Morphological Characterization and Transcriptome Analysis of New Dwarf and Narrow-Leaf (dnl2) Mutant in Maize.

Lodging is the primary factor limiting high yield under a high plant density. However, an optimal plant height and leaf shape can effectively decrease the lodging risk. Here we studied an ethyl methanesulfonate (EMS)-induced dwarf and a narrow-leaf mutant, dnl2. Gene mapping indicated that the mutant was controlled by a gene located on chromosome nine. Phenotypic and cytological observations revealed that dnl2 showed inhibited cell growth, altered vascular bundle patterning, and disrupted secondary cell wall structure when compared with the wild-type, which could be the direct cause of the dwarf and narrow-leaf phenotype. The phytohormone levels, especially auxin and gibberellin, were significantly decreased in dnl2 compared to the wild-type plants. Transcriptome profiling of the internodes of the dnl2 mutant and wild-type revealed a large number of differentially expressed genes enriched in the cell wall biosynthesis, remodeling, and hormone biosynthesis and signaling pathways. Therefore, we suggest that crosstalk between hormones (the altered vascular bundle and secondary cell wall structure) may contribute to the dwarf and narrow-leaf phenotype by influencing cell growth. These results provide a foundation for DNL2 gene cloning and further elucidation of the molecular mechanism of the regulation of plant height and leaf shape in maize.

Comparative proteomic analysis reveals that the Heterosis of two maize hybrids is related to enhancement of stress response and photosynthesis respectively.

BACKGROUND: Heterosis refers to superior traits exhibiting in a hybrid when compared with both parents. Generally, the hybridization between parents can change the expression pattern of some proteins such as non-additive proteins (NAPs) which might lead to heterosis. 'Zhongdan808' (ZD808) and 'Zhongdan909' (ZD909) are excellent maize hybrids in China, however, the heterosis mechanism of them are not clear. Proteomics has been wildly used in many filed, and comparative proteomic analysis of hybrid and its parents is helpful for understanding the mechanism of heterosis in the two maize hybrids. RESULTS: Over 2000 protein groups were quantitatively identified from second seedling leaves of two hybrids and their parents by label-free quantification. Statistical analysis of total identified proteins, differentially accumulated proteins (DAPs) and NAPs of the two hybrids revealed that both of them were more similar to their female parents. In addition, most of DAPs were up-regulated and most of NAPs were high parent abundance or above-high parent abundance in ZD808, while in ZD909, most of DAPs were down-regulated and most of NAPs were low parent abundance or below-low parent abundance. Pathway enrichment analysis showed that more of stress response-related NAPs in ZD808 were high parent abundance or above-high parent abundance, and most of PS related NAPs in ZD909 were high parent abundance or above-high parent abundance. Finally, four stress response-related proteins and eight proteins related to PS were verified by PRM, ten of them had significant differences between hybrid and midparent value. CONCLUSIONS: Even though every one of the two hybrids were more similar to its female parent at proteome level, the biological basis of heterosis is different in the two maize hybrids. In comparison with their parents, the excellent agronomic traits of hybrid ZD808 is mainly correlated with the high expression levels of some proteins related to stress responses and metabolic functions, while traits of ZD909 is mainly correlated with high expressed proteins related to photosynthesis. Our proteomics results support previous physiological and morphological research and have provided useful information in understanding the reason of valuable agronomic traits.

Identifying quantitative trait loci for the general combining ability of yield-relevant traits in maize.

Maize is the most important staple crop worldwide. Many of its agronomic traits present with a high level of heterosis. Combining ability was proposed to exploit the rule of heterosis, and general combining ability (GCA) is a crucial measure of parental performance. In this study, a recombinant inbred line population was used to construct testcross populations by crossing with four testers based on North Carolina design II. Six yield-relevant traits were investigated as phenotypic data. GCA effects were estimated for three scenarios based on the heterotic group and the number of tester lines. These estimates were then used to identify quantitative trait loci (QTL) and dissect genetic basis of GCA. A higher heritability of GCA was obtained for each trait. Thus, testing in early generation of breeding may effectively select candidate lines with relatively superior GCA performance. The GCA QTL detected in each scenario was slightly different according to the linkage mapping. Most of the GCA-relevant loci were simultaneously detected in all three datasets. Therefore, the genetic basis of GCA was nearly constant although discrepant inbred lines were appointed as testers. In addition, favorable alleles corresponding to GCA could be pyramided via marker-assisted selection and made available for maize hybrid breeding.

Genetic mapping and genomic selection for maize stalk strength.

BACKGROUND: Maize is one of the most important staple crops and is widely grown throughout the world. Stalk lodging can cause enormous yield losses in maize production. However, rind penetrometer resistance (RPR), which is recognized as a reliable measurement to evaluate stalk strength, has been shown to be efficient and useful for improving stalk lodging-resistance. Linkage mapping is an acknowledged approach for exploring the genetic architecture of target traits. In addition, genomic selection (GS) using whole genome markers enhances selection efficiency for genetically complex traits. In the present study, two recombinant inbred line (RIL) populations were utilized to dissect the genetic basis of RPR, which was evaluated in seven growth stages. RESULTS: The optimal stages to measure stalk strength are the silking phase and stages after silking. A total of 66 and 45 quantitative trait loci (QTL) were identified in each RIL population. Several potential candidate genes were predicted according to the maize gene annotation database and were closely associated with the biosynthesis of cell wall components. Moreover, analysis of gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway further indicated that genes related to cell wall formation were involved in the determination of RPR. In addition, a multivariate model of genomic selection efficiently improved the prediction accuracy relative to a univariate model and a model considering RPR-relevant loci as fixed effects. CONCLUSIONS: The genetic architecture of RPR is highly genetically complex. Multiple minor effect QTL are jointly involved in controlling phenotypic variation in RPR. Several pleiotropic QTL identified in multiple stages may contain reliable genes and can be used to develop functional markers for improving the selection efficiency of stalk strength. The application of genomic selection to RPR may be a promising approach to accelerate breeding process for improving stalk strength and enhancing lodging-resistance.

Maize WI5 encodes an endo-1,4-ß-xylanase required for secondary cell wall synthesis and water transport in xylem.

Water transport from roots to leaves through xylem is important for plant growth and development. Defects in water transport can cause drought stress, even when there is adequate water in the soil. Here, we identified the maize (Zea mays) wilty5 (wi5) mutant, which exhibits marked dwarfing and leaf wilting throughout most of its life cycle under normal growth conditions. wilty5 seedlings exhibited lower xylem conductivity and wilted more rapidly under drought, NaCl, and high temperature treatments than wild-type plants. Map-based cloning revealed that WI5 encodes an active endo-1,4-ß-xylanase from glycosyl dehydration family 10, which mainly functions in degrading and reorganizing cell wall xylan. Reverse-transcription polymerase chain reaction and ß-glucuronidase assays revealed that WI5 is highly expressed in stems, especially in internodes undergoing secondary wall assembly. RNA sequencing suggested that WI5 plays a unique role in internode growth. Immunohistochemistry and electron microscopy confirmed that wi5 is defective in xylan deposition and secondary cell wall thickening. Lignin deposition and xylan content were markedly reduced in wi5 compared to the wild-type plants. Our results suggest that WI5 functions in xylem cell wall thickening through its xylanase activity and thereby regulates xylem water transport, the drought stress response, and plant growth in maize.

Transcriptome profiling and comparison of maize ear heterosis during the spikelet and floret differentiation stages.

BACKGROUND: Hybridization is a prominent process in the evolution of crop plants that can give rise to gene expression variation, phenotypic novelty and heterosis. Maize is the most successful crop in utilizing heterosis. The development of hybrid maize ears exhibits strong heterotic vigor and greatly affects maize yield. However, a comprehensive perspective on transcriptional variation and its correlation with heterosis during maize ear development is not available. RESULTS: Using RNA sequencing technology, we investigated the transcriptome profiles of maize ears in the spikelet and floret differentiation stages of hybrid ZD808 and its parents CL11 and NG5. Our results revealed that 53.9% (21,258) of maize protein-coding genes were transcribed in at least one genotype. In both development stages, significant numbers of genes were differentially expressed between the hybrid and its parents. Gene expression inheritance analysis revealed approximately 80% of genes were expressed additively, which suggested that the complementary effect may play a foundation role in maize ear heterosis. Among non-additively expressed genes, NG5-dominant genes were predominant. Analyses of the allele-specific gene expression in hybrid identified pervasive allelic imbalance and significant preferential expression of NG5 alleles in both developmental stages. The results implied that NG5 may provide beneficial alleles that contribute greatly to heterosis. Further comparison of parental and hybrid allele-specific expression suggested that gene expression variation is largely attributable to cis-regulatory variation in maize. The cis-regulatory variations tend to preserve the allelic expression levels in hybrid and result in additive expression. Comparison between the two development stages revealed that allele-specific expression and cis-/trans-regulatory variations responded differently to developmental cues, which may lead to stage-specific vigor phenotype during maize ear development. CONCLUSION: Our research suggests that cis-regulated additive expression may fine-tune gene expression level into an optimal status and play a foundation role in maize ear heterosis. Our work provides a comprehensive insight into transcriptional variation and its correlation with heterosis during maize ear development. The knowledge gained from this study presents novel opportunity to improve our maize varieties.

Genome-wide association analysis of forage quality in maize mature stalk.

BACKGROUND: Plant digestibility of silage maize (Zea mays L.) has a large influence on nutrition intake for animal feeding. Improving forage quality will enhance the utilization efficiency and feeding value of forage maize. Dissecting the genetic basis of forage quality will improve our understanding of the complex nature of cell wall biosynthesis and degradation, which is also helpful for breeding good quality silage maize. RESULTS: Acid detergent fiber (ADF), neutral detergent fiber (NDF) and in vitro dry matter digestibility (IVDMD) of stalk were evaluated in a diverse maize population, which is comprised of 368 inbred lines and planted across seven environments. Using a mixed model accounting for population structure and polygenic background effects, a genome-wide association study was conducted to identify single nucleotide polymorphisms (SNPs) significantly associated with forage quality. Scanning 559,285 SNPs across the whole genome, 73, 41 and 82 SNPs were found to be associated with ADF, NDF, and IVDMD, respectively. Each significant SNP explained 4.2 %-6.2 % of the phenotypic variation. Underlying these associated loci, 56 genes were proposed as candidate genes for forage quality. CONCLUSIONS: Of all the candidate genes proposed by GWAS, we only found a C3H gene (ZmC3H2) that is directly involved in cell wall component biosynthesis. The candidate genes found in this study are mainly involved in signal transduction, stress resistance, and transcriptional regulation of cell wall biosynthetic gene expression. Adding high digestibility maize into the association panel would be helpful for increasing genetic variability and identifying more genes associated with forage quality traits. Cloning and functional validation of these genes would be helpful for understanding the molecular mechanism of the fiber content and digestibility. These findings provide us new insights into cell wall formation and deposition.

Extensive, clustered parental imprinting of protein-coding and noncoding RNAs in developing maize endosperm.

Although genetic imprinting was discovered in maize 40 years ago, its exact extent in the triploid endosperm remains unknown. Here, we have analyzed global patterns of allelic gene expression in developing maize endosperms from reciprocal crosses between inbreds B73 and Mo17. We have defined an imprinted gene as one in which the relative expression of the maternal and paternal alleles differ at least fivefold in both hybrids of the reciprocal crosses. We found that at least 179 genes (1.6% of protein-coding genes) expressed in the endosperm are imprinted, with 68 of them showing maternal preferential expression and 111 paternal preferential expression. Additionally, 38 long noncoding RNAs were imprinted. The latter are transcribed in either sense or antisense orientation from intronic regions of normal protein-coding genes or from intergenic regions. Imprinted genes show a clear pattern of clustering around the genome, with a number of imprinted genes being adjacent to each other. Analysis of allele-specific methylation patterns of imprinted loci in the hybrid endosperm identified 21 differentially methylated regions (DMRs) of several hundred base pairs in length, corresponding to both imprinted genes and noncoding transcripts. All DMRs identified are uniformly hypomethylated in maternal alleles and hypermethylated in paternal alleles, regardless of the imprinting direction of their corresponding loci. Our study indicates highly extensive and complex regulation of genetic imprinting in maize endosperm, a mechanism that can potentially function in the balancing of the gene dosage of this triploid tissue.

Doxorubicin-Loaded Liposome with the Function of "Killing Two Birds with One Stone" against Glioma.

The blood-brain barrier (BBB) continues to be one of the main clinical obstacles in the treatment of glioma. Current chemotherapies always bring many different side effects, some even permanent. To date, nanomaterial-based vehicles have shown great potential in treating glioma. Herein, we developed a dual targeting liposomal delivery vector loaded with the anticancer drug doxorubicin (DOX) to treat glioma. SS31, a small peptide, has shown dual targeting effects of penetrating the BBB and specifically targeting mitochondria. In this study, a new liposomal delivery system, LS-DOX, was prepared by modifying DOX-loaded liposomes with SS31 for the treatment of in situ glioma. The liposomes demonstrated a high drug encapsulation rate and drug-loading capacity, satisfactory biocompatibility, high glioma accumulation ability, and good stability in vitro. Experimental results showed that the liposomes could effectively cross the BBB and target gliomas, and mitochondria-targeting of SS31 enhances cell uptake. In addition, the liposomes showed a good therapeutic effect on nude mice with glioma in situ with no obvious toxicity and side effects. Therefore, the present research will provide a novel alternative and reference for the effective treatment of glioma.

Characterization and Transcriptome Analysis of Maize Small-Kernel Mutant smk7a in Different Development Stages.

The kernel serves as a storage organ for various nutrients and determines the yield and quality of maize. Understanding the mechanisms regulating kernel development is important for maize production. In this study, a small-kernel mutant smk7a of maize was characterized. Cytological observation suggested that the development of the endosperm and embryo was arrested in smk7a in the early development stage. Biochemical tests revealed that the starch, zein protein, and indole-3-acetic acid (IAA) contents were significantly lower in smk7a compared with wild-type (WT). Consistent with the defective development phenotype, transcriptome analysis of the kernels 12 and 20 days after pollination (DAP) revealed that the starch, zein, and auxin biosynthesis-related genes were dramatically downregulated in smk7a. Genetic mapping indicated that the mutant was controlled by a recessive gene located on chromosome 2. Our results suggest that disrupted nutrition accumulation and auxin synthesis cause the defective endosperm and embryo development of smk7a.

Improving Genomic Selection With Quantitative Trait Loci and Nonadditive Effects Revealed by Empirical Evidence in Maize.

Genomic selection (GS), a tool developed for molecular breeding, is used by plant breeders to improve breeding efficacy by shortening the breeding cycle and to facilitate the selection of candidate lines for creating hybrids without phenotyping in various environments. Association and linkage mapping have been widely used to explore and detect candidate genes in order to understand the genetic mechanisms of quantitative traits. In the current study, phenotypic and genotypic data from three experimental populations, including data on six agronomic traits (e.g., plant height, ear height, ear length, ear diameter, grain yield per plant, and hundred-kernel weight), were used to evaluate the effect of trait-relevant markers (TRMs) on prediction accuracy estimation. Integrating information from mapping into a statistical model can efficiently improve prediction performance compared with using stochastically selected markers to perform GS. The prediction accuracy can reach plateau when a total of 500-1,000 TRMs are utilized in GS. The prediction accuracy can be significantly enhanced by including nonadditive effects and TRMs in the GS model when genotypic data with high proportions of heterozygous alleles and complex agronomic traits with high proportion of nonadditive variancein phenotypic variance are used to perform GS. In addition, taking information on population structure into account can slightly improve prediction performance when the genetic relationship between the training and testing sets is influenced by population stratification due to different allele frequencies. In conclusion, GS is a useful approach for prescreening candidate lines, and the empirical evidence provided by the current study for TRMs and nonadditive effects can inform plant breeding and in turn contribute to the improvement of selection efficiency in practical GS-assisted breeding programs.

Disrupted Brain Entropy And Functional Connectivity Patterns Of Thalamic Subregions In Major Depressive Disorder.

PURPOSE: Entropy analysis of resting-state functional magnetic resonance imaging (R-fMRI) has recently been adopted to characterize brain temporal dynamics in some neuropsychological or psychiatric diseases. Thalamus-related dysfunction might be a potential trait marker of major depressive disorder (MDD), but the abnormal changes in the thalamus based on R-fMRI are still unclear from the perspective of brain temporal dynamics. The aim of this study was to identify local entropy changes and subregional connectivity patterns of the thalamus in MDD patients. PATIENTS AND METHODS: We measured the sample entropy of the R-fMRI data from 46 MDD patients and 32 matched healthy controls. We employed the Louvain method for the module detection algorithm to automatically identify a functional parcellation of the thalamus and then examined the whole-brain subregional connectivity patterns. RESULTS: The results indicated that the MDD patients had decreased entropy in the bilateral thalami compared with healthy controls. Increased functional connectivity between the thalamic subregions and the medial part of the superior frontal gyrus (mSFG) was found in MDD patients. CONCLUSION: This study showed new evidence about sample entropy changes in MDD patients. The functional connectivity alterations that were widely distributed across almost all the thalamic subregions with the mSFG in MDD suggest a general involvement independent of the location and function of the subregions.

Genome-wide proteomic profiling reveals the role of dominance protein expression in heterosis in immature maize ears.

Heterosis refers to the phenomenon in which hybrid progeny show superior performance relative to their parents. Early maize ear development shows strong heterosis in ear architecture traits and greatly affects grain yield. To explore the underlying molecular mechanisms, genome-wide proteomics of immature ears of maize hybrid ZD909 and its parents were analyzed using tandem mass tag (TMT) technology. A total of 9,713 proteins were identified in all three genotypes. Among them, 3,752 (38.6%) proteins were differentially expressed between ZD909 and its parents. Multiple modes of protein action were discovered in the hybrid, while dominance expression patterns accounted for 63.6% of the total differentially expressed proteins (DEPs). Protein pathway enrichment analysis revealed that high parent dominance proteins mainly participated in carbon metabolism and nitrogen assimilation processes. Our results suggested that the dominant expression of favorable alleles related to C/N metabolism in the hybrid may be essential for ZD909 ear growth and heterosis formation. Integrated analysis of proteomic and quantitative trait locus (QTL) data further support our DEP identification and provide useful information for the discovery of genes associated with ear development. Our study provides comprehensive insight into the molecular mechanisms underlying heterosis in immature maize ears from a proteomic perspective.

Genetic and Quantitative Trait Locus Analysis of Cell Wall Components and Forage Digestibility in the Zheng58 × HD568 Maize RIL Population at Anthesis Stage.

The plant cell wall plays vital roles in various aspects of the plant life cycle. It provides a basic structure for cells and gives mechanical rigidity to the whole plant. Some complex cell wall components are involved in signal transduction during pathogenic infection and pest infestations. Moreover, the lignification level of cell walls strongly influences the digestibility of forage plants. To determine the genetic bases of cell wall components and digestibility, quantitative trait locus (QTL) analyses for six related traits were performed using a recombinant inbred line (RIL) population from a cross between Zheng58 and HD568. Eight QTL for in vitro neutral detergent fiber (NDF) digestibility were observed, out of which only two increasing alleles came from HD568. Three QTL out of ten with alleles increasing in vitro dry matter digestibility also originated from HD568. Five-ten QTL were detected for lignin, cellulose content, acid detergent fiber, and NDF content. Among these results, 29.8% (14/47) of QTL explained >10% of the phenotypic variation in the RIL population, whereas 70.2% (33/47) explained ≤10%. These results revealed that in maize stalks, a few large-effect QTL and a number of minor-effect QTL contributed to most of the genetic components involved in cell wall biosynthesis and digestibility.

Genome-Wide Association Study Reveals the Genetic Basis of Stalk Cell Wall Components in Maize.

Lignin, cellulose and hemicellulose are the three main components of the plant cell wall and can impact stalk quality by affecting cell wall structure and strength. In this study, we evaluated the lignin (LIG), cellulose (CEL) and hemicellulose (HC) contents in maize using an association mapping panel that included 368 inbred lines in seven environments. A genome-wide association study using approximately 0.56 million SNPs with a minor allele frequency of 0.05 identified 22, 18 and 24 loci significantly associated with LIG, CEL and HC at P < 1.0×10-4, respectively. The allelic variation of each significant association contributed 4 to 7% of the phenotypic variation. Candidate genes identified by GWAS mainly encode enzymes involved in cell wall metabolism, transcription factors, protein kinase and protein related to other biological processes. Among the association signals, six candidate genes had pleiotropic effects on lignin and cellulose content. These results provide valuable information for better understanding the genetic basis of stalk cell wall components in maize.

Map-based cloning of zb7 encoding an IPP and DMAPP synthase in the MEP pathway of maize.

IspH is a key enzyme in the last step of the methyl-D-erythritol-4-phosphate (MEP) pathway. Loss of function of IspH can often result in complete yellow or albino phenotype in many plants. Here, we report the characterization of a recessive mutant of maize, zebra7 (zb7), showing transverse green/yellow striped leaves in young plants. The yellow bands of the mutant have decreased levels of chlorophylls and carotenoids with delayed chloroplast development. Low temperature suppressed mutant phenotype, while alternate light/dark cycle or high temperature enlarged the yellow section. Map-based cloning demonstrated that zb7 encodes the IspH protein with a mis-sense mutation in a conserved region. Transgenic silencing of Zb7 in maize resulted in complete albino plantlets that are aborted in a few weeks, confirming that Zb7 is important in the early stages of maize chloroplast development. Zb7 is constitutively expressed and its expression subject to a 16-h light/8-h dark cycle regulation. Our results suggest that the less effective or unstable IspH in zb7 mutant, together with its diurnal expression, are mechanistically accounted for the zebra phenotype. The increased IspH mRNA in the leaves of zb7 at the late development stage may explain the restoration of mutant phenotype in mature stages.