Genetic analysis and fine mapping of a grain size QTL in the smallgrain sterile rice line Zhuo201S

The development and application of the small-grain rice sterile line Zhuo201S(Z201S)has demonstrated its potential for mechanized hybrid rice seed production,leading to significant cost reductions.However,the molecular mechanism responsible for the small-grain size characteristic of Z201S remains unclear.In this study,we conducted a genetic analysis using near-isogenic lines constructed from Z210S,a small-grain rice sterile line,and R2115,a normal-grain variety.The results revealed that the small-grain trait in Z201S is governed by a single partially dominant gene which also enhances grain number.Through mapping,we localized the causal gene to the short arm of chromosome 2,within a 113 kb physical region delimited by the molecular markers S2-4-1 and LB63.Transgenic analysis and gene expression assays indicated LOC_Os02g14760 as the most likely candidate gene,suggesting that the small-grain size trait of Z201S is controlled by a novel locus that has not been previously identified.

Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci （QTLs）. We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

Identification and fine mapping of two blast resistance genes in rice cultivar 93-11

Rice blast, caused by Magnaporthe oryzae, is a major disease of rice almost worldwide. The Chinese indica cultivar 93-11 is resistant to numerous isolates of the blast fungus in China, and can be used as broad-spectrum resistance resource, particularly in japonica rice breeding programs. In this study, we identified and mapped two blast resistance genes, Pi60(t) and Pi61(t), in cv. 93-11 using F2 and F3 populations derived from a cross between the susceptible cv. Lijiangxintuanheigu(LTH) and resistant cv. 93-11 and inoculated with M. oryzae isolates from different geographic origins. Pi60(t) was delimited to a 274 kb region on the short arm of chromosome 11, flanked by InDel markers K1-4 and E12 and cosegregated with InDel markers B1 and Y10. Pi61(t) was mapped to a 200 kb region on the short arm(near the centromere) of chromosome 12, flanked by InDel markers M2 and S29 and cosegregating with InDel marker M9. In the 274 kb region of Pi60(t), 93-11 contains six NBS-LRR genes including the two Pia/ PiCO39 alleles(BGIOSGA034263 and BGIOSGA035032) which are quite close to the two Pia/ PiCO39 alleles(SasRGA4 and SasRGA5) in Sasanishiki and CO39, with only nine amino acids differing in the protein sequences of BGIOSGA035032 and SasRGA5. In the 200 kb region of Pi61(t), 93-11 contains four NBS-LRR genes, all of which show high identities in protein sequence with their corresponding NBS-LRR alleles in susceptible cv. Nipponbare. Comparison of the response spectra and physical positions between the target genes and other R genes in the same chromosome regions indicated that Pi60(t) could be Pia/PiCO39 or its allele, whereas Pi61(t) appears to be different from Pita, Pita-2, Pi19(t), Pi39(t) and Pi42(t) in the same R gene cluster. DNA markers tightly linked to Pi60(t) and Pi61(t) will enable marker-assisted breeding and map-based cloning.

Breeding by design for future rice:Genes and genome technologies

1.Introduction Rice is a staple food for 3.2 billion people.The food security threat that shook many Asian countries in 2008 still looms,because farmers are facing the challenge of producing more rice with fewer resources of water,land,and inputs.

Wheat functional genomics research in China:A decade of development

In the past decade we witnessed a revolutionary development of wheat genomics and functional genomics, thanks to the development of next generation sequencing (NSG) technology.Wheat, as one of the most important crops in China and the world and with a huge, repetitive, and polyploid genome,was unconquerable in the past and is now catching up with other crops due to the availability of an increasing number of resources and platforms.Wheat researchers in China have worked unostentatiously during the last decade after The National High Technology Research and Development Program of China first set up the wheat functional genomics program in 2005. Since then many papers on wheat were published in a wide range of international journals demonstrating significant progress in wheat functional genomics.

Mitochondrion-targeted PENTATRICOPEPTIDE REPEAT5 is required for cis-splicing of nad4 intron 3 and endosperm development in rice

Endosperm as the storage organ of starch and protein in cereal crops largely determines grain yield and quality.Despite the fact that several pentatricopeptide repeat(PPR)proteins required for endosperm development have been identified in rice,the molecular mechanisms of many P-type PPR proteins in endosperm development remains unclear.Here,we isolated a rice floury endosperm mutant ppr5 that developed small starch grains and an abnormal aleurone layer,accompanied by decreased starch,protein,and amylose contents.Map-based cloning combined with a complementation test demonstrated that PPR5 encodes a P-type PPR protein that is localized to the mitochondria.The mutation in PPR5 caused reduced splicing efficiency of mitochondrial NADH dehydrogenase 4(nad4)gene intron 3 and reduced complex I assembly and activity.Loss of PPR5 function greatly upregulated expression of alternative oxidases(AOXs),reduced ATP production,and affected mitochondrial morphology.We demonstrate that PPR5,as a P-type PPR protein,is required for mitochondrial function and endosperm development by controlling the cis-splicing of mitochondrial nad4 intron 3.

Crop genome editing: A way to breeding by design

Increasing population and consumption in our planet is placing unprecedented challenges on agriculture for meeting food security and sustainability needs[1].Meanwhile,the adaptation of modern agricultural techniques[2]is central to minimize extensive losses due to abiotic stresses[3]under global climate change.

Agriculture and crop science in China:Innovation and sustainability

The International Crop Science Congress(ICSC) is a regularly held event allowing crop scientists to integrate current knowledge into a global context and international applications.The 7th ICSC was held on August 14–19,2016 in Beijing,China,with the theme "Crop Science:Innovation and Sustainability".As a companion production for this great congress,the nine papers collected in this special issue feature important fields of crop science in China.This editorial first briefly introduces the 7th ICSC,followed by a brief discussion of the current status of,constraints to,and innovations in Chinese agriculture and crop science.Finally,the main scientific points of the papers published in this special issue are surveyed,covering important advances in hybrid rice breeding,minor cereals,food legumes,rapeseed,crop systems,crop management,cotton,genomics-based germplasm research,and QTL mapping.In a section describing future prospects,it is indicated that China faces a full transition from traditional to modern agriculture and crop science.

Genetic Crop Improvement： A Guarantee for Sustainable Agricultural Production

Crop production provides food, feed, and other nutrients that support our everyday lives. Cereal crops, which include wheat （Triticum aestivurn）, rice （Oryza sativa）, maize （Zea mays）, barley （Hordeum vulgate）, millet （Setaria spp.）, sorghum （Sorghum bicolor）, rye （Secale cereale）, oats （Avena sativa）, and so forth, are the most important source of food for human consumption.

The Crop Journal:A new scientific journal for the global crop science community

As global population increases and demands for food supplies become greater,we face great challenges in providing more products and in larger quantities from less arable land.Crop science has gained increasing importance in meeting these challenges and results of scientific research must be communicated worldwide on a regular basis.In many countries,however,crop scientists have to publish the results of their investigations in national journals with heterogeneous con-

Breeding to Optimize Agriculture in a Changing World

Breeding to Optimize Chinese Agriculture(OPTICHINA) was a three-year EU–China project launched in June of 2011. As designed, the project acted as a new strategic model to reinforce systematic cooperation on agricultural research between Europe and China. The OPTICHINA International Conference "Breeding to Optimize Agriculture in a Changing World" was held in Beijing, May 26–29, 2014. The conference included six thematic areas:(1) defining and protecting the yield potential of traits and genes;(2) high-throughput precision phenotyping in the field;(3) molecular technologies in modern breeding;(4) plant ideotype;(5) data analysis,data management, and bioinformatics; and(6) national challenges and opportunities for China. The 10 articles collected in this special issue represent key contributions and topics of this conference. This editorial provides a brief introduction to the OPTICHINA project, followed by the main scientific points of articles published in this special issue. Finally, outcomes from a brainstorming discussion at the end of the conference are summarized, representing the authors' opinions on trends in breeding for a changing world.

Clock component OsPRR59 delays heading date by repressing transcription of Ehd3 in rice

Heading date(or flowering time),an important agronomic trait in crop species,is closely associated with regional adaptation and yield.Members of the Pseudo-Response Regulator(PRR)family play key roles in regulating flowering.However,their role and molecular mechanism controlling heading date in rice is not very clear.Here,we identified rice OsPRR protein,OsPRR59,which delayed heading under longday conditions.OsPRR59 positively regulates yield by affecting plant height,secondary branches number per panicle,grain number per panicle,seed setting rate,and grain weight per plant.OsPRR59 is expressed in most tissues and its protein is localized to the nucleus.We also found that OsPRR59 directly binds to the promoter of Ehd3 to inhibit its expression.Compared with the WT,osprr59 ehd3 showed a significantly delayed heading phenotype,as did the ehd3 mutant.This was opposite to the phenotype of the osprr59 mutant,confirming that Ehd3 acted downstream of OsPRR59 in regulating rice flowering.Our results identified a direct regulator of Ehd3,and revealed a novel molecular mechanism of clock component OsPRR proteins in regulating heading date and provide a new genetic resource for fine-tuning heading date in rice.

The RNA binding protein EHD6 recruits the m^(6)A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering

N6-Methyladenosine(m^(6)A)is one of the most abundant modifications of eukaryotic mRNA,but its comprehensive biological functionality remains further exploration.In this study,we identified and characterized a new flowering-promoting gene,EARLY HEADING DATE6(EHD6),in rice.EHD6 encodes an RNA recognition motif(RRM)-containing RNA binding protein that is localized in the non-membranous cytoplasm ribonucleoprotein(RNP)granules and can bind both m^(6)A-modified RNA and unmodified RNA indiscriminately.We found that EHD6 can physically interact with YTH07,a YTH(YT521-B homology)domain-containing m^(6)A reader.We showed that their interaction enhances the binding of an m^(6)A-modified RNA and triggers relocation of a portion of YTH07 from the cytoplasm into RNP granules through phase-separated condensation.Within these condensates,the mRNA of a rice flowering repressor,CONSTANS-like 4(OsCOL4),becomes sequestered,leading to a reduction in its protein abundance and thus accelerated flowering through the Early heading date 1 pathway.Taken together,these results not only shed new light on the molecular mechanism of efficient m^(6)A recognition by the collaboration between an RNA binding protein and YTH family m^(6)A reader,but also uncover the potential for m^(6)A-mediated translation regulation through phaseseparated ribonucleoprotein condensation in rice.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice

Although both protein arginine methylation(PRMT)and jasmonate(JA)signaling are crucial for regulating plant development,the relationship between these processes in the control of spikelet development remains unclear.In this study,we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures.Interestingly,we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7.We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs,thereby promoting the ubiquitination of OsJAZ1 by the SCF^(OsCOI1a/OsCOI1b) complex and degradation via the 26S proteasome.This process ultimately releases OsMYC2,a core transcriptional regulator in the JA signaling pathway,to activate or repress JA-responsive genes,thereby maintaining normal plant(spikelet)development.However,in the osprmt6a-1 mutant,reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs.As a result,OsJAZ1 proteins become more stable,repressing JA responses,thus causing the formation of abnormal spikelet structures.Moreover,we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner,thereby establishing a negative feedback loop to balance JA signaling.We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures.Collectively,our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Efficient genome editing in rice with miniature Cas12f variants

Genome editing,particularly using the CRISPR/Cas system,has revolutionized biological research and crop improvement.Despite the widespread use of CRISPR/Cas9,it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells.The hypercompact Cas12f,derived from Acidibacillus sulfuroxidans(AsCas12f),stands out due to its small size of only 422 amino acids and its preference for a T-rich motif,presenting advantageous features over SpCas9.However,its editing efficiency is extremely low in plants.Recent studies have generated two AsCas12f variants,AsCas12f-YHAM and AsCas12f-HKRA,demonstrating higher editing efficiencies in mammalian cells,yet their performance in plants remains unexplored.In this study,through a systematic investigation of genome cleavage activity in rice,we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants,particularly for AsCas12f-HKRA,which achieved an editing efficiency of up to 53%.Furthermore,our analysis revealed that AsCas12f predominantly induces deletion in the target DNA,displaying a unique deletion pattern primarily concentrated at positions 12,13,23,and 24,resulting in deletion size mainly of 10 and 11 bp,suggesting significant potential for targeted DNA deletion using AsCas12f.These findings expand the toolbox for efficient genome editing in plants,offering promising prospects for precise genetic modifications in agriculture.

Exploiting viral vectors to deliver genome editing reagents in plants

Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture,a labor-intensive and technically challenging process for many elite crop cultivars.In this review,we describe various virus-based methods that have been employed to deliver genome editing reagents,including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants.We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species,highlight the advantages and limitations of these delivery approaches,and discuss the remaining challenges.

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.

Engineer and split an efficient hypercompact CRISPR–CasΦ genome editor in plants

The programmable CRISPR-Cas genome editing technology,adopted from prokaryotic adaptive immune systems,has revolutionized genome engineering in plants(Liu et al.,2022a).Many efforts have been made to improve the activity,specificity,and protospacer adjacent motif(PAM)variants of Class 2 Cas nucleases,such as Cas9,Cas12a,and Cas12b(Liu et al.,2022a).However,their large size(∼1000–1400 amino acids)poses a challenge in scenarios requiring a compact Cas nuclease,particularly in urgent situations like plant virus-induced genome editing(Cheuk and Houde,2018;Li et al.,2021;Varanda et al.,2021).

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development

Most of the reported P-type pentatricopeptide repeat(PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22(flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein.Mutation of FLO22 resulting in defective transsplicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly upregulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development

Pentatricopeptide repeat(PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice(Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear.Here, we characterized a rice young leaf white stripe(ylws) mutant, which has defective chloroplast development during early seedling growth.Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear-and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2,and rps12, and editing of ndhA, ndhB, and rps14transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.