Breeding herbicide-resistant rice (Oryza sativa) using CRISPR/Cas gene editing and other technologies.

The emergence of herbicide-resistant weeds in crop fields and the extensive use of herbicides have led to a decrease in rice (Oryza sativa) yields and an increase in production costs. To address these challenges, researchers have focused on discovering new germplasm resources with herbicide resistance. The most promising candidates among these genes have been functionally studied and applied in rice breeding. Here, we review recent progress in breeding herbicide-resistant rice. We provide examples of breeding herbicide-resistant rice using various techniques, such as physical and chemical mutagenesis, genetic transformation, and CRISPR/Cas-mediated gene editing. Moreover, we highlight factors involved in breeding herbicide-resistant rice, including target genes, rice varieties, degrees of herbicide resistance, and the tools employed. Finally, we suggest methods for breeding herbicide-resistant rice that could potentially be applied for weed management in direct seeding farming systems.

Rice seed storability: From molecular mechanisms to agricultural practices.

The storability of rice seeds is crucial for ensuring flexible planting options, agricultural seed security, and global food safety. With the intensification of global climate change and the constant fluctuations in agricultural production conditions, enhancing the storability of rice seeds has become particularly important. Seed storability is a complex quantitative trait regulated by both genetic and environmental factors. This article reviews the main regulatory mechanisms of rice seed storability, including the accumulation of seed storage proteins, late embryogenesis abundant (LEA) proteins, heat shock proteins, sugar signaling, hormonal regulation by gibberellins and abscisic acid, and the role of the ubiquitination pathway. Additionally, this article explores the improvement of storability using wild rice genes, molecular marker-assisted selection, and gene editing techniques such as CRISPR/Cas9 in rice breeding. By providing a comprehensive scientific foundation and practical guidance, this review aims to promote the development of rice varieties with enhanced storability to meet evolving agricultural demands.

Wild rice: unlocking the future of rice breeding.

Germplasm resources serve as the foundations of advancements in breeding and are crucial for maintaining food security. Wild rice species of the genus Oryza include rich sources of genetic diversity and high adaptability, making them a substantial resource for rice breeding. The discovery of wild-type cytoplasmic male sterility resources enabled the achievement of the 'three lines' goal in hybrid rice, significantly increasing rice yields. The application of resistance alleles from wild rice enables rice production to withstand losses caused by stress. Reduced genetic diversity due to rice breeding poses a significant limitation to further advances and can be alleviated through a systematic use of wild genetic resources that integrate geographic, climatic and environmental data of the original habitat, along with extensive germplasm collection and identification using advanced methods. Leveraging technological advancements in plant genomics, the understanding of genetic mechanisms and the application of artificial intelligence and gene editing can further enhance the efficiency and accuracy of this process. These advancements facilitate rapid isolation and functional studies of genes, and precise genome manipulation. This review systematically summarizes the utilization of superior genes and germplasm resources derived from wild rice sources, while also exploring the collection, conservation, identification and utilization of further wild rice germplasm resources. A focus on genome sequencing and biotechnology developments is leading to new breeding and biotechnology opportunities. These new opportunities will not only promote the development of rice varieties that exhibit high yields, superior stress resistance and high quality but also expand the genetic diversity among rice cultivars.

Origins of Susceptibility to Insect Herbivores in High-Yielding Hybrid and Inbred Rice Genotypes.

Several studies have reported higher damage from insect herbivores to hybrid compared to inbred (pure line) rice. We used a collection of 20 hybrid and 12 inbred genotypes from diverse origins to test the hypotheses that hybrid rice susceptibility is due to (a) the hybrid plant type and/or (b) rice phylogeny. We challenged the genotypes with Nilaparvata lugans (BPH), Sogatella furcifera (WBPH) and Scirpophaga incertulas (YSB) in greenhouse and screenhouse bioassays and monitored herbivores in field plots. We used single nucleotide polymorphic (SNP) markers to assess genetic similarities between the genotypes and found that the hybrids and inbreds formed two distinct clusters regardless of origin. In the screenhouse, hybrids were more susceptible than inbreds to YSB; however, resistant hybrids and susceptible inbreds were also apparent from both the screenhouse and field plots. Plant biomass was the best predictor of susceptibility to YSB. Plant origin had a greater effect than plant type on susceptibility to BPH and WBPH. WBPH was the most abundant planthopper in the field plots where numbers were highly correlated with planthopper fitness in the greenhouse bioassays. Our results provide evidence that high-yielding hybrids that are relatively resistant to herbivores can be achieved through careful breeding. The avoidance of susceptible genotypes during breeding should remain a key element of integrated rice pest management.

GWAS analysis reveals the genetic basis of blast resistance associated with heading date in rice.

Rice blast is a destructive fungal disease affecting rice plants at various growth stages, significantly threatening global yield stability. Development of resistant rice cultivars stands as a practical means of disease control. Generally, association mapping with a diversity panel powerfully identifies new alleles controlling trait of interest. On the other hand, utilization of a breeding panel has its advantage that can be directly applied in a breeding program. In this study, we conducted a genome-wide association study (GWAS) for blast resistance using 296 commercial rice cultivars with low population structure but large phenotypic diversity. We attempt to answer the genetic basis behind rice blast resistance among early maturing cultivars by subdividing the population based on its Heading date 1 (Hd1) functionality. Subpopulation-specific GWAS using the mixed linear model (MLM) based on blast nursery screening conducted in three years revealed a total of 26 significant signals, including three nucleotide-binding site leucine-rich repeat (NBS-LRR) genes (Os06g0286500, Os06g0286700, and Os06g0287500) located at Piz locus on chromosome 6, and one at the Pi-ta locus (Os12g0281300) on chromosome 12. Haplotype analysis revealed blast resistance associated with Piz locus was exclusively specific to Type 14 hd1 among japonica rice. Our findings provide valuable insights for breeding blast resistant rice and highlight the applicability of our elite cultivar panel to detect superior alleles associated with important agronomic traits.

Mapping and Omics Integration: Towards Precise Rice Disease Resistance Breeding.

Rice (Oryza sativa), as a staple crop feeding a significant portion of the global population, particularly in Asian countries, faces constant threats from various diseases jeopardizing global food security. A precise understanding of disease resistance mechanisms is crucial for developing resilient rice varieties. Traditional genetic mapping methods, such as QTL mapping, provide valuable insights into the genetic basis of diseases. However, the complex nature of rice diseases demands a holistic approach to gain an accurate knowledge of it. Omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, enable a comprehensive analysis of biological molecules, uncovering intricate molecular interactions within the rice plant. The integration of various mapping techniques using multi-omics data has revolutionized our understanding of rice disease resistance. By overlaying genetic maps with high-throughput omics datasets, researchers can pinpoint specific genes, proteins, or metabolites associated with disease resistance. This integration enhances the precision of disease-related biomarkers with a better understanding of their functional roles in disease resistance. The improvement of rice breeding for disease resistance through this integration represents a significant stride in agricultural science because a better understanding of the molecular intricacies and interactions underlying disease resistance architecture leads to a more precise and efficient development of resilient and productive rice varieties. In this review, we explore how the integration of mapping and omics data can result in a transformative impact on rice breeding for enhancing disease resistance.

PlantMine: A Machine-Learning Framework to Detect Core SNPs in Rice Genomics.

As a fundamental global staple crop, rice plays a pivotal role in human nutrition and agricultural production systems. However, its complex genetic architecture and extensive trait variability pose challenges for breeders and researchers in optimizing yield and quality. Particularly to expedite breeding methods like genomic selection, isolating core SNPs related to target traits from genome-wide data reduces irrelevant mutation noise, enhancing computational precision and efficiency. Thus, exploring efficient computational approaches to mine core SNPs is of great importance. This study introduces PlantMine, an innovative computational framework that integrates feature selection and machine learning techniques to effectively identify core SNPs critical for the improvement of rice traits. Utilizing the dataset from the 3000 Rice Genomes Project, we applied different algorithms for analysis. The findings underscore the effectiveness of combining feature selection with machine learning in accurately identifying core SNPs, offering a promising avenue to expedite rice breeding efforts and improve crop productivity and resilience to stress.

[Experimental teaching design of CRISPR/Cas9 technology in rice breeding application].

The CRISPR/Cas9 gene editing technology has proven to be valuable in crop breeding applications. Understanding and mastering this technology will provide a strong foundation for students majoring in biology, agronomy, and related fields to engage in scientific research and work. To incorporate CRISPR/Cas9 technology into experimental teaching courses at colleges, an innovative teaching experiment entitled "Enhancing the resistance of rice plants to bacterial blight disease using CRISPR/Cas9 technology" was designed. The experiment allows students to deepen their understanding of the basic principles of CRISPR/Cas technology, acquire proficiency in its protocol, and learn to apply the technology for targeted molecular breeding of rice. It not only expands students' knowledge and skills, but also promotes the reform and innovation of experimental teaching methods.

Hybrid Rice Production: A Worldwide Review of Floral Traits and Breeding Technology, with Special Emphasis on China.

Rice is an important diet source for the majority of the world's population, and meeting the growing need for rice requires significant improvements at the production level. Hybrid rice production has been a significant breakthrough in this regard, and the floral traits play a major role in the development of hybrid rice. In grass species, rice has structural units called florets and spikelets and contains different floret organs such as lemma, palea, style length, anther, and stigma exsertion. These floral organs are crucial in enhancing rice production and uplifting rice cultivation at a broader level. Recent advances in breeding techniques also provide knowledge about different floral organs and how they can be improved by using biotechnological techniques for better production of rice. The rice flower holds immense significance and is the primary focal point for researchers working on rice molecular biology. Furthermore, the unique genetics of rice play a significant role in maintaining its floral structure. However, to improve rice varieties further, we need to identify the genomic regions through mapping of QTLs (quantitative trait loci) or by using GWAS (genome-wide association studies) and their validation should be performed by developing user-friendly molecular markers, such as Kompetitive allele-specific PCR (KASP). This review outlines the role of different floral traits and the benefits of using modern biotechnological approaches to improve hybrid rice production. It focuses on how floral traits are interrelated and their possible contribution to hybrid rice production to satisfy future rice demand. We discuss the significance of different floral traits, techniques, and breeding approaches in hybrid rice production. We provide a historical perspective of hybrid rice production and its current status and outline the challenges and opportunities in this field.

Genetic and molecular mechanisms of reproductive isolation in the utilization of heterosis for breeding hybrid rice.

Heterosis, also known as hybrid vigor, is commonly observed in rice crosses. The hybridization of rice species or subspecies exhibits robust hybrid vigor, however, the direct harnessing of this vigor is hindered by reproductive isolation. Here, we review recent advances in the understanding of the molecular mechanisms governing reproductive isolation in inter-subspecific and inter-specific hybrids. This review encompasses the genetic model of reproductive isolation within and among Oryza sativa species, emphasizing the essential role of mitochondria in this process. Additionally, we delve into the molecular intricacies governing the interaction between mitochondria and autophagosomes, elucidating their significant contribution to reproductive isolation. Furthermore, our exploration extends to comprehending the evolutionary dynamics of reproductive isolation and speciation in rice. Building on these advances, we offer a forward-looking perspective on how to overcome the challenges of reproductive isolation and facilitate the utilization of heterosis in future hybrid rice breeding endeavors.

Multi-Omics Approach Reveals OsPIL1 as a Regulator Promotes Rice Growth, Grain Development, and Blast Resistance.

Rice (Oryza sativa) is a crucial crop, achieving high yield concurrent pathogen resistance remains a challenge. Transcription factors play roles in growth and abiotic tolerance. However, rice phytochrome-interacting factor-like 1 (OsPIL1) in pathogen resistance and agronomic traits remains unexplored. We generated OsPIL1 overexpressing (OsPIL1 OE) rice lines and evaluated their impact on growth, grain development, and resistance to Magnaporthe oryzae. Multiomics analysis (RNA-seq, metabolomics, and CUT&Tag) and RT-qPCR validated OsPIL1 target genes and key metabolites. In the results, OsPIL1 OE rice lines exhibited robust growth, longer grains, and enhanced resistance to M. oryzae without compromising growth. Integrative multiomics analysis revealed a coordinated regulatory network centered on OsPIL1, explaining these desirable traits. OsPIL1 likely acts as a positive regulator, targeting transcriptional elements or specific genes with direct functions in several biological programs. In particular, a range of key signaling genes (phosphatases, kinases, plant hormone genes, transcription factors), and metabolites (linolenic acid, vitamin E, trigonelline, d-glucose, serotonin, choline, genistein, riboflavin) contributed to enhanced rice growth, grain size, pathogen resistance, or a combination of these traits. These findings highlight OsPIL1's regulatory role in promoting important traits and provide insights into potential strategies for rice breeding.

A historical review of hybrid rice breeding.

The development of germplasm resources and advances in breeding methods have led to steady increases in yield and quality of rice (Oryza sativa L.). Three milestones in the recent history of rice breeding have contributed to these increases: dwarf rice breeding, hybrid rice breeding, and super rice breeding. On the 50th anniversary of the success of three-line hybrid rice, we highlight important scientific discoveries in rice breeding that were made by Chinese scientists and summarize the broader history of the field. We discuss the strategies that could be used in the future to optimize rice breeding further in the hope that China will continue to play a leading role in international rice breeding.

An efficient method for screening rice breeding lines against races of Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae, is the most destructive rice disease worldwide. The disease symptoms are usually expressed on the leaf and panicle. The leaf disease intensity in controlled environmental conditions is frequently quantified using a 0-5 scale, where 0 represents the absence of symptoms and 5 represents large eyespot lesions. However, this scale restricts the qualitative classification of the varieties into intermediate resistant and susceptible categories. Here we develop a 0-6 scale for blast disease that allows proper assignment of rice breeding lines and varieties into six resistance levels (highly resistant, resistant, moderate resistant, moderate susceptible, susceptible, and highly susceptible). We evaluated 41 common rice varieties against four major blast races (IB1, IB17, IB49, and IE1-K). Varieties carrying the Pi-ta gene were either highly resistant, resistant, or moderate resistant to IB17. The IE1-K race was able to break Pi-ta-mediate resistance of the rice varieties. The Pi-z gene conferred resistance to the IB17 and IE1-K races. The varieties M201, Cheniere, and Frontier were highly susceptible (score 6; 100% disease) to the race IE1-K. Moreover, varieties that were resistant or susceptible to all four blast races also showed similar levels of resistance/susceptibility to blast disease in the field. Taken together, our data proved that the 0-6 blast scale can efficiently determine the resistance levels of rice varieties against major blast races. This robust method will assist rice breeding programs to incorporate durable resistance against major and emerging blast races.

Photoperiod Genes Contribute to Daylength-Sensing and Breeding in Rice.

Rice (Oryza sativa L.), one of the most important food crops worldwide, is a facultative short-day (SD) plant in which flowering is modulated by seasonal and temperature cues. The photoperiodic molecular network is the core network for regulating flowering in rice, and is composed of photoreceptors, a circadian clock, a photoperiodic flowering core module, and florigen genes. The Hd1-DTH8-Ghd7-PRR37 module, a photoperiodic flowering core module, improves the latitude adaptation through mediating the multiple daylength-sensing processes in rice. However, how the other photoperiod-related genes regulate daylength-sensing and latitude adaptation remains largely unknown. Here, we determined that mutations in the photoreceptor and circadian clock genes can generate different daylength-sensing processes. Furthermore, we measured the yield-related traits in various mutants, including the main panicle length, grains per panicle, seed-setting rate, hundred-grain weight, and yield per panicle. Our results showed that the prr37, elf3-1 and ehd1 mutants can change the daylength-sensing processes and exhibit longer main panicle lengths and more grains per panicle. Hence, the PRR37, ELF3-1 and Ehd1 locus has excellent potential for latitude adaptation and production improvement in rice breeding. In summary, this study systematically explored how vital elements of the photoperiod network regulate daylength sensing and yield traits, providing critical information for their breeding applications.

Identification of a Major Locus for Lodging Resistance to Typhoons Using QTL Analysis in Rice.

We detected a new target quantitative trait locus (QTL) for lodging resistance in rice by analyzing lodging resistance to typhoons (Maysak and Haishen) using a scale from 0 (no prostrating) to 1 (little prostrating or prostrating) to record the resistance score in a Cheongcheong/Nagdong double haploid rice population. Five quantitative trait loci for lodging resistance to typhoons were detected. Among them, qTyM6 and qTyH6 exhibited crucial effects of locus RM3343-RM20318 on chromosome 6, which overlaps with our previous rice lodging studies for the loci qPSLSA6-2, qPSLSB6-5, and qLTI6-2. Within the target locus RM3343-RM20318, 12 related genes belonging to the cytochrome P450 protein family were screened through annotation. Os06g0599200 (OsTyM/Hq6) was selected for further analysis. We observed that the culm and panicle lengths were positively correlated with lodging resistance to typhoons. However, the yield was negatively correlated with lodging resistance to typhoons. The findings of this study improve an understanding of rice breeding, particularly the culm length, early maturing, and heavy panicle varieties, and the mechanisms by which the plant's architecture can resist natural disasters such as typhoons to ensure food safety. These results also provide the insight that lodging resistance in rice may be associated with major traits such as panicle length, culm length, tiller number, and heading date, and thereby improvements in these traits can increase lodging resistance to typhoons. Moreover, rice breeding should focus on maintaining suitable varieties that can withstand the adverse effects of climate change in the future and provide better food security.

Potentially Useful Dwarfing or Semi-dwarfing Genes in Rice Breeding in Addition to the sd1 Gene.

The "Green revolution" gene sd1 has been used widely in the breeding of modern rice varieties for over half a century. The application of this gene has increased rice yields and thereby supported a significant proportion of the global population. The use of a single gene, however, has raised concerns in the scientific community regarding its durability, especially given the bottleneck in genetic background and the need for large input of fertilizer. New dwarfing or semi-dwarfing genes are needed to alleviate our dependence on the sole "Green revolution" gene. In the past few years, several new dwarfing and semi-dwarfing genes as well as their mutants have been reported. Here, we provide an extensive review of the recent discoveries concerning newly identified genes that are potentially useful in rice breeding, including methods employed to create and effectively screen new rice mutants, the phenotypic characteristics of the new dwarfing and semi-dwarfing mutants, potential values of the new dwarfing and semi-dwarfing genes in rice breeding, and potential molecular mechanisms associated with the newly identified genes.

Improvement of Rice Agronomic Traits by Editing Type-B Response Regulators.

Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T0 transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by Cas9 specific primer in the T1 generation, and homozygous mutants without Cas9 were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 OsRRs were obtained, except for RR24. Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The osrr30 mutant in the T2 generation was then further examined. As a result, the heading date of the osrr30 mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that osrr30 has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.

Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice.

Cytoplasmic male sterility (CMS) determined by mitochondrial genes and restorer of fertility (Rf) controlled by nuclear-encoded genes provide the breeding systems of many hybrid crops for the utilization of heterosis. Although several CMS/Rf systems have been widely exploited in rice, hybrid breeding using these systems has encountered difficulties due to either fertility instability or complications of two-locus inheritance or both. In this work, we characterized a type of CMS, Fujian Abortive cytoplasmic male sterility (CMS-FA), with stable sporophytic male sterility and a nuclear restorer gene that completely restores hybrid fertility. CMS is caused by the chimeric open reading frame FA182 that specifically occurs in the mitochondrial genome of CMS-FA rice. The restorer gene OsRf19 encodes a pentatricopeptide repeat (PPR) protein targeted to mitochondria, where it mediates the cleavage of FA182 transcripts, thus restoring male fertility. Comparative sequence analysis revealed that OsRf19 originated through a recent duplication in wild rice relatives, sharing a common ancestor with OsRf1a/OsRf5, a fertility restorer gene for Boro II and Hong-Lian CMS. We developed six restorer lines by introgressing OsRf19 into parental lines of elite CMS-WA hybrids; hybrids produced from these lines showed equivalent or better agronomic performance relative to their counterparts based on the CMS-WA system. These results demonstrate that CMS-FA/OsRf19 provides a highly promising system for future hybrid rice breeding.