Genetic characterization and linkage disequilibrium mapping of resistance to gray leaf spot in maize(Zea mays L.)

Gray leaf spot(GLS),caused by Cercospora zeae-maydis,is an important foliar disease of maize(Zea mays L.)worldwide,resistance to which is controlled by multiple quantitative trait loci(QTL).To gain insights into the genetic architecture underlying the resistance to this disease,an association mapping population consisting of 161 inbred lines was evaluated for resistance to GLS in a plant pathology nursery at Shenyang in 2010 and 2011.Subsequently,a genome-wide association study,using 41,101 single-nucleotide polymorphisms(SNPs),identified 51 SNPs significantly(P<0.001)associated with GLS resistance,which could be converted into 31 QTL.In addition,three candidate genes related to plant defense were identified,including nucleotidebinding-site/leucine-rich repeat,receptor-like kinase genes similar to those involved in basal defense.Two genic SNPs,PZE-103142893 and PZE-109119001,associated with GLS resistance in chromosome bins 3.07 and 9.07,can be used for marker-assisted selection(MAS)of GLS resistance.These results provide an important resource for developing molecular markers closely linked with the target trait,enhancing breeding efficiency.

Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize

Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Metabolomics provide insights into the endogenous mechanism of strobilation in the scyphozoan jellyfi sh Rhopilema esculentum

Strobilation is a well-organized metamorphosis process in scyphozoan jellyfi sh,through which the benthic polyp develops into the pelagic medusa.So far,knowledge on the molecular mechanisms behind the drastic morphological and physiological changes remains limited.In the present study,the metabolic profi les in polyp and strobila stages of jellyfi sh Rhopilema esculentum were examined using ultra-performance liquid chromatography coupled to a mass spectrometer.A total of 3071 metabolites with putative annotation were detected,of which 167 were identifi ed as diff erential metabolites between the polyp and strobila.Among the metabolites,31 signifi cantly decreased and 136 signifi cantly increased in abundance in the strobila.Thyroxin,one of the previously proposed strobilation inducer of Aurelia,was not detected in this study.The indole-containing compounds are known for triggering strobilation in Discomedusae.Two indole derivatives and an indole-isomer containing metabolite were detected among the most signifi cantly increased metabolites,which may be potential endogenous molecules for strobilation initiation.The membrane-associated phospholipids and the infl ammation and oxidative stress-related eicosanoids were found to have signifi cant changes.Although the functions of these metabolites in strobilation are not yet completely known,they provide some clues to the induction of the process of strobilation and metabolic responses that take place during strobilation.

Multi-factor Analysis Model for Improving Profit Management Using Excel in Shellfish Farming Projects

By using a farm's data in Yantai City and the theory of Cost-Volume-Profit analysis and the financial management methods,this paper construct a multi-factor analysis model for improving profit management using Excel 2007 in Shellfish farming projects and describes the procedures to construct a multi-factor analysis model.The model can quickly calculate the profit,improve the level of profit management,find out the breakeven point and enhance the decision-making efficiency of businesses etc.It is also a thought of the application to offer suggestions for government decisions and economic decisions for corporations as a simple analysis tool.While effort has been exerted to construct a four-variable model,some equally important variables may not be discussed sufficiently due to limitation of the paper's space and the authors'knowledge.All variables can be listed in EXCEL 2007 and can be associated in a logical way to manage the profit of shellfish farming projects more efficiently and more practically.

快充型锂离子电池电极材料与电解液结构调控及设计

实现锂离子电池的快速充电是促进电动汽车普及、解决环境和能源问题的有效途径。然而,常规锂离子电池体系在快速充电条件下的缓慢动力学和安全风险的增加严重阻碍了该技术的实际应用。本文综述了快充型锂离子电池电极材料和电解液在结构调控与设计方面的研究进展。首先,详细介绍了通过电极材料的结构调控来提高锂离子在电极材料中扩散速度的方法。然后,阐述了通过对锂离子溶剂化结构的调控来提高锂离子在电解液中的传输和相界面处转移速度的方法。最后,对快充型锂离子电池所面临的关键科学问题进行了总结以及对未来的研究方向进行了展望。

亲社会风险行为及其理论模型与神经机制

亲社会风险行为(prosocial risky behavior)是指为了他人利益或社会福利做出的冒险行为.因兼具亲社会性和风险性,且经常与消极的风险行为交替发生,亲社会风险行为在日常生活中难以被识别,但在一定程度上表现出了人类积极健康发展的特点.在明确亲社会风险行为概念的来源、界定及其测量方法的基础上,本文借鉴了亲社会行为和风险行为的解释路径,从风险偏好和社会偏好整合加工的视角对人类亲社会风险行为的前因研究成果进行了归纳,进而构建了一个新颖的亲社会风险行为理论模型,并系统阐述了其大脑神经基础和时间加工进程特点.未来的研究应进一步明确亲社会风险行为的心理结构,优化其研究范式,比较经济与社会决策领域下框架效应的特点及其认知神经机制,并探索不同方面的因素对亲社会风险行为的影响作用.

Micro-flower-like MoS_(2)-modified Co_(9)S_(8)heterostructure as anode material for sodium-ion batteries with superior reversibility and rate capacity

Designing and fabricating of heterostructured materials with long-term cycling stability and high-rate capacity for the anode of sodium-ion batteries(SIBs)still remain a great challenge.Herein,micro-flower-like MoS_(2)-modified Co_(9)S_(8)(Co_(9)S_(8)/MoS_(2))with a three-dimensional(3D)heterostructure was first obtained via a simple solvothermal synthesis followed by a solid sulfidation treatment process.As a material for the anode of SIBs,the Co_(9)S_(8)/MoS_(2)-based electrode with an initial Co/Mo molar ratio of 1/1(denoted as CM55-S)exhibits the best sodium storage performance with a boosted capacity,superior reversibility(424.5 mAh g^(-1)@2 A g^(-1)at the 1600th cycle,401.1 mAh g^(-1)@5 A g^(-1)at the 800th cycle),and an excellent rate capacity(210.1 mAh g^(-1)@20 A g^(-1)).Density functional theory(DFT)calculations confirm that the Co_(9)S_(8)/MoS_(2)heterostructure has a lower energy barrier(0.30 eV)than the pure Co_(9)S_(8)(0.53 eV).It is expected that such a heterostructured material could be an attractive candidate as the material of the anode for SIBs.

Pollen self-elimination CRISPR–Cas genome editing prevents transgenic pollen dispersal in maize

Dear Editor,Genome editing with clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated nuclease(Cas)-mediated technologies have revolutionized basic plant science and crop genetic improvement(Chen et al.,2019).Stable genetic transformation of CRISPR–Cas cassette(s)is the main approach to genome editing in planta.In many sexually reproducing plants,a major concern is the dispersal of genetically modified elements through pollen(Devos et al.,2005).Maize(Zea mays L.).

Genome Editing Enables Next-Generation Hybrid Seed Production Technology

The next-generation hybrid seed technology enables the successful production of sortable hybrid seeds from genic male sterile(GMS)lines and maintainers;however,it requires multiple laborious and complicated steps.Here,we designed a simple next-generation hybrid seed production strategy that takes advantage of the CRISPR/Cas9 technology to create a Manipulated GMS Maintainer(MGM)system via a single transformation.Under this schema,the maize male fertility gene ZmMS26 was nullified by removal of its fifth exon using the CRISPR/Cas9 system on a vector,and a second vector carrying a functional ZmMS26 cDNA was co-transformed to restore fertility.The second vector also contains a male gametophyte inactivation gene(ZmAA1)encoding maizeα-amylase driven by the pollen-specific promoter PG47 and an endosperm fluorescent marker(DsRED)driven by the barley endosperm aleurone-specific promoter Ltp2.The derived single-copy hemizygous MGM lines bore a mutated MS26 gene,leading to complete male sterility but normal vegetative growth and grain yield.The MGM system could prevent genetic transmission of the MGM elements via male gametophytes,providing an efficient method for sorting maintainer seeds labeled by DsRED.This strategy can be extended to any GMS gene and to hybrid crops other than maize.

Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize

Genome editing by clustered regularly interspaced short palindromic sequences(CRISPR)/CRISPRassociated protein 9(Cas9)has revolutionized functional gene analysis and genetic improvement.While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation,this approach has not been well established in seed crops.Here,we established the seed fluorescence reporter(SFR)-assisted CRISPR/Cas9 systems in maize(Zea mays L.),using the red fluorescent Ds RED protein expressed in the endosperm(En-SFR/Cas9),embryos(Em-SFR/Cas9),or both tissues(Em/En-SFR/Cas9).All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out.We describe several case studies of the implementation of En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9 to identify plants not harboring the genomeediting cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries,and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9,Em-SFR/Cas9,and Em/En-SFR/Cas9.SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture.Here,we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool.We systematically characterized AsCas12a,LbCas12a,LwaCas13a,and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection.Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input.Using this tool,we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field.Our method,from sample preparation to result readout,could be rapidly and easily deployed in the field.This system could be extended to other crop pathogens,including those that currently lack a detection method and have metabolite profiles that make detection challenging.This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping,transgene detection,and qualitative detection of gene expression in the field.

A DNA G-quadruplex converts SOD1 into fibrillar aggregates

Nucleic acids with G4 elements play a role in the formation of aggregates involved in intracellular phase transitions.Our previous studies suggest that different forms of DNA could act as an accelerating template in Cu/Zn superoxide dismutase(SOD1)aggregation.Here,we examined the regulation of fo rmation and cytotoxicity of the SOD1 aggregates by single-stranded 12-merdeoxynucleotide oligomers(dN)_(12)(N=A,T,G,C;ssDNAs)under acidic conditions.The ssDNAs can be divided into two groups based on their roles in SOD1 binding,exposure of hydrophobic clusters in SOD1,accelerated formation,morphology and cytotoxicity of SOD1 aggregates.G-quadruplexes convert SOD1 into fibrillar aggregates as a template,a fact which was observed for the first time in the nucleic acid regulation of protein aggregation.Moreover,the fibrillar or fibril-like SOD1 species with a G-quadruplex provided by(dG)_(12)were less toxic than the amorphous species with(dN)_(12)(N=A,T).This study not only indicates that both morphology and cytotoxicity of protein aggregates can be regulated by the protein-bound DNAs,but also help us understand roles of nucleic aid G-quadruplexes in the formation of aggregates and membrane less organelles involved in intracellular phase transitions.