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.

The cuticle plays important roles in plant development, growth and defense against biotic and abiotic attacks. Crystallized epicuticular wax, the outermost layer of cuticle, is visible as white-bluish glaucousness. In crops like barley and wheat, glaucousness is trait of adaption to the dry and hot cultivation conditions, and hentriacontane-14,16-dione (β-diketone) and its hydroxy derivatives are the major and unique components of cuticular wax in the upper parts of adult plants. But their biosynthetic pathway and physiological role largely remain unknown. In the present research, we identified a novel wax mutant in wheat cultivar Bobwhite. The mutation is not allelic to the known wax production gene loci W1 and W2, and designated as W3 accordingly. Genetic analysis localized W3 on chromosome arm 2BS. The w3 mutation reduced 99% of β-diketones, which account for 63.3% of the total wax load of the wild-type. W3 is necessary for β-diketone synthesis, but has a different effect on β-diketone hydroxylation because the hydroxy-β-diketones to β-diketone ratio increased 11-fold in the w3 mutant. Loss of β-diketones caused failure to form glaucousness and significant increase of cuticle permeability in terms of water loss and chlorophyll efflux in the w3 mutant. Transcription of 23 cuticle genes from five functional groups was altered in the w3 mutant, 19 down-regulated and four up-regulated, suggesting a possibility that W3 encodes a transcription regulator coordinating expression of cuticle genes. Biosynthesis of β-diketones in wheat and their implications in glaucousness formation and drought and heat tolerance were discussed.Key Message W3 is essential for β-diketone biosynthesis but suppresses its hydroxylation. Loss-of-function mutation w3 significantly increased cuticle permeability in terms of water loss and chlorophyll efflux.

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Ear length (EL), which is controlled by quantitative trait loci (QTLs), is an important component of grain yield and as such is a key target trait in maize breeding. However, very few EL QTLs have been cloned, and their molecular mechanisms are largely unknown.Here, using a genome wide association study (GWAS), we identified a QTL, YIGE1, which encodes an unknown protein that regulates EL by affecting pistillate floret number. Overexpression of YIGE1 increased female inflorescence meristem (IM) size, increased EL and kernel number per row (KNPR), and thus enhanced grain yield. By contrast, CRISPR/Cas9 knockout and Mutator insertion mutant lines of YIGE1 displayed decreased IM size and EL.A single-nucleotide polymorphism (SNP) located in the regulatory region of YIGE1 had a large effect on its promoter strength, which positively affected EL by increasing gene expression. Further analysis shows that YIGE1 may be involved in sugar and auxin signal pathways to regulate maize ear development, thus affecting IM activity and floret production in maize inflorescence morphogenesis.These findings provide new insights into ear development and will ultimately facilitate maize molecular breeding.

Plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPCp) are ubiquitous proteins that play pivotal roles in plant metabolism and are involved in stress response. However, the mechanism of GAPCp’s function in plant stress resistance process remains unclear. Here we isolated, identified, and characterized the TaGAPCp1 gene from Chinese Spring wheat for further investigation. Subcellular localization assay indicated that the TaGAPCp1 protein was localized in the plastid of tobacco (Nicotiana tobacum) protoplast. In addition, quantitative real-time PCR (qRT-PCR) unraveled that the expression of TaGAPCp1 (GenBank: MF477938.1) was evidently induced by osmotic stress and abscisic acid (ABA). This experiment also screened its interaction protein, cytochrome b6-f complex iron sulfite subunit (Cyt b6f), from the wheat cDNA library using TaGAPCp1 protein as a bait via the yeast two-hybrid system (Y2H) and the interaction between Cyt b6f and TaGAPCp1 was verified by bimolecular fluorescence complementation assay (BiFC). Moreover, H2O2 could also be used as a signal molecule to participate in the process of Cyt b6f response to abiotic stress. Subsequently, we found that the chlorophyll content in OE-TaGAPCp1 plants was significantly higher than that in wild type (WT) plants. In conclusion, our data revealed that TaGAPCp1 plays an important role in abiotic stress response in wheat and this stress resistance process may be completed by H2O2-mediated ABA signaling pathway.

Chromosomal rearrangements (CRs) play important roles in karyotype diversity and speciation. While many CR breakpoints have been characterized at the sequence level in yeast, insects, and primates, little is known about the structure of evolutionary CR breakpoints in plant genomes, which are much more dynamic in genome size and sequence organization. Here, we report identification of breakpoints of a translocation between chromosome arms 4L and 5L of Triticeae, which is fixed in several species, including diploid wheat and rye, by comparative mapping and analysis of the draft genome and chromosome survey sequences of the Triticeae species. The wheat translocation joined the ends of breakpoints downstream of a WD40 gene on 4AL and a gene of the PMEI family on 5AL. A basic helix-loop-helix transcription factor gene in 5AL junction was significantly restructured. Rye and wheat share the same position for the 4L breakpoint, but the 5L breakpoint positions are not identical, although very close in these two species, indicating the recurrence of 4L/5L translocations in the Triticeae. Although barley does not carry the translocation, collinearity across the breakpoints was violated by putative inversions and/or transpositions. Alignment with model grass genomes indicated that the translocation breakpoints coincided with ancient inversion junctions in the Triticeae ancestor. Our results show that the 4L/5L translocation breakpoints represent two CR hotspots reused during Triticeae evolution, and support breakpoint reuse as a widespread mechanism in all eukaryotes. The mechanisms of the recurrent translocation and its role in Triticeae evolution are also discussed.

Background Maize (Zea mays L.) is at the vanguard facing the upcoming breeding challenges. However, both a super pan-genome for the Zea genus and a comprehensive genetic variation map for maize breeding are still lacking. Results Here, we construct an approximately 6.71-Gb pan-Zea genome that contains around 4.57-Gb non-B73 reference sequences from fragmented de novo assemblies of 721 pan-Zea individuals. We annotate a total of 58,944 pan-Zea genes and find around 44.34% of them are dispensable in the pan-Zea population. Moreover, 255,821 common structural variations are identified and genotyped in a maize association mapping panel. Further analyses reveal gene presence/absence variants and their potential roles during domestication of maize. Combining genetic analyses with multi-omics data, we demonstrate how structural variants are associated with complex agronomic traits. Conclusions Our results highlight the underexplored role of the pan-Zea genome and structural variations to further understand domestication of maize and explore their potential utilization in crop improvement.

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