One-third of the global population is affected by micronutrient deficiency, particularly folate. Although folate synthesis has been relatively well characterized, few folate-related genes in maize have been cloned, and the molecular mechanism regulating folate synthesis in maize remains unclear. In this study, transcriptome and proteome analyses of three waxy maize inbred lines with high, medium, and low folate contents were performed to identify key genes controlling folate biosynthesis. Pairwise comparisons revealed 21 differentially expressed genes and 20 differentially expressed proteins potentially associated with folate biosynthesis in the three lines. Six key folate-associated genes, ZmMocos2, ZmGGH, ZmADCL2, ZmCBR1, ZmSHMT, and ZmPurH, were identified. These genes encode enzymes that potentially function in folate biosynthesis. Functional validation of one of these genes, ZmADCL2, using an EMS mutant (Mut9264) showed that a 4-base insertion in an exon increased the folate content of fresh maize kernels 1.37-fold that of the wild type. ZmADCL2 was considered a potential target for generating maize lines with higher folate content. KEGG enrichment analysis of differentially expressed genes and proteins showed that several pathways in addition to folate biosynthesis were likely indirectly involved in folate metabolism and content (e.g., glycine, serine, and threonine metabolism; purine metabolism; cysteine and methionine metabolism; alanine, aspartate and glutamate metabolism; glutathione metabolism; and pyruvate metabolism. The transcriptome and proteomic data generated in this study will help to clarify the mechanisms underlying folate accumulation and aid breeding efforts to biofortify maize with folate.

Maize is the most widely cultivated, used and highest yield crop in the world and China. Southern corn rust (SCR) is an air borne disease caused by Puccinia polysora Underw., which mainly occurs in tropical and subtropical maize growing areas. In recent years, SCR has become one of the major diseases in the Huang-Huai-hai maize production region due to the climate change, which directly leads to compromised grain quality and poor yields in maize and significantly jeopardizes maize production in China. At present, SCR usually spreads in a large area within a short period of time once occurred because most maize varieties promoted in China are susceptible, and conventional chemical measures is usually in vain. Therefore, cultivating resistant cultivars by exploiting resistance genes in maize germplasm resources is the most effective and economical strategy for controlling SCR. The highly resistant germplasm is scarce in maize resources, mainly from tropical and subtropical regions, and barely no temperate germplasm can be directly used in breeding practice. Compared with foreign maize germplasm, the highly resistant maize germplasms of China were much less, mainly from local landraces or P group materials containing tropical origins with relatively limited genetic variation. The identification and cloning of SCR resistance genes in maize is essential for promoting molecular marker-assisted breeding, as well as accelerating the breeding process of new varieties with desired resistance. At present, several SCR resistance genes have been identified and cloned, laying a foundation for molecular marker-assisted selection. Over the years, Chinese breeders have developed a number of elite maize inbred lines resistant to SCR with limited resistance germplasm resources, and successfully created disease-resistant hybrids. Recent studies on the genome of SCR pathogens revealed that pathogens have differentiated into highly toxic lineages in China, thus escaping the recognition of resistance genes. Therefore, the exploration and utilization of extensive genetic resources in resistant germplasm still need to be further strengthened. In this paper, we outlined the biological characteristics and hazards of SCR, systematically summarized the research progresses in the identification and utilization of maize germplasm resources resistant to SCR, the mapping and cloning of SCR resistant genes and the breeding of resistant varieties, and prospect the future research direction of SCR. This review will provide references for the prevention and control of SCR, as well as the breeding of resistant maize varieties.

Doubled haploid (DH) technology is an efficient method used in commercial maize breeding. Chromosome doubling is a vital step of DH technology; however, the underlying processes regulating chromosome doubling of haploid is still not well understood, which is key to optimize the technology. In this study, the immature haploid embryos of the maize inbred line Zheng58 treated with amiprophos-methyl (APM) or colchicine were used to analyze transcriptomic and metabolomic changes, 75 and 60 differential expressed metabolites (DEMs) were identified between control treatment, respectively. Most differentially expressed genes (DEGs) related to artificial chromosome doubling were down regulated; these were mainly involved in mitosis process. Both DEMs and DEGs co-expression analyses showed that, compared to controls, zeatin biosynthesis and cofactor and vitamin metabolism were significantly enriched in both APM and colchicine treatments. In a parallel experiment, exogenous vitamins including thiamine, nicotinic acid, vitamin B6, or trans-zeatin were added to colchicine treatment; there were synergistic effects between vitamins or zeatin and colchicine in haploid artificial chromosome doubling. These results provide novel insights in exploring the molecular responses to antimitotic reagents at both the transcriptomic and metabolomic levels. In addition, the application efficiency of haploid breeding will be greatly improved by the key factors for artificial chromosome doubling.

To reduce the cost and increase the efficiency of plant genetic marker fingerprinting for variety discrimination, it is desirable to identify the optimal marker combinations. We describe a marker combination screening model based on the genetic algorithm (GA) and implemented in a software tool, LociScan. Ratio-based variety discrimination power provided the largest optimization space among multiple fitness functions. Among GA parameters, an increase in population size and generation number enlarged optimization depth but also calculation workload. Exhaustive algorithm afforded the same optimization depth as GA but vastly increased calculation time. In comparison with two other software tools, LociScan accommodated missing data, reduced calculation time, and offered more fitness functions. In large datasets, the sample size of training data exerted the strongest influence on calculation time, whereas the marker size of training data showed no effect, and target marker number had limited effect on analysis speed.

Genic male sterility (GMS) is one of the most important resources for exploiting heterosis in crop breeding, so that identifying genomic loci regulating GMS is desirable. However, many regulatory genes controlling GMS have not yet been characterized in maize, owing partly to a lack of genetic materials. We generated a recessive male-sterile maize mutant in the Jing 724 genetic background via ethyl methanesulfonate treatment, and found the male sterility to be due to a single gene mutation. Bulk-segregant RNA sequencing of three replicates indicated that one genomic region located at the end of chromosome 4 was associated with the observed mutant phenotype. Among genes with nonsynonymous mutations, Zm00001d053895 (bHLH51) showed abolished expression in the sterile bulks and was annotated as a bHLH transcription factor orthologous to Arabidopsis AMS, suggesting an association with the male sterility of the mutant. Kompetitive Allele-Specific PCR assays further validated the exclusive correlation of male sterility with the single C-to-T mutation in the fifth exon. The new maize mutant and the potential SNP locus provide novel genetic material for investigating the molecular mechanism underlying tapetal development and may facilitate the improvement of hybrid production systems.

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has been widely used for genome editing. In this system, the cytosine base editor (CBE) and adenine base editor (ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine (C) or adenine (A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE (sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal (NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS (F4NLS^r2) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE (sCBE) and F4NLS^r2 involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.