Genome-wide association study of quality traits and starch pasting properties of maize kernels.

BACKGROUND: Starch are the main nutritional components of maize (Zea mays L.), and starch pasting properties are widely used as essential indicators for quality estimation. Based on the previous studies, various genes related to pasting properties have been identified in maize. However, the loci underlying variations in starch pasting properties in maize inbred lines remain to be identified. RESULTS: To investigate the genetic architecture of these traits, the starch pasting properties were examined based on 292 maize inbred lines, which were genotyped with the MaizeSNP50 BeadChip composed of 55,126 evenly spaced, random SNPs. A genome-wide association study (GWAS) implemented in the software package FarmCPU was employed to identify genomic loci for the starch pasting properties. 48 SNPs were found to be associated with pasting properties. Moreover, 37 candidate genes were correlated with pasting properties. Among the candidate genes, GRMZM2G143646 and GRMZM2G166407 were associated with breakdown and final viscosity significantly, and both genes encode PPR (Pentatricopeptide repeat) protein. We used GWAS to explore candidate genes of maize starch pasting properties in this study. The identified candidate genes will be useful for further understanding of the genetic architecture of starch pasting properties in maize. CONCLUSION: This study showed a complex regulation network about maize quality trait and starch pasting properties. It may provide some useful markers for marker assisted selection and a basis for cloning the genes behind these SNPs.

Multiomics comparative analysis of the maize large grain mutant tc19 identified pathways related to kernel development.

BACKGROUND: The mechanism of grain development in elite maize breeding lines has not been fully elucidated. Grain length, grain width and grain weight are key components of maize grain yield. Previously, using the Chinese elite maize breeding line Chang7-2 and its large grain mutant tc19, we characterized the grain size developmental difference between Chang7-2 and tc19 and performed transcriptomic analysis. RESULTS: In this paper, using Chang7-2 and tc19, we performed comparative transcriptomic, proteomic and metabolomic analyses at different grain development stages. Through proteomics analyses, we found 2884, 505 and 126 differentially expressed proteins (DEPs) at 14, 21 and 28 days after pollination, respectively. Through metabolomics analysis, we identified 51, 32 and 36 differentially accumulated metabolites (DAMs) at 14, 21 and 28 days after pollination, respectively. Through multiomics comparative analysis, we showed that the phenylpropanoid pathways are influenced at transcriptomic, proteomic and metabolomic levels in all the three grain developmental stages. CONCLUSION: We identified several genes in phenylpropanoid biosynthesis, which may be related to the large grain phenotype of tc19. In summary, our results provided new insights into maize grain development.

Transcriptomic analysis of the maize inbred line Chang7-2 and a large-grain mutant tc19.

BACKGROUNDS: Grain size is a key factor in crop yield that gradually develops after pollination. However, few studies have reported gene expression patterns in maize grain development using large-grain mutants. To investigate the developmental mechanisms of grain size, we analyzed a large-grain mutant, named tc19, at the morphological and transcriptome level at five stages corresponding to days after pollination (DAP). RESULTS: After maturation, the grain length, width, and thickness in tc19 were greater than that in Chang7-2 (control) and increased by 3.57, 8.80, and 3.88%, respectively. Further analysis showed that grain width and 100-kernel weight in tc19 was lower than in Chang7-2 at 14 and 21 DAP, but greater than that in Chang7-2 at 28 DAP, indicating that 21 to 28 DAP was the critical stage for kernel width and weight development. For all five stages, the concentrations of auxin and brassinosteroids were significantly higher in tc19 than in Chang7-2. Gibberellin was higher at 7, 14, and 21 DAP, and cytokinin was higher at 21 and 35 DAP, in tc19 than in Chang7-2. Through transcriptome analysis at 14, 21, and 28 DAP, we identified 2987, 2647 and 3209 differentially expressed genes (DEGs) between tc19 and Chang7-2. By using KEGG analysis, 556, 500 and 633 DEGs at 14, 21 and 28 DAP were pathway annotated, respectively, 77 of them are related to plant hormone signal transduction pathway. ARF3, AO2, DWF4 and XTH are higher expressed in tc19 than that in Chang7-2. CONCLUSIONS: We found some DEGs in maize grain development by using Chang7-2 and a large-grain mutant tc19. These DEGs have potential application value in improving maize performance.

Exogenous Sorbitol Application Confers Drought Tolerance to Maize Seedlings through Up-Regulating Antioxidant System and Endogenous Sorbitol Biosynthesis.

This study aims to explore the impacts of exogenous sorbitol on maize seedlings under polyethylene glycol (PEG)-simulated drought stress. Six treatments were set: normal condition (CK), PEG (P), 10 mM sorbitol (10S), PEG plus 10 mM sorbitol (10SP), 100 mM sorbitol (100S) and PEG plus 100 mM sorbitol (100SP). Maize seedlings' growth under PEG-simulated drought stress was significantly inhibited and exogenous sorbitol largely alleviated this growth inhibition. The seedlings under 10SP treatment grew much better than those under P, 100S and 100SP treatments and no significant difference in growth parameters was observed between the control and 10S treatment. The seedlings treated with 10SP had higher contents of soluble sugar, soluble protein, proline, ascorbic acid (AsA), reduced glutathione (GSH), sorbitol and relative water content, higher activities of antioxidant enzymes and aldose reductase, but lower contents of malondialdehyde (MDA), H2O2 and relative electrical conductivity than those treated with P, 100S and 100SP. qRT-PCR analysis showed that the transcript levels of genes encoding putative aldose reductase (AR) under P treatment were significantly up-regulated in sorbitol-applied treatments. Taken together, the results demonstrated that exogenous sorbitol application conferred drought tolerance to maize seedlings by up-regulating the expression levels of AR-related genes to enhance the accumulation of intracellular osmotic substances such as sorbitol and improve antioxidant systems to tone down the damage caused by drought stress.

Transcriptional and Metabolic Responses of Maize Shoots to Long-Term Potassium Deficiency.

Potassium is important for plant growth and crop yield. However, the effects of potassium (K+) deficiency on silage maize biomass yield and how maize shoot feedback mechanisms of K+ deficiency regulate whole plant growth remains largely unknown. Here, the study aims to explore the maize growth, transcriptional and metabolic responses of shoots to long-term potassium deficiency. Under the K+ insufficiency condition, the biomass yield of silage maize decreased. The transcriptome data showed that there were 922 and 1,107 differential expression genes in DH605 and Z58, respectively. In the two varieties, 390 differently expressed overlapping genes were similarly regulated. These genes were considered the fundamental responses to K+ deficiency in maize shoots. Many stress-induced genes are involved in transport, primary and secondary metabolism, regulation, and other processes, which are involved in K+ acquisition and homeostasis. Metabolic profiles indicated that most amino acids, phenolic acids, organic acids, and alkaloids were accumulated in shoots under K+ deficiency conditions and part of the sugars and sugar alcohols also increased. It revealed that putrescine and putrescine derivatives were specifically accumulated under the K+ deficiency condition, which may play a role in the feedback regulation of shoot growth. These results confirmed the importance of K+ on silage maize production and provided a deeper insight into the responses to K+ deficiency in maize shoots.

Identification of genetic loci associated with rough dwarf disease resistance in maize by integrating GWAS and linkage mapping.

Maize rough dwarf disease (MRDD) is a viral disease that causes substantial yield loss, especially in China's summer planted maize area. Discovery of resistance genes would help in developing high-yielding resistant maize hybrids. Genome-wide association studies (GWASs) have advanced quickly and are now a powerful tool for dissecting complex genetic architectures. In this study, the disease severity index (DSI) of 292 maize inbred lines and an F6 linkage population were investigated across multiple environments for two years. Using the genotypes obtained from the Maize SNP 50K chip, a GWAS was performed with four analytical models. The results showed that 22 SNPs distributed on chromosomes 1, 3, 4, 6, 7 and 8 were significantly associated with resistance to MRDD (P<0.0001). The SNPs on chromosomes 3, 6 and 8 were consistent with the quantitative trait locus (QTL) regions from linkage mapping in an RIL population. Candidate genes identified by GWAS included an LRR receptor-like serine/threonine-protein kinase (GRMZM2G141288), and a DRE-binding protein (GRMZM2G006745). In addition, we performed an allele variation analysis of the SNP loci selected by GWAS and linkage mapping and found that the main alleles of the two SNP loci PZE_101170408 and PZE_106082685 on chromosome 1 differed in terms of disease-resistant materials and disease-susceptible materials. The identified SNPs and genes provide useful information for MRDD-related gene cloning and insights on the underlying disease resistance mechanisms, and they can be used in marker-assisted breeding to develop MRDD-resistant maize.

ZmACY-1 Antagonistically Regulates Growth and Stress Responses in Nicotiana benthamiana.

Aminoacylase-1 is a zinc-binding enzyme that is important in urea cycling, ammonia scavenging, and oxidative stress responses in animals. Aminoacylase-1 (ACY-1) has been reported to play a role in resistance to pathogen infection in the model plant Nicotiana benthamiana. However, little is known about its function in plant growth and abiotic stress responses. In this study, we cloned and analyzed expression patterns of ZmACY-1 in Zea mays under different conditions. We also functionally characterized ZmACY-1 in N. benthamiana. We found that ZmACY-1 is expressed specifically in mature shoots compared with other tissues. ZmACY-1 is repressed by salt, drought, jasmonic acid, and salicylic acid, but is induced by abscisic acid and ethylene, indicating a potential role in stress responses and plant growth. The overexpression of ZmACY-1 in N. benthamiana promoted growth rate by promoting growth-related genes, such as NbEXPA1 and NbEIN2. At the same time, the overexpression of ZmACY-1 in N. benthamiana reduced tolerance to drought and salt stress. With drought and salt stress, the activity of protective enzymes, such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) from micrococcus lysodeikticus was lower; while the content of malondialdehyde (MDA) and relative electrolytic leakage was higher in ZmACY-1 overexpression lines than that in wild-type lines. The results indicate that ZmACY-1 plays an important role in the balance of plant growth and defense and can be used to assist plant breeding under abiotic stress conditions.

Constitutive expression of aldose reductase 1 from Zea mays exacerbates salt and drought sensitivity of transgenic Escherichia coli and Arabidopsis.

Aldose reductases (ARs) have been considered to play important roles in sorbitol biosynthesis, cellular detoxification and stress response in some plants. ARs from maize are capable of catalyzing the oxidation of sorbitol to glucose. However, little is known how maize ARs response to abiotic stresses. In this work, we cloned one isoform of maize ARs (ZmAR1), and furthermore we analyzed the roles of ZmAR1 in response to salt and drought stresses at both prokaryotic and eukaryotic levels. ZmAR1 encodes a putative 35 kDa protein that contains 310 amino acids. Under normal growth conditions, ZmAR1 was expressed in maize seedlings, and the highest expression level was found in leaves. But when seedlings were subjected to drought or salt treatment, the expression levels of ZmAR1 were significantly reduced. The constitutive expression of ZmAR1 increased the sensitivity of recombinant E. coli cells to drought and salt stresses compared with the control. Under salt and drought stresses, transgenic Arabidopsis lines displayed lower seed germination rate, shorter seedling root length, lower chlorophyll content, lower survival rate and lower antioxidant enzyme activity than wild type (WT) plants, but transgenic Arabidopsis had higher relative conductivity, higher water loss rate, and more MDA content than WT. Meanwhile, the introduction of ZmAR1 into Arabidopsis changed the expression levels of some stress-related genes. Taken together, our results suggested that ZmAR1 might act as a negative regulator in response to salt and drought stresses in Arabidopsis by reducing the sorbitol content and modulating the expression levels of some stress-related genes.

Novel x-type high-molecular-weight glutenin genes from Aegilops tauschii and their implications on the wheat origin and evolution mechanism of Glu-D1-1 proteins.

Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5*t and 1Dx5.1*t in Aegilops tauschii, were identified by SDS-PAGE, RP-HPLC, and MALDI-TOF-MS. The coding sequences were isolated by AS-PCR and the complete ORFs were obtained. Allele 1Dx5*t consists of 2481 bp encoding a mature protein of 827 residues with deduced Mr of 85,782 Da whereas 1Dx5.1*t comprises 2526 bp encoding 842 residues with Mr of 87,663 Da. The deduced Mr's of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5*t were correspondingly closer to the consensus compared to 1Dx5.1*t and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5*t and 8 in 1Dx5.1*t) and two indels in 1Dx5*t were identified, among which 8 SNPs were due to C-T or A-G transitions (an average of 73%). Expression of the cloned ORFs and N-terminal sequencing confirmed the authenticities of the two genes. Interestingly, several hybrid clones of 1Dx5*t expressed a slightly smaller protein relative to the authentic subunit present in seed proteins; this was confirmed to result from a deletion of 180 bp through illegitimate recombination as well as an in-frame stop codon. Network analysis demonstrated that 1Dx5*t, 1Dx2t, 1Dx1.6t, and 1Dx2.2* represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in an associated star-like phylogeny, suggesting that there were at least four independent origins of hexaploid wheat. In addition to unequal homologous recombination, duplication and deletion of large fragments occurring in Glu-D-1-1 alleles were attributed to illegitimate recombination.

Molecular cloning and characterization of four novel LMW glutenin subunit genes from Aegilops longissima, Triticum dicoccoides and T. zhukovskyi.

This paper reports cloning and characterisation of four novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2) from the genomic DNA of Triticum dicoccoides, T. zhukovskyi and Aegilops longissima. The coding regions of TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2 were 1056 bp, 903 bp, 1056 bp and 1050 bp in length, encoding 350, 300, 350 and 348 amino acid residues, respectively. The deduced amino acid sequences showed that the four novel genes were classified as LMW-m types and the comparison results indicated that the four genes had a more similar structure and a higher level of homology with the LMW-m genes than the LMW-s and -i types genes. However, the first cysteine residue's positions of TzLMW-m2, TdLMW-m1 and AlLMW-m2 were different from the others. Moreover, AlLMW-m2, TdLMW-m1 and TzLMW-m2 all possessed a longer repetitive domain, which was considered to be associated with good quality of wheat. The secondary structure prediction revealed that the content of beta-strand in AlLMW-m2 and TdLMW-m1 exceeded the positive control, suggesting that AlLMW-m2 and TdLMW-m1 should be considered as candidate genes that may have positive effect on dough quality. In order to investigate the evolutionary relationship of the novel genes with the other LMW-GSs, a phylogenetic tree was constructed. The results lead to a speculation that AlLMW-m2, TdLMW-m1 and TzLMW-m2 may be the middle types during the evolution of LMW-m and LMW-s.

Genetic Diversity and Genome-Wide Association Study of Major Ear Quantitative Traits Using High-Density SNPs in Maize.

Kernel and ear traits are key components of grain yield in maize (Zea mays L.). Investigation of these traits would help to develop high-yield varieties in maize. Genome-wide association study (GWAS) uses the linkage disequilibrium (LD) in the whole genome to determine the genes affecting certain phenotype. In this study, five ear traits (kernel length and width, ear length and diameter, cob diameter) were investigated across multi-environments for 2 years. Combining with the genotype obtained from Maize SNP50 chip, genetic diversity and association mapping in a set of 292 inbred lines were performed. Results showed that maize lines were clustered into seven subgroups and a total of 20 SNPs were found to be associated with ear traits significantly (P < 3.95E-05). The candidate genes identified by GWAS mainly encoded ubiquitin-activation enzymes (GRMZM2G015287), carotenoid cleavage dioxygenase (GRMZM2G446858), MYB-CC type transfactor, and phosphate starvation response protein 3, and they were associated with kernel length (KL) and ear diameter (ED), respectively. Moreover, two novel genes corresponding to RNA processing and fructose metabolism were found. Further, the SNPs detected by GWAS were confirmed by meta-QTL analysis. These genes and SNPs identified in the study would offer essential information for yield-related genes clone and breeding program in maize.

Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) from Aegilops tauschii.

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS-PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The gene encoding the HMM subunit Dy10.1t was amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1t subunit gene consisted of 1980 bp encoding a protein of 658 residues with an M rs of 68 611 Da, which was similar to the M rs determined by MALDI-TOF-MS. The deduced amino acid sequence indicated that Dy10.1t subunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1t gene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.