Loss-of-function of LIGULELESS1 activates the jasmonate pathway and promotes maize resistance to corn leaf aphids.

Corn leaf aphids (Rhopalosiphum maidis) are highly destructive pests of maize (Zea mays) that threaten growth and seed yield, but resources for aphid resistance are scarce. Here, we identified an aphid-resistant maize mutant, resistance to aphids 1 (rta1), which is allelic to LIGULELESS1 (LG1). We confirmed LG1's role in aphid resistance using the independent allele lg1-2, allelism tests and LG1 overexpression lines. LG1 interacts with, and increases the stability of ZINC-FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM (ZIM1), a central component of the jasmonic acid (JA) signalling pathway, by disturbing its interaction with the F-box protein CORONATINE INSENSITIVE 1a (COI1a). Natural variation in the LG1 promoter was associated with aphid resistance among inbred lines. Moreover, a loss-of-function mutant in the LG1-related gene SPL8 in the dicot Arabidopsis thaliana conferred aphid resistance. This study revealed the aphid resistance mechanism of lg1, providing a theoretical basis and germplasm for breeding aphid-resistant crops.

Expediting Next-Generation Hybrid Technology in Recalcitrant Maize Inbreds through In Vivo Targeted Activity of CRISPR/Cas9.

The Manipulated Genic Male Sterile Maintainer (MGM) system, a next-generation hybrid seed technology, enables efficient production of sortable seeds from genic male sterile (GMS) lines. However, implementing robust MGM systems in commercial maize inbred lines requires stable transformation, a genotype-specific and laborious process. This study aimed to integrate MGM technology into the commercial maize inbred line Z372, developing both GMS and MGM lines. We utilized the MGM line ZC01-3A-7, which contains the MS26ΔE5 editor T-DNA and MGM T-DNA, previously established in the highly transformable ZC01 recipient plants. Through a combination of crossing and backcrossing with Z372, we targeted the fertility gene Ms26 within the Z372 genome for mutation using the in vivo CRISPR/Cas9 activity within the MS26ΔE5 editor T-DNA construct. This approach facilitated precise editing of the Ms26 locus, minimizing linkage drag associated with the Ms26 mutation. Whole-genome SNP analysis achieved a 98.74% recovery rate for GMS and 96.32% for MGM in the BC2F2 generation. Importantly, the Z372-GMS line with the ms26ΔE5 mutation is non-transgenic, avoiding linkage drag and demonstrating production readiness. This study represents a significant advancement in maize breeding, enabling the rapid generation of GMS and MGM lines for efficient hybrid seed production.

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.

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.

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.

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-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.

CRISPR/Cas9-mediated genome editing in plants.

The increasing burden of the world's population on agriculture necessitates the development of more robust crops. As the amount of information from sequenced crop genomes increases, technology can be used to investigate the function of genes in detail and to design improved crops at the molecular level. Recently, an RNA-programmed genome-editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9 has provided a powerful platform to achieve these goals. By combining versatile tools to study and modify plants at different molecular levels, the CRISPR/Cas9 system is paving the way towards a new horizon for basic research and crop development. In this review, the accomplishments, problems and improvements of this technology in plants, including target sequence cleavage, knock-in/gene replacement, transcriptional regulation, epigenetic modification, off-target effects, delivery system and potential applications, will be highlighted.

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.

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.

Genetic architecture of the maize kernel row number revealed by combining QTL mapping using a high-density genetic map and bulked segregant RNA sequencing.

BACKGROUND: The maize kernel row number (KRN) is a key component that contributes to grain yield and has high broad-sense heritability (H 2 ). Quantitative trait locus/loci (QTL) mapping using a high-density genetic map is a powerful approach to detecting loci that are responsible for traits of interest. Bulked segregant ribonucleic acid (RNA) sequencing (BSR-seq) is another rapid and cost-effective strategy to identify QTL. Combining QTL mapping using a high-density genetic map and BSR-seq may dissect comprehensively the genetic architecture underlying the maize KRN. RESULTS: A panel of 300 F2 individuals derived from inbred lines abe2 and B73 were genotyped using the specific-locus amplified fragment sequencing (SLAF-seq) method. A total of 4,579 high-quality polymorphic SLAF markers were obtained and used to construct a high-density genetic map with a total length of 2,123 centimorgan (cM) and an average distance between adjacent markers of 0.46 cM. Combining the genetic map and KRN of F2 individuals, four QTL (qKRN1, qKRN2, qKRN5, and qKRN8-1) were identified on chromosomes 1, 2, 5, and 8, respectively. The physical intervals of these four QTL ranged from 4.36 Mb for qKRN8-1 to 7.11 Mb for qKRN1 with an average value of 6.08 Mb. Based on high-throughput sequencing of two RNA pools bulked from leaves of plants with extremely high and low KRNs, two QTL were detected on chromosome 8 in the 10-25 Mb (BSR_QTL1) and 60-150 Mb (BSR_QTL2) intervals. According to the physical positions of these QTL, qKRN8-1 was included by BSR_QTL2. In addition, qKRN8-1 was validated using QTL mapping with a recombinant inbred lines population that was derived from inbred lines abe2 and B73. CONCLUSIONS: In this study, we proved that combining QTL mapping using a high-density genetic map and BSR-seq is a powerful and cost-effective approach to comprehensively revealing genetic architecture underlying traits of interest. The QTL for the KRN detected in this study, especially qKRN8-1, can be used for performing fine mapping experiments and marker-assisted selection in maize breeding.

Strand-specific RNA-Seq transcriptome analysis of genotypes with and without low-phosphorus tolerance provides novel insights into phosphorus-use efficiency in maize.

BACKGROUND: Phosphorus (P) stress is a global problem in maize production. Although macro/microarray technologies have greatly increased our general knowledge of maize responses to P stress, a greater understanding of the diversity of responses in maize genotypes is still needed. RESULTS: In this study, we first evaluated the tolerance to low P of 560 accessions under field conditions, and selected the low P-tolerant line CCM454 and the low P-sensitive line 31778 for further research. We then generated 24 strand-specific RNA libraries from shoots and roots of CCM454 and 31778 that had been subjected to P stress for 2 and 8 days. The P deficiency-responsive genes common to CCM454 and 31778 were involved in various metabolic processes, including acid phosphatase (APase) activity. Determination of root-secretory APase activities showed that the induction of APase by P stress occurred much earlier in CCM454 than that in 31778. Gene Ontology analysis of differentially expressed genes (DEGs) and CAT/POD activities between CCM454 and 31778 under P-sufficient and -deficient conditions demonstrated that CCM454 has a greater ability to eliminate reactive oxygen species (ROS) than 31778. In addition, 16 miRNAs in roots and 12 miRNAs in shoots, including miRNA399s, were identified as DEGs between CCM454 and 31778. CONCLUSIONS: The results indicate that the tolerance to low P of CCM454 is mainly due to the rapid responsiveness to P stress and efficient elimination of ROS. Our findings increase the understanding of the molecular events involved in the diversity of responses to P stress among maize accessions.

Fine mapping of a quantitative trait locus conferring resistance to maize rough dwarf disease.

KEY MESSAGE: A QTL qMrdd8 that confers resistance to MRDD was fine mapped into an interval of 347 kb; one SNP and two InDels identified in the interval were significantly associated with resistance to MRDD. Maize rough dwarf disease (MRDD) is highly prevalent in the summer maize-growing areas in China, and leads to significant yield losses in maize (Zea mays L.). In this study, the quantitative trait locus (QTL) qMrdd8, which confers resistance to MRDD, was fine mapped. Initially, qMrdd8 was consistently identified in the interval between the simple sequence repeat markers umc1617 and phi121 in three F2 sub-populations derived from a cross between the resistant recombinant inbred line NL203 and the susceptible line B73. Subsequently, qMrdd8 was fine mapped into an interval of 347 kb defined by the markers IDRQ2 and IDRQ20 using a recombinant-derived progeny test strategy. Based on single nucleotide polymorphism (SNP) genotypes identified using the MaizeSNP50 BeadChip, a long haplotype including qMrdd8 was identified in four resistant inbred lines. One SNP, the 2549-bp insertion/deletion polymorphism (InDel) InDel25, and the 2761-bp InDel27, which all were significantly associated with resistance to MRDD in a set of 226 maize inbred lines (P < 0.05), were detected within qMrdd8. Furthermore, two candidate genes, CG1 and CG2, were detected in the interval using RNA sequencing (RNA-Seq), and InDel25 was localized within the candidate gene CG1. In conclusion, the fine mapping of qMrdd8 will be helpful in cloning the resistance gene, and the three polymorphic markers identified in this study could be used to improve MRDD resistance via a marker-assisted selection approach.

Identification and functional characterization of the AGO1 ortholog in maize.

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1-27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild-type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.

bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism.

• Although proteins in the basic helix-loop-helix (bHLH) family are universal transcription factors in eukaryotes, the biological roles of most bHLH family members are not well understood in plants. • The Arabidopsis thaliana bHLH122 transcripts were strongly induced by drought, NaCl and osmotic stresses, but not by ABA treatment. Promoter::GUS analysis showed that bHLH122 was highly expressed in vascular tissues and guard cells. Compared with wild-type (WT) plants, transgenic plants overexpressing bHLH122 displayed greater resistance to drought, NaCl and osmotic stresses. In contrast, the bhlh122 loss-of-function mutant was more sensitive to NaCl and osmotic stresses than were WT plants. • Microarray analysis indicated that bHLH122 was important for the expression of a number of abiotic stress-responsive genes. In electrophoretic mobility shift assay and chromatin immunoprecipitation assays, bHLH122 could bind directly to the G-box/E-box cis-elements in the CYP707A3 promoter, and repress its expression. Further, up-regulation of bHLH122 substantially increased cellular ABA levels. • These results suggest that bHLH122 functions as a positive regulator of drought, NaCl and osmotic signaling.

Kernel number as a positive target trait for prediction of hybrid performance under low-nitrogen stress as revealed by diallel analysis under contrasting nitrogen conditions.

Environmental sustainability concerns make improving yield under lower N input a desirable breeding goal. To evaluate genetic variation and heterosis for low-N tolerance breeding, 28 F1 hybrids from a diallel scheme, along with their eight parental lines, were tested for agronomic traits including kernel number per ear (KNE) and grain yield per plant (GY), in replicated plots over two years under low-nitrogen (LN, without nitrogen application) and normal-nitrogen (NN, 220 kg N ha(-1)) conditions. Taken together the heritability in this and our previous studies, the correlation with grain yield, and the sensitivity to the stress for target trait selection, KNE was a good secondary target trait for LN selection in maize breeding. KNE also showed much higher mid-parent heterosis than hundred-kernel weight under both nitrogen levels, particularly under LN, indicating that KNE contributed the majority of GY heterosis, particularly under LN. Therefore, KNE can be used as a positive target trait for hybrid performance prediction in LN tolerance breeding. Our results also suggest that breeding hybrids for LN tolerance largely relies on phenotypic evaluation of hybrids under LN condition and yield under LN might be improved more by selection for KNE than by direct selection for GY per se.