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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 maize α-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.

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.

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-DTMwx. 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-DTMwx. The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged 94.9%, while those of the wild-type controls were significantly (P

Establishing a rapid, accurate, high-throughput and easily implementable genotyping method is highly desirable for functional genomics, genetic improvement and mutant screening. Here, we describe a convenient and inexpensive technique for genotyping using the targeted DNA endonuclease activity of Cas9 or Cas9NG ribonucleoproteins complex (sgRNA/Cas9-RNP or sgRNA/Cas9NG-RNP). In this study, Cas9 and Cas9NG protein purified from E. coli extract was assembled with in vitro transcribed single guide RNA (sgRNA)or enhanced sgRNA (esgRNA) as an assembled ribonucleoprotein (RNP) complex to fulfill the targeted endonuclease activity on the PCR amplicons of ZmWx exon 7. The restriction profiles can be converted into genotyping results of wildtype, homozygous or heterozygous mutant, respectively. Our data showed that ZmWx gene-edited mutants can be genotyped rapidly and efficiently by the sgRNA-optimized system esgRNA/Cas9. The reaction component optimization data suggested that 500 ng of DNA substrates could be cleavaged completely by incubating with 1 µg 1:1 molar ratio of esgRNA/Cas9 ribonucleoproteins for 30 minutes at 37°C, or by 4 µg 1:1 molar ratio of esgRNA/Cas9NG ribonucleoproteins for 4 hours at 37°C. Expanding the targeting flexibility of mutant detection via esgRNA/Cas9NG indicated that Cas9NG variant might recognize relax NG PAM (protospacer-adjacent-motif, PAM) at the expense of decreasing restriction activity, which is necessary to improve the activity of esgRNA/Cas9NG by further optimization. Therefore, the establishment and application of esgRNA/Cas9 based PCR/RNP technique provides an easy, simple and low-cost approach to genotyping in functional genomics, molecular breeding and mutant screening. In addition, our in vitro data on esgRNA/Cas9NG has certain and significant reference value for developing it into in vivo genome editing studies.

Algal blooms bring massive amounts of algal organic matter (AOM) into eutrophic lakes, which influences microbial methylmercury (MeHg) production. However, because of the complexity of AOM and its dynamic changes during algal decomposition, the relationship between AOM and microbial Hg methylators remains poorly understood, which hinders predicting MeHg production and its bioaccumulation in eutrophic shallow lakes. To address that, we explored the impacts of AOM on microbial Hg methylators and MeHg production by characterizing dissolved organic matter with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy and quantifying the microbial Hg methylation gene hgcA. We first reveal that the predominance of methanogens, facilitated by eutrophication-induced carbon input, could drive MeHg production in lake water. Specifically, bioavailable components of AOM (i.e., CHONs such as aromatic proteins and soluble microbial byproduct-like materials) increased the abundances (Archaea-hgcA gene: 438-2240% higher) and activities (net CH4 production: 16.0-44.4% higher) of Archaea (e.g., methanogens). These in turn led to enhanced dissolved MeHg levels (24.3-15,918% higher) for three major eutrophic shallow lakes in China. Nevertheless, our model results indicate that AOM-facilitated MeHg production could be offset by AOM-induced MeHg biodilution under eutrophication. Our study would help reduce uncertainties in predicting MeHg production, providing a basis for mitigating the MeHg risk in eutrophic lakes.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated 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 DsRED 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 genome-editing 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.

Uncovering the genetic basis of seed development will provide useful tools for improving both crop yield and nutritional value. However, the genetic regulatory networks of maize (Zea mays) seed development remain largely unknown. The maize opaque endosperm and small germ 1 (os1) mutant has opaque endosperm and a small embryo. Here, we cloned OS1 and show that it encodes a putative transcription factor containing an RWP-RK domain. Transcriptional analysis indicated that OS1 expression is elevated in early endosperm development, especially in the basal endosperm transfer layer (BETL), conducting zone (CZ), and central starch endosperm (CSE) cells. RNA sequencing (RNA-Seq) analysis of the os1 mutant revealed sharp downregulation of certain genes in specific cell types, including ZmMRP-1 and Meg1 in BETL cells and a majority of zein- and starch-related genes in CSE cells. Using a haploid induction system, we show that wild-type endosperm could rescue the smaller size of os1 embryo, which suggests that nutrients are allocated by the wild-type endosperm. Therefore, our data imply that the network regulated by OS1 accomplishes a key step in nutrient allocation between endosperm and embryo within maize seeds. Identification of this network will help uncover the mechanisms regulating the nutritional balance between endosperm and embryo.

Aims 14,15-Epoxyeicosatrienoic acid (14,15-EET) is abundantly expressed in brain and exerts protective effects against ischaemia. 14,15-EET is hydrolysed by soluble epoxide hydrolase (sEH). sEH−/− mice show a higher level of 14,15-EET in the brain. Astrocytes play a pivotal role in neuronal survival under ischaemic conditions. However, it is unclear whether the neuroprotective effect of 14,15-EET is associated with astrocytes. Methods A mouse model of focal cerebral ischaemia was induced by middle cerebral artery occlusion. Oxygen–glucose deprivation/reoxygenation (OGD/R) was performed on cultured murine astrocytes, neurons and a human cell line. Cell viabilities were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The mRNA expressions were quantified by real-time PCR. Brain derived neurotrophic factor (BDNF) concentration was measured by ELISA. Protein expressions were quantified by Western blotting. BDNF and peroxisome proliferators-activated receptor gamma (PPAR-γ) expressions were analysed by confocal microscopy. Results Decreased infarct volumes, elevated BDNF expression and increased numbers of BDNF/GFAP Glial Fibrillary Acidic Protein double-positive cells were observed in the ischaemic penumbra of sEH−/− mice. The decreased infarct volumes of sEH−/− mice were diminished by intracerebroventricular injection of a blocker of BDNF receptor. 14,15-EET increases BDNF expression and cell viability of murine astrocytes and U251 cells by BDNF-TrkB Tyrosine receptor kinase-B-extracellular signal-regulated kinase 1/2 signalling during OGD/R. 14,15-EET protects neurons from OGD/R by stimulating the production of astrocyte-derived BDNF. 14,15-EET stimulates the production of astrocyte-derived BDNF through PPAR-γ/p-cAMP-response element binding protein signal pathways. Conclusions Our study demonstrates the importance of 14,15-EET-mediated production of astrocyte-derived BDNF for enhancing viability of astrocytes and protecting neurons from the ischaemic injury and provides insights into the mechanism by which 14,15-EET is involved in neuroprotection.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.

Leaf width (LW) influences canopy architecture of population-cultured maize and can thus contribute to density breeding. In previous studies, almost all maize LW-related mutants have extreme effect on leaf development or accompanied unfavorable phenotypes. In addition, the identification of quantitative trait loci (QTLs) has been resolution-limited, with cloning and fine-mapping rarely performed. Here, we constructed a bin map for 670 recombinant inbred lines (RILs) using ∼1.2 billion 100-bp re-sequencing reads. QTL analysis of the LW trait directly narrowed the major effect QTL, qLW4, to a ∼270-kb interval. A fine-mapping population and near-isogenic lines (NILs) were quickly constructed using a key RIL harboring heterozygous genotypes across the qLW4 region. A recombinant-derived progeny testing strategy was subsequently used to further fine-map qLW4 to a 55-kb interval. Examination of NILs revealed that qLW4 has a completely dominant effect on LW, with no additional effect on leaf length. Candidate gene analysis suggested that this locus may be a novel LW controlling allele in maize. Our findings demonstrate the advantage of large-population high-density bin mapping, and suggest a strategy for efficiently fine-mapping or even cloning of QTLs. These results should also be helpful for further dissection of the genetic mechanism of LW variation, and benefit maize density breeding.