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Abstract Background Gibberella ear rot (GER) is one of the most devastating diseases in maize growing areas, which directly reduces grain yield and quality. However, the underlying defense response of maize to pathogens infection is largely unknown. Results To gain a comprehensive understanding of the defense response in GER resistance, two contrasting inbred lines ‘Nov-82’ and ‘H10’ were used to explore transcriptomic profiles and defense-related phytohormonal alterations during Fusarium graminearum infection. Transcriptomic analysis revealed 4,417 and 4,313 differentially expressed genes (DEGs) from the Nov-82 and H10, respectively, and 647 common DEGs between the two lines. More DEGs were obviously enriched in phenylpropanoid biosynthesis, secondary metabolites biosynthesis, metabolic process and defense-related pathways. In addition, the concentration of the defense-related phytohormones, jasmonates (JAs) and salicylates (SAs), was greatly induced after the pathogen infection. The level of JAs in H10 was more higher than in Nov-82, whereas an opposite pattern for the SA between the both lines. Integrated analysis of the DEGs and the phytohormones revealed five vital modules based on co-expression network analysis according to their correlation. A total of 12 hub genes encoding fatty acid desaturase, subtilisin-like protease, ethylene-responsive transcription factor, 1-aminocyclopropane-1-carboxylate oxidase, and sugar transport protein were captured from the key modules, indicating that these genes might play unique roles in response to pathogen infection, Conclusions Overall, our results indicate that large number DEGs related to plant disease resistance and different alteration of defensive phytohormones were activated during F. graminearum infection, providing new insight into the defense response against pathogen invasion, in addition to the identified hub genes that can be further investigated for enhancing maize GER resistance.

The study of yield traits can reveal the genetic architecture of grain yield for improving maize production. In this study, an association panel comprising 362 inbred lines and a recombinant inbred line population derived from X178 × 9782 were used to identify candidate genes for nine yield traits. High-priority overlap (HPO) genes, which are genes prioritized in a genome-wide association study (GWAS), were investigated using coexpression networks. The GWAS identified 51 environmentally stable SNPs in two environments and 36 pleiotropic SNPs, including three SNPs with both attributes. Seven hotspots containing 41 trait-associated SNPs were identified on six chromosomes by permutation. Pyramiding of superior alleles showed a highly positive effect on all traits, and the phenotypic values of ear diameter and ear weight consistently corresponded with the number of superior alleles in tropical and temperate germplasm. A total of 61 HPO genes were detected after trait-associated SNPs were combined with the coexpression networks. Linkage mapping identified 16 environmentally stable and 16 pleiotropic QTL. Seven SNPs that were located in QTL intervals were assigned as consensus SNPs for the yield traits. Among the candidate genes predicted by our study, some genes were confirmed to function in seed development. The gene Zm00001d016656 encoding a serine/threonine protein kinase was associated with five different traits across multiple environments. Some genes were uniquely expressed in specific tissues and at certain stages of seed development. These findings will provide genetic information and resources for molecular breeding of maize grain yield.

The current assembled maize genomes cannot represent the broad genetic diversity of maize germplasms. Acquiring more genome sequences is critical for constructing a pan-genome and elucidating the linkage between genotype and phenotype in maize. Here we describe the genome sequence and annotation of A188, a maize inbred line with high phenotypic variation relative to other lines, acquired by single-molecule sequencing and optical genome mapping. We assembled a 2210-Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to 9.73 Mb for B73 and 10.2 Mb for Mo17. Based on the B73_RefGen_V4 genome, 295 scaffolds (2084.35 Mb, 94.30% of the final genome assembly) were anchored and oriented on ten chromosomes. Comparative analysis revealed that ~30% of the predicted A188 genes showed large structural divergence from B73, Mo17, and W22 genomes, which causes high protein divergence and may lead to phenotypic variation among the four inbred lines. As a line with high embryonic callus (EC) induction capacity, A188 provides a convenient tool for elucidating the molecular mechanism underlying the formation of EC in maize. Combining our new A188 genome with previously reported QTL and RNA sequencing data revealed eight genes with large structural variation and two differentially expressed genes playing potential roles in maize EC induction.

Genetic engineering technology offers opportunities to improve many important agronomic traits in crops, including insect-resistance. However, genetically modified (GM) exogenous proteins in edible tissues of transgenic crops has become an issue of intense public concern. To advance the application of GM techniques in maize, a Cre/loxP-based strategy was developed for manipulating the transgenes in green tissues while locking them in non-green tissues. In the strategy, the site-specific excision can be used to switch on or off the expression of transgenes at specific tissues. In this work, two basic transgenic maize, named KEY, carrying the Cre gene, and LOCK, containing the Vip3A gene with a blocked element, were obtained based on their separate fusion gene cassettes. The expression level and concentration of Vip3A were observed with a high specific accumulation in the green tissues (leaf and stem), and only a small amount was observed in the root and kernel tissues in the KEY × LOCK hybrids. The insect resistance of transgenic maize against two common lepidopteran pests, Ostrinia furnacalis and Spodoptera frugiperda, was assessed in the laboratory and field. The results indicate that the hybrids possessed high resistance levels against the two pests, with mortality rates above 73.6% and damage scales below 2.4 compared with the control group. Our results suggest that the Cre/loxP-mediated genetic engineering approach has a competitive advantage in GM maize. Overall, the findings from this study are significant for providing a feasible strategy for transgenes avoiding expression in edible parts and exploring novel techniques toward the biosafety of GM plants.

Abstract Background Long non-coding RNAs (lncRNAs) play important roles in response to abiotic stresses in plants, by acting as cis- or trans-acting regulators of protein-coding genes. As a widely cultivated crop worldwide, maize is sensitive to salt stress particularly at the seedling stage. However, it is unclear how the expressions of protein-coding genes are affected by non-coding RNAs in maize responding to salt tolerance. Results The whole transcriptome sequencing was employed to investigate the differential lncRNAs and target transcripts responding to salt stress between two maize inbred lines with contrasting salt tolerance. We developed a flexible, user-friendly, and modular RNA analysis workflow, which facilitated the identification of lncRNAs and novel mRNAs from whole transcriptome data. Using the workflow, 12,817 lncRNAs and 8,320 novel mRNAs in maize seedling roots were identified and characterized. A total of 742 lncRNAs and 7,835 mRNAs were identified as salt stress-responsive transcripts. Moreover, we obtained 41 cis- and 81 trans-target mRNA for 88 of the lncRNAs. Among these target transcripts, 11 belonged to 7 transcription factor (TF) families including bHLH, C2H2, Hap3/NF-YB, HAS, MYB, WD40, and WRKY. The above 8,577 salt stress-responsive transcripts were further classified into 28 modules by weighted gene co-expression network analysis. In the salt-tolerant module, we constructed an interaction network containing 79 nodes and 3081 edges, which included 5 lncRNAs, 18 TFs and 56 functional transcripts (FTs). As a trans-acting regulator, the lncRNA MSTRG.8888.1 affected the expressions of some salt tolerance-relative FTs, including protein-serine/threonine phosphatase 2C and galactinol synthase 1, by regulating the expression of the bHLH TF. Conclusions The contrasting genetic backgrounds of the two inbred lines generated considerable variations in the expression abundance of lncRNAs and protein-coding transcripts. In the co-expression networks responding to salt stress, some TFs were targeted by the lncRNAs, which further regulated the salt tolerance-related functional transcripts. We constructed a regulatory pathway of maize seedlings to salt stress, which was mediated by the hub lncRNA MSTRG.8888.1 and participated by the bHLH TF and its downstream target transcripts. Future work will be focused on the functional revelation of the regulatory pathway.

Lead (Pb) is a highly toxic contaminant to living organisms and the environment. Excessive Pb in soils affects crop yield and quality, thus threatening human health via the food chain. Herein, we investigated Pb tolerance among a maize association panel using root bushiness (BSH) under Pb treatment as an indicator. Through a genome-wide association study of relative BSH, we identified four single nucleotide polymorphisms (SNPs) and 30 candidate genes associated with Pb tolerance in maize seedlings. Transcriptome analysis showed that four of the 30 genes were differentially responsive to Pb treatment between two maize lines with contrasting Pb tolerance. Among these, the ZmbZIP107 transcription factor was confirmed as the key gene controlling maize tolerance to Pb by using gene-based association studies. Two 5’ UTR_variants in ZmbZIP107 affected its expression level and Pb tolerance among different maize lines. ZmbZIP107 protein was specifically targeted to the nucleus and ZmbZIP107 mRNA showed the highest expression in maize seedling roots among different tissues. Heterologous expression of ZmbZIP107 enhanced rice tolerance to Pb stress and decreased Pb absorption in the roots. Our study provided the basis for revelation of the molecular mechanism underlying Pb tolerance and contributed to cultivation of Pb-tolerant varieties in maize.

Plant regeneration occurs when plants repair or replace damaged structures based on the totipotency and pluripotency of their cells. Tissue culture is one of the most widely used regenerative technologies. Recently, a series of breakthroughs were made in the study of plant regeneration. This review summarizes two regenerative pathways in tissue culture: somatic embryogenesis and de novo organogenesis. Furthermore, we review the environmental factors influencing plant regeneration from explant sources, basal culture medium, plant growth regulators, and light/dark treatment. Additionally, we analyse the molecular mechanisms underlying two pathways. This knowledge will promote an understanding of the fundamental principles of plant regeneration from precursor cells and lay a solid foundation for applying plant micropropagation and genetic modification.

Root system architecture (RSA) contributes to nitrogen (N) uptake and utilization in maize. In this study, a germplasm enhancement of maize double haploid population of 226 lines genotyped with 61,634 SNPs was used to investigate the genetic basis of RSA under two N levels using a genome-wide association study (GWAS). GLM + PCA, FarmCPU, and MLM models were utilized to balance false positives and false negatives. In total, 33 and 51 significant SNP-trait associations were detected under high and low N conditions, respectively. Under high N, SNP S9_2483543 was detected by all models. Linkage disequilibrium (LD) regions of some SNPs overlapped with the intervals of QTL for RSA and N response that were detected in previous studies. In particular, several known genes, Rtcs, Rtcl, Rtcl, and Ms44, were located in the LD regions of S1_9992325, S9_151726472, S9_154381179, and S4_197073985, respectively. Among the candidate genes identified by this study, GRMZM2G139811, GRMZM2G314898, GRMZM2G054050, GRMZM2G173682, GRMZM2G470914, GRMZM2G462325, GRMZM2G416184, and GRMZM2G064302 were involved in seedling, seed, and root system development or N metabolism in Arabidopsis or rice. The markers identified in this study can be used for marker-assisted selection of RSA traits to improve nitrogen use efficiency in maize breeding, and the candidate genes will contribute to further understanding of the genetic basis of RSA under diverse N conditions. Keywords: Maize, Root system architecture, Different nitrogen conditions, Genome-wide association study, Genetic basis

Ear shank length (ESL) has significant effects on grain yield and kernel dehydration rate in maize. Herein, linkage mapping and genome-wide association study were combined to reveal the genetic architecture of maize ESL. Sixteen quantitative trait loci (QTL) were identified in the segregation population, among which five were repeatedly detected across multiple environments. Meanwhile, 23 single nucleotide polymorphisms were associated with the ESL in the association panel, of which four were located in the QTL identified by linkage mapping and were designated as the population-common loci. A total of 42 genes residing in the linkage disequilibrium regions of these common variants and 12 of them were responsive to ear shank elongation. Of the 12 genes, five encode leucine-rich repeat receptor-like protein kinases, proline-rich proteins, and cyclin11, respectively, which were previously shown to regulate cell division, expansion, and elongation. Gene-based association analyses revealed that the variant located in Cyclin11 promoter affected the ESL among different lines. Cyclin11 showed the highest expression in the ear shank 15 days after silking among diverse tissues of maize, suggesting its role in modulating ESL. Our study contributes to the understanding of the genetic mechanism underlying maize ESL and genetic modification of maize dehydration rate and kernel yield.

The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including qLS1-1 (11.54%), qLS1-3 (10.68%), and qREC4-1 (11.45%). Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes (Zm00001d028477, Zm00001d047896, Zm00001d034388, and Zm00001d022542). Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize.

Salinization seriously threatens the normal growth of maize, especially at the seedling stage. Recent studies have demonstrated that circular RNAs (circRNAs) play vital roles in the regulation of plant stress resistance. Here, we performed a genome-wide association study (GWAS) on the survival rate of 300 maize accessions under a salt stress treatment. A total of 5 trait-associated SNPs and 86 candidate genes were obtained by the GWAS. We performed RNA sequencing for 28 transcriptome libraries derived from 2 maize lines with contrasting salt tolerance under normal and salt treatment conditions. A total of 1217 highly expressed circRNAs were identified, of which 371 were responsive to a salt treatment. Using PCR and Sanger sequencing, we verified the reliability of these differentially expressed circRNAs. An integration of the GWAS and RNA-Seq analyses uncovered two differentially expressed hub genes (Zm00001eb013650 and Zm00001eb198930), which were regulated by four circRNAs. Based on these results, we constructed a regulation model of circRNA/miRNA/mRNA that mediated salt stress tolerance in maize. By conducting hub gene-based association analyses, we detected a favorable haplotype in Zm00001eb198930, which was responsible for high salt tolerance. These results help to clarify the regulatory relationship between circRNAs and their target genes as well as to develop salt-tolerant lines for maize breeding.

As one of the major crops, maize (Zea mays L.) is mainly distributed in tropical and temperate regions. However, with the changes of the environments, chilling stress has become a significantly abiotic stress affecting seed germination and thus the reproductive and biomass accumulation of maize. Herein, we investigated five seed germination-related phenotypes among 300 inbred lines under low-temperature condition (10 °C). By combining 43,943 single nucleotide polymorphisms (SNPs), a total of 15 significant (P

Abstract Background Maize is one of the primary crops of genetic manipulation, which provides an excellent means of promoting stress resistance and increasing yield. However, the differences in induction and regeneration capacity of embryonic callus (EC) among various genotypes result in genotypic dependence in genetic transformation. Results In this study, embryonic calli of two maize inbred lines with strong redifferentiation capacity and two lines with weak redifferentiation capability were separately subjected to transcriptome sequencing analysis during the early redifferentiation stages (stage I, 1–3 d; stage II, 4–6 d; stage III, 7–9 d) along with their corresponding controls. A total of ~ 654.72 million cDNA clean reads were yielded, and 62.64%~ 69.21% clean reads were mapped to the reference genome for each library. In comparison with the control, the numbers of differentially expressed genes (DEGs) for the four inbred lines identified in the three stages ranged from 1694 to 7193. By analyzing the common and specific DEGs of the four materials, we found that there were 321 upregulated genes and 386 downregulated genes identified in the high-regeneration lines (141 and DH40), whereas 611 upregulated genes and 500 downregulated genes were specifically expressed in the low-regeneration lines (ZYDH381–1 and DH3732). Analysis of the DEG expression patterns indicated a sharp change at stage I in both the high- and low-regeneration lines, which suggested that stage I constitutes a crucial period for EC regeneration. Notably, the specific common DEGs of 141 and DH40 were mainly associated with photosynthesis, porphyrin and chlorophyll metabolism, ribosomes, and plant hormone signal transduction. In contrast, the DEGs in ZYDH381–1 and DH3732 were mainly related to taurine and hypotaurine metabolism, nitrogen metabolism, fatty acid elongation, starch and sucrose metabolism, phenylpropanoid biosynthesis, and plant circadian rhythm. More importantly, WOX genes, which have an ancestral role in embryo development in seed plants and promote the regeneration of transformed calli, were specifically upregulated in the two high-regeneration lines. Conclusions Our research contributes to the elucidation of molecular regulation during early redifferentiation in the maize embryonic callus.

Low moisture content (MC) and high dehydration rate (DR) at physiological maturity affect grain mechanical harvest, transport, and storage. In this study, we used an association panel composed of 241 maize inbred lines to analyze ear moisture changes at physiological maturity stage. A genome-wide association study revealed nine significant SNPs and 91 candidate genes. One SNP (SYN38588) was repeatedly detected for two traits, and 15 candidate genes were scanned in the linkage disequilibrium regions of this SNP. Of these, genes Zm00001d020615 and Zm00001d020623 were individually annotated as a polygalacturonase (PG) and a copper transporter 5.1 (COPT5.1), respectively. Candidate gene association analysis showed that three SNPs located in the exons of Zm00001d020615 were significantly associated with the dehydration rate, and AATTAA was determined as the superior haplotype. All these findings suggested that Zm00001d020615 was a key gene affecting moisture changes of maize at the physiological maturity stage. These results have demonstrated the genetic basis of ear moisture changes in maize and indicated a superior haplotype for cultivating maize varieties with low moisture content and high dehydration rates.

Salt stress affects crop yield by limiting growth and delaying development. In this study, we constructed 16 transcriptome libraries from maize seedling roots using two maize lines, with contrasting salt tolerance, that were exposed to salt stress for 0, 6, 18 and 36 h. In total, 6,584 differential expression genes (DEGs; 3,669 upregulated, 2,915 downregulated) were induced in the salt-sensitive line and 6,419 DEGs (3,876 upregulated, 2,543 downregulated) were induced in the salt-tolerant line. Several DEGs common to both lines were enriched in the ABA signaling pathway, which was presumed to coordinate the process of maize salt response. A total of 459 DEGs were specifically induced in the salt-tolerant line and represented candidate genes responsible for high salt-tolerance. Expression pattern analysis for these DEGs indicated that the period between 0 and 6 h was a crucial period for the rapid response of the tolerant genes under salt stress. Among these DEGs, several genes, Aux/IAA, SAUR, and CBL-interacting kinase have been reported to regulate salt tolerance. In addition, the transcription factors WRKY, bZIP and MYB acted as regulators in the salt-responsive regulatory network of maize roots. Our findings will contribute to understanding of the mechanism on salt response and provide references for functional gene revelation in plants.

Lead-based liquid metals (LLMs) such as lead–bismuth eutectic (LBE) and lead, are currently the most interesting candidate coolants for fast reactors because of their excellent physical properties, which can improve safety and reduce costs. However, in comparison to other liquid metals, previous research on the flow and heat-transfer characteristics of LLMs has been limited. Therefore, this work carried out flow and heat-transfer experiments in LBE flowing through a circular tube in the Natural Circulation Capability Loop (NCCL) facility. The results show a significantly higher friction factor than that of water flowing in a smooth pipe. Furthermore, the Nusselt numbers were found to be lower than those found in data in the literature for experiments carried out in a smooth tube at low Péclet numbers, while they were higher at high Péclet numbers. Therefore, theoretical analyses were performed for LLMs flowing in both smooth and rough pipes, and the impacts of roughness on the heat transfer of an LLM were examined. The theoretical relations for a smooth pipe and a rough pipe were validated using experimental data from the literature and the results of the NCCL experiments, respectively. The results of the theoretical relation for a smooth pipe fitted the literature data well. The derived theoretical relation for a rough pipe with a relative roughness of 0.004 fitted the NCCL data best. Moreover, it was established from the theoretical analysis that roughness has two competitive impacts on the heat transfer of an LLM: it reduces conductive heat transfer while enhancing convective heat transfer. Because conductive heat transfer is important for liquid metals, even with turbulent flow, a small roughness will lead to heat-transfer deterioration at low Péclet numbers, and it may even deteriorate across the whole typical Péclet-number range. This discovery has important implications for the thermal–hydraulic design of LLM reactors, because corrosion and erosion by an LLM will lead to a rough surface after long operating times.

Abstract Background Accumulation of cadmium (Cd) in maize (Zea mays L.) poses a significant risk to human health as it is ingested via the food chain. A genome-wide association study (GWAS) was conducted in a population of 269 maize accessions with 43,737 single nucleotide polymorphisms (SNPs) to identify candidate genes and favorable alleles for controlling Cd accumulation in maize. Results When grown in contaminated soil, accessions varied significantly in leaf Cd concentration at both the seeding and maturing stages with phenotypic variation and the coefficient of variation all above 48%. The co-localized region between SYN27837 (147,034,650 bp) and SYN36598 (168,551,327 bp) on chromosome 2 was associated with leaf Cd under three soil conditions varying in Cd content in 2015 and 2016. The significant SNP (SYN25051) at position 161,275,547 could explained 27.1% of the phenotype variation. Through QTL mapping using the IBMSyn10 double haploid (DH) population, we validated the existence of a major QTL identified by GWAS; qLCd2 could explain the 39.8% average phenotype variation across the experiments. Expression of GRMZM2G175576 encoding a cadmium/zinc-transporting ATPase underlying the QTL was significantly increased in roots, stems and leaves of B73, a low Cd accumulation line in response to Cd stress. Conclusions Our findings provide new insights into the genetic control of Cd accumulation and could aid rapid development of maize genotypes with low-Cd accumulation by manipulation of the favorable alleles.

Abstract Background Maize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. Results We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. Conclusions These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

Abstract The low ratio of embryonic callus (EC) induction has inhibited the rapid development of maize genetic engineering. Still, little is known to explain the genotype-dependence of EC induction. Here, we performed a large-scale, quantitative analysis of the maize EC metabolome and proteome at three typical induction stages in two inbred lines with a range of EC induction capabilities. Comparison of the metabolomes and proteomes suggests that the differential molecular responses begin at an early stage of development and continue throughout the process of EC formation. The two inbred lines show different responses under various conditions, such as metal ion binding, cell enlargement, stem cell formation, meristematic activity maintenance, somatic embryogenesis, cell wall synthesis, and hormone signal transduction. Furthermore, the differences in hormone (auxin, cytokinin, gibberellin, salicylic acid, jasmonic acid, brassinosteroid and ethylene) synthesis and transduction ability could partially explain the higher EC induction ratio in the inbred line 18-599R. During EC formation, repression of the “histone deacetylase 2 and ERF transcription factors” complex in 18-599R activated the expression of downstream genes, which further promoted EC induction. Together, our data provide new insights into the molecular regulatory mechanism responsible for efficient EC induction in maize.

P1B-type ATPases, known as heavy metal ATPases (HMAs), play an important role in the control of cadmium (Cd) accumulation in plants. In this study, a total of 12 ZmHMA genes were identified in the maize genome and particularly classified into six clusters based on their phylogenetic relationship and motif compositions. Furthermore, the expression patterns of different ZmHMA genes varied with developmental stages, and were tissue specific under normal conditions. ZmHMA2 and ZmHMA3 genes exhibited significant up-regulation under Cd treatment. Eventually, the association analysis between 103 inbred lines and alleles in ZmHMA2 and ZmHMA3 revealed that one insertion–deletion (InDel) in the intron from ZmHMA2 was associated with leaf Cd concentration under low Cd condition at the seedling stage. Twenty polymorphisms in ZmHMA3 were significantly associated with leaf Cd concentration under various Cd levels at seedling and maturing stages. Five single nucleotide polymorphisms (SNPs) and two InDels of these significantly associated polymorphic loci from ZmHMA3 caused the amino acid substitutions and insertion or deletion events. Importantly, the proteins encoded by ZmHMA2 and ZmHMA3 genes were located in the plasma membrane. This comprehensive analysis will provide an important theoretical basis for future functional verification of ZmHMA genes to unravel the mechanisms of Cd accumulation in leaves of maize. Additionally, the favorable alleles in ZmHMA3 will lay a foundation for the marker-assisted selection of low Cd accumulation in maize.

Stalk lodging resistance, which is mainly measured by stem diameter (SD), stalk bending strength (SBS), and rind penetrometer resistance (RPR) in maize, seriously affects the yield and quality of maize (Zea mays L.). To dissect its genetic architecture, in this study multi-locus genome-wide association studies for stalk lodging resistance-related traits were conducted in a population of 257 inbred lines, with tropical, subtropical, and temperate backgrounds, genotyped with 48,193 high-quality single nucleotide polymorphisms. The analyses of phenotypic variations for the above traits in three environments showed high broad-sense heritability (0.679, 0.720, and 0.854, respectively). In total, 423 significant Quantitative Trait Nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB methods to be associated with the above traits. Among these QTNs, 29, 34, and 48 were commonly detected by multiple methods or across multiple environments to be related to SD, SBS, and RPR, respectively. The superior allele analyses in 30 elite lines showed that only eight lines contained more than 50% of the superior alleles, indicating that stalk lodging resistance can be improved by the integration of more superior alleles. Among sixty-three candidate genes of the consistently expressed QTNs, GRMZM5G856734 and GRMZM2G116885, encoding membrane steroid-binding protein 1 and cyclin-dependent kinase inhibitor 1, respectively, possibly inhibit cell elongation and division, which regulates lodging resistance. Our results provide the further understanding of the genetic foundation of maize lodging resistance.

The regenerative capacity of the embryonic callus, a complex quantitative trait, is one of the main limiting factors for maize transformation. This trait was decomposed into five traits, namely, green callus rate (GCR), callus differentiating rate (CDR), callus plantlet number (CPN), callus rooting rate (CRR), and callus browning rate (CBR). To dissect the genetic foundation of maize transformation, in this study multi-locus genome-wide association studies (GWAS) for the five traits were performed in a population of 144 inbred lines genotyped with 43,427 SNPs. Using the phenotypic values in three environments and best linear unbiased prediction (BLUP) values, as a result, a total of 127, 56, 160, and 130 significant quantitative trait nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB, respectively. Of these QTNs, 63 QTNs were commonly detected, including 15 across multiple environments and 58 across multiple methods. Allele distribution analysis showed that the proportion of superior alleles for 36 QTNs was

Drought stress response is a complex trait regulated at transcriptional and post-transcriptional levels in tobacco. Since the 1990s, many studies have shown that miRNAs act in many ways to regulate target expression in plant growth, development and stress response. The recent draft genome sequence of Nicotiana benthamiana has provided a framework for Digital Gene Expression (DGE) and small RNA sequencing to understand patterns of transcription in the context of plant response to environmental stress. We sequenced and analyzed three Digital Gene Expression (DGE) libraries from roots of normal and drought-stressed tobacco plants, and four small RNA populations from roots, stems and leaves of control or drought-treated tobacco plants, respectively. We identified 276 candidate drought responsive genes (DRGs) with sequence similarities to 64 known DRGs from other model plant crops, 82 were transcription factors (TFs) including WRKY, NAC, ERF and bZIP families. Of these tobacco DRGs, 54 differentially expressed DRGs included 21 TFs, which belonged to 4 TF families such as NAC (6), MYB (4), ERF (10), and bZIP (1). Additionally, we confirmed expression of 39 known miRNA families (122 members) and five conserved miRNA families, which showed differential regulation under drought stress. Targets of miRNAs were further surveyed based on a recently published study, of which ten targets were DRGs. An integrated gene regulatory network is proposed for the molecular mechanisms of tobacco root response to drought stress using differentially expressed DRGs, the changed expression profiles of miRNAs and their target transcripts. This network analysis serves as a reference for future studies on tobacco response stresses such as drought, cold and heavy metals.

The induction efficiency of maize embryonic callus is highly dependent on the genotype, and only a few lines possess a high capacity for callus formation. Although certain genes and pathways have been reported to contribute to the regulation of callus induction, to the best of our knowledge, the functions of the small interfering RNAs (siRNAs) involved in this process remain unknown. In this study, we identified 861 differentially expressed siRNAs and 576 target genes in the callus induction process. These target genes were classified into 3 clusters, and their functions involve controlling metalloexopeptidase activity, catalase activity, transcription regulation, and O-methyltransferase activity. In addition, certain genes related to auxin transport and stem cell or meristem development (e.g., PLT5-like, ARF15, SAUR-like, FAS1-like, Fea3, SCL5, and Zmwox2A) were regulated by the differentially expressed siRNAs. Moreover, zma-siR004119-2 directly cleaves the 5' UTR of Homeobox-transcription factor 25, which further leads to the down-regulation of its expression. Twelve 24-nt-siRNAs led to the hyper-methylation of GRMZM2G013465, which further decreases its expression. These results suggest that differentially expressed siRNAs regulate callus formation by controlling the expression of their target genes.

Heterosis, or hybrid vigor, contributes to superior agronomic performance of hybrids compared to their inbred parents. Despite its importance, little is known about the genetic and molecular basis of heterosis. Early maize ear inflorescences formation affects grain yield, and are thus an excellent model for molecular mechanisms involved in heterosis. To determine the parental contributions and their regulation during maize ear-development-genesis, we analyzed genome-wide digital gene expression profiles in two maize elite inbred lines (B73 and Mo17) and their F1 hybrid using deep sequencing technology. Our analysis revealed 17,128 genes expressed in these three genotypes and 22,789 genes expressed collectively in the present study. Approximately 38% of the genes were differentially expressed in early maize ear inflorescences from heterotic cross, including many transcription factor genes and some presence/absence variations (PAVs) genes, and exhibited multiple modes of gene action. These different genes showing differential expression patterns were mainly enriched in five cellular component categories (organelle, cell, cell part, organelle part and macromolecular complex), five molecular function categories (structural molecule activity, binding, transporter activity, nucleic acid binding transcription factor activity and catalytic activity), and eight biological process categories (cellular process, metabolic process, biological regulation, regulation of biological process, establishment of localization, cellular component organization or biogenesis, response to stimulus and localization). Additionally, a significant number of genes were expressed in only one inbred line or absent in both inbred lines. Comparison of the differences of modes of gene action between previous studies and the present study revealed only a small number of different genes had the same modes of gene action in both maize seedlings and ear inflorescences. This might be an indication that in different tissues or developmental stages, different global expression patterns prevail, which might nevertheless be related to heterosis. Our results support the hypotheses that multiple molecular mechanisms (dominance and overdominance modes) contribute to heterosis.

Given the important roles of miRNAs in post-transcriptional gene regulation, identification of differentially expressed miRNAs will facilitate the elucidation of molecular mechanisms underlying kernel development. In this study, we constructed a small RNA library to comprehensively represent the full complement of individual small RNAs and to characterize miRNA expression profiles in pooled ears of maize (Zea mays L.) at 10, 15, 20, 22, 25 and 30 days after pollination (DAP). At least 21 miRNAs were differentially expressed. The differential expression of three of these miRNAs, i.e., miR528a, miR167a and miR160b, at each stage was verified by qRT-PCR. The results indicated that these miRNAs might be involved in kernel development. In addition, the predicted functions of target genes indicated that most of the target genes are involved in signal transduction and cell communication pathways, particularly the auxin signaling pathway. The expression of candidate germination-associated miRNAs was analyzed by hybridization to a maize genome microarray, and revealed differential expression of genes involved in plant hormone signaling pathways. This finding suggests that phytohormones play a critical role in the development of maize kernels. We found that in combination with other miRNAs, miR528a regulated a putative laccase, a Ring-H2 zinc finger protein and a MADS box-like protein, whereas miR167a and miR160b regulated multiple target genes, including ARF (auxin response factor), a member of the B3 transcription factor family. All three miRNAs are important for ear germination, development and physiology. The small RNA transcriptomes and mRNA obtained in this study will help us gain a better understanding of the expression and function of small RNAs in the development of maize kernel.

Nitrogen (N) is an essential nutrient for plants, and it directly affects grain yield and protein content in cereal crops. Plant root systems are not only critical for anchorage in the soil, but also for N acquisition. Therefore, genes controlling root development might also affect N uptake by plants. In this study, the responses of nitrogen on root architecture of mutant rtcs and wild-type of maize were investigated by morphological and physiological analysis. Subsequently, we performed a comparative RNA-Seq analysis to compare gene expression profiles between mutant rtcs roots and wild-type roots under different N conditions. We identified 786 co-modulated differentially expressed genes (DEGs) related to root development. These genes participated in various metabolic processes. A co-expression cluster analysis and a cis-regulatory motifs analysis revealed the importance of the AP2-EREBP transcription factor family in the rtcs-dependent regulatory network. Some genotype-specific DEGs contained at least one LBD motif in their promoter region. Further analyses of the differences in gene transcript levels between rtcs and wild-type under different N conditions revealed 403 co-modulated DEGs with distinct functions. A comparative analysis revealed that the regulatory network controlling root development also controlled gene expression in response to N-deficiency. Several AP2-EREBP family members involved in multiple hormone signaling pathways were among the DEGs. These transcription factors might play important roles in the rtcs-dependent regulatory network related to root development and the N-deficiency response. Genes encoding the nitrate transporters NRT2-1, NAR2.1, NAR2.2, and NAR2.3 showed much higher transcript levels in rtcs than in wild-type under normal-N conditions. This result indicated that the LBD gene family mainly functions as transcriptional repressors, as noted in other studies. In summary, using a comparative RNA-Seq-based approach, we identified DEGs related to root development that also participated in the N-deficiency response in maize. These findings will increase our understanding of the molecular regulatory networks controlling root development and N-stress responses.

[This corrects the article DOI: 10.1371/journal.pone.0132379.].

Genomic selection is a promising research area due to its practical application in breeding. In this study, impact of realized genetic relationship and linkage disequilibrium (LD) on marker density and training population size required was investigated and their impact on practical application was further discussed. This study is based on experimental data of two populations derived from the same two founder lines (B73, Mo17). Two populations were genotyped with different marker sets at different density: IBM Syn4 and IBM Syn10. A high-density marker set in Syn10 was imputed into the Syn4 population with low marker density. Seven different prediction scenarios were carried out with a random regression best linear unbiased prediction (RR-BLUP) model. The result showed that the closer the real genetic relationship between training and validation population, the fewer markers were required to reach a good prediction accuracy. Taken the short-term cost for consideration, relationship information is more valuable than LD information. Meanwhile, the result indicated that accuracies based on high LD between QTL and markers were more stable over generations, thus LD information would provide more robust prediction capacity in practical applications.

Pb can damage the physiological function of human organs by entering the human body via food-chain enrichment. Revealing the mechanisms of maize tolerance to Pb is critical for preventing this. In this study, a Pb-tolerant maize inbred line, 178, was used to analyse transcription factors (TFs) expressed under Pb stress based on RNA sequencing data. A total of 464 genes expressed in control check (CK) or Pb treatment samples were annotated as TFs. Among them, 262 differentially expressed transcription factors (DETs) were identified that responded to Pb treatment. Furthermore, the DETs were classified into 4 classes according to their expression patterns, and 17, 12 and 2 DETs were significantly annotated to plant hormone signal transduction, basal transcription factors and base excision repair, respectively. Seventeen DETs were found to participate in the plant hormone signal transduction pathway, where basic leucine zippers (bZIPs) were the most significantly enriched TFs, with 12 members involved. We further obtained 5 Arabidopsis transfer DNA (T-DNA) mutants for 6 of the maize bZIPs, among which the mutants atbzip20 and atbzip47, representing ZmbZIP54 and ZmbZIP107, showed obviously inhibited growth of roots and above-ground parts, compared with wild type. Five highly Pb-tolerant and 5 highly Pb-sensitive in maize lines were subjected to DNA polymorphism and expression level analysis of ZmbZIP54 and ZmbZIP107. The results suggested that differences in bZIPs expression partially accounted for the differences in Pb-tolerance among the maize lines. Our results contribute to the understanding of the molecular regulation mechanisms of TFs in maize under Pb stress.

Maize is one of the most important cereal crops worldwide and one of the primary targets of genetic manipulation, which provides an excellent way to promote its production. However, the obvious difference of the dedifferentiation frequency of immature maize embryo among various genotypes indicates that its genetic transformation is dependence on genotype and immature embryo-derived undifferentiated cells. To identify important genes and metabolic pathways involved in forming of embryo-derived embryonic calli, in this study, DGE (differential gene expression) analysis was performed on stages I, II, and III of maize inbred line 18-599R and corresponding control during the process of immature embryo dedifferentiation. A total of ∼21 million cDNA tags were sequenced, and 4,849,453, 5,076,030, 4,931,339, and 5,130,573 clean tags were obtained in the libraries of the samples and the control, respectively. In comparison with the control, 251, 324 and 313 differentially expressed genes (DEGs) were identified in the three stages with more than five folds, respectively. Interestingly, it is revealed that all the DEGs are related to metabolism, cellular process, and signaling and information storage and processing functions. Particularly, the genes involved in amino acid and carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis and signal transduction mechanism have been significantly changed during the dedifferentiation. To our best knowledge, this study is the first genome-wide effort to investigate the transcriptional changes in dedifferentiation immature maize embryos and the identified DEGs can serve as a basis for further functional characterization.