Publications

Extensive lead (Pb) absorption by plants affects their growth and development and causes damage to the human body by entering the food chain. In this study, we cloned ZmHIPP, a gene associated with Pb tolerance and accumulation in maize, using combined linkage mapping and weighted gene co-expression network analysis. We show that ZmHIPP, which encodes a heavy metal-associated isoprenylated plant protein, positively modulated Pb tolerance and accumulation in maize seedlings, Arabidopsis, and yeast. The genetic variation locus (A/G) in the promoter of ZmHIPP contributed to the phenotypic disparity in Pb tolerance among different maize inbred lines by altering the expression abundance of ZmHIPP. Knockdown of ZmHIPP significantly inhibited growth and decreased Pb accumulation in maize seedlings under Pb stress. ZmHIPP facilitated Pb deposition in the cell wall and prevented it from entering the intracellular organelles, thereby alleviating Pb toxicity in maize seedlings. Compared to that in the mutant zmhipp, the accumulated Pb in the wild-type line mainly consisted of the low-toxicity forms of Pb. Our study increases the understanding of the mechanism underlying Pb tolerance in maize and provides new insights into the bioremediation of Pb-polluted soil.

The ability of immature maize (Zea mays) embryos to form embryonic calluses (ECs) is highly genotype dependent, which limits transgenic breeding development in maize. Here, we report the association map-based cloning of ZmSAUR15 using an association panel (AP) consisting of 309 inbred lines with diverse formation abilities for ECs. We demonstrated that ZmSAUR15, which encodes a small auxin-upregulated RNA, acts as a negative effector in maize EC induction. Polymorphisms in the ZmSAUR15 promoter that influence the expression of ZmSAUR15 transcripts modulate the EC induction capacity in maize. ZmSAUR15 is involved in indole-3-acetic acid biosynthesis and cell division in immature embryo-derived callus. The ability of immature embryos to induce EC formation can be improved by the knockout of ZmSAUR15, which consequently increases the callus regeneration efficiency. Our study provides new insights into overcoming the genotypic limitations associated with EC formation and improving genetic transformation in maize.

MADS26 affecting maize seed germination was identified by GWAS and transcriptomics. Gene-based association analyses revealed three variations within MADS26 regulating seed germination traits. Overexpressed MADS26 in Arabidopsis improved seed germination.

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.

Soil salinization is an important factor threatening the yield and quality of maize. Ca2+ plays a considerable role in regulating plant growth under salt stress. Herein, we examined the shoot Ca2+ concentrations, root Ca2+ concentrations, and transport coefficients of seedlings in an association panel composed of 305 maize inbred lines under normal and salt conditions. A genome-wide association study was conducted by using the investigated phenotypes and 46,408 single-nucleotide polymorphisms of the panel. As a result, 53 significant SNPs were specifically detected under salt treatment, and 544 genes were identified in the linkage disequilibrium regions of these SNPs. According to the expression data of the 544 genes, we carried out a weighted coexpression network analysis. Combining the enrichment analyses and functional annotations, four hub genes (GRMZM2G051032, GRMZM2G004314, GRMZM2G421669, and GRMZM2G123314) were finally determined, which were then used to evaluate the genetic variation effects by gene-based association analysis. Only GRMZM2G123314, which encodes a pentatricopeptide repeat protein, was significantly associated with Ca2+ transport and the haplotype G-CT was identified as the superior haplotype. Our study brings novel insights into the genetic and molecular mechanisms of salt stress response and contributes to the development of salt-tolerant varieties in maize.

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.

Two hub genesGRMZM2G075104 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.

Microelements are necessary for plant growth and development, they control key processes of physiological metabolism. Herein, we evaluated three accumulation-related performances for each of the four microelements (Fe, Zn, Cu, and Mn) among 305 inbred maize lines. Quantification of these microelements in maize roots and shoots revealed abundant phenotypic variations in the association panel, with the variation coefficients ranging from 0.31 to 0.76. Principal component analysis (PCA) of the three related traits (concentration in root, concentration in shoot, and transport coefficient) showed that PC1 and PC2 explained \textgreater95% of phenotypic variations for each element. The scores of PC1 and PC2 were thereby used for a genome-wide association study by combining 44,134 SNPs of this panel. A total of 27, 1, 5, and 3 SNPs were significantly (P \textless .05) associated with Zn-PC1, Zn-PC2, Cu-PC1, and Mn-PC2, respectively, with 11 genes closely linked (r2 \textgreater 0.8) to these SNPs. Of them, GRMZM2G142870, GRMZM2G045531, and GRMZM2G143512 were individually annotated as ABC transporter C family member 14, zinc transporter 3, and heavy metal ATPase10. A candidate gene association analysis further verified that GRMZM2G142870 and GRMZM2G045531 affect Zn and Mn accumulations, respectively. Evaluation of contrasting allele ratios in elite lines indicated that the majority of the alleles correlating with higher Zn or Cu had not been utilized in maize breeding. Integration of more “higher-accumulation” alleles in the elite lines will be practical for improving Zn and Cu accumulations in maize. Our findings contribute to genetic revelation and molecular marker-assisted selection of microelement accumulations in maize.

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 \textless 2.03 × 10-6) 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.

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 metals lead and cadmium have become important pollutants in the environment, which exert negative effects on plant morphology, growth and photosynthesis. It is particularly significant to uncover the genetic loci and the causal genes for lead and cadmium tolerance in plants. This study used an IBM Syn10 DH population to identify the quantitative trait loci (QTL) controlling maize seedling tolerance to lead and cadmium by linkage mapping. The broad-sense heritability of these seedling traits ranged from 65.8–97.3% and 32.0–98.8% under control (CK) and treatment (T) conditions, respectively. A total of 53 and 64 QTL were detected under CK and T conditions, respectively. Moreover, 42 QTL were identified using lead and cadmium tolerance coefficient (LCTC). Among these QTL, five and two major QTL that explained \textgreater 10% of phenotypic variation were identified under T condition and using LCTC, respectively. Furthermore, eight QTL were simultaneously identified by T and LCTC, explaining 5.23% to 9.21% of the phenotypic variations. Within these major and common QTL responsible for the combined heavy metal tolerance, four candidate genes (Zm00001d048759, Zm00001d004689, Zm00001d004843, Zm00001d033527) were previously reported to correlate with heavy metal transport and tolerance. These findings will contribute to functional gene identification and molecular marker-assisted breeding for improving heavy metal tolerance in maize.

Kernel size-related traits are the most direct traits correlating with grain yield. The genetic basis of three kernel traits of maize, kernel length (KL), kernel width (KW) and kernel thickness (KT), was investigated in an association panel and a biparental population. A total of 21 single nucleotide polymorphisms (SNPs) were detected to be most significantly (P \textless 2.25 × 10−6) associated with these three traits in the association panel under four environments. Furthermore, 50 quantitative trait loci (QTL) controlling these traits were detected in seven environments in the intermated B73 × Mo17 (IBM) Syn10 doubled haploid (DH) population, of which eight were repetitively identified in at least three environments. Combining the two mapping populations revealed that 56 SNPs (P \textless 1 × 10−3) fell within 18 of the QTL confidence intervals. According to the top significant SNPs, stable-effect SNPs and the co-localized SNPs by association analysis and linkage mapping, a total of 73 candidate genes were identified, regulating seed development. Additionally, seven miRNAs were found to situate within the linkage disequilibrium (LD) regions of the co-localized SNPs, of which zma-miR164e was demonstrated to cleave the mRNAs of Arabidopsis CUC1, CUC2 and NAC6 in vitro. Overexpression of zma-miR164e resulted in the down-regulation of these genes above and the failure of seed formation in Arabidopsis pods, with the increased branch number. These findings provide insights into the mechanism of seed development and the improvement of molecular marker-assisted selection (MAS) for high-yield breeding in maize.

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.

Coix is a grass crop domesticated as early as the Neolithic era. It is still widely cultivated for both highly nutritional food and medicinal use. However, the genetic study and breeding of this crop are hindered by the lack of a sequenced genome. Here, we report de novo sequencing and assembly of the 1619-Mb genome of Coix, and annotation of 75.39% repeats and 39 629 protein-coding genes. Comparative genomics analysis showed that Coix is more closely related to sorghum than maize, but intriguingly only Coix and maize had a recent genome duplication event, which was not detected in sorghum. We further constructed a genetic map and mapped several important traits, especially the strength of hull. Selection of papery hull (thin: easy dehulling) from the stony hull (thick: difficult dehulling) in wild progenitors was a key step in Coix domestication. The papery hull makes seed easier to process and germinate. Anatomic and global transcriptome analysis revealed that the papery hull is a result of inhibition of cell division and wall biogenesis. We also successfully demonstrated that seed hull pressure resistance is controlled by two major quantitative trait loci (QTLs), which are associated with hull thickness and color, respectively. The two QTLs were further fine mapped within intervals of 250 kb and 146 kb, respectively. These resources provide a platform for evolutionary studies and will facilitate molecular breeding of this important crop.

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.

Ear tip-barrenness (ETB) phenotype threatens crop yield, because it reduces the kernel number per ear. The genetic basis of ETB in maize remains largely unknown. Herein, a genome-wide association study (GWAS) and quantitative trait loci (QTL) mapping were jointly applied to identify the significant genetic loci interrelated with ETB. Six significant SNPs were detected at a stringent P-value threshold (1.95 × 10−6). Additionally, four environment-stable SNPs were co-detected across a single environment and best linear unbiased prediction (BLUP) model at a less stringent P-value threshold (1 × 10−4). The above 10 SNPs were closely linked to 6 candidate genes, which mainly involved seed development, photosynthesis and ethylene response. Moreover, the ratio of superior allele at each significant SNP ranged from 0 to 83.33% in 30 investigated maize elite lines. QTL mapping identified 14 QTL with phenotypic variation explained (PVE) ranging from 3.64 to 7.09%, of which one QTL (qETB2-1) was repeatedly identified in two environments. Combined analysis of GWAS and QTL mapping showed that one SNP (PZE-102175229, chromosome 2: 217 66 Mb) was located in the QTL (qETB2-2, chromosome 2: 215 90–217 82 Mb). Eighteen gene models situated in the linkage disequilibrium (LD) region of the co-localized SNP were further used to evaluate their correlation with ETB by candidate gene association analysis. Two superior haplotypes and two superior alleles were detected among 74 lines for Zm00001d007195, Zm00001d007197 and Zm00001d007201. These results provide more information for clarifying the molecular mechanism of ETB and for speeding up the genetic improvement of maize varieties.

The free moisture in crop kernels after being naturally dried is referred to as kernel moisture content (KMC). Maize KMC reflects grain quality and influences transportation and storage of seeds. We used an IBM Syn10 DH maize population consisting of 249 lines and an association panel comprising 310 maize inbred lines to identify the genetic loci affecting maize KMC in three environments. Using the IBM population detected 13 QTL on seven chromosomes, which were clustered into nine common QTL. Genome-wide association analysis (GWAS) identified 16 significant SNPs across the 3 environments, which were linked to 158 genes across the three environments. Combined QTL mapping and GWAS found two SNPs that were located in two of the mapped QTL, respectively. Twenty-three genes were linked with the loci co-localized in both populations. Of these 181 genes, five have previously been reported to be associated with KMC or to regulate seed development. These associations were verified by candidate gene association analysis. Two superior alleles and one favorable haplotype for Zm00001d007774 and Zm00001d047868 were found to influence KMC. These findings provide insights into molecular mechanisms underlying maize KMC and contribute to the use of marker-assisted selection for breeding low-KMC maize.

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize.

Kernel weight in a unit volume is referred to as kernel test weight (KTW) that directly reflects maize (Zea mays L.) grain quality. In this study, an inter-mated B73 × Mo17 (IBM) Syn10 doubled haploid (DH) population and an association panel were used to identify loci responsible for KTW of maize across multiple environments. A total of 18 significant KTW-related single-nucleotide polymorphisms (SNPs) were identified using genome-wide association study (GWAS); they were closely linked to 12 candidate genes. In the IBM Syn10 DH population, linkage analysis detected 19 common quantitative trait loci (QTL), five of which were repeatedly detected among multiple environments. Several verified genes that regulate maize seed development were found in the confidence intervals of the mapped QTL and the LD regions of GWAS, such as ZmYUC1, BAP2, ZmTCRR-1, dek36 and ZmSWEET4c. Combined QTL mapping and GWAS identified one significant SNP that was co-identified in the both populations. Based on the co-localized SNP across the both populations, 17 candidate genes were identified. Of them, Zm00001d044075, Zm00001d044086, and Zm00001d044081 were further identified by candidate gene association study for KTW. Zm00001d044081 encodes homeobox-leucine zipper protein ATHB-4, which has been demonstrated to control apical embryo development in Arabidopsis. Our findings provided insights into the mechanism underlying maize KTW and contributed to the application of molecular-assisted selection of high KTW breeding in maize.

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.

Maize tassel architecture is a complex quantitative trait that is significantly correlated with biomass yield and grain yield. The present study evaluated the major trait of maize tassel architecture, namely, tassel branch number (TBN), in an association population of 359 inbred lines and an IBM Syn 10 population of 273 doubled haploid lines across three environments. Approximately 43,958 high-quality single nucleotide polymorphisms were utilized to detect significant QTNs associated with TBN based on new multi-locus genome-wide association study methods. There were 30, 38, 73, 40, 47, and 53 QTNs associated with tassel architecture that were detected using the FastmrEMMA, FastmrMLM, EM-BLASSO, mrMLM, pkWMEB, and pLARmEB models, respectively. Among these QTNs, 51 were co-identified by at least two of these methods. In addition, 12 QTNs were consistently detected across multiple environments. Furthermore, 19 QTLs distributed on chromosomes 1, 2, 3, 4, 6, and 7 were detected in 3 environments and the BLUP model based on 6618 bin markers, which explained 3.64–10.96% of the observed phenotypic variations in TBN. Of these, three QTLs were co-detected in two environments. One QTN associated with TBN was localized to one QTL. Approximately 55 candidate genes were detected by common QTNs and LD criteria. One candidate gene, Zm00001d016615, was identified as a putative target of the RA1 gene. Meanwhile, RA1 was previously validated to plays an important role in tassel development. In addition, the newly identified candidate genes Zm00001d003939, Zm00001d030212, Zm00001d011189, and Zm00001d042794 have been reported to involve in a spikelet meristem identity module. The findings of the present study improve our understanding of the genetic basis of tassel architecture in maize.

Stalk lodging severely limits the grain yield of maize (Zea mays L.). Mechanical stalk strength can be reflected by the traits of stalk diameter (SD), stalk bending strength (SBS), and lodging rind penetrometer resistance (RPR). To determine the genetic basis of maize stalk lodging, quantitative trait loci (QTLs) were mapped for these three traits using the IBM Syn10 DH population in three environments. The results indicated that there were strong genetic correlations among the three traits, and the analyses of phenotypic variations for SD, SBS, and RPR across the three environments showed high broad-sense heritability (0.6843, 0.5175, and 0.7379, respectively). In total, 44 significant QTLs were identified control the above traits across the 3 environments. A total of 14, 14, and 16 QTLs were identified for SD, SBS, and RPR across single-environment mapping, respectively. Notably, ten QTLs were stably expressed across multiple-environments, including two QTLs for SD, three for SBS, and five for RPR. Three major QTLs each accounting for over 10% of the phenotypic variation were qSD6-2 (10.03%), qSD8-2 (13.73%), and qSBS1-2 (11.89%). Comprehensive analysis of all QTLs in this study revealed that 5 QTL clusters including 12 QTLs were located on chromosomes 1, 3, 7, and 8, respectively. Among these 44 QTLs, 9 harbored 13 stalk lodging-associated SNPs that were detected by our recently published work, with 1 SNP successfully validated in the IBM Syn10 DH population. These chromosomal regions will be useful for marker-assisted selection and fine mapping of stalk lodging-related traits in maize.

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.

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 \textless50% in 31 elite inbred lines. Meanwhile, these superior alleles had obviously additive effect on the regenerative capacity. This indicates that the regenerative capacity-related traits can be improved by proper integration of the superior alleles using marker-assisted selection. Moreover, a total of 40 candidate genes were found based on these common QTNs. Some annotated genes were previously reported to relate with auxin transport, cell fate, seed germination, or embryo development, especially, GRMZM2G108933 (WOX2) was found to promote maize transgenic embryonic callus regeneration. These identified candidate genes will contribute to a further understanding of the genetic foundation of maize embryonic callus regeneration.

Leaf-related traits (leaf length, leaf width, leaf area and leaf angle) are very important for the yield of maize (Zea mays L) due to their influence on plant type. Therefore, it is necessary to identify quantitative trait loci (QTLs) for leaf-related traits. In this report, 221 doubled haploid lines (DHLs) of an IBM Syn10 DH population were provided for QTL mapping. In total, 54 QTLs were detected for leaf-related traits in single environments using a high-density genetic linkage map. Among them, only eight common QTLs were identified across two or three environments, and the common QTLs for the four traits explained 4.38%–19.99% of the phenotypic variation. qLL-2-1 (bin 2.09), qLW-2-2 (bin 2.09), qLW-6-3 (bin 6.07) and qLA-5-2 (bin 2.09) were detected in previous studies, and qLL-1-1, qLAr-1-1, qLAr-2-1 and qLA-7-1 may be new QTLs. Notably, qLW-6-3 and qLA-5-2 were found to be major QTLs explaining 19.99% and 10.96% of the phenotypic variation, respectively. Interestingly, we found three pairs of QTLs (qLW-2-2 and qLAr-2-1, qLW-8-1 and qLL-8-2, qLL-3-3 and qLAr-3-3) that control different traits and that were located on the same chromosome or in a nearby location. Moreover, nine pairs of loci with epistatic effects were identified for the four traits. These results may provide the foundation for QTL fine mapping and for an understanding of the genetic basis of variation in leaf-related traits.

Leucine-rich repeat disease resistance (LRRDR) genes are important for defending plants from a range of pathogens. However, little information has been reported on the systematic analysis of LRRDR genes in maize. In this study, 235 LRRDR genes were identified in the complete genome sequence of maize (Zea mays cv. B73), classified as six different structural types, and then characterized based on conserved protein motifs, chromo- somal locations and gene duplications. Subsequent phylogenetic comparisons indicated that ~20 pairs of maize LRRDR proteins possessed high similarities to LRRDR proteins with known functions. Analyses of the physical locations and duplications of LRRDR genes indicated that gene duplication events involving LRRDR genes were high in maize and 84% occurred between chromosomes, which may ensure the functional performance and en- hancement of maize LRRDR genes. Meanwhile, the functions and expression patterns of the LRRDR genes were associated with their conserved protein secondary structures, suggesting that different conserved domains might distinguish their biological functions. Transcripts of 13 genes were regulated by two or more fungal pathogens, respectively, indicating that one LRRDR gene might mediate resistance to multiple fungal pathogens, suggest- ing that the signal networks of the maize-fungal pathogen interactions were partially crossed. Additionally, we screened five candidate LRRDR genes for ear rot resistance. The results reported in this study contribute to an improved understanding of the LRRDR gene family in maize.

Large phenotypic variations in the lead (Pb) concentration were observed in grains and leaves of maize plants. A further understanding of inheritance of Pb accumulation may facilitate improvement of low-Pb-accumulating cultivars in maize. A genome-wide association study was conducted in a population of 269 maize accessions with 43,737 single-nucleotide polymorphisms (SNPs). The Pb concentrations in leaves and kernels of 269 accessions were collected in pot-culture and field experiments in years of 2015 and 2016. Significant differences in Pb accumulation were found among individuals under different environments. Using the structure and kinship model, a total of 21 SNPs significantly associated with the Pb accumulation were identified with P \textless 2.28 × 10−5 and FDR \textless 0.05 in the pot-culture and field experiments across 2 years. Three SNPs on chromosome 4 had significant associations simultaneously with the Pb concentrations of kernels and leaves and were co-localized with the previously detected quantitative trait loci. Through ridge regression best linear unbiased prediction Pb accumulation in the association population, the prediction accuracies by cross validation were 0.18–0.59 and 0.17–0.64, depending on the k-fold and the size of the training population. The results are helpful for genetic improvement and genomic prediction of Pb 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.

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.

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.

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.

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.

We carried out a study of regeneration capacities of embryonic callus from maize immature embryo culture with 144 different inbred lines of natural groups from different countries, and found that the regeneration capacity was affected by three factors: environment, genotype and the interaction between the environment and genotype. We found that green embryonic callus rate (GCR), embryonic callus differentiating rate (CDR) and the plantlet number of embryonic callus regeneration (CPN) have significant positive correlations with each other, and they all have significant negative correlations with embryonic callus browning rate (CBR). Moreover, embryonic callus cloning index for the first subculture (CCI1) and embryonic callus cloning index for the second subculture (CCI2) have a significant positive correlation with each other, and CCI2 is positively correlated with green GCR, and is negatively correlated with CBR. Embryonic callus rooting rate (CRR) is positively correlated with GCR, CDR and CPN to some degree. Furthermore, we calculated Broad-Sense Heritability of each trait, and uncovered that the heritability index of CCI1, CCI2 and CRR was lower, and the heritability index of others was higher. In addition, by using the Ward method for two-way cluster analysis, we found eleven inbred lines with high regenerating abilities, and the rooting situation of regenerating plantlet was excellent by rooting culture, which could be used as the elite inbred lines of the maize transgenic receptor.

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.

In this study, a maize inbred line with a strong capacity to induce embryonic callus, 18-599R, was used to analyze the transcription factors expressed during embryonic callus formation. A total of 1180 transcription factors were found to be expressed during three key stages of callus induction. Of these, compared with control, 361, 346 and 328 transcription factors were significantly downregulated during stages I, II and III, respectively. In contrast, 355, 372 and 401 transcription factors (TFs) were upregulated during the respective stages. We constructed a transcription factor-mediated regulatory network and found that plant hormone signal transduction was the pathway most significantly enriched among TFs. This pathway includes 48 TFs regulating cell enlargement, cell differentiation, cell division and cell dedifferentiation via the response to plant hormones. Through real-time polymerase chain reaction (PCR) and degradome sequencing, we identified 23 transcription factors that are regulated by miRNA. Through further analysis, ZmMYB138, a member of the MYB transcription factor family localized in the nucleus, was verified to promote embryonic callus formation in the maize embryo through GA signal transduction.

Increasing knowledge of plant genome sequences requires the development of more reliable and efficient genetic approaches for genotype–phenotype validation. Functional identification of plant genes is generally achieved by a combination of creating genetic modifications and observing the according phenotype, which begins with forward-genetic methods represented by random physical and chemical mutagenesis and move towards reverse-genetic tools as targeted genome editing. A major bottleneck is time need to produce modified homozygous genotypes that can actually be used for phenotypic validation. Herein, we comprehensively address and compare available experimental approaches for functional validation of plant genes, and propose haploid strategies to reduce the time needed and cost consumed for establishing gene function.

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.

To safeguard the food supply for the growing human population, it is important to understand and exploit the genetic basis of quantitative traits. Next-generation sequencing technology performs advantageously and effectively in genetic mapping and genome analysis of diverse genetic resources. Hence, we combined re-sequencing technology and a bin map strategy to construct an ultra-high-density bin map with thousands of bin markers to precisely map a quantitative trait locus.

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.

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.

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.

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.

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.

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.

Currently, the molecular regulation mechanisms of disease-resistant involved in maize leaf sheaths infected by banded leaf and sheath blight (BLSB) are poorly known. To gain insight into the transcriptome dynamics that are associated with their disease-resistant, genome-wide gene expression profiling was conducted by Solexa sequencing. More than four million tags were generated from sheath tissues without any leaf or development leaf, including 193,222 and 204,824 clean tags in the two libraries, respectively. Of these, 82,864 (55.4 %) and 91,678 (51.5 %) tags were matched to the reference genes. The most differentially expressed tags with log2 ratio \textgreater2 or \textless−2 (P \textless 0.001) were further analyzed, representing 1,476 up-regulated and 1,754 down-regulated genes, except for unknown transcripts, which were classified into 11 functional categories. The most enriched categories were those of metabolism, signal transduction and cellular transport. Next, the expression patterns of 12 genes were assessed by quantitative real-time PCR, and it is showed the results were general agreement with the Solexa analysis, although the degree of change was lower in amplitude. In conclusion, we first reveal the complex changes in the transcriptome during the early development of maize sheath infected by BLSB and provide a comprehensive set of data that are essential for understanding its molecular regulation mechanism.

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.

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.

The fungus Rhizoctonia solani Kuhn is a widespread and destructive plant pathogen with a very broad host range. Although various pathogens, including R. solani, have been traditionally controlled using chemical pesticides, their use faces drawbacks such as environmental pollution, development of pesticide resistance, and other negative effects. Carbendazim is a well-known antifungal agent capable of controlling a broad range of plant diseases, but its use is hampered by its poor aqueous solubility. In this study, we describe an environmentally friendly pharmaceutical microemulsion system using carbendazim as the active ingredient, chloroform and acetic acid as solvents, and the surfactants HSH and 0204 as emulsifiers. This system increased the solubility of carbendazim to 30 g/L. The optimal microemulsion formulation was determined based on a pseudo-ternary phase diagram; its physicochemical characteristics were also tested. The cloud point was greater than 90°C and it was resistant to freezing down to −18°C, both of which are improvements over the temperature range in which pure carbendazim can be used. This microemulsion meets the standard for pesticide microemulsions and demonstrated better activity against R. solani AG1-IA, relative to an aqueous solution of pure carbendazim (0.2 g/L). The mechanism of activity was reflected in the inhibition of against R. solani AG1-IA including mycelium growth, and sclerotia formation and germination were significantly better than that of 0.2 g/L carbendazim water solution according to the results of t-test done by SPSS 19.

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 30days 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.

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 186million 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.

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.