Adjusting the amylose content of semi-glutinous japonica rice by genome editing of uORF6 in the Wx gene

Amylose content,the key determinant of rice eating and cooking quality,is regulated primarily by the Waxy(Wx)gene.We adjusted the amylose content and transparency of semi-glutinous japonica rice carrying the Wxmp allele by genome editing of upstream open reading frame 6(uORF6)of Wx.

Introgression of lac6/tl1/du13 improves the palatability of japonica rice

Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable in specific culinary contexts.To harness these traits,significant efforts have been made to manipulate AC to improve rice ECQ.Our research utilized the MutMap+approach to identify LAC6/TL1,a gene that is an allele of Du13,responsible for low AC.LAC6 encodes a C2H2 zinc finger protein,which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele.Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype,whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight.Despite these drawbacks,such mutations positively impact rice palatability,presenting a trade-off between grain size and eating quality.To address the challenges posed by the reduced grain weight associated with LAC6 mutations,we developed a specific molecular marker for LAC6,which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size.Our findings demonstrate that the“small grain”trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection.This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel,high-quality soft rice varieties through targeted breeding strategies.

Brassinosteroid and gibberellin coordinate rice seed germination and embryo growth by regulating glutelin mobilization

Seed germination is the beginning of a new lifecycle,and involves many complex physiological and biochemical reactions including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo.Although glutelin is a dominant storage protein in rice,its contribution to seed germination and its regulatory mechanisms are mostly unknown.Gibberellin (GA) and brassinosteroid (BR),two major growth-promoting phytohormones,also play positive roles in controlling seed germination.However,how GA and BR interact and coordinate seed germination and facilitate glutelin mobilization remains unclear.In the present study,biochemical and physiological analyses of seed germination indicated that both GA and BR promote seed germination and post-germination growth.Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity.Proteomic and q RT-PCR results showed that the expression of several glutelin proteins and their encoding genes was induced by BR and GA in the embryo.Expression assays suggested that the increased accumulation of glutelin protein in the embryo was due to the accelerated degradation of glutelin by a cysteine proteinase (REP-1) in the endosperm.The breakdown of glutelin in the endosperm showed a strict positive correspondence with the length of the shoot.The GluA2 mutation led to reduced degradation rate of glutelin and defects in seed germination,and the promotion effect of GA on seed germination was weakened in the glua2mutant.In vitro culture assay of rice embryos showed that glutelin mobilization functioned downstream of the GA and BR pathways to promote shoot elongation.These findings suggest a mechanism that mediates crosstalk between BR and GA in co-regulating rice seed germination and embryo growth.

水稻分子设计育种启示:传统与现代相结合

水稻是世界上最重要的粮食作物之一,养育了全球超过1/2的人口。随着人口增加及气候条件变化,当前对水稻品种产量及其他综合性状表现提出了更高要求。分子设计育种概念的提出为快速突破现有品种的局限提供了契机,然而如何在育种实践中有效实现不同基因的组合利用是需要关注的重点。本文结合近年来水稻分子标记辅助选择(maker assisted selection,MAS)育种及重要性状基因编辑育种研究进展,对分子设计育种可能涉及的不同方面进行了归纳总结,既包括关键育种基因及其组合的遗传效应、不同回交世代遗传背景恢复特点、连锁累赘及重组筛选等经典MAS遗传规律总结,也涵盖高通量基因分型技术利用、基因编辑创制有利性状变异的实现途径及其效果等前沿技术应用评价。最后,结合当前水稻育种现状及实际需求,对传统育种资源及现代分子技术的综合利用策略进行了展望,以期为今后进一步优化分子设计育种流程提供思路。

Generation of selectable marker-free soft transgenic rice with transparent kernels by downregulation of SSSⅡ-2

The amylose content(AC) of rice endosperm starch varies from 0 to 35%,and is associated with rice cooking and eating quality.Soft rice has low AC,generally between 6% and 15%,and its eating quality is high whether it is consumed hot or cold.However,the appearance quality of current soft rice cultivars needs to be improved,especially opaque endosperm.Conventional genetic engineering has improved some agronomic traits of soft rice varieties,but not endosperm appearance.In the present study,a RNAi construct of the soluble starch synthase Ⅱ-2(SSSⅡ-2) and the hygromycin phosphotransferase(HPT) gene were introduced into an elite japonica rice variety,Kangtiaowuyunjing(KWY8) by co-transformation.Several selectable marker-free(SMF) transgenic lines were obtained,and SSSⅡ-2 expression was significantly downregulated in selected transgenic lines,resulting in lower AC of the endosperm.The physicochemical properties of the transgenic rice kernels,including gel consistency(GC) and rapid visco analyzer(RVA) profile,differed significantly from those of wild-type rice and were similar to those of a soft rice variety,Nanjing 46(NJ46).These findings indicate that the cooking,eating,and processing qualities of transgenic rice are comparable to those of NJ46.However,the transgenic rice endosperm retained a transparent appearance under low-moisture conditions.Thus,SMF SSSⅡ-2 RNAi rice provides a resource for breeding soft rice with transparent endosperm.

DNA methylation modification in heterosis initiation through analyzing rice hybrid contemporary seeds

Heterosis is an important biological phenomenon and widely applied in agriculture.Although many studies have been performed by using vegetative organs of F_(1) hybrid plants,how heterosis (or hybrid vigor) is initiated and formed,particularly the underlying molecular mechanism,remain elusive.Hybrid contemporary seeds of rice indica varieties 9311 and PA64 were innovatively used and analysis of DNA methylome of embryo and endosperm at early developing stages revealed the globally decreased DNA methylation.Genes,especially those relate to hormones function and transcriptional regulation present non-additive methylation.Previously identified heterosis-related superior genes are non-additively methylated in early developing hybrid contemporary seeds,suggesting that key genes/loci responsible for heterosis are epigenetically modified even in early developing hybrid seeds and hypomethylation of hybrid seeds after cross-pollination finally result in the long-term transcriptional change of F_(1) hybrid vegetative tissues after germination,demonstrating that altered DNA methylation in hybrid seeds is essential for initiation regulation and maintenance of heterosis exhibiting in F_(1) hybrid plants.Notably,a large number of genes show non-additive methylation in the endosperm of reciprocal hybrids,suggesting that endosperm might also contribute to heterosis.

Fine Mapping and Candidate Gene Prediction of the Quantitative Trait Locus qPL8 for Panicle Length in Rice

Rice panicle is the sink organ where assimilation product accumulates,and its morphology determines the rice yield.Panicle length has been suggested as a yield-related trait,but the genetic factor for its control is still limited.In this study,we carried out fine-mapping of qPL8,a QTL identified for panicle length in our previous work.Near isogenic line(NIL)with qPL8 exhibited elongated panicle without obvious effect on other panicle elements.With five key recombinants from NIL population,the locus was finally narrowed down to a 278-kb region,where 44 genes are annotated.By comparing the genomic sequence of two parents,17 genes were identified with SNPs or InDels variations in the coding region.Expression analysis showed that eight genes were up-regulated in the NIL with qPL8.Considering both the coding variation and expression status,several candidate genes for the locus were identified,and OsMADS37 was raised as the most possible candidate.Interestingly,an expression QTL(eQTL)also resides in the locus,leading to a cluster of gene expression variation in the region.This study will facilitate the application of qPL8 locus in rice breeding for yield potential.

Identification and fine mapping of a fertility restorer gene for wild abortive cytoplasmic male sterility in the elite indica rice non-restorer line 9311

The wild abortive(WA)-type cytoplasmic male sterility(CMS)derived from the wild rice species Oryza rufipogon Griff.is used widely in three-line indica hybrids.The identification and mapping of restorer of fertility(Rf)genes aided in the development of WA-type hybrids.Here we report that testcross F1 plants from the WA-type CMS line and 9311 exhibited stainable pollen grains with no seed set,indicating that 9311 carries minor-effect Rfs for WA-type CMS.We developed an advanced backcross population consisting of plants harboring small regions of donor chromosomal segments from 9311 in the WATianfeng A genetic background with moderate seed setting rates.Genetic analysis showed that the pollen fertility levels of the backcross individuals are governed by a single gene from 9311 that we named Rf19(t).By use of the RICE 40 K gene chip,three introduced segments were identified in the fertile lines,and a candidate region spanning 4.37–8.29 Mb on chromosome 1 was identified for Rf19(t).Finally,Rf19(t)was fine-mapped to a region of 90 kb between the DNA marker loci STS1-163 and STS1-183,in which eight ORFs were predicted.Also,using relative expression analyses,comparative sequence analyses and functional domain analyses,we identified LOC_Os01g10530 as the most likely candidate gene for Rf19(t).Furthermore,Rf19(t)was found to function in fertility restoration,most probably by regulating the degradation of m RNA transcribed from the mitochondrial gene WA352.These results increase our knowledge of fertility restoration in WA-type CMS lines and will facilitate the development of high-quality pairs of WAtype CMS and maintainer lines.

Serotonin enrichment of rice endosperm by metabolic engineering

In animals,serotonin is a neurotransmitter and mood regulator.In plants,serotonin functions in energy acquisition,tissue maintenance,delay of senescence,and response to biotic and abiotic stresses.In this study,we examined the effect of serotonin enrichment of rice endosperm on plant growth,endosperm development,and grain quality.To do so,TDCs and T5H were selected as targets for serotonin fortification.Overexpression of TDC1 or TDC3 increased serotonin accumulation relative to overexpression of T5H in rice grain.Transgenic lines of target genes driven by the Gt1 promoter showed better field performance than those driven by the Ubi promoter.Overexpression of T5H showed little effect on plant growth or grain physicochemical quality.In neuronal cell culture assays,serotonin induced neuroprotective action against apoptosis.Breeding of rice cultivars with high serotonin content may be beneficial for health and nutrition.

Exploration and Validation of the Potential Downstream Genes Underlying ipa1-2D Locus for Rice Panicle Branching

In recent years,some super hybrid rice varieties were bred with strong culms and large panicles,which are mainly contributed by the ipa1-2D locus.A gain-of-function allele of OsSPL14 is the ipa1-2D and it can greatly increase the panicle primary branch number.However,the key downstream genes mediating this trait variation are not fully explored.In this study,we developed high-quality near-isogenic lines(NILs)with a difference of only 30 kb chromosomal segment covering the ipa1-2D locus.Using the NILs,we explored the impact of ipa1-2D on five sequential stages of early inflorescence development,and found that the locus can greatly enhance the initiation of primary branch meristems.A transcriptomic analysis was performed to unveil the downstream molecular network of ipa1-2D,and 87 genes were found differentially expressed,many of which are involved in metabolism and catalysis processes.In addition,transgenic lines of overexpression and RNA interference were generated to shape different levels of OsSPL14.They were also used to validate the expression variation explored by transcriptome.Based on the gene annotation,twelve potential downstream targets of ipa1-2D were selected,and their expression variation was confirmed by qRT-PCR analysis both in NILs and transgenic lines.This research expands the molecular network underlying ipa1-2D and provides novel gene information which might be involved in the control of panicle branching.We discussed the potential function of identified genes and highlighted their values for future function exploration and breeding application.

Mapping QTLs for Heat Tolerance in Rice (Oryza sativa L. ) at Heading Stage Using Chromosome Segment Substitution Lines

In the present study, a japonica rice （ Oryza sativa L. ） variety Nipponbare, an indica variety 9311 and a set of chromosome segment substitution lines （CSSLs） which were generated using Nipponbare as the recipient parent and 9311 as the donor parent were used as the experimental materials. The CSSLs were grown in 2012 （normal temperature condition） and 2013 （high temperature condition） in Yangzhou, Jiangsu, and were used to map the quantitative trait loci （QTLs） for heat tolerance, based on the heat tolerance index [ （The seed setting rate under normal temperature condition -The seed setting rate under high temper- ature condition） / The seed setting rate under normal temperature condition]. As a result, three QTLs related to heat tolerance in rice were mapped on chromo- somes 2, 4 and 12, respectively. They had LOD （logarithm of rntds） scores of 2.56, 4.02 and 2.79, and contributian rates of 4.95%, 7.99% and 5.44%. Among them, qHT12.1 showed positive effect, while qHT2.1 and qHT4. t showed negative effect on heat tolerance. The results lay a foundation for the fine mapping and cloning of the QTLs and genes related to heat tolerance, and for the breeding of heat-tolerant rice varieties.

Effect and Its Mechanism of Brassinolide-regulated Starch Metabolism in Rice Germination

The study was aimed to explore the effect and mechanism of brassinolide（ BL） regulated starch metabolism in rice endosperm during seed germination.The radicle elongation of rice seeds treated with 1 μmol/L BL was inhibited during germination. The analysis on rice seeds with a GUS-fused promoter showed that BL had different regulatory effects on Wx,SBEI,and AGPS1. The effect of BL treatment on the physicochemical properties of rice starch was further investigated by scanning electron microscopy,Fourier transform infrared spectroscopy,and X-ray diffraction. The results showed that the starch treated with BL maintained better crystallinity and orderly structure,indicating that during the germination process,the degradation of starch in endosperm was slow,which might be one of the reasons for the slow radicle growth in the early stages of seed germination. This study provided important clues for further analysis of the molecular mechanisms underlying BR-regulated rice seed germination.

Two ABCI family transporters,OsABCI15 and OsABCI16,are involved in grain-filling in rice

Seed development is critical for plant reproduction and crop yield,with panicle seed-setting rate,grain-filling,and grain weight being key seed characteristics for yield improvement.However,few genes are known to regulate grain filling.Here,we identify two adenosine triphosphate(ATP)-binding cassette(ABC)I-type transporter genes,OsABCI15 and OsABCI16,involved in rice grain-filling.Both genes are highly expressed in developing seeds,and their proteins are localized to the plasma membrane and cytosol.Interestingly,knockout of OsABCI15 and OsABCI16 results in a significant reduction in seed-setting rate,caused predominantly by the severe empty pericarp phenotype,which differs from the previously reported low seed-setting phenotype resulting from failed pollination.Further analysis indicates that OsABCI15 and OsABCI16 participate in ion homeostasis and likely export ions between filial tissues and maternal tissues during grain filling.Importantly,overexpression of OsABCI15 and OsABCI16 enhances the seed-setting rate and grain yield in transgenic plants and decreases ion accumulation in brown rice.Moreover,the OsABCI15/16 orthologues in maize exhibit a similar role in kernel development,as demonstrated by their disruption in transgenic maize.Therefore,ourfindings reveal the important roles of two ABC transporters in cereal grain filling,highlighting their value in crop yield improvement.

Exploring the Trait Plasticity of ipa1-2D and qPL6 under Different Nitrogen Treatments and Heading Periods

Panicle size is one of the important factors in shaping yield potential in rice,but it shows plasticity in different environments,which leads to yieldfluctuation.Variations in panicle size among varieties are largely determined by quantitative trait loci(QTLs).QTL analysis could elaborate on the environmental impact on trait plasticity using nearly isogenic lines(NILs)of different QTLs.Two QTLs,ipa1-2D and qPL6 are identified to have pleio-tropic contributions to panicle size and plant architecture,but their responses to different growth conditions are still unclear.In this study,we developed NILs harboring a single locus or both loci of ipa1-2D and qPL6 and sub-sequently evaluated these QTL effects under different nitrogen treatments or heading periods.Trait comparison showed that panicle length was highly responsive to the high nitrogen treatment independent of qPL6.At the same time,ipa1-2D reduced the response of plant height,panicle number,and grain yield to the treatment.The background of long heading periods decreased the stem diameter for any genotype combinations but enhanced the performance of ipa1-2D for the panicle primary branch number.Moreover,the middle heading background could better balance the pleiotropic effect of the two QTLs and showed the highest yield potential.In-parallel analysis of the QTL contributions under different nitrogen treatments or heading periods confirmed the significant effect of ipa1-2D in increasing stem diameter,panicle primary branch number,and spikelet number per panicle.We proved that the two individual QTLs had a stable effect in increasing the yield potential but com-peted to decrease the panicle secondary branch number,panicle number,and yield potential when they were pyr-amided.This work provides a full view of the plasticity of two QTLs in shaping yield-related traits and lays the foundation for the rational design of rice breeding in the future.

Wx^lv、the Ancestral Allele of Rice Waxy Gene

In rice grains,the Waxy (Wx) gene is responsible for the synthesis of amylose,the most important determinant for eating and cooking quality.The effects of several Wx alleles on amylose content and the taste of cooked rice have been elucidated.However,the relationship between artificial selection and the evolution of various Wx alleles as well as their distribution remain unclear.Here we report the identification of an ancestral allele,Wx^lv,which dramatically affects the mouthfeel of rice grains by modulating the size of amylose molecules.We demonstrated that WF originated directly from wild rice,and the three major Wx alleles in cultivated rice (Wx^b,Wx^a,and Wx^in) differentiated after the substitution of one base pair at the functional sites.These data indicate that the Wx^lv allele played an important role in artificial selection and domestication.The findings also shed light on the evolution of various Wx alleles,which have greatly contributed to improving the eating and cooking quality of rice.

A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

In rice(Oryza sativa), amylose content(AC) is the major factor that determines eating and cooking quality(ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy(Wx)locus. Here we identified a rare Wx allele, Wx^(mw) ,which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wx^(mw) may have derived from recombination between two important natural Wx alleles, Wx^(in) and Wx^(b). We validated the effects of Wx^(mw) on rice grain quality using both transgenic lines and near-isogenic lines(NILs). When introgressed into the japonica Nipponbare(NIP) background, Wx^(mw) resulted in a moderate AC that was intermediate between that of NILs carrying the Wx^(b)allele and NILs with the Wx^(mp) allele. Notably, mature grains of NILs fixed for Wx^(mw) had an improved transparent endosperm relative to soft rice. Further, we introduced Wx^(mw) into a high-yielding japonica cultivar via molecular marker-assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wx^(mw) is a promising allele to improve grain quality, especially ECQ and grain transparency of high-yielding japonica cultivars, in rice breeding programs.

Starch biosynthesis in cereal endosperms: An updated review over the last decade

Starch is a vital energy source for living organisms and is a key raw material and additive in the food and non-food industries.Starch has received continuous attention in multiple research fields.The endosperm of cereals(e.g.,rice,corn,wheat,and barley)is the most important site for the synthesis of storage starch.Around 2010,several excellent reviews summarized key progress in various fields of starch research,serving as important references for subsequent research.In the past 10 years,many achievements have been made in the study of starch synthesis and regulation in cereals.The present review provides an update on research progress in starch synthesis of cereal endosperms over the past decade,focusing on new enzymes and non-enzymatic proteins involved in starch synthesis,regulatory networks of starch synthesis,and the use of elite alleles of starch synthesis-related genes in cereal breeding programs.We also provide perspectives on future research directions that will further our understanding of cereal starch biosynthesis and regulation to support the rational design of ideal quality grain.

Post-genomics revolution in the design of premiumquality rice in a high-yieldingbackground to meet consumer demands in the 21st century

The eating and cooking quality(ECQ)of rice is critical for determining its economic value in the marketplace and promoting consumer acceptance.It has therefore been of paramount importance in rice breeding programs.Here,we highlight advances in genetic studies of ECQ and discuss prospects for further enhancement of ECQ in rice.Innovations in gene-and genome-editing techniques have enabled improvements in rice ECQ.Significant genes and quantitative trait loci(QTLs)have been shown to regulate starch composition,thereby affecting amylose content and thermal and pasting properties.A limited number of genes/QTLs have been identified for other ECQ properties such as protein content and aroma.Marker-assisted breeding has identified rare alleles in diverse genetic resources that are associated with superior ECQ properties.The post-genomics-driven information summarized in this review is relevant for augmenting current breeding strategies to meet consumer preferences and growing population demands.

Sake or supper? Breeding rice for culinary excellence and optimal brewing

Improving grain quality is a paramount objective within modern rice breeding frameworks.The concept of rice grain quality is intricate,comprising aspects related to milling,appearance,cooking,nutrition,and hygiene(Custodio et al.,2019).It is well established that the quality of rice grain is a sophisticated quantitative trait governed by multiple genes(Ren et al.,2023).Over the past decade,tremendous efforts have been made to breed elite rice varieties with desired grain quality traits.For instance,the culinary quality of contemporary indica rice has undergone significant enhancements through the substitution of the Wxb allele in place of the Wxa allele,a pivotal domestication-related gene controlling rice grain amylose content,affecting both its cooking and consumption qualities(Zhang et al.,2019;Gu et al.,2023).Recent genomic studies on various modern cultivars and landraces have pinpointed multiple domesticated genes,as well as specific genomic regions,that contribute to distinct agronomic traits and environmental adaptations(Liu et al.,2023).Nevertheless,the identification of genes related to grain quality that have been selected through breeding in modern cultivars continues to be constrained because of the complex regulatory networks implicated.