Genome-wide association study of drought tolerance in wheat (Triticum aestivum L.) identifies SNP markers and candidate genes.

Drought stress poses a severe threat to global wheat production, necessitating an in-depth exploration of the genetic basis for drought tolerance associated traits. This study employed a 90 K SNP array to conduct a genome-wide association analysis, unravelling genetic determinants of key traits related to drought tolerance in wheat, namely plant height, root length, and root and shoot dry weight. Using the mixed linear model (MLM) method on 125 wheat accessions subjected to both well-watered and drought stress treatments, we identified 53 SNPs significantly associated with stress susceptibility (SSI) and tolerance indices (STI) for the targeted traits. Notably, chromosomes 2A and 3B stood out with ten and nine associated markers, respectively. Across 17 chromosomes, 44 unique candidate genes were pinpointed, predominantly located on the distal ends of 1A, 1B, 1D, 2A, 3A, 3B, 4A, 6A, 6B, 7A, 7B, and 7D chromosomes. These genes, implicated in diverse functions related to plant growth, development, and stress responses, offer a rich resource for future investigation. A clustering pattern emerged, notably with seven genes associated with SSI for plant height and four genes linked to both STI of plant height and shoot dry weight, converging on specific regions of chromosome arms of 2AS and 3BL. Additionally, shared genes encoding polygalacturonase, auxilin-related protein 1, peptide deformylase, and receptor-like kinase underscored the interconnectedness between plant height and shoot dry weight. In conclusion, our findings provide insights into the molecular mechanisms governing wheat drought tolerance, identifying promising genomic loci for further exploration and crop improvement strategies.

Identification of KASP markers and candidate genes for drought tolerance in wheat using 90K SNP array genotyping of near-isogenic lines targeting a 4BS quantitative trait locus.

KEY MESSAGE: This study identified a novel SNP and developed a highly efficient KASP marker for drought tolerance in wheat by genotyping NILs targeting a major QTL for drought tolerance using an SNP array and validation with commercial varieties. Common wheat (Triticum aestivum L.) is an important winter crop worldwide and a typical allopolyploid with a large and complex genome. With global warming, the environmental volatility and incidence of drought in wheat-producing areas will increase. Molecular markers for drought tolerance are urgently needed to enhance drought tolerance breeding. Here, we genotyped four near-isogenic line (NIL) pairs targeting a major QTL qDSI.4B.1 on wheat chromosome arm 4BS for drought tolerance using the 90K SNP Illumina iSelect array and discovered a single nucleotide polymorphism (SNP) (Excalibur_c100336_106) with consistent genotype-phenotype associations among all four NIL pairs and their parents. Then, we converted the SNP into a Kompetitive Allele-Specific PCR (KASP) marker, with an accuracy of 100% for the four NIL pairs and their parents and as high as 81.8% for the 44 tested wheat lines with known phenotypes collected from Australia and China. Two genes near this SNP were suggested as candidate genes for drought tolerance in wheat after checking the Chinese Spring reference genome annotation version 1.1. One gene, TraesCS4B02G085300, encodes an F-box protein reportedly related to the ABA network, a main pathway for drought tolerance, and another gene, TraesCS4B02G085400, encodes a calcineurin-like metallophos-phoesterase transmembrane protein, which participates in Ca2+-dependent phosphorylation regulatory system. Based on this work and previous research on pre-harvest sprouting, we established a quick and efficient general SQV-based approach for KASP marker development, integrating genotyping by SNP arrays (S) using NILs targeting major QTL for a specific trait (Q) and validating them with commercial varieties (V). The identified SNP and developed KASP marker could be applied to marker-assisted selection in drought breeding, and further study of the candidate genes may improve our understanding of drought tolerance in wheat.

Proteomic analysis of near-isogenic lines reveals key biomarkers on wheat chromosome 4B conferring drought tolerance.

Drought is a major constraint for wheat production that is receiving increased attention due to global climate change. This study conducted isobaric tags for relative and absolute quantitation proteomic analysis on near-isogenic lines to shed light on the underlying mechanism of qDSI.4B.1 quantitative trait loci (QTL) on the short arm of chromosome 4B conferring drought tolerance in wheat. Comparing tolerant with susceptible isolines, 41 differentially expressed proteins were identified to be responsible for drought tolerance with a p-value of < 0.05 and fold change >1.3 or <0.7. These proteins were mainly enriched in hydrogen peroxide metabolic activity, reactive oxygen species metabolic activity, photosynthetic activity, intracellular protein transport, cellular macromolecule localization, and response to oxidative stress. Prediction of protein interactions and pathways analysis revealed the interaction between transcription, translation, protein export, photosynthesis, and carbohydrate metabolism as the most important pathways responsible for drought tolerance. The five proteins, including 30S ribosomal protein S15, SRP54 domain-containing protein, auxin-repressed protein, serine hydroxymethyltransferase, and an uncharacterized protein with encoding genes on 4BS, were suggested as candidate proteins responsible for drought tolerance in qDSI.4B.1 QTL. The gene coding SRP54 protein was also one of the differentially expressed genes in our previous transcriptomic study.

Genomic Regions, Molecular Markers, and Flanking Genes of Metribuzin Tolerance in Wheat (Triticum aestivum L.).

Understanding the genetics of metribuzin (a group C herbicide) tolerance in wheat is vital in developing tolerant cultivars to improve wheat productivity in dryland farming systems. This study investigated metribuzin tolerance in wheat by conducting a Genome-wide Association Studies (GWAS) with a panel of 150 wheat genotypes of diverse genetic backgrounds and genotyped them with the wheat 90 K SNP genotyping assay. The phenotyping was conducted in a temperature-controlled glasshouse at the University of Western Australia (UWA). Genotypes were sprayed with a metribuzin dose of 400 grams of active ingredient (g. a.i.) ha-1 as pre-emergent in a specialized spraying cabinet and transferred to the glasshouse where the tolerance level of the genotypes was assessed by measuring the relative reduction in chlorophyll content of the leaves. The decrease in chlorophyll content of the treated plants compared to the control was regarded as the phytotoxic effects of metribuzin. GWAS analysis following a mixed linear model revealed 19 genomic regions with significant marker-trait associations (MTAs), including ten on chromosome 6A, three on chromosome 2B, and one on chromosomes 3A, 5B, 6B 6D, 7A, and 7B, respectively. Sequences of the significant markers were blasted against the wheat genome, IWGSC RefSeq V1.0, and candidate genes having annotations related to herbicide tolerance in wheat, especially in pathways reported to be involved in metribuzin tolerance, such as cytochrome P450 pathways and ATP Binding Cassette (ABC) superfamilies, were identified in these genomic regions. These included TraesCS6A01G028800, TraesCS6A02G353700, TraesCS6A01G326200, TraesCS7A02G331000, and TraesCS2B01G465200. These genomic regions were validated on 30 top tolerant and 30 most susceptible genotypes using the five closest SSR makers to the flanked SNPs. Sufficient polymorphism was detected on two markers (wms193 and barc1036) that were found to differentiate between the susceptible and tolerant alleles and a t-test analysis of the phenotypic data shows a significant (value of p < 0.001) difference suggesting that these markers can be used for marker-assisted selection (MAS) in metribuzin studies and wheat breeding programs.

Transcriptome Analyses of Near Isogenic Lines Reveal Putative Drought Tolerance Controlling Genes in Wheat.

Drought stress, especially at the grain-filling stage, is a major constraint for wheat production. Drought tolerance is a complex trait controlled by a large array of genes and pathways. This study conducted gene expression profiling on two pairs of near-isogenic lines (NILs) for an important qDSI.4B.1 QTL conferring drought tolerance on the short arm of chromosome 4B in wheat. Analysis showed 1,614 genome-wide differentially expressed genes (DEGs) between the tolerant and susceptible isolines in both NIL pairs. Six common DEGs were found between NIL1 and NIL2 at both 7 and 14 days after stress induction, with two of them having single nucleotide polymorphism (SNP) variants. These six genes that were confirmed by quantitative real-time PCR (qRT-PCR) expression analysis are considered candidate genes for drought tolerance mediated by qDSI.4B.1 QTL with their main contributions to gene regulation, cell elongation, protein quality control, secondary metabolism, and hormone signaling. These six candidate genes and the highest number of DEGs and variants (SNPs/indels) were located between 49 and 137 Mbp of 4BS, making this interval the most probable location for the qDSI.4B.1 locus. Additionally, 765 and 84 DEGs were detected as responsive genes to drought stress in tolerant and susceptible isolines, respectively. According to gene ontology (GO), protein phosphorylation, oxidation reduction, and regulation of transcription were top biological processes involved in the drought response and tolerance. These results provide insights into stress responses regulated by the 4BS locus and have identified candidate genes and genetic markers that can be used for fine mapping of the qDSI.4B.1 locus and, ultimately, in wheat breeding programs for drought tolerance.

Root transcriptome profiling of contrasting wheat genotypes provides an insight to their adaptive strategies to water deficit.

Water deficit limits plant growth and productivity in wheat. The effect of water deficit varies considerably in the contrasting genotypes. This study attempted comparative transcriptome profiling of the tolerant (Abura) and susceptible (AUS12671) genotypes under PEG-simulated water stress via genome-wide RNA-seq technology to understand the dynamics of tolerance mechanism. Morphological and physiological analyses indicated that the tolerant genotype Abura had a higher root growth and net photosynthesis, which accounted for its higher root biomass than AUS12671 under stress. Transcriptomic analysis revealed a total of 924 differentially expressed genes (DEGs) that were unique in the contrasting genotypes under stress across time points. The susceptible genotype AUS12671 had slightly more abundant DEGs (505) than the tolerant genotype Abura (419). Gene ontology enrichment and pathway analyses of these DEGs suggested that the two genotypes differed significantly in terms of adaptive mechanism. Predominant upregulation of genes involved in various metabolic pathways was the key adaptive feature of the susceptive genotype AUS12671 indicating its energy-consuming approach in adaptation to water deficit. In contrast, downregulation the expression of genes of key pathways, such as global and overview maps, carbohydrate metabolism, and genetic information processing was the main strategy for the tolerant genotype Abura. Besides, significantly higher number of genes encoding transcription factors (TF) families like MYB and NAC, which were reported to be associated with stress defense, were differentially expressed in the tolerant genotype Abura. Gene encoding transcription factors TIFY were only differentially expressed between stressed and non-stressed conditions in the sensitive genotype. The identified DEGs and the suggested differential adaptive strategies of the contrasting genotypes provided an insight for improving water deficit tolerance in wheat.

Phenotypic and genotypic characterization of near-isogenic lines targeting a major 4BL QTL responsible for pre-harvest sprouting in wheat.

BACKGROUND: Resistance to pre-harvest sprouting (PHS) is one of the major objectives in wheat breeding programs. However, the complex quantitative nature of this trait presents challenges when breeding for PHS resistance. Characterization of PHS using near-isogenic lines (NILs) targeting major quantitative trait locus/loci (QTL/QTLs) can be an effective strategy for the identification of responsible genes and underlying mechanisms. RESULTS: In this study, multiple pairs of NILs were developed and confirmed for a major QTL located on the 4BL chromosome arm that contributes to PHS resistance in wheat, using a combined heterogeneous inbred family method and a fast generation cycling system. Phenotypic characterization of these confirmed NILs revealed significant differences in PHS resistance between the isolines, where the presence of the resistant allele increased the resistance to sprouting on spikes by 54.0-81.9% (average 70.8%) and reduced seed germination by 59.4-70.5% (average 66.2%). The 90 K SNP genotyping assay on the confirmed NIL pairs identified eight SNPs on 4BL, associated with five candidate genes; two of the candidate genes were related to seed dormancy. Agronomic traits in the NIL pairs were investigated; both plant height and grain number per spike were positively correlated with PHS susceptibility. CONCLUSIONS: This study confirmed multiple pairs of NILs and identified SNPs between PHS isolines, which are valuable resources for further fine-mapping of this locus to clone the major genes for PHS resistance and investigate the possible functional regulation of these genes on important agronomic traits, such as plant height and grain number per spike.

Multiple Near-Isogenic Lines Targeting a QTL Hotspot of Drought Tolerance Showed Contrasting Performance Under Post-anthesis Water Stress.

The complex quantitative nature of drought-related traits is a major constraint to breed tolerant wheat varieties. Pairs of near-isogenic lines (NILs) with a common genetic background but differing in a particular locus could turn quantitative traits into a Mendelian factor facilitating our understanding of genotype and phenotype interactions. In this study, we report our fast track development and evaluation of NILs from C306 × Dharwar Dry targeting a wheat 4BS QTL hotspot in C306, which confers drought tolerance following the heterogeneous inbreed family (HIF) analysis coupled with immature embryo culture-based fast generation technique. Molecular marker screening and phenotyping for grain yield and related traits under post-anthesis water stress (WS) confirmed four isoline pairs, viz., qDSI.4B.1-2, qDSI.4B.1-3, qDSI.4B.1-6, and qDSI.4B.1-8. There were significant contrasts of responses between the NILs with C306 QTL (+NILs) and the NILs without C306 QTL (-NILs). Among the four confirmed NIL pairs, mean grain yield per plant of the +NILs and -NILs showed significant differences ranging from 9.61 to 10.81 and 6.30 to 7.56 g, respectively, under WS condition, whereas a similar grain yield was recorded between the +NILs and -NILs under well-watered condition. Isolines of +NIL and -NIL pairs showed similar chlorophyll content (SPAD), assimilation rate (A), and transpiration rate (Tr) at the beginning of the stress. However, the +NILs showed significantly higher SPAD (12%), A (66%), stomatal conductance (75%), and Tr (97%) than the -NILs at the seventh day of stress. Quantitative RT-PCR analysis targeting the MYB transcription factor gene Triticum aestivum MYB 82 (TaMYB82), within this genomic region which was retrieved from the wheat reference genome TGACv1, also revealed differential expression in +NILs and -NILs under stress. These results confirmed that the NILs can be invaluable resources for fine mapping of this QTL, and also for cloning and functional characterization of the gene(s) responsible for drought tolerance in wheat.