Characterization and Evaluation of OsLCT1 and OsN ramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice

To explore how rice(Oryza sativa L.) can be safely produced in Cd-polluted soil, OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis. One of OsLCT1 mutant(lct1×1) and two of OsNramp5 mutants(nramp5×7 and nramp5×9) were evaluated for grain Cd accumulation and agronomic performances. In paddy field soil containing approximately 0.9 mg/kg Cd, lct1×1 grains contained approximately 40%(0.17 mg/kg) of the Cd concentration of the wild type parental line, less than the China National Food Safety Standard(0.20 mg/kg). Both OsNramp5 mutants showed low grain Cd accumulation(< 0.06 mg/kg) in the paddy(approximately 0.9 mg/kg Cd) or in pots in soil spiked with 2 mg/kg Cd. However, only nramp5×7 showed normal growth and yield, whereas the growth of nramp5×9 was severely impaired. The study showed that lct1×1 could be used to produce rice grains safe for human consumption in lightly contaminated paddy soils and nramp5×7 used in soils contaminated by much higher levels of Cd.

Thermal Resistance of Common Rice Maintainer and Restorer Lines to High Temperature During Flowering and Early Grain Filling Stages

Fifteen common rice maintainer lines and 26 high-yielding restorer lines were used to evaluate their thermal resistance and fertility during flowering and early grain filling stages.The rice plants were subjected to high temperature stress(39-43 ℃) for 1-15 d from main stem flowering.Based on the heat stress index,they were divided into thermal resistant lines,semi-thermal resistant lines,semi-thermal sensitive lines and thermal sensitive lines.Therefore,the maintainer lines K22B,Bobai B and V20B belonged to thermal resistant lines,whereas II-32B,Zhongzhe B and Zhong 9B belonged to thermal sensitive lines.For rice restorer lines,Minghui 63 had the highest thermal resistance,followed by R207,P32,P929,and the lowest thermal resistant lines P62-2-2,R8006 and P51.The correlation analysis indicated that the heat stress index was significantly correlated with seed-setting rate and abortive grain rate under heat stress,but not under natural conditions.This indicated that heat stress occurred during flowering and early grain filling stages mainly decreased the seedsetting rate and significantly increased the abortive grain rate in both rice maintainer and restorer lines.

Identification of a major quantitative trait locus and its candidate underlying genetic variation for rice stigma exsertion rate

Stigma exsertion in male sterile lines of hybrid rice is important for seed yield.In the present study, ZS616 [Oryza sativa subsp.Xian(indica)], a male sterile line with a stigma exsertion rate(SER) as high as 94.5%, was crossed to DS552, a japonica line with almost no exserted stigmas.F3 plants with extremely low and high SER were sequenced to identify SER-associated quantitative trait loci(QTL).A major QTL for SER, qSER-3.1, was identified along with other QTL on chromosome 3 in a 3.9 Mb region.A total of 307 nonsynonymous single-nucleotide polymorphisms(SNPs) and 27 frame-shift insertion/deletions(InDels)differentiating ZS616 and DS552 were identified in the region containing qSER-3.1.Most SNPs(294) and InDels(25) were excluded after further analysis because they were shared by ZS616 and low(<2.0%) SER accessions in the Huazhong Agricultural University(HAU) core rice collection.Association analysis using the full HAU collection identified a 17-bp InDel in OS03 G0689400 as the most likely causal genetic variant underlying qSER-3.1.ZS616-type accessions(n = 54, with the 17-bp insertion) in the HAU collection had minimum(16.5%)and mean(39.6%) SERs significantly greater than those(n = 424) without the insertion(with minimum and mean SERs of 0.2% and 20.6%, respectively).Thus, this study identified a major QTL for stigma exsertion and revealed the mutation in a candidate gene for the QTL.

Brassinosteroids Mediate Endogenous Phytohormone Metabolism to Alleviate High Temperature Injury at Panicle Initiation Stage in Rice

High temperatures cause physiological and biochemical changes and significantly affect young panicle development of rice(Oryza sativa L.).Brassinosteroids play important roles in enhancing crop stress resistance.In this study,we subjected rice cultivars Huanghuazhan(heat-resistant)and IR36(heat-sensitive)to high temperature(HT,40 oC)or normal temperature(NT,33 oC)for 7 d at the panicle initiation stage,in conjunction with application of 24-epibrassinolide[EBR,a synthetic brassinolide(BR)]or brassinazole(BRZ,a BR biosynthesis inhibitor)at the beginning of the treatments.HT exacerbated spikelet degeneration and inhibited young panicle growth,which were partially prevented by EBR application,while BRZ application aggravated the reduction in spikelet number.HT decreased the contents of BR,active cytokinins(aCTK),active gibberellins(aGA)and indole-3-acetic acid(IAA),but increased the content of abscisic acid(ABA)in young panicles.The activities of key enzymes involved in sucrose hydrolysis,glycolysis and the tricarboxylic acid cycle in young panicles were decreased with the change of endogenous hormone levels under HT.In addition,the contents of H2O2 and malondialdehyde(MDA)were increased and the activities of antioxidant enzymes were decreased in young panicles.Exogenous application of EBR induced the expression of phytohormone biosynthesis-related genes and down-regulated the expression of phytohormone catabolism-related genes to increase the contents of endogenous BR,aCTK,aGA and ABA,thus promoting the decomposition and utilization of sucrose in young panicles,enhancing the activities of superoxide dismutase,catalase and peroxidase,and reducing the accumulation of H2O2 and MDA in young panicles,whereas application of BRZ had the opposite physiological effects.These results showed that brassinosteroids mediate endogenous phytohormone metabolism to alleviate HT injury at the panicle initiation stage in rice.

Functions of Nitrogen,Phosphorus and Potassium in Energy Status and Their Influences on Rice Growth and Development

Nitrogen(N),phosphorus(P)and potassium(K)are important essential nutrients for plant growth and development,but their functions in energy status remains unclear.Here,we grew Nipponbare rice seedlings in a growth chamber for 20 d at 30℃/24℃day/night)under natural sunlight conditions with different nutrient regimes.The results showed that N had the strongest influence on the plant growth and development,followed by P and K.The highest nonstructural carbohydrate content,dry matter weight,net photosynthetic rate(Pn),ATP content,as well as NADH dehydrogenase,cytochrome oxidase and ATPase activities were found in the plants that received sufficient N,P and K.The lowest values of these parameters were detected in the N-deficient plants.Higher dry matter accumulation was observed in the K-deficient than in the P-deficient treatments,but there was no significant difference in the ratio of respiration rate to Pn between these two treatments,suggesting that differences in energy production efficiency may have accounted for this result.This hypothesis was confirmed by higher ATP contents and activities of NADH dehydrogenase,cytochrome oxidase and ATPase in the K-deficient plants than in the P-deficient plants.We therefore inferred different abilities in energy production efficiency among N,P and K in rice seedlings,which determined rice plant growth and development.

Rice Aroma: A Natural Gift Comes with Price and the Way Forward

Aromatic rice belongs to a small but important sub-group of rice,which is highly regarded for its excellent aroma and superior grain quality.Aromatic rice,especially Basmati-and Jasmine-type rice,is being traded at a high price in the local and global markets.Genetically,rice aroma is a phenotypical expression of spontaneous recessive mutations of the OsBadh2 gene(also known as fgr/badh2/osbadh2/os2AP gene).These mutations inhibit the flow ofγ-aminobutyraldehyde(GAB-ald)toγ-aminobutyric acid(GABA),and consequently,the accumulated GAB-ald is diverted to a potent flavour component 2-acetyl-1-pyrroline(2AP)by a non-enzymatic reaction with methylglyoxal.The natural incidence of non-functional osbadh2 mutation along with selection and nursing by the farmer from the ancient time makes rice aroma as a prominent natural gift.As GABA and methylglyoxal play significant roles in stress tolerance,and their biosynthesis is strictly regulated in rice plants,the accumulation of 2AP in aromatic rice depends on the interaction of various genetic and environmental factors,and its production may come at some costs of sacrificing tolerance.This review focused on some potential underlying genes in the 2AP and GABA biosynthesis pathways,and analyzed most aspects of aroma formation in rice,and summarized the molecular mechanism of aroma production together with its genetic and non-genetic influencing factors.The present review also stated approaches to produce high-quality aromatic rice via developing novel cultivars and with good agronomic knowledge-based practice.

Male Parent Plays More Important Role in Heat Tolerance in Three-Line Hybrid Rice

Ten F1 combinations with their male and female parents were employed to evaluate their heat tolerance during the flowering and early grain filling stages. The rice plants were subjected to heat stress(39 °C–43 °C) for 1–15 d during flowering. Based on the heat stress index, heat tolerance was only observed in the F1 combinations H2(K22A × R207), H3(Bobai A × R207) and H4(Bobai A × Minghui 63), whereas the others received above 0.5000 of heat stress index. Both parents of the tolerant combination(heat-tolerant × heat-tolerant) possessed heat tolerance, whereas the susceptible combinations were crossed by heat-tolerant(sterile lines) × heat-susceptible(restorer lines), heat-susceptible × heat-tolerant, or heat-susceptible × heat-susceptible parents. This result indicated that heat tolerance in rice was controlled by recessive genes. Thus, both parents should possess high temperature tolerance to develop heat-tolerant F1 combinations. Furthermore, the heat stress index of F1 combinations was significantly correlated with the heat stress index of restorer lines but not with the heat stress index of maintainer lines. This result suggested that male parents play a more important role in heat-tolerant combinations than female parents. Therefore, the heat susceptibility of the hybrid rice in China is mainly due to the wide application of low-heat-tolerant restorer lines with high yield in three-line hybrid rice breeding.

Current insights on rice(Oryza sativa L.)bakanae disease and exploration of its management strategies

Bakanae is an emerging rice disease caused by the seed-and soil-borne pathogen Fusarium fujikuroi.It is becoming a more serious threat to sustainable rice production throughout rice-growing regions.Bakanae disease infection is responsible for high yield losses ranging from 3%to 95%,and disease incidence varies based on the region and cultivars.Hence,understanding the nature of the pathogen,its pathogenicity,disease epidemiology,symptoms,host–pathogen interaction,and the role of secondary metabolites in the disease cycle will be helpful in the development of effective and sustainable management strategies.However,very few comprehensive studies have described the details of rice bakanae disease.Thus,in this review we summarize and discuss in detail the information available from 1898 to 2023 on various critical facets of bakanae disease,and provide perspectives on future research.

Fine mapping and candidate gene analysis of purple pericarp gene Pb in rice (Oryza sativa L.)

Purple rice is a type of rice with anthocyanins deposited in its grain pericarp. The rice Pb gene control-ling purple pericarp character is known to be on chromosome 4,and the purple color is dominant over white color. In this study,we fine mapped the Pb gene using two F2 segregating populations,i.e. Pei'ai 64S(white) × Yunanheixiannuo(purple) and Pei'ai 64S × Chuanheinuo(purple) . In the first-pass map-ping,the Pb gene was located in the region downstream the SSR marker RM3820. In the fine mapping,the candidate region was saturated with InDel and CAPS markers developed specifically for this study. Eventually,the Pb gene was mapped within the 25-kb region delimited by the upstream marker RID3 and the downstream marker RID4. The delimited region contained two annotated genes,Ra and bhlh16(TIGR Rice Genome,R.5) . The former is a homologue of the Myc transcription factor Lc controlling anthocyanin biosynthesis in maize,and the latter is a homologue of the TT8 gene,which is also an Myc transcription factor gene controlling the pericarp pigmentation in Arabidopsis thaliana. Sequence analysis showed that the exon 7 of the Ra gene of Yunanheixiannuo and Chuanheinuo had a 2-bp(GT) deletion compared with those of the white rice varieties Pei'ai 64S,9311 and Nipponbare. A CAPS marker,CAPSRa,was developed according to the GT deletion for analysis of the two F2 segregating populations and 106 rice lines. The results showed that all F2 plants with white pericarp,and all non-purple rice lines(63 white and 22 red) contained no GT deletion,but all 20 purple rice lines con-tained the GT deletion. These results suggested that the Ra gene may be the Pb gene and the purple pericarp characteristic of rice is caused by the GT deletion within exon 7 of the Ra gene.

Dissecting the Genetic Basis of Extremely Large Grain Shape in Rice Cultivar 'JZ1560'

Rice grain shape,grain length(GL),width(GW),thickness(GT)and length-to-width ratio(LWR),are usually controlled by multiple quantitative trait locus(QTL).To elucidate the genetic basis of extremely large grain shape,QTL analysis was performed using an F2 population derived from a cross between a japonica cultivar 'JZ1560'(extremely large grain)and a contrasting indica cultivar 'FAZ1'(small grain).A total number of 24 QTLs were detected on seven different chromosomes.QTLs for GL,GW,GT and LWR explained 11.6%,95.62%,91.5%and 89.9%of total phenotypic variation,respectively.Many QTLs pleiotropically controlled different grain traits,contributing complex traits correlation.GW2 and qSW5/GW5,which have been cloned previously to control GW,showed similar chromosomal locations with qGW2-I/qGT2-I/qLWR2-2 and qGW5-2/qLWR5-l and should be the right candidate genes.Plants pyramiding GW2 and qSW5/GW5 showed a significant increase in GW compared with those carrying one of the two major QTLs.Furthermore,no significant QTL interaction was observed between GW2 and qSW5/GW5.These results suggested that GW2 and qSW5/GW5 might work in independent pathways to regulate grain traits.'JZ1560' alleles underlying all QTLs contributed an increase in GW and GT and the accumulation of additive effects generates the extremely large grain shape in 'JZ1560'.

Salicylic acid and ethylene signaling pathways are involved in production of rice trypsin proteinase inhibitors induced by the leaf folder Cnaphalocrocis medinalis(Guenée)

The roles of signaling pathways in the production of trypsin proteinase inhibitors(TrypPIs)in rice infested by the leaf folder(LF) Cnaphalocrocis medinalis were studied.Infestation by LF increased TrypPI levels in the leaves of rice plants at the tillering,booting and flowering stages but decreased TrypPI levels at the ripening stage;TrypPI levels in rice stems did not increase at any developmental stage.Infestation by LF at the tillering stage systemically increased TrypPI levels in leaves but not in stems;it also enhanced salicylic acid(SA)levels in leaves and stems,and the ethylene level released from plants.However,LF infestation did not increase JA concentrations.Exogenous application of SA or ethylene enhanced TrypPI levels in the leaves and stems of plants at the tillering stage,whereas treatment with both SA and ethylene induced lower levels of TrypPIs than treatment with SA or ethylene alone,suggesting an antagonistic effect of SA and ethylene on TrypPIs induction.The results suggest that both SA and ethylene signaling pathways are involved in the production of TrypPIs in rice induced by LF;moreover,the antagonistic effect of SA and ethylene may explain the changes in TrypPI levels seen at different plant developmental stages and in different organs.

Identification of Quantitative Trait Loci for Lipid Metabolism in Rice Seeds

植物种子油为人的饮食的消费和工业申请是重要的。油特点被量的特点 loci (QTL ) 控制，但是为丰满的酸作文的 QTL 都不在米饭被知道， monocot 模型植物。QTL 分析从一个 indica 变化和一个装饰用的梨树变化的一个十字用 F2 和 F2:3 子孙被执行。煤气的 chromatographymass spectrometry (GCMS ) 分析在稻谷油的丰满的酸作文揭示了父母线之间的重要差别，并且 29 在 F2 联系了 QTL 或 F2:3 人口在整个米饭染色体被识别，除了染色体 9 和 10。八 QTL 反复越过不同环境在两张人口被识别。五 loci 多种地控制了不同特点，贡献有丰满的酸并且在丰满的酸之间的油的复杂相互作用。把关键酶编码为与 11 co 局部性的类脂化合物新陈代谢的 Arabidopsis 基因的九米饭 orthologs 印射 QTL。强壮的 QTL 为油(18:1 ) 并且罂酸(18:2 ) 酸与编码 acylCoA:diacylglycerol acyltransferase (DGAT ) 的基因的米饭 ortholog 被联系，并且为十六酸酸(16:0 ) 的另外一个同样印射到 acylACP thioesterase (FatB ) 基因 ortholog。我们的途径很快并且高效地为控制丰满的酸作文和油集中的印射的 QTL 识别了候选人基因，提供信息因为由标记的改善米饭谷物质量帮助了选择。

OsNAC5 orchestrates OsABI5 to fine-tune cold tolerance in rice

Due to its tropical origins,rice(Oryza sativa)is susceptible to cold stress,which poses severe threats to production.OsNAC5,a NAC-type transcription factor,participates in the cold stress response of rice,but the detailed mechanisms remain poorly understood.Here,we demonstrate that OsNAC5 positively regulates cold tolerance at germination and in seedlings by directly activating the expression of ABSCISIC ACID INSENSITIVE 5(OsABI5).Haplotype analysis indicated that single nucleotide polymorphisms in a NAC-binding site in the OsABI5 promoter are strongly associated with cold tolerance.OsNAC5 also enhanced OsABI5 stability,thus regulating the expression of cold-responsive(COR)genes,enabling fine-tuned control of OsABI5 action for rapid,precise plant responses to cold stress.DNA affinity purification sequencing coupled with transcriptome deep sequencing identified several OsABI5 target genes involved in COR expression,including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1A(OsDREB1A),OsMYB20,and PEROXIDASE 70(OsPRX70).In vivo and in vitro analyses suggested that OsABI5 positively regulates COR gene transcription,with marked COR upregulation in OsNAC5-overexpressing lines and downregulation in osnac5 and/or osabi5 knockout mutants.This study extends our understanding of cold tolerance regulation via OsNAC5 through the OsABI5-CORs transcription module,which may be used to ameliorate cold tolerance in rice via advanced breeding.

基于高分辨率熔解曲线技术的水稻伽玛射线诱发突变的TILLIN体系（英文）

目的:建立适用于筛选伽马射线诱发突变的、基于高分辨率熔解曲线(high-resolution melting,HRM)技术的高通量定向诱导基因组局部突变技术(Targeting Induced Local Lesions IN Genomes,TILLING)体系。创新点:建立起了基于HRM技术、适用于伽玛射线诱发的小片段插入/缺失突变的高通量TILLING体系(HRM-TILLING)。方法:通过不同野生型/突变型比例混池DNA的HRM分析,确定HRM检测不同类型插入/缺失突变的能力,确定M2植株突变检测的适宜混池比例,并用一个伽玛诱变M2群体(n=4560)筛选Os LCT1和SPDT两个基因的突变体,确定实际效果。结论:以4株M2植株混样,采用HRM可以有效检出突变。建立的基于HRM的TILLING体系适用于伽玛射线诱发突变的高通量筛选。

基因组重测序分析γ射线诱发水稻可遗传变异的频率与特征（英文）

目的:研究γ射线对水稻基因组的诱变效应,明确其诱发突变的类型、分布和频率。创新点:首次针对种子繁殖植物在全基因组范围及单核苷酸水平上揭示了γ射线诱发可遗传变异的频率与特征。方法:利用Illumina Hiseq2000对三种γ射线剂量辐照培育的6株水稻(日本晴)M2植株进行基因组重测序,生物信息学分析确定单碱基替换(SBS)和插入缺失(Indel)突变,以及结构变异和拷贝数等变异的频率和基因组分布。利用Sanger测序、目标片段扩增或定量多聚酶链反应(qP CR)对各类突变进行验证。综合重测序和验证结果估算诱发突变频率。结论:结果表明,γ射线既可以诱发单碱基替换,也可以诱发插入缺失突变和结构变异;水稻M2代植株中的平均突变频率达到7.5×10^(-6)~9.8×10^(-6);Indel突变频率约为SBS变异的1/4,而结构变异频率更低;SBS和Indel突变随机分布在12条染色体上,无明显的突变热点。

基因编辑:将基因生物学用于植物育种的工具

人口不断增长给世界粮食安全带来了严峻的挑战,植物育种是应对这一挑战的最重要手段之一。过去三十年来,性状形成和调控的新知识(如功能基因组学)和新技术(如生物信息学和表型组学)极大地支持了植物育种的发展。基因编辑,特别是基于CRISPR/Cas技术和其衍生技术,已成为强有力的植物研究技术,可能直接改变植物育种的方法和策略。植物表型性状受编码基因和非编码基因的控制,在本文中,我们提出了编辑这两类基因的不同策略。对于编码基因,其编码蛋白的活性和数量可在转录和转录后水平以及翻译和翻译后水平加以调节,我们由此提出了创造基因功能性变异从而改变性状表型的基因编辑策略。对于非编码基因,则可以采用基因编辑技术对其转录水平或对靶基因的目标序列加以改造,达到产生新的性状的目的。此外,我们还提出了一种基于原生质体的基因编辑方案,使基因编辑技术更适合于植物育种。总之,本文提出了一系列可供植物育种者选择的将基因生物学知识转化为实用育种策略的方案,即基因编辑技术成为将基因生物学知识用于植物育种的技术。