Natural variation in ZmNAC087 contributes to total root length regulation in maize seedlings under salt stress.

Soil salinity poses a significant challenge to crop growth and productivity, particularly affecting the root system, which is vital for water and nutrient uptake. To identify genetic factors that influence root elongation in stressful environments, we conducted a genome-wide association study (GWAS) to investigate the natural variation associated with total root length (TRL) under salt stress and normal conditions in maize seedlings. Our study identified 69 genetic variants associated with 38 candidate genes, among which a specific single nucleotide polymorphism (SNP) in ZmNAC087 was significantly associated with TRL under salt stress. Transient expression and transactivation assays revealed that ZmNAC087 encodes a nuclear-localized protein with transactivation activity. Further candidate gene association analysis showed that non-coding variations in ZmNAC087 promoter contribute to differential ZmNAC087 expression among maize inbred lines, potentially influencing the variation in salt-regulated TRL. In addition, through nucleotide diversity analysis, neutrality tests, and coalescent simulation, we demonstrated that ZmNAC087 underwent selection during maize domestication and improvement. These findings highlight the significance of natural variation in ZmNAC087, particularly the favorable allele, in maize salt tolerance, providing theoretical basis and valuable genetic resources for the development of salt-tolerant maize germplasm.

Genetic Diversification of Starch Branching Enzymes during Maize Domestication and Improvement.

Elucidating the genetic basis of starch pasting and gelatinization properties is crucial for enhancing the quality of maize and its utility as feed and industrial raw material. In maize, ZmSBE genes encode important starch branching enzymes in the starch biosynthesis pathway. In this study, we re-sequenced the genomic sequences of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII in three lines called 335 inbred lines, 68 landrace lines, and 32 teosinte lines. Analyses of nucleotide polymorphisms and haplotype diversity revealed differences in the selection patterns of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII during maize domestication and improvement. A marker-trait association analysis of inbred lines detected 22 significant loci, including 18 SNPs and 4 indels significantly associated with three maize starch physicochemical properties. The allele frequencies of two variants (SNP17249C and SNP5055G) were examined in three lines. The frequency of SNP17249C in ZmSBEIIb was highest in teosinte lines, followed by landrace lines, and inbred lines, whereas there were no significant differences in the frequency of SNP5055G in ZmSBEIII among the three lines. These results suggest that ZmSBE genes play an important role in the phenotypic variations in the starch physicochemical properties in maize. The genetic variants detected in this study may be used to develop functional markers for improving maize starch quality.

GWAS and Transcriptome Analysis Reveal Key Genes Affecting Root Growth under Low Nitrogen Supply in Maize.

Nitrogen (N) is one of the most important factors affecting crop production. Root morphology exhibits a high degree of plasticity to nitrogen deficiency. However, the mechanisms underlying the root foraging response under low-N conditions remain poorly understood. In this study, we analyzed 213 maize inbred lines using hydroponic systems and regarding their natural variations in 22 root traits and 6 shoot traits under normal (2 mM nitrate) and low-N (0 mM nitrate) conditions. Substantial phenotypic variations were detected for all traits. N deficiency increased the root length and decreased the root diameter and shoot related traits. A total of 297 significant marker-trait associations were identified by a genome-wide association study involving different N levels and the N response value. A total of 51 candidate genes with amino acid variations in coding regions or differentially expressed under low nitrogen conditions were identified. Furthermore, a candidate gene ZmNAC36 was resequenced in all tested lines. A total of 38 single nucleotide polymorphisms and 12 insertions and deletions were significantly associated with lateral root length of primary root, primary root length between 0 and 0.5 mm in diameter, primary root surface area, and total length of primary root under a low-N condition. These findings help us to improve our understanding of the genetic mechanism of root plasticity to N deficiency, and the identified loci and candidate genes will be useful for the genetic improvement of maize tolerance cultivars to N deficiency.

Primary root response to combined drought and heat stress is regulated via salicylic acid metabolism in maize.

The primary root is the first organ to perceive the stress signals for abiotic stress. In this study, maize plants subjected to drought, heat and combined stresses displayed a significantly reduced primary root length. Metabolic and transcriptional analyses detected 72 and 5,469 differentially expressed metabolites and genes in response to stress conditions, respectively. The functional annotation of differentially expressed metabolites and genes indicated that primary root development was mediated by pathways involving phenylalanine metabolism, hormone metabolism and signaling under stress conditions. Furthermore, we found that the concentration of salicylic acid and two precursors, shikimic acid and phenylalanine, showed rapid negative accumulation after all three stresses. The expression levels of some key genes involved in salicylic acid metabolism and signal transduction were differentially expressed under stress conditions. This study extends our understanding of the mechanism of primary root responses to abiotic stress tolerance in maize.

Exploiting natural variation in crown root traits via genome-wide association studies in maize.

BACKGROUND: Root system architecture (RSA), which is determined by the crown root angle (CRA), crown root diameter (CRD), and crown root number (CRN), is an important factor affecting the ability of plants to obtain nutrients and water from the soil. However, the genetic mechanisms regulating crown root traits in the field remain unclear. METHODS: In this study, the CRA, CRD, and CRN of 316 diverse maize inbred lines were analysed in three field trials. Substantial phenotypic variations were observed for the three crown root traits in all environments. A genome-wide association study was conducted using two single-locus methods (GLM and MLM) and three multi-locus methods (FarmCPU, FASTmrMLM, and FASTmrEMMA) with 140,421 SNP. RESULTS: A total of 38 QTL including 126 SNPs were detected for CRA, CRD, and CRN. Additionally, 113 candidate genes within 50 kb of the significant SNPs were identified. Combining the gene annotation information and the expression profiles, 3 genes including GRMZM2G141205 (IAA), GRMZM2G138511 (HSP) and GRMZM2G175910 (cytokinin-O-glucosyltransferase) were selected as potentially candidate genes related to crown root development. Moreover, GRMZM2G141205, encoding an AUX/IAA transcriptional regulator, was resequenced in all tested lines. Five variants were identified as significantly associated with CRN in different environments. Four haplotypes were detected based on these significant variants, and Hap1 has more CRN. CONCLUSIONS: These findings may be useful for clarifying the genetic basis of maize root system architecture. Furthermore, the identified candidate genes and variants may be relevant for breeding new maize varieties with root traits suitable for diverse environmental conditions.

Integrating transcriptome, co-expression and QTL-seq analysis reveals that primary root growth in maize is regulated via flavonoid biosynthesis and auxin signal transduction.

The primary root is critical for early seedling growth and survival. To understand the molecular mechanisms governing primary root development, we performed a dynamic transcriptome analysis of two maize (Zea mays) inbred lines with contrasting primary root length at nine time points over a 12-day period. A total of 18 702 genes were differentially expressed between two lines or different time points. Gene enrichment, phytohormone content determination, and metabolomics analysis showed that auxin biosynthesis and signal transduction, as well as the phenylpropanoid and flavonoid biosynthesis pathways, were associated with root development. Co-expression network analysis revealed that eight modules were associated with lines/stages, as well as primary or lateral root length. In root-related modules, flavonoid metabolism accompanied by auxin biosynthesis and signal transduction constituted a complex gene regulatory network during primary root development. Two candidate genes (rootless concerning crown and seminal roots, rtcs and Zm00001d012781) involved in auxin signaling and flavonoid biosynthesis were identified by co-expression network analysis, QTL-seq and functional annotation. These results increase our understanding of the regulatory network controlling the development of primary and lateral root length, and provide a valuable genetic resource for improvement of root performance in maize.

Genetic control of root plasticity in response to salt stress in maize.

KEY MESSAGE: GWAS identified 559 significant SNPs associated with the remodelling of the root architecture in response to salt, and 168 candidate genes were prioritized by integrating RNA-seq, DEG and WGCNA data. Salinity is a major environmental factor limiting crop growth and productivity. The root is the first plant organ to encounter salt stress, yet the effects of salinity on maize root development remain unclear. In this study, the natural variations in 14 root and 4 shoot traits were evaluated in 319 maize inbred lines under control and saline conditions. Considerable phenotypic variations were observed for all traits, with high salt concentrations decreasing the root length, but increasing the root diameter. A genome-wide association study was conducted to analyse these traits and their plasticity (relative variation). We detected 559 significant single nucleotide polymorphisms, of which 125, 181 and 253 were associated with the control condition, stress condition and trait plasticity, respectively. A total of 168 of 587 candidate genes identified by genome-wide association study were supported by the differentially expressed genes or co-expression networks. Two candidate genes ZmIAA1 and ZmGRAS43 were validated by resequencing. Among these genes, 130 were detected under stress condition or trait plasticity that involved in diverse biological processes including plant hormone signal transduction, phenylpropanoid biosynthesis and fatty acid biosynthesis. Our findings clarify the root remodelling to salinity, and the identified loci and candidate genes may be important for the genetic improvement of root traits and salt tolerance in maize.

Natural Variation and Domestication Selection of ZmCKX5 with Root Morphological Traits at the Seedling Stage in Maize.

Root system architecture plays a crucial role in water and nutrient acquisition in maize. Cytokinins, which can be irreversibly degraded by the cytokinin oxidase/dehydrogenase (CKX), are important hormones that regulate root development in plants. In this study, ZmCKX5 was resequenced in 285 inbred lines, 68 landraces, and 32 teosintes to identify the significant variants associated with root traits in maize. Sequence polymorphisms and nucleotide diversity revealed that ZmCKX5 might be selected during domestication and improvement processes. Marker-trait association analysis in inbred lines identified 12 variants of ZmCKX5 that were significantly associated with six root traits, including seed root number (SRN), lateral root length (LRL), total root area (RA), root length in 0 to 0.5 mm diameter class (RL005), total root volume (RV), and total root length (TRL). SNP-1195 explained the most (6.01%) phenotypic variation of SRN, and the frequency of this allele G increased from 6.25% and 1.47% in teosintes and landraces, respectively, to 17.39% in inbred lines. Another significant variant, SNP-1406, with a pleiotropic effect, is strongly associated with five root traits, with the frequency of T allele increased from 25.00% and 23.73% in teosintes and landraces, respectively, to 35.00% in inbred lines. These results indicate that ZmCKX5 may be involved in the development of the maize root system and that the significant variants can be used to develop functional markers to accelerate the improvement in the maize root system.

Integrating GWAS and Gene Expression Analysis Identifies Candidate Genes for Root Morphology Traits in Maize at the Seedling Stage.

Root system plays an essential role in water and nutrient acquisition in plants. Understanding the genetic basis of root development will be beneficial for breeding new cultivars with efficient root system to enhance resource use efficiency in maize. Here, the natural variation of 13 root and 3 shoot traits was evaluated in 297 maize inbred lines and genome-wide association mapping was conducted to identify SNPs associated with target traits. All measured traits exhibited 2.02- to 21.36-fold variations. A total of 34 quantitative trait loci (QTLs) were detected for 13 traits, and each individual QTL explained 5.7% to 15.9% of the phenotypic variance. Three pleiotropic QTLs involving five root traits were identified; SNP_2_104416607 was associated with lateral root length (LRL), root surface area (RA), root length between 0 and 0.5mm in diameter (RL005), and total root length (TRL); SNP_2_184016997 was associated with RV and RA, and SNP_4_168917747 was associated with LRL, RA and TRL. The expression levels of candidate genes in root QTLs were evaluated by RNA-seq among three long-root lines and three short-root lines. A total of five genes that showed differential expression between the long- and short-root lines were identified as promising candidate genes for the target traits. These QTLs and the potential candidate genes are important source data to understand root development and genetic improvement of root traits in maize.

Natural Variation and Domestication Selection of ZmPGP1 Affects Plant Architecture and Yield-Related Traits in Maize.

ZmPGP1, involved in the polar auxin transport, has been shown to be associated with plant height, leaf angle, yield traits, and root development in maize. To explore natural variation and domestication selection of ZmPGP1, we re-sequenced the ZmPGP1 gene in 349 inbred lines, 68 landraces, and 32 teosintes. Sequence polymorphisms, nucleotide diversity, and neutral tests revealed that ZmPGP1 might be selected during domestication and improvement processes. Marker-trait association analysis in inbred lines identified 11 variants significantly associated with 4 plant architecture and 5 ear traits. SNP1473 was the most significant variant for kernel length and ear grain weight. The frequency of an increased allele T was 40.6% in teosintes, and it was enriched to 60.3% and 89.1% during maize domestication and improvement. This result revealed that ZmPGP1 may be selected in the domestication and improvement process, and significant variants could be used to develop functional markers to improve plant architecture and ear traits in maize.

Natural variation of ZmHKT1 affects root morphology in maize at the seedling stage.

MAIN CONCLUSION: Eight variants in ZmHKT1 promoter were significantly associated with root diameter, four haplotypes based on these significant variants were found, and Hap2 has the largest root diameter. Roots play an important role in uptake of water, nutrients and plant anchorage. Identification of gene and corresponding SNPs associated with root traits would enable develop maize lines with better root traits that might help to improve capacity for absorbing nutrients and water acquisition. The genomic sequences of a salt tolerance gene ZmHKT1 was resequenced in 349 maize inbred lines, and the association between nucleotide polymorphisms and seedling root traits was detected. A total of 269 variants in ZmHKT1 were identified, including 226 single nucleotide polymorphisms and 43 insertions and deletions. The gene displayed high level of nucleotide diversity, especially in non-genic regions. A total of 19 variations in untranslated region of ZmHKT1 were found to be associated with six seedling traits. Eight variants in promoter region were significantly associated with average root diameter (ARD), four haplotypes were found based on these significant variants, and Hap2 has the largest ARD. Two SNPs in high-linkage disequilibrium (SNP-415 and SNP 2169) with pleiotropic effects were significantly associated with plant height, root surface area, root volume, and shoot dry weight. This result revealed that ZmHKT1 was an important contributor to the phenotypic variations of seedling root traits in maize, these significant variants could use to develop functional markers to improve root traits.

Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus.

BACKGROUND: Aflatoxin contamination caused by Aspergillus flavus in peanut (Arachis hypogaea) including in pre- and post-harvest stages seriously affects industry development and human health. Even though resistance to aflatoxin production in post-harvest peanut has been identified, its molecular mechanism has been poorly understood. To understand the mechanism of peanut response to aflatoxin production by A. flavus, RNA-seq was used for global transcriptome profiling of post-harvest seed of resistant (Zhonghua 6) and susceptible (Zhonghua 12) peanut genotypes under the fungus infection and aflatoxin production stress. RESULT: A total of 128.72 Gb of high-quality bases were generated and assembled into 128, 725 unigenes (average length 765 bp). About 62, 352 unigenes (48.43%) were annotated in the NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Swiss-Prot, KEGG Ortholog, Protein family, Gene Ontology, or eukaryotic Ortholog Groups database and more than 93% of the unigenes were expressed in the samples. Among obtained 30, 143 differentially expressed unigenes (DEGs), 842 potential defense-related genes, including nucleotide binding site-leucine-rich repeat proteins, polygalacturonase inhibitor proteins, leucine-rich repeat receptor-like kinases, mitogen-activated protein kinase, transcription factors, ADP-ribosylation factors, pathogenesis-related proteins and crucial factors of other defense-related pathways, might contribute to peanut response to aflatoxin production. Notably, DEGs involved in phenylpropanoid-derived compounds biosynthetic pathway were induced to higher levels in the resistant genotype than in the susceptible one. Flavonoid, stilbenoid and phenylpropanoid biosynthesis pathways were enriched only in the resistant genotype. CONCLUSIONS: This study provided the first comprehensive analysis of transcriptome of post-harvest peanut seeds in response to aflatoxin production, and would contribute to better understanding of molecular interaction between peanut and A. flavus. The data generated in this study would be a valuable resource for genetic and genomic studies on crops resistance to aflatoxin contamination.

Functional Genomic Analysis of Aspergillus flavus Interacting with Resistant and Susceptible Peanut.

In the Aspergillus flavus (A. flavus)-peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with resistant and susceptible peanut genotypes. In total, 67.46 Gb of high-quality bases were generated for A. flavus-resistant (af_R) and -susceptible peanut (af_S) at one (T1), three (T2) and seven (T3) days post-inoculation. The uniquely mapped reads to A. flavus reference genome in the libraries of af_R and af_S at T2 and T3 were subjected to further analysis, with more than 72% of all obtained genes expressed in the eight libraries. Comparison of expression levels both af_R vs. af_S and T2 vs. T3 uncovered 1926 differentially expressed genes (DEGs). DEGs associated with mycelial growth, conidial development and aflatoxin biosynthesis were up-regulated in af_S compared with af_R, implying that A. flavus mycelia more easily penetrate and produce much more aflatoxin in susceptible than in resistant peanut. Our results serve as a foundation for understanding the molecular mechanisms of aflatoxin production differences between A. flavus-R and -S peanut, and offer new clues to manage aflatoxin contamination in crops.

Deep sequencing analysis of transcriptomes in Aspergillus flavus in response to resveratrol.

BACKGROUND: Resveratrol has been reported as a natural phytoalexin that inhibits infection or the growth of certain fungi including Aspergillus flavus. Our previous research revealed that aflatoxin production in A. flavus was reduced in medium with resveratrol. To understand the molecular mechanism of the A. flavus response to resveratrol treatment, the high-throughput paired-end RNA-Seq was applied to analyze the transcriptomic profiles of A. flavus. RESULTS: In total, 366 and 87 genes of A. flavus were significantly up- and down- regulated, respectively, when the fungus was treated with resveratrol. Gene Ontology (GO) functional enrichment analysis revealed that 48 significantly differentially expressed genes were involved in 6 different terms. Most genes in the aflatoxin biosynthetic pathway genes cluster (#54) did not show a significant change when A. flavus was treated with resveratrol, but 23 of the 30 genes in the #54 cluster were down-regulated. The transcription of aflA and aflB was significantly suppressed under resveratrol treatment, resulting in an insufficient amount of the starter unit hexanoate for aflatoxin biosynthesis. In addition, resveratrol significantly increased the activity of antioxidative enzymes that destroy radicals, leading to decreased aflatoxin production. Moreover, stuA, fluG, flbC, and others genes involved in mycelial and conidial development were down-regulated, which disrupted the cell's orderly differentiation and blocked conidia formation and mycelia development. The transcripts of laeA and veA were slightly inhibited by resveratrol, which may partly decrease aflatoxin production and depress conidia formation. CONCLUSIONS: Resveratrol can affect the expression of A. flavus genes that are related to developmental and secondary metabolic processes, resulting in decreased aflatoxin production and conidia formation and could also cause abnormal mycelia development. These results provide insight into the transcriptome of A. flavus in response to resveratrol and a new clew for further study in regulation of aflatoxin biosynthesis in A. flavus.