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

This study reports the development of a programmed pollen self-elimination CRISPR-Cas (PSEC) system in which the pollen is infertile when PSEC is present in haploid pollen. PSEC can be inherited through the female gametophyte and retains genome editing activity in vivo across generations. This system could greatly alleviate serious concerns about the widespread diffusion of genetically modified (GM) elements into natural and agricultural environments via outcrossing.

A straight-forward seed production technology system for foxtail millet (Setaria italica).

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility (CMS) and environment-sensitive genic male sterility (EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet (Setaria italica). Here, we report on the establishment and application of a seed production technology (SPT) system for this crop. First, we established a DsRed-based SPT system, but found that it was unsuitable because it required the use of a fluorescent device for seed sorting. Instead, we constructed an SPT system with de novo betalain biosynthesis as the selection marker. This allowed us to distinguish transgenic seeds with the naked eye, thereby facilitating the identification of SPT maintainer line seeds. In this system, a seed sorter was not required to obtain sufficient seeds. The key point of the strategy is that the seed pool of the SPT maintainer line is propagated by artificial identification and harvesting of male-fertile individuals in the field, and the male-sterile line seed pool for hybrid production is produced and propagated by free pollination of male-sterile plants with the SPT maintainer line. In a field experiment, we obtained 423.96 kg male-sterile line seeds per acre, which is sufficient to plant 700.18 acres of farmland for hybrid seed production or male-sterile line reproduction. Our study therefore describes a powerful tool for hybrid seed production in foxtail millet, and demonstrates how the SPT system can be used for a small-grained crop with high reproduction efficiency.

Combining quantitative trait locus mapping with multiomics profiling reveals genetic control of corn leaf aphid (Rhopalosiphum maidis) resistance in maize.

The corn leaf aphid (Rhopalosiphum maidis) is a major maize pest that frequently causes substantial yield losses. Exploring the genetic basis of resistance to aphids is important for improving maize yield and quality. Here, we used a maize recombinant inbred line population derived from two parents with different susceptibility to aphids, B73 (susceptible) and Abe2 (resistant), and performed quantitative trait locus (QTL) mapping using aphid resistance scores as an indicator. We mapped a stable QTL, qRTA6, to chromosome 6 using data from 2 years of field trials, which explained 40.12-55.17% of the phenotypic variation. To further investigate the mechanism of aphid resistance in Abe2, we constructed transcriptome and metabolome libraries from Abe2 and B73 leaves with or without aphid infestation at different time points. Integrating QTL mapping and transcriptome data revealed three aphid resistance candidate genes (Zm00001d035736, Zm00001d035751, and Zm00001d035767) associated with the hypersensitive response, the jasmonic acid pathway, and protein ubiquitination. Integrated transcriptomic and metabolomic analysis revealed that the differentially expressed genes and metabolites were enriched in flavonoid biosynthesis. These findings extend our understanding of the molecular mechanisms controlling aphid resistance in maize, and the QTL and candidate genes are valuable resources for increasing this resistance.

CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate (CRISPRa) or inhibit transcription. Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds. We report here a CRISPR/dCas9-based toolkit equipped with dCas9-VP64 and MS2-p65-HSF1 effectors that may specifically target genes with high activation capability. We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2 (ZmBBM2), acting as a fertilization checkpoint, as a means to engineer parthenogenesis. We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells. Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds. Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.

Linkage mapping combined with GWAS revealed the genetic structural relationship and candidate genes of maize flowering time-related traits.

BACKGROUND: Flowering time is an important agronomic trait of crops and significantly affects plant adaptation and seed production. Flowering time varies greatly among maize (Zea mays) inbred lines, but the genetic basis of this variation is not well understood. Here, we report the comprehensive genetic architecture of six flowering time-related traits using a recombinant inbred line (RIL) population obtained from a cross between two maize genotypes, B73 and Abe2, and combined with genome-wide association studies to identify candidate genes that affect flowering time. RESULTS: Our results indicate that these six traits showed extensive phenotypic variation and high heritability in the RIL population. The flowering time of this RIL population showed little correlation with the leaf number under different environmental conditions. A genetic linkage map was constructed by 10,114 polymorphic markers covering the whole maize genome, which was applied to QTL mapping for these traits, and identified a total of 82 QTLs that contain 13 flowering genes. Furthermore, a combined genome-wide association study and linkage mapping analysis revealed 17 new candidate genes associated with flowering time. CONCLUSIONS: In the present study, by using genetic mapping and GWAS approaches with the RIL population, we revealed a list of genomic regions and candidate genes that were significantly associated with flowering time. This work provides an important resource for the breeding of flowering time traits in maize.

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.

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

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.

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 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.

[Regulatory framework of genome-edited products - a review].

Genome editing is a genetic engineering technique that uses site-directed cleavage activity of specific artificial nucleases and endogenous DNA damage repair activity to generate insertions, deletions or substitutions in the targeted genomic loci. As the accuracy and efficiency of genome editing is improving and the operation is simple, the application of genome editing is expanding. This article provides an overview of the three major genome editing technologies and genome editing types, and the regulatory frameworks for genome-edited products were summarized in the United States, the European Union, and other countries. At the same time, based on the Chinese safety management principles and systems for genetically modified organisms (GMOs), the authors proposed a regulatory framework for genome-edited products. Genome-edited products should first be classified according to whether containing exogenous genetic components such as Cas9 editing enzymes or not. They should be regulated as traditional genetically modified organisms if they do. Otherwise, the regulation of genome-edited products depends on targeted modifications.

Genome-wide association study dissects yield components associated with low-phosphorus stress tolerance in maize.

KEY MESSAGE: Phosphorus deficiency in soil is a worldwide constraint threatening maize production. Through a genome-wide association study, we identified molecular markers and associated candidate genes and molecular pathways for low-phosphorus stress tolerance. Phosphorus deficiency in soils will severely affect maize (Zea mays L.) growth and development, thus decreasing the final yield. Deciphering the genetic basis of yield-related traits can benefit our understanding of maize tolerance to low-phosphorus stress. However, considering that yield-related traits should be evaluated under field condition with large populations rather than under hydroponic condition at a single-plant level, searching for appropriate field experimental sites and target traits for low-phosphorus stress tolerance is still very challenging. In this study, a genome-wide association analysis using two natural populations was performed to detect candidate genes in response to low-phosphorus stress at two experimental sites representative of different climate and soil types. In total, 259 candidate genes were identified and these candidate genes are mainly involved in four major pathways: transcriptional regulation, reactive oxygen scavenging, hormone regulation, and remodeling of cell wall. Among these candidate genes, 98 showed differential expression by transcriptome data. Based on a haplotype analysis of grain number under phosphorus deficiency condition, the positive haplotypes with favorable alleles across five loci increased grain number by 42% than those without favorable alleles. For further verifying the feasibility of genomic selection for improving maize low-phosphorus tolerance, we also validated the predictive ability of five genomic selection methods and suggested that moderate-density SNPs were sufficient to make accurate predictions for low-phosphorus tolerance traits. All these results will facilitate elucidating genetic basis of maize tolerance to low-phosphorus stress and improving marker-assisted selection efficiency in breeding process.

Construction of the third-generation Zea mays haplotype map.

Background: Characterization of genetic variations in maize has been challenging, mainly due to deterioration of collinearity between individual genomes in the species. An international consortium of maize research groups combined resources to develop the maize haplotype version 3 (HapMap 3), built from whole-genome sequencing data from 1218 maize lines, covering predomestication and domesticated Zea mays varieties across the world. Results: A new computational pipeline was set up to process more than 12 trillion bp of sequencing data, and a set of population genetics filters was applied to identify more than 83 million variant sites. Conclusions: We identified polymorphisms in regions where collinearity is largely preserved in the maize species. However, the fact that the B73 genome used as the reference only represents a fraction of all haplotypes is still an important limiting factor.

[Advances in targeted replacement genome editing in plants].

Targeted replacement genome editing refers to DNA modification and engineering technology that could induce and achieve mutations of targeted gene replacement or knockin at a target gene or DNA region. In this review, the principles, implementation methods, factors that influence efficiency and accuracy, and applications of gene replacement editing were summarized and discussed. It provides the reference for gene functional characterization and genetic improvements through gene replacement strategies in higher plant especially crops.

Enhancing genetic gain in the era of molecular breeding.

As one of the important concepts in conventional quantitative genetics and breeding, genetic gain can be defined as the amount of increase in performance that is achieved annually through artificial selection. To develop pro ducts that meet the increasing demand of mankind, especially for food and feed, in addition to various industrial uses, breeders are challenged to enhance the potential of genetic gain continuously, at ever higher rates, while they close the gaps that remain between the yield potential in breeders' demonstration trials and the actual yield in farmers' fields. Factors affecting genetic gain include genetic variation available in breeding materials, heritability for traits of interest, selection intensity, and the time required to complete a breeding cycle. Genetic gain can be improved through enhancing the potential and closing the gaps, which has been evolving and complemented with modern breeding techniques and platforms, mainly driven by molecular and genomic tools, combined with improved agronomic practice. Several key strategies are reviewed in this article. Favorable genetic variation can be unlocked and created through molecular and genomic approaches including mutation, gene mapping and discovery, and transgene and genome editing. Estimation of heritability can be improved by refining field experiments through well-controlled and precisely assayed environmental factors or envirotyping, particularly for understanding and controlling spatial heterogeneity at the field level. Selection intensity can be significantly heightened through improvements in the scale and precision of genotyping and phenotyping. The breeding cycle time can be shortened by accelerating breeding procedures through integrated breeding approaches such as marker-assisted selection and doubled haploid development. All the strategies can be integrated with other widely used conventional approaches in breeding programs to enhance genetic gain. More transdisciplinary approaches, team breeding, will be required to address the challenge of maintaining a plentiful and safe food supply for future generations. New opportunities for enhancing genetic gain, a high efficiency breeding pipeline, and broad-sense genetic gain are also discussed prospectively.

Fast diffusion of domesticated maize to temperate zones.

Adaptation to a temperate climate was a prerequisite for the spread of maize across a broad geographical range. To explicitly explore the demographic process underlying maize adaptation, we used a diffusion-based method to model the differentiation between temperate and tropical populations using the Non-Stiff Stalk group as a proxy for temperate maize. Based on multiple sequential Markovian coalescent approaches, we estimate that tropical and temperate maize diverged approximately 3'000 to 5'000 years ago and the population size shrank after the split. Using composite likelihood approaches, we identified a distinct tropical-temperate divergence event initiated 4'958 years ago (95% confidence interval (CI): 4'877-5'039) from an ancestral population whose effective size was 24,162 (95% CI: 23,914-24,409). We found that continuous gene flow between tropical and temperate maize accompanied the differentiation of temperate maize. Long identical-by-descent tracts shared by tropical and temperate inbred lines have been identified, which might be the result of gene flow between tropical and temperate maize or artificial selection during domestication and crop improvement. Understanding the demographic history of maize diffusion not only provides evidence for population dynamics of maize, but will also assist the identification of regions under selection and the genetic basis of complex traits of agronomic importance.

RNA-guided Cas9 as an in vivo desired-target mutator in maize.

The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.