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Using GWAS and QTL mapping identified 100 QTL and 138 SNPs, which control yield-related traits in maize. The candidate gene GRMZM2G098557 was further validated to regulate ear row number by using a segregation population. Understanding the genetic basis of yield-related traits contributes to the improvement of grain yield in maize. This study used an inter-mated B73 × Mo17 (IBM) Syn10 doubled-haploid (DH) population and an association panel to identify the genetic loci responsible for nine yield-related traits in maize. Using quantitative trait loci (QTL) mapping, 100 QTL influencing these traits were detected across different environments in the IBM Syn10 DH population, with 25 co-detected in multiple environments. Using a genome-wide association study (GWAS), 138 single-nucleotide polymorphisms (SNPs) were identified as correlated with these traits (P

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 -6) SNPs were identified to correlate with seed germination under cold stress based on the FarmCPU model in GWAS, among which three loci were repeatedly associated with multiple traits. Ten gene models were closely linked to these three variations, among which Zm00001d010454, Zm00001d010458, Zm00001d010459, and Zm00001d050021 were further verified by candidate gene association study and expression pattern analysis. Importantly, these candidate genes were previously reported to involve plant tolerance to chilling stress and other abiotic stress. Our findings contribute to the understanding of the genetic and molecular mechanisms underlying chilling germination in maize.

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.

SUMMARY Inorganic phosphorus (Pi) is an essential element in numerous metabolic reactions and signaling pathways, but the molecular details of these pathways remain largely unknown. In this study, metabolite profiles of maize (Zea mays L.) leaves and roots were compared between six low‐Pi‐sensitive lines and six low‐Pi‐tolerant lines under Pi‐sufficient and Pi‐deficient conditions to identify pathways and genes associated with the low‐Pi stress response. Results showed that under Pi deprivation the concentrations of nucleic acids, organic acids and sugars were increased, but that the concentrations of phosphorylated metabolites, certain amino acids, lipid metabolites and nitrogenous compounds were decreased. The levels of secondary metabolites involved in plant immune reactions, including benzoxazinoids and flavonoids, were significantly different in plants grown under Pi‐deficient conditions. Among them, the 11 most stable metabolites showed significant differences under low‐ and normal‐Pi conditions based on the coefficient of variation (CV). Isoleucine and alanine were the most stable metabolites for the identification of Pi‐sensitive and Pi‐resistant maize inbred lines. With the significant correlation between morphological traits and metabolites, five low‐Pi‐responding consensus genes associated with morphological traits and simultaneously involved in metabolic pathways were mined by combining metabolites profiles and genome‐wide association study (GWAS). The consensus genes induced by Pi deficiency in maize seedlings were also validated by reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR). Moreover, these genes were further validated in a recombinant inbred line (RIL) population, in which the glucose‐6‐phosphate‐1‐epimerase encoding gene mediated yield and correlated traits to phosphorus availability. Together, our results provide a framework for understanding the metabolic processes underlying Pi‐deficient responses and give multiple insights into improving the efficiency of Pi use in maize., Significance statement Metabolite profiling and GWAS were applied to reveal maize low‐Pi response mechanisms.

Heretofore, little is known about the mechanism underlying the genotype-dependence of embryonic callus (EC) induction, which has severely inhibited the development of maize genetic engineering. Here, we report the genome sequence and annotation of a maize inbred line with high EC induction ratio, A188, which is assembled from single-molecule sequencing and optical genome mapping. We assembled a 2,210 Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to those of 9.73 Mb for B73 and 10.2 Mb for Mo17. Comparative analysis revealed that ∼30% of the predicted A188 genes had large structural variations to B73, Mo17 and W22 genomes, which caused considerable protein divergence and might lead to phenotypic variations between the four inbred lines. Combining our new A188 genome, previously reported QTLs and RNA sequencing data, we reveal 8 large structural variation genes and 4 differentially expressed genes playing potential roles in EC induction.HighlightOur manuscript presents a high-quality reference genome of the inbred line A188, and provides new insights into candidate genes underlying maize embryonic callus induction and other maize agronomic traits.

Two hub genes GRMZM2G075104 and GRMZM2G333183 involved in salt tolerance were identified by GWAS and WGCNA. Furthermore, they were verified to affect salt tolerance by candidate gene association analysis. Salt stress influences maize growth and development. To decode the genetic basis and hub genes controlling salt tolerance is a meaningful exploration for cultivating salt-tolerant maize varieties. Herein, we used an association panel consisting of 305 lines to identify the genetic loci responsible for Na+- and K+-related traits in maize seedlings. Under the salt stress, seven significant single nucleotide polymorphisms were identified using a genome-wide association study, and 120 genes were obtained by scanning the linkage disequilibrium regions of these loci. According to the transcriptome data of the above 120 genes under salinity treatment, we conducted a weighted gene co-expression network analysis. Combined the gene annotations, two SNaC/SKC (shoot Na+ content/shoot K+ content)-associated genes GRMZM2G075104 and GRMZM2G333183 were finally identified as the hub genes involved in salt tolerance. Subsequently, these two genes were verified to affect salt tolerance of maize seedlings by candidate gene association analysis. Haplotypes TTGTCCG-CT and CTT were determined as favorable/salt-tolerance haplotypes for GRMZM2G075104 and GRMZM2G333183, respectively. These findings provide novel insights into genetic architectures underlying maize salt tolerance and contribute to the cultivation of salt-tolerant varieties 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.

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.

The heavy metal cadmium (Cd), acts as a widespread environmental contaminant, which has shown to adversely affect human health, food safety and ecosystem safety in recent years. However, research on how plant respond to various kinds of heavy metal stress is scarcely reported, especially for understanding of complex molecular regulatory mechanisms and elucidating the gene networks of plant respond to Cd stress. Here, transcriptomic changes during Mo17 and B73 seedlings development responsive to Cd pollution were investigated and comparative RNAseq-based approach in both genotypes were performed. 115 differential expression genes (DEGs) with significant alteration in expression were found co-modulated in both genotypes during the maize seedling development; of those, most of DGEs were found comprised of stress and defense responses proteins, transporters, as well as transcription factors, such as thaumatin-like protein, ZmOPR2 and ZmOPR5. More interestingly, genotype-specific transcriptional factors changes induced by Cd stress were found contributed to the regulatory mechanism of Cd sensitivity in both different genotypes. Moreover, 12 co-expression modules associated with specific biological processes or pathways (M1 to M12) were identified by consensus co-expression network. These results will expand our understanding of complex molecular mechanism of response and defense to Cd exposure in maize seedling roots.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that, in most cases, negatively regulate gene expression at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptation to environmental stress including biotic and abiotic stresses. Here, we report a comprehensive analysis of miRNAs and associated target genes under banded leaf and sheath blight (BLSB) stress caused by R. solani in maize. Eight differentially expressed miRNAs were randomly selected from deep sequencing results and validated by qRT-PCR together with their putative target genes, most of which are transcription factors as well as metabolic genes involved in auxin signaling. The results revealed that majorities of the analyzed miRNAs show an inverse correlation with their corresponding predicted target genes. In addition, a putative regulatory network of miRNAs-mRNAs responsive to R. solani was constructed. This study provides insight into the regulatory functions of miRNAs, thereby expanding our knowledge of the molecular mechanisms of pathogen resistance.

The present study profiled and analyzed gene expression of the maize ear at four key developmental stages. Based on genome-wide profile analysis, we detected differential mRNA of maize genes. Some of the differentially expressed genes (DEGs) were predicted to be potential candidates of maize ear development. Several well-known genes were found with reported mutant analyses, such as, compact plant2 (ct2), zea AGAMOUS homolog1 (zag1), bearded ear (bde), and silky1 (si1). MicroRNAs such as microRNA156 were predicted to target genes involved in maize ear development. Antisense transcripts were widespread throughout all the four stages, and are suspected to play important roles in maize ear development. Thus, identification and characterization of important genes and regulators at all the four developmental stages will contribute to an improved understanding of the molecular mechanisms responsible for maize ear development.

Lead (Pb), as a heavy metal element, has become the most important metal pollutant of the environment. With allocating a relatively higher proportion of its biomass in roots, maize could be a potential important model to study the phytoremediation of Pb-contaminated soil. Here we analyzed the maize root transcriptome of inbred lines 9782 under heavy metal lead (Pb) pollution, which was identified as a non-hyperaccumulator for Pb in roots. In the present study, more than 98 millions reads were mapped to define gene structure and detect polymorphism, thereby to qualify transcript abundance along roots development under Pb treatment. A total of 17,707, 17,440, 16,998 and 16,586 genes were identified in maize roots at four developmental stages (0, 12 h, 24 h and 48 h) respectively and 2,825, 2,626, 2161 and 2260 stage-specifically expressed genes were also identified respectively. In addition, based on our RNA-Seq data, transcriptomic changes during maize root development responsive to Pb were investigated. A total of 384 differentially expressed genes (DEGs) (log2Ratio ≥ 1, FDR ≤ 0.001) were identified, of which, 36 genes with significant alteration in expression were detected in four developmental stages; 12 DEGs were randomly selected and successful validated by qRT-PCR. Additionally, many transcription factor families might act as the important regulators at different developmental stages, such as bZIP, ERF and GARP et al. These results will expand our understanding of the complex molecular and cellular events in maize root development and provide a foundation for future study on root development in maize under heavy metal pollution and other cereal crops.

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.

Phosphorus (P) is one of the three primary macronutrients that are required in large amounts for plant growth and development. To better understand molecular mechanism of maize and identify relevant genes in response to phosphorus deficiency, we used Solexa/Illumina's digital gene expression (DGE) technology to investigate six genome-wide expression profiles of seedling roots of the low-P tolerant maize inbred line 178. DGE studies were conducted at 6, 24 and 72 h under both phosphorus deficient and sufficient conditions. Approximately 3.93 million raw reads for each sample were sequenced and 6 816 genes exhibited significant levels of differential expressions in at least one of three time points in response to P starvation. The number of genes with increased expression increased over time from 6 to 24 h, whereas genes with decreased expression were more abundant at 72 h, suggesting a gradual response process for P deficiency at different stages. Gene annotations illustrated that most of differentially expressed genes (DEGs) are involved in different cellular and molecular processes such as environmental adaptation and carbohydrate metabolism. The expression of some known genes identified in other plants, such as those involved in root architecture, P metabolism and transport were found to be altered at least two folds, indicating that the mechanisms of molecular and morphological adaptation to P starvation are conserved in plants. This study provides insight into the general molecular mechanisms underlying plant adaptation to low-P stress and thus may facilitate molecular breeding for improving P utilization in maize.

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.

Ear rot, caused by Fusarium verticillioides (FV), is a destructive disease of maize as it reduces grain yield and increases risks of mycotoxin production, thus endangering livestock. To identify genes differentially expressed during FV infection, four cDNA libraries were constructed for suppression subtractive hybridization using RNA isolated from bracts of an FV-resistant inbred maize line, Bt-1, as well as an FV-susceptible maize inbred line, Ye478. A total of 145 clones were obtained following reverse dot-blot hybridization, and these were sequenced from these libraries. Similarity analysis revealed that of these genes, 93 were unique, including 68 of known function, 24 of unknown function, and a single novel gene. Most genes of known function were predominantly involved in plant defense such as cell defense, transcription regulation, signal transduction, and metabolism. Expression profiles of eight representative genes, using semiquantitative reverse transcription-polymerase chain reaction, confirmed that differential gene regulation during FV infection was involved. These findings suggested that these differentially expressed genes might be involved in FV defense responses in maize.

Lead (Pb) has become one of the most abundant heavy metal pollutants of the environment. With its large biomass, maize could be an important object for studying the phytoremediation of Pb-contaminated soil. In our previous research, we screened 19 inbred lines of maize for Pb concentration, and line 178 was identified to be a hyperaccumulator for Pb in both the roots and aboveground parts. To identify important genes and metabolic pathways related to Pb accumulation and tolerance, line 178 was underwent genome expression profile under Pb stress and a control (CK). A total of approximately 11 million cDNA tags were sequenced and 4 665 539 and 4 936 038 clean tags were obtained from the libraries of the test and CK, respectively. In comparison to CK, 2379 and 1832 genes were identified up- or downregulated, respectively, more than fivefolds under Pb stress. Interestingly, all the genes were related to cellular processes and signaling, information storage and processing or metabolism functions. Particularly, the genes involved in posttranslational modification, protein turnover and chaperones; signal transduction, carbohydrate transport and metabolism; and lipid transport and metabolism significantly changed under the treatment. In addition, seven pathways including ribosome, photosynthesis, and carbon fixation were affected significantly, with 118, 12, 34, 21, 18, 72 and 43 differentially expressed genes involved. The significant upregulation of the ribosome pathway may reveal an important secret for Pb tolerance of line 178. And the sharp increase of laccase transcripts and metal ion transporters were suggested to account in part for Pb hyperaccumulation in the line.

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress. Electronic supplementary material The online version of this article (doi:10.1007/s11033-012-1661-5) contains supplementary material, which is available to authorized users.

As introns are vulnerable to changes such as insertions and deletions when exposed to various evolutionary forces, they constitute a repository for developing genetic markers based on intron length polymorphisms (ILP). This study developed a set of genetic markers that use the potential intron length polymorphism in resistance gene analogs (RGAs) in Zea mays. By searching the genome of Zea mays B73 for the homologs of 73 R genes which have already been identified in plants, we found 861 RGAs, 632 of which have at least one intron that can serve as putative markers targeting the intron length polymorphism in RGAs (RGA-ILP). We developed 1972 candidate markers via electronic PCR (e-PCR) with primer pairs designed in each pair of exonic regions that flank an intron. Furthermore, the performance of RGA-ILP among four maize inbred lines (Huangzao4, B73, Mo17, and Dan340) was evaluated with 69 pairs of randomly selected primers. Of them, 46.4% showed bands that had discriminating length polymorphism, and between any two of the inbred lines the proportion of polymorphism ranged from 23.2 to 31.9%. To make it convenient to use these markers for those interested in molecular breeding of disease-resistant maize, we provide all related information in a web-based database named MaizeRGA, which is available at http://www.sicau.edu.cn/web/yms/rga/maizeRGA.html.

Different stressed conditions impair plant growth and further, cause great loss of crop yield and even lead to lose production completely. Increasing resistance/tolerance of crops under stressed conditions is a major goal of numerous plant breeders, and many elegant works are focusing on this area to uncover these complicated mechanisms underlying it. However, the traditional strategies including physiological and biochemical methods, as well as studies on a few genes, can not well understand the overall biological mechanism. Microarray analysis opens a door to uncover these cryptic mechanisms, and has the ability of detecting gene transcription and regulation at genomic level in different plant tissues. And works in association with related methods of proteomics and metabolomics. Therefore, it is possible to locate genes in certain key metabolism pathways. Through these procedures, it is also possible to look for critical genes in the pathway and to well understand the molecular mechanism of resistance/tolerance. These results can be as a guidance for increasing the resistance/tolerance of stressed conditions using biotechnology methods in future. This paper mainly focused on and discussed the advances of microarray analysis of stressed conditions-related genes in plants.

Biotic and abiotic stresses influence plant growth and cause great loss to crop yield. In the long course of evo-lution, plants have developed intricate biological mechanism to resist stressed conditions. Under various stressed conditions, not only the protein-coding genes, but also the non-protein-coding genes were induced for response. More and more re-searches showed that the transcripts of these non-protein-coding genes played important role in regulation of gene expres-sion. miRNA is one of the groups in these no-coding regulatory small RNAs. Recent findings showed that in order to resist the biotic and abiotic stresses, expression of microRNA (miRNA) genes will be induced and their transcripts (miRNAs) can regulate gene expression by guiding target mRNA cleavage or translation inhibition. This paper focused on the advances of plant miRNAs research in stressed conditions, especially induced expression of miRNA and target gene regulation and its role on adaptation under stressed conditions. Then, the methods of miRNA researches in stressed environments are dis-cussed.

MicroRNAs (miRNAs) are a set of small, non-coding RNAs that negatively and post-transcriptionally mediate their respective target mRNAs by directing the target mRNA cleavage or translational repression. Plant miRNAs have been involved in developmental processes and adaption to biotic and abiotic stresses in their environment. The banded leaf and sheath blight (BLSB) caused by Rhizoctonia solani is extremely harmful to maize. To investigate the functions of miRNAs under R. solani inoculation, miRNA expression in R. solani infected maize (Zea mays L.) was profiled using deep sequencing. In total, 41 significantly differentially expressed known miRNAs and 39 novel R. solani-responsive miRNAs were identified, of which 9 identified miRNAs were further validated by qRT-PCR, and 2 important miRNAs were analyzed by in situ hybridization. Target genes were also predicted for these R. solani-responsive miRNAs; most of these putative target genes encoded transcription factors and proteins associated with metabolic processes or stress responses. In addition, the mRNA expression levels of several target genes that negatively correlated with the levels of corresponding miRNAs under R. solani inoculation were validated by qRT-PCR. These findings hypothesized that these miRNAs play an important role in R. solani resistance in maize, highlighting novel molecular mechanisms of R. solani resistance in plants.

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.

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.

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.

As an economic crop, pepper satisfies people's spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded similar to 0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of similar to 81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.

Background In plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages. Results We used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed. Conclusions This study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.

MicroRNAs (miRNAs) play a pivotal role in post-transcriptional regulation of gene expression in plants. In this study, we investigate miRNAs in an agronomically important common tobacco in China, named Honghua Dajinyuan (a drought-tolerant cultivar). Here, we report a comprehensive analysis of miRNA expression profiles in mock-treat grown (CK) and 20 % polyethylene glycol-grown (PEG-grown) tobacco roots using a high-throughput sequencing approach. A total of 656 unique miRNAs representing 53 miRNA families were identified in the two libraries, of which 286 unique miRNAs representing 162 microRNAs were differentially expressed. In addition, nine differentially expressed microRNAs selected from different expressed miRNA family with high abundance were subjected to further analysis and validated by quantitative real-time PCR (Q-PCR). In addition, the expression pattern of these identified candidate conserved miRNA and target genes of three identified miRNA (nta-miR172b, nta-miR156i, and nta-miR160a) were also validated by Q-PCR. Gene ontology (GO) enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in metabolic process and response to stimulus. In particular, 25 target genes are involved in regulating plant hormone signal transduction and metabolism, indicating that these association microRNAs may play important regulatory roles in responding to PEG resistance. Moreover, this study adds a significant number of novel miRNAs to the tobacco miRNome.

Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.

Maize (Zea mays) is the most widely cultivated crop around the world, however, it is commonly affected by phosphate (Pi) deficiency and the underlying molecular basis of responses mechanism is still unknown. In this study, the transcriptional response of maize roots to Pi starvation at 3 days after the onset of Pi deprivation was assessed. The investigation revealed a total of 283 Pi-responsive genes, of which 199 and 84 genes were found to be either up- or down-regulated respectively, by 2-fold or more. Pi-responsive genes were found to be involved in sugar and nitrogen metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. In addition, the expression patterns of maize inorganic phosphorus transporters, acid phosphatase, phytase, 2-deoxymugineic acid synthase1, POD and MYB transcription factor were validated in 178 roots response to low phosphorus stress. of which, two genes encoding phytase and acid phosphatase were significantly induced by Pi deficiency and may play a pivotal role in the process of absorption and re-utilization of Pi in Maize. These results not only enhance our knowledge about molecular processes associated with Pi deficiency, but also facilitate the identification of key molecular determinants for improving Pi use in maize. Moreover, this work sets a framework to produce Pi-specific maize microarrays to study the changes in global gene expression between Pi-efficient and Pi-inefficient maize genotypes.

Banded leaf and sheath blight (BLSB) caused by Rhizoctonia solani Kuhn is an emerging problem in maize worldwide. To study gene expression in maize plants infected by R. solani Kuhn, we constructed two suppression subtractive hybridization (SSH) library using complementary DNA (cDNA) from uninoculated plants as a driver and those from inoculated plants as a tester. After two cycles of hybridization, 84 cDNA clones from the forward and reverse SSH library were obtained and sequenced. Gene analysis indicated that these clones represented 51 single genes, and the functions of 35 genes could be assigned using existing databases. Thirty-five expression sequences tags (ESTs) were classified in fuctional categories, such as: transcription, regulation, protein processing, metabolism, defense, disease response and other functions. The expression of 15 genes was analyzed by semi-quantitative reverse-transcription-polymerase chain reaction (RT-PCR) and found to be up-regulated or down-regulated in response to R. solani Kuhn re-infection. In conclusion, this study provides a basis for a molecular understanding of host reaction in response to pathogen. Key words: Banded leaf and sheath blight, expressed sequence tag, gene expression, maize, suppression subtractive hybridization.

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.