The availability of the B73 inbred reference genome sets the stage for high-throughput functional characterization of maize genes on a whole-genome scale. Among the 39 324 protein-coding genes predicted, the vast majority are untapped due to the lack of suitable high-throughput reverse genetic resources. We have generated a gene-indexed maize mutant collection through ethyl methanesulfonate mutagenesis and detected the mutations by combining exome capture and next-generation sequencing. A total of 1086 mutated M plants were sequenced, and 195 268 CG>TA-type point mutations, including stop gain/loss, missplice, start gain/loss, and various non-synonymous protein mutations as well as 4610 InDel mutations, were identified. These mutations were distributed on 32 069 genes, representing 82% of the predicted protein-coding genes in the maize genome. We detected an average of 180 mutations per mutant line and 6.1 mutations per gene. As many as 27 214 mutations of start codons, stop codons, or missplice sites were identified in 14 101 genes, among which 6232 individual genes harbored more than two such mutations. Application of this mutant collection is exemplified by the identification of the ent-kaurene synthase gene, which encodes a key enzyme in the gibberellin biosynthesis pathway. This gene-indexed genome-wide mutation collection provides an important resource for functional analysis of maize genes and may bring desirable allelic variants for genetic breeding in maize.

SummaryAlthough hundreds of genetic male sterility (GMS) mutants have been identified in maize, few are commercially used due to a lack of effective methods to produce large quantities of pure male‐sterile seeds. Here, we develop a multicontrol sterility (MCS) system based on the maize male sterility 7 (ms7) mutant and its wild‐type Zea mays Male sterility 7 (ZmMs7) gene via a transgenic strategy, leading to the utilization of GMS in hybrid seed production. ZmMs7 is isolated by a map‐based cloning approach and encodes a PHD‐finger transcription factor orthologous to rice PTC1 and Arabidopsis MS1. The MCS transgenic maintainer lines are developed based on the ms7‐6007 mutant transformed with MCS constructs containing the (i) ZmMs7 gene to restore fertility, (ii) α‐amylase gene ZmAA and/or (iii) DNA adenine methylase gene Dam to devitalize transgenic pollen, (iv) red fluorescence protein gene DsRed2 or mCherry to mark transgenic seeds and (v) herbicide‐resistant gene Bar for transgenic seed selection. Self‐pollination of the MCS transgenic maintainer line produces transgenic red fluorescent seeds and nontransgenic normal colour seeds at a 1:1 ratio. Among them, all the fluorescent seeds are male fertile, but the seeds with a normal colour are male sterile. Cross‐pollination of the transgenic plants to male‐sterile plants propagates male‐sterile seeds with high purity. Moreover, the transgene transmission rate through pollen of transgenic plants harbouring two pollen‐disrupted genes is lower than that containing one pollen‐disrupted gene. The MCS system has great potential to enhance the efficiency of maize male‐sterile line propagation and commercial hybrid seed production.

Maize (Zea mays) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants.

Neonicotinoid insecticides are widely used for controlling sucking pests, and sublethal effects can be expected in beneficial arthropods like natural enemies. Serangium japonicum is an important predator in many agricultural systems in China, and a potential biological control agent against Bemisia tabaci. We evaluated the toxicity of imidacloprid to S. japonicum and its impact on the functional response to B. tabaci eggs. S. japonicum adults exposed through contact to dried residues of imidacloprid at the recommended field rate on cotton against B. tabaci (4 g active ingredient per 100 l, i.e. 40 ppm [part per million]), and reduced rates (25, 20, 15 and 10 ppm) for 24 h showed high mortality rates. The mortality induced by a lowest rate, 5 ppm, was not significantly different than the control group and thus it was considered as a sublethal rate. The lethal rate 50 and hazard quotient (HQ) were estimated to be 11.54 ppm and 3.47 respectively, indicating a risk for S. japonicum in treated fields (HQ > 2). When exposed to dried residues of imidacloprid at the sublethal rate (5 ppm) on cotton leaves, functional response of S. japonicum to B. tabaci eggs was affected with an increase in handling time and a reduction in peak consumption of eggs. Imidacloprid residues also disturbed predator voracity, the number of B. tabaci eggs consumed on treated leaves being significantly lower than on untreated leaves. All effects disappeared within a few hours after transfer to untreated cotton leaves. Imidacloprid systemically applied at the recommended field rate (for cotton) showed no toxicity to S. japonicum, nor affected the functional response of the predator. Sublethal effects of imidacloprid on S. japonicum observed in our study likely negatively affect S. japonicum development and reproductive capacity and may ultimately reduce predator population growth. These results hint at the importance of assessing potential effects of imidacloprid on S. japonicum for developing effective integrated pest management programs of B. tabaci in China.