Background: Maize (Zea maysL.) 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 ofr² ≥ 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) (|log₂fold change (FC)| ≥ 0.585,P < 0.05) were found within the set distance of LD decay (r² ≥ 0.2). Among these genes, the expression of six DEGs was verified using qRT-PCR.Zm00001d039219andZm00001d034319were 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.

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.

Understanding combining ability and heterosis among diverse maize germplasm resources is important for breeding hybrid maize (Zea mays L.). Using 28 temperate and 23 tropical maize inbreds that represent different ecotypes and worldwide diversity of maize germplasm, we first developed a large-scale multiple-hybrid population (MHP) with 724 hybrids, which could be divided into three subsets, 325 temperate diallel hybrids and 136 tropical diallel hybrids generated in Griffing IV, and 263 temperate by tropical hybrids generated in NCD II. All the parental lines and hybrids were evaluated for 11 traits in replicated tests across two locations and three years. Several widely used inbreds showed strong general combining ability (GCA), and their derived hybrids showed strong specific combining ability (SCA). Heterosis is a quantifiable, trait-dependent and environment-specific phenotype, and the response of parental lines and their hybrids to environments resulted in various levels of heterosis. For all the tested traits except plant height and hundred grain weight (HGW), NCD II (temperate × tropical) hybrids showed higher average heterosis than the temperate and tropical diallel hybrids, with higher hybrid performance for ear length, ear diameter, and HGW. Tropical maize germplasm can be used to improve the yield potential for temperate lines. Grain number per row and grain number per ear were two most important traits that determined yield heterosis, which can be used as direct selection criteria for yield heterosis. The hybrids from heterotic groups, Reid × SPT, Reid × LRC, SPT × PA, and Lancaster × LRC, contributed highly significant positive SCA effects and strong heterosis to yield-related traits, and the heterotic patterns identified in this study were potentially useful for commercial maize breeding. Heterosis was more significantly and positively correlated with SCA than GCA, indicating that SCA can be used in heterosis prediction to develop potential hybrids in commercial maize breeding. The results of the present study not only contribute to developing breeding strategies, but also improve targeted breeding efficiency by using both temperate and tropical maize to broaden genetic basis. Large sets of parental lines with available genotypic information can be shared and used in worldwide hybrid breeding programs through an open-source breeding strategy. Potential applications of the reported results in developing hybrid maize breeding strategies were also discussed.

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.

Plant specific protein kinase SnRK2s play crucial roles in response to various environmental stimuli. TaSnRK2.3, a SnRK2 member, was involved in the response to multiple abiotic stresses in wheat. To facilitate the use of TaSnRK2.3 in wheat breeding, the three genomic sequences of TaSnRK2.3, originating from the A, B, and D genomes of hexaploid wheat, were obtained. Sequence polymorphism assays showed four and ten variations were detected at TaSnRK2.3-1A and at TaSnRK2.3-1B, respectively, yet no variation was identified at TaSnRK2.3-1D. Three haplotypes for A genome, and two main haplotypes for B genome of TaSnRK2.3 were identified in 32 genotypes. Functional markers (2.3AM1, 2.3AM2, 2.3BM1, 2.3BM2) were successfully developed to distinguish different haplotypes. Association analysis was performed with the general linear model in TASSEL 2.1. The results showed that both TaSnRK2.3-1A and TaSnRK2.3-1B were significantly associated with plant height (PH), length of peduncle and penultimate node, as well as 1,000-grain weight (TGW) under different environments. Additionally, TaSnRK2.3-1B was significantly associated with stem water-soluble carbohydrates at flowering and mid-grain filling stages. Hap-1A-1 had higher TGW and lower PH; Hap-1B-1 had higher TGW and stem water-soluble carbohydrates, as well as lower PH, thus the two haplotypes were considered as elite haplotypes. Geographic distribution and allelic frequencies indicated that the two preferred haplotypes Hap-1A-1 and Hap-1B-1 were positively selected in the process of Chinese wheat breeding. These results could be highly valuable for genetic improvement and germplasm enhancement by molecular marker assisted selection in wheat breeding.