Plant architecture attributes, such as plant height, ear height, and internode number, have played an important role in the historical increases in grain yield, lodging resistance, and biomass in maize (Zea mays L.). Analyzing the genetic basis of variation in plant architecture using high density QTL mapping will be of benefit for the breeding of maize for many traits. However, the low density of molecular markers in existing genetic maps has limited the efficiency and accuracy of QTL mapping. Genotyping by sequencing (GBS) is an improved strategy for addressing a complex genome via next-generation sequencing technology. GBS has been a powerful tool for SNP discovery and high-density genetic map construction. The creation of ultra-high density genetic maps using large populations of advanced recombinant inbred lines (RILs) is an efficient way to identify QTL for complex agronomic traits. A set of 314 RILs derived from inbreds Ye478 and Qi319 were generated and subjected to GBS. A total of 137,699,000 reads with an average of 357,376 reads per individual RIL were generated, which is equivalent to approximately 0.07-fold coverage of the maize B73 RefGen_V3 genome for each individual RIL. A high-density genetic map was constructed using 4183 bin markers (100-Kb intervals with no recombination events). The total genetic distance covered by the linkage map was 1545.65 cM and the average distance between adjacent markers was 0.37 cM with a physical distance of about 0.51 Mb. Our results demonstrated a relatively high degree of collinearity between the genetic map and the B73 reference genome. The quality and accuracy of the bin map for QTL detection was verified by the mapping of a known gene, pericarp color 1 (P1), which controls the color of the cob, with a high LOD value of 80.78 on chromosome 1. Using this high-density bin map, 35 QTL affecting plant architecture, including 14 for plant height, 14 for ear height, and seven for internode number were detected across three environments. Interestingly, pQTL10, which influences all three of these traits, was stably detected in three environments on chromosome 10 within an interval of 14.6 Mb. Two MYB transcription factor genes, GRMZM2G325907 and GRMZM2G108892, which might regulate plant cell wall metabolism are the candidate genes for qPH10. Here, an ultra-high density accurate linkage map for a set of maize RILs was constructed using a GBS strategy. This map will facilitate identification of genes and exploration of QTL for plant architecture in maize. It will also be helpful for further research into the mechanisms that control plant architecture while also providing a basis for marker-assisted selection.

Soil salt-alkalization is a common yet critical environmental stress factor for plant growth and development. Discovering and exploiting genes associated with alkaline tolerance in maize (Zea mays L.) is helpful for improving alkaline resistance. Here, an association panel consisting of 200 maize lines was used to identify the genetic loci responsible for alkaline tolerance-related traits in maize seedlings. A total of nine single-nucleotide polymorphisms (SNPs) and their associated candidate genes were found to be significantly associated with alkaline tolerance using a genome-wide association study (GWAS). An additional 200 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r² ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then conducted to confirm the linkage between the candidate genes and alkali tolerance. From these data, a total of five differentially expressed genes (DEGs; |log2FC| ≥ 0.585, p < 0.05) were verified as the hub genes involved in alkaline tolerance. Subsequently, two candidate genes, Zm00001d038250 and Zm00001d001960, were verified to affect the alkaline tolerance of maize seedlings by qRT-PCR analysis. These genes were putatively involved protein binding and “flavonoid biosynthesis process,” respectively, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. Gene promoter region contains elements related to stress and metabolism. The results of this study will help further elucidate the mechanisms of alkaline tolerance in maize, which will provide the groundwork for future breeding projects.

Rice black-streaked dwarf virus (RBSDV) causes maize rough dwarf disease (MRDD), which is a viral disease that significantly affects maize yields worldwide. Plants tolerate stress through transcriptional reprogramming at the alternative splicing (AS), transcriptional, and fusion gene (FG) levels. However, it is unclear whether and how AS and FG interfere with transcriptional reprogramming in MRDD. In this study, we performed global profiling of AS and FG on maize response to RBSDV and compared it with transcriptional changes. There are approximately 1.43 to 2.25 AS events per gene in maize infected with RBSDV. GRMZM2G438622 was only detected in four AS modes (A3SS, A5SS, RI, and SE), whereas GRMZM2G059392 showed downregulated expression and four AS events. A total of 106 and 176 FGs were detected at two time points, respectively, including six differentially expressed genes and five differentially spliced genes. The gene GRMZM2G076798 was the only FG that occurred at two time points and was involved in two FG events. Among these, 104 GOs were enriched, indicating that nodulin-, disease resistance-, and chloroplastic-related genes respond to RBSDV stress in maize. These results provide new insights into the mechanisms underlying post-transcriptional and transcriptional regulation of maize response to RBSDV stress.

Seed storability is an important trait for improving grain quality and germplasm conservation, but little is known about the regulatory mechanisms and gene networks involved. MicroRNAs (miRNAs) are small non-coding RNAs regulating the translation and accumulation of their target mRNAs by means of sequence complementarity and have recently emerged as critical regulators of seed germination. Here, we used the germinating embryos of two maize inbred lines with significant differences in seed storability to identify the miRNAs and target genes involved. We identified a total of 218 previously known and 448 novel miRNAs by miRNA sequencing and degradome analysis, of which 27 known and 11 newly predicted miRNAs are differentially expressed in two maize inbred lines, as measured by Gene Ontology (GO) enrichment analysis. We then combined transcriptome sequencing and real-time quantitative polymerase chain reaction (RT-PCR) to screen and confirm six pairs of differentially expressed miRNAs associated with seed storability, along with their negative regulatory target genes. The enrichment analysis suggested that the miRNAs/target gene mediation of seed storability occurs via the ethylene activation signaling pathway, hormone synthesis and signal transduction, as well as plant organ morphogenesis. Our results should help elucidate the mechanisms through which miRNAs are involved in seed storability in maize.

Cytokinins (CTKs) are a major phytohormone group that are significant in the promotion of cellular division, growth, and divergence. Isopentenyl transferase (IPT) regulates a rate-limiting step in plant CTK synthesis, promotes the synthesis of isopentenyl adenonucleotides from 5-AMP and isopentenyl pyrophosphate, and then converts both these chemicals into various CTKs. Here, the full-length cDNA of ZmIPT2, which encodes 322 amino acids, was isolated and was introduced into a maize inbred line by Agrobacterium-mediated transformation. In both controlled environments and field experiments, the overexpression of ZmIPT2 gene in the transformed plants delayed leaf senescence. Compared to the receptor line, the transgenic maize lines retained higher chlorophyll levels, photosynthetic rates, and cytokinin content for an extended period of time, and produced significantly higher grain yield by a margin of 17.71–20.29% under normal field planting conditions. Subsequently, ten possible genes that interacted with ZmIPT2 were analyzed by qRT-PCR, showing that the expression pattern of GRMZM2G022904 was consistent with ZmIPT2 expression. Through comprehensive analysis, we screened for transgenic lines with stable inheritance of ZmIPT2 gene, clear functional efficiency, and significant yield improvement, in order to provide theoretical basis and material support for the breeding of new high-yield transgenic maize varieties.

The NAC gene family has transcription factors specific to plants, which are involved in development and stress response and adaptation. In this study, ZmNAC89, an NAC gene in maize that plays a role in saline–alkaline tolerance, was isolated and characterized. ZmNAC89 was localized in the nucleus and had transcriptional activation activity during in vitro experiments. The expression of ZmNAC89 was strongly upregulated under saline–alkaline, drought and ABA treatments. Overexpression of the ZmNAC89 gene in transgenic Arabidopsis and maize enhanced salt tolerance at the seedling stage. Differentially expressed genes (DEGs) were then confirmed via RNA-sequencing analysis with the transgenic maize line. GO analyses showed that oxidation–reduction process-regulated genes were involved in ZmNAC89-mediated salt–alkaline stress. ZmNAC89 may regulate maize saline–alkali tolerance through the REDOX pathway and ABA signal transduction pathway. From 140 inbred maize lines, 20 haplotypes and 16 SNPs were found in the coding region of the ZmNAC89 gene, including the excellent haplotype HAP20. These results contribute to a better understanding of the response mechanism of maize to salt–alkali stress and marker-assisted selection during maize breeding.