Background Maize ( Zea mays L.) is one of the main agricultural crops with the largest yield and acreage in the world. However, maize germplasm is very sensitive to low temperatures, mainly during germination, and low temperatures significantly affect plant growth and crop yield. Therefore, the identification of genes capable of increasing tolerance to low temperature has become necessary. Results In this study, fourteen phenotypic traits related to seed germination were used to assess the genetic diversity of maize through genome-wide association study (GWAS). A total of 30 single-nucleotide polymorphisms (SNPs) linked to low-temperature tolerance were detected (−log10( P ) > 4), fourteen candidate genes were found to be directly related to the SNPs, further additional 68 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r 2 ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then used to confirm the linkage between the candidate gene and low-temperature tolerance. A total of ten differentially expressed genes (DEGs) (|log 2 fold change (FC)| ≥ 0.585, P < 0.05) were found within the set distance of LD decay ( r 2 ≥ 0.2). Among these genes, the expression of six DEGs was verified using qRT-PCR. Zm00001d039219 and Zm00001d034319 were putatively involved in ‘mitogen activated protein kinase (MAPK) signal transduction’ and ‘fatty acid metabolic process’, respectively, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Thus, these genes appeared to be related to low-temperature signal transduction and cell membrane fluidity. Conclusion Overall, by integrating the results of our GWAS and DEG analysis of low-temperature tolerance during germination in maize, we were able to identify a total of 30 SNPs and 82 related candidate genes, including 10 DEGs, two of which were involved in the response to tolerance to low temperature. Functional analysis will provide valuable information for understanding the genetic mechanism of low-temperature tolerance during germination in maize.

Head smut, caused by the fungus Sporisorium reilianum, is a devastating global disease of maize (Zea mays). In the present study, maize seedlings were artificially inoculated with compatible mating-type strains of S. reilianum by needle inoculation of mesocotyls (NIM) or by soaking inoculation of radicles (SIR). After NIM or SIR, Huangzao4 mesocotyls exhibited severe damage with brownish discoloration and necrosis, whereas Mo17 mesocotyls exhibited few lesions. Fluorescence and electron microscopy showed that S. reilianum infected maize within 0.5 day after SIR and mainly colonized the phloem. With longer incubation, the density of S. reilianum hyphae increased in the vascular bundles, concentrated mainly in the phloem. In Mo17, infected cells exhibited apoptosis-like features, and hyphae became sequestered within dead cells. In contrast, in Huangzao4, pathogen invasion resulted in autophagy that failed to prevent hyphal spreading. The growth of S. reilianum hyphae diminished at 6 days after inoculation when expression of the R genes ZmWAK and ZmNL peaked. Thus, 6 days after SIR inoculation might be an important time for inhibiting the progress of S. reilianum infection in maize. The results of this study will provide a basis for further analysis of the mechanisms of maize resistance to S. reilianum.

Rice black-streaked dwarf virus (RBSDV), a ds-RNA virus in Fijivirus genus with family Reoviridae, which is transmitted by the small brown planthopper, is responsible for incidence of maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD). To understand the variation and evolution of S5, a unique fragment in the genome of RBSDV which encodes two partially overlapping ORFs (ORF5-1 and ORF5-2), we analyzed 127 sequences from maize and rice exhibiting symptoms of dwarfism. The nucleotide diversity of both ORF5-1 (π = 0.039) and ORF5-2 (π = 0.027) was higher than that of the overlapping region (π = 0.011) (P < 0.05). ORF5-2 was under the greatest selection pressure based on codon bias analysis, and its activation was possibly influenced by the overlapping region. The recombinant fragments of three recombinant events (14NM23, 14BM20, and 14NM17) cross the overlapping region. Based on neighbor-joining tree analysis, the overlapping region could represent the evolutionary basis of the full-length S5, which was classified into three main groups. RBSDV populations were expanding and haplotype diversity resulted mainly from the overlapping region. The genetic differentiation of combinations (T127-B35, T127-J34, A58-B35, A58-J34, and B35-J34) reached significant or extremely significant levels. Gene flow was most frequent between subpopulations A58 and B35, with the smallest |Fst| (0.02930). We investigated interactions between 13 RBSDV proteins by two-hybrid screening assays and identified interactions between P5-1/P6, P6/P9-1, and P3/P6. We also observed self-interactive effects of P3, P6, P7-1, and P10. In short, we have proven that RBSDV populations were expanding and the overlapping region plays an important role in the genetic variation and evolution of RBSDV S5. Our results enable ongoing research into the evolutionary history of RBSDV-S5 with two partly overlapping ORFs.

Maize (Zea mays L.) is an important food crop planted across the world, and low-temperature stress can affect maize germination. Alternative splicing (AS) is widely present in plants under abiotic stress; however, the response of AS to lowtemperature stress in maize remains unclear. In this study, a genome-wide analysis of AS during maize response to low temperatures was performed. AS events were distributed on each chromosome, approximately 2.05−2.09 AS events per gene. Seven genes only had AS in low-temperature-resistant inbred lines. A total of 278 KEGGs and 46 GOs were enriched based on overlapping AS genes, which were associated with hormone and oxidoreductase activity. The mutant was used to verify the function of AS gene ZmWRKY48, and the RGR, RSL, RRL, and RRSA of the mutant decreased by 15.16%−19.87% compared with the normal line. These results contribute to subsequent analysis of the regulatory mechanism of maize in response to low-temperature stress.

THICK TASSEL DWARF1 (TD1) is a critical gene that negatively modulates meristem size during maize inflorescence development and may also regulate ear-related traits. In the present study, the whole genomic DNA sequences and the promoter regions of TD1 were analyzed in 165 diverse maize inbred lines. Polymorphism analysis identified 39 SNPs and five InDels in the genic region of TD1 and allowed 23 haplotypes to be classified. Among these sites, eight SNPs and one InDel were significantly associated with kernel number (KN) ( p < 0.05), seven SNPs and one InDel were significantly associated with kernel number per row (KNPR) ( p < 0.05), and three SNPs were significantly associated with kernel row number (KRN) ( p < 0.05). In addition, 21 SNPs and 14 InDels were identified in the promoter regions of TD1 , and two SNPs and seven InDels of these sites were found to be significantly associated with KRN ( p < 0.05). The results denote that Hap_7 was the favorable haplotype for both KN and KNPR, and Hap_2 was the elite haplotype for KRN. These favorable haplotypes could be utilized in molecular marker-assisted selection (MAS) to improve KN, KNPR, or KRN, and thereby further increase grain yield in maize breeding programs.