus to Allium , identified 814 simple sequence repeats, 131 tandem repeats, 154 dispersed repeats and 109 palindromic repeats, and found six highly variable regions. The phylogenetic relationships of the Allium species inferred from the chloroplast genomes obtained high support, indicating that chloroplast genome data will be useful for further resolution of the phylogeny of the genus Allium .

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Leaves, the collective organ produced by the shoot apical meristem (SAM), are polarized along their adaxial-abaxial axis. In this study, we characterized two rice (Oryza sativa) allelic rolled-leaf mutants, rolled leaf 9-1 (rl9-1) and rl9-2, which display very similar phenotypes with completely adaxialized leaves and malformed spikelets. We cloned the RL9 gene by way of a map-based cloning strategy. Molecular studies have revealed that RL9 encodes a GARP protein, an orthologue of Arabidopsis KANADIs. RL9 is mainly expressed in roots, leaves, and flowers. The transient expression of a RL9-GFP (green fluorescent protein) fusion protein has indicated that RL9 protein is localized in the nucleus, suggesting that RL9 acts as a putative transcription factor.

Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2· -is involved in the process.