Identification of Candidate Genes for Drought Tolerance at Maize Seedlings Using Genome-Wide Association.

BACKGROUND: Drought stress is a serious threat that limit maize growth and production. OBJECTIVES: The assessment tolerance level of maize by measuring changes in the main biochemical and physiological indicators under drought stress. MATERIAL AND METHODS: We performed a genome-wide association analysis of biochemical and physiological indicators using an elite association panel. RESULTS: The results revealed that eight significant SNPs (p<0.05/N) located in eight genes that are distributed on different chromosomes were associated with drought resistance indices under drought stress. Among these genes, four genes were linked via the associated SNPs with drought-resistance indices of the malondialdehyde activity (MDA), three genes were linked with drought resistance indexes of the superoxide dismutase activity (SOD), and one gene was linked with drought resistance indexes of relative conductivity (REC). The candidate genes functioned as transcription factors, enzymes, and transporters, which included trehalase, the AP2/EREB160 transcription factor, and glutathione S-transferase and also encoded a gene of unknown function. These genes may be directly or indirectly involved in drought resistance. The expression levels of ZmEREB160 responded to ABA and drought stress. CONCLUSIONS: These results provided good information to understand the genetic basis of variation in drought resistance indices of biochemical and physiological indicators during drought stress.

Genome-wide identification and expression analysis of the GA2ox gene family in maize (Zea mays L.) under various abiotic stress conditions.

GA 2-oxidases (GA2oxs) are a class of enzymes that inhibit the biosynthesis of bioactive GAs in plants. Although GA2oxs have clear roles in the development and defence responses in Arabidopsis, rice, and wheat, their potential effects on maize remain unclear. This study identified thirteen ZmGA2ox genes in maize and further characterized them using phylogenetic, gene structure, genomic locus, expression pattern analyses and GA content determination. Phylogenetic relationship analysis clearly divided the ZmGA2ox family into three groups-seven in C19-GA2ox class I, three in C19-GA2ox class II, and three in C20-GA2ox class. Evolutionary analysis suggested that ZmGA2ox1;1 and ZmGA2ox1;2, ZmGA2ox3;1 and ZmGA2ox3;2, and ZmGA2ox7;1 and ZmGA2ox7;2 are three pairs of segmental duplicated genes. Prediction of cis-regulatory elements in promoters suggested that ZmGA2ox genes were mainly associated with growth, development, hormones, and biotic/abiotic stress. Therefore, their spatial and temporal expression patterns and responses to various stress treatments were analysed on the basis of published RNA-seq data. Moreover, the changes of ZmGA2ox expression in leaves and roots of maize seedlings was detected under salt, alkali, dehydration, and cold stresses by qRT-PCR. The ZmGA2oxs exhibited obvious expression tendencies or characteristics in various organs under different abiotic stresses. The variations in the expression of three ZmGA2ox genes in the C20-GA2ox class in maize seedling roots showed significant regularity and a clear negative correlation with bioactive GA contents under cold and drought conditions, indicating that these three genes might exert key effects on the regulation of GA synthesis and the response to drought and cold stress. Taken together, this study is useful for further dissection of the effect of ZmGA2oxs on abiotic stress responses and might provide potential targets for the genetic improvement of maize.

Detection of QTNs for kernel moisture concentration and kernel dehydration rate before physiological maturity in maize using multi-locus GWAS.

Maize is China's largest grain crop. Mechanical grain harvesting is the key technology in maize production, and the kernel moisture concentration (KMC) is the main controlling factor in mechanical maize harvesting in China. The kernel dehydration rate (KDR) is closely related to the KMC. Thus, it is important to conduct genome-wide association studies (GWAS) of the KMC and KDR in maize, detect relevant quantitative trait nucleotides (QTNs), and mine relevant candidate genes. Here, 132 maize inbred lines were used to measure the KMC every 5 days from 10 to 40 days after pollination (DAP) in order to calculate the KDR. These lines were genotyped using a maize 55K single-nucleotide polymorphism array. QTNs for the KMC and KDR were detected based on five methods (mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, and ISIS EM-BLASSO) in the package mrMLM. A total of 334 significant QTNs were found for both the KMC and KDR, including 175 QTNs unique to the KMC and 178 QTNs unique to the KDR; 116 and 58 QTNs were detected among the 334 QTNs by two and more than two methods, respectively; and 9 and 5 QTNs among 58 QTNs were detected in 2 and 3 years, respectively. A significant enrichment in cellular component was revealed by Gene Ontology enrichment analysis of candidate genes in the intervals adjacent to the 14 QTNs and this category contained five genes. The information provided in this study may be useful for further mining of genes associated with the KMC and KDR in maize.

QTL mapping and GWAS for field kernel water content and kernel dehydration rate before physiological maturity in maize.

Kernel water content (KWC) and kernel dehydration rate (KDR) are two main factors affecting maize seed quality and have a decisive influence on the mechanical harvest. It is of great importance to map and mine candidate genes related to KWCs and KDRs before physiological maturity in maize. 120 double-haploid (DH) lines constructed from Si287 with low KWC and JiA512 with high KWC were used as the mapping population. KWCs were measured every 5 days from 10 to 40 days after pollination, and KDRs were calculated. A total of 1702 SNP markers were used to construct a linkage map, with a total length of 1,309.02 cM and an average map distance of 0.77 cM. 10 quantitative trait loci (QTLs) and 27 quantitative trait nucleotides (QTNs) were detected by genome-wide composite interval mapping (GCIM) and multi-locus random-SNP-effect mixed linear model (mrMLM), respectively. One and two QTL hotspot regions were found on Chromosome 3 and 7, respectively. Analysis of the Gene Ontology showed that 2 GO terms of biological processes (BP) were significantly enriched (P ≤ 0.05) and 6 candidate genes were obtained. This study provides theoretical support for marker-assisted breeding of mechanical harvest variety in maize.

Function analysis of ZmNAC33, a positive regulator in drought stress response in Arabidopsis.

Drought significantly affects plant growth and has devastating effects on crop production, NAC transcription factors respond to abiotic stresses by activating gene expression. In this study, a maize NAC transcription factor, ZmNAC33, was cloned and characterized its function in Arabidopsis. Transient transformation in Arabidopsis leaves mesophyll protoplasts and trans-activation assays in yeast showed that ZmNAC33 was localized in the nucleus and had transactivation activity. qRT-PCR analysis showed that ZmNAC33 in maize was induced by drought, high salinity and abscisic acid (ABA) stress. Promoter analysis identified multiple stress-related cis-acting elements in the promoter region of ZmNAC33. In ZmNAC33 transgenic Arabidopsis, germination rates were higher than in wild type plants under ABA and osmotic stress at the germination stage, and overexpression lines exhibited higher survival rates and higher antioxidant enzyme activities compared with wild type under drought stress. These results indicate that ZmNAC33 actes as a positive regulator in drought tolerance in plants.

iTRAQ-Based Proteomic Analysis Reveals Several Strategies to Cope with Drought Stress in Maize Seedlings.

Drought stress, especially during the seedling stage, seriously limits the growth of maize and reduces production in the northeast of China. To investigate the molecular mechanisms of drought response in maize seedlings, proteome changes were analyzed. Using an isotopic tagging relative quantitation (iTRAQ) based method, a total of 207 differentially accumulated protein species (DAPS) were identified under drought stress in maize seedlings. The DAPS were classified into ten essential groups and analyzed thoroughly, which involved in signaling, osmotic regulation, protein synthesis and turnover, reactive oxygen species (ROS) scavenging, membrane trafficking, transcription related, cell structure and cell cycle, fatty acid metabolism, carbohydrate and energy metabolism, as well as photosynthesis and photorespiration. The enhancements of ROS scavenging, osmotic regulation, protein turnover, membrane trafficking, and photosynthesis may play important roles in improving drought tolerance of maize seedlings. Besides, the inhibitions of some protein synthesis and slowdown of cell division could reduce the growth rate and avoid excessive water loss, which is possible to be the main reasons for enhancing drought avoidance of maize seedlings. The incongruence between protein and transcript levels was expectedly observed in the process of confirming iTRAQ data by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, which further indicated that the multiplex post-transcriptional regulation and post-translational modification occurred in drought-stressed maize seedlings. Finally, a hypothetical strategy was proposed that maize seedlings coped with drought stress by improving drought tolerance (via. promoting osmotic adjustment and antioxidant capacity) and enhancing drought avoidance (via. reducing water loss). Our study provides valuable insight to mechanisms underlying drought response in maize seedlings.

Association Analysis and Identification of ZmHKT1;5 Variation With Salt-Stress Tolerance.

The high-affinity potassium transporter (HKT) genes are essential for plant salt stress tolerance. However, there were limited studies on HKTs in maize (Zea mays), and it is basically unknown whether natural sequence variations in these genes are associated with the phenotypic variability of salt tolerance. Here, the characterization of ZmHKT1;5 was reported. Under salt stress, ZmHKT1;5 expression increased strongly in salt-tolerant inbred lines, which accompanied a better-balanced Na+/K+ ratio and preferable plant growth. The association between sequence variations in ZmHKT1;5 and salt tolerance was evaluated in a diverse population comprising 54 maize varieties from different maize production regions of China. Two SNPs (A134G and A511G) in the coding region of ZmHKT1;5 were significantly associated with different salt tolerance levels in maize varieties. In addition, the favorable allele of ZmHKT1; 5 identified in salt tolerant maize varieties effectively endowed plant salt tolerance. Transgenic tobacco plants of overexpressing the favorable allele displayed enhanced tolerance to salt stress better than overexpressing the wild type ZmHKT1;5. Our research showed that ZmHKT1;5 expression could effectively enhance salt tolerance by maintaining an optimal Na+/K+ balance and increasing the antioxidant activity that keeps reactive oxygen species (ROS) at a low accumulation level. Especially, the two SNPs in ZmHKT1;5 might be related with new amino acid residues to confer salt tolerance in maize. Key Message: Two SNPs of ZmHKT1;5 related with salt tolerance were identified by association analysis. Overexpressing ZmHKT1;5 in tobaccos showed that the SNPs might enhance its ability to regulating Na+/K+ homeostasis.

Fine mapping of a yield-enhancing QTL cluster associated with transgressive variation in an Oryza sativa x O. rufipogon cross.

A high-resolution physical map targeting a cluster of yield-related QTLs on the long arm of rice chromosome 9 has been constructed across a 37.4 kb region containing seven predicted genes. Using a series of BC3F4 nearly isogenic lines (NILs) derived from a cross between the Korean japonica cultivar Hwaseongbyeo and Oryza rufipogon (IRGC 105491), a total of seven QTLs for 1,000-grain weight, spikelets per panicle, grains per panicle, panicle length, spikelet density, heading date and plant height were identified in the cluster (P

Fine mapping of a grain weight quantitative trait locus on rice chromosome 8 using near-isogenic lines derived from a cross between Oryza sativa and Oryza rufipogon.

A quantitative trait locus (QTL) for grain weight (GW) was detected near SSR marker RM210 on chromosome 8 in backcross populations derived from a cross between the Korean japonica cultivar Hwaseongbyeo and Oryza rufipogon (IRGC 105491). The O. rufipogon allele increased GW in the Hwaseongbyeo background despite the fact that O. rufipogon was the small-seeded parent. Using sister BC(3)F(3) near-isogenic lines (NILs), gw8.1 was validated and mapped to a 6.1 cM region in the interval between RM42 and RM210 (P < or = 0.0001). Substitution mapping with eight BC(3)F(4) sub-NILs further narrowed the interval containing gw8.1 to about 306.4 kb between markers RM23201.CNR151 and RM30000.CNR99. A yield trial using homozygous BC(3)F(4) sister sub-NILs and the Hwaseongbyeo recurrent parent indicated that the NIL carrying an O. rufipogon chromosome segment across the entire gw8.1 target region out-yielded its sister NIL (containing Hwaseongbyeo chromosome in the RM42-RM210 interval) by 9% (P=0.029). The higher-yielding NIL produced 19.3% more grain than the Hwaseongbyeo recurrent parent (P=0.018). Analysis of a BC(3)F(4) NIL indicated that the variation for GW is associated with variation in grain shape, specifically grain length. The locus, gw8.1 is of particular interest because of its independence from undesirable height and grain quality traits. SSR markers tightly linked to the GW QTL will facilitate cloning of the gene underlying this QTL as well as marker-assisted selection for variation in GW in an applied breeding program.