High-LD SNP markers exhibiting pleiotropic effects on salt tolerance at germination and seedlings stages in spring wheat.

KEY MESSAGE: Salt tolerance at germination and seedling growth stages was investigated. GWAS revealed nine genomic regions with pleiotropic effects on salt tolerance. Salt tolerant genotypes were identified for future breeding program. With 20% of the irrigated land worldwide affected by it, salinity is a serious threat to plant development and crop production. While wheat is the most stable food source worldwide, it has been classified as moderately tolerant to salinity. In several crop plants; such as barley, maize and rice, it has been shown that salinity tolerance at seed germination and seedling establishment is under polygenic control. As yield was the ultimate goal of breeders and geneticists, less attention has been paid to understanding the genetic architecture of salt tolerance at early stages. Thus, the genetic control of salt tolerance at these stages is poorly understood relative to the late stages. In the current study, 176 genotypes of spring wheat were tested for salinity tolerance at seed germination and seedling establishment. Genome-Wide Association Study (GWAS) has been used to identify the genomic regions/genes conferring salt tolerance at seed germination and seedling establishment. Salinity stress negatively impacted all germination and seedling development parameters. A set of 137 SNPs showed significant association with the traits of interest. Across the whole genome, 33 regions showed high linkage disequilibrium (LD). These high LD regions harbored 15 SNPs with pleiotropic effect (i.e. SNPs that control more than one trait). Nine genes belonging to different functional groups were found to be associated with the pleiotropic SNPs. Noteworthy, chromosome 2B harbored the gene TraesCS2B02G135900 that acts as a potassium transporter. Remarkably, one SNP marker, reported in an early study, associated with salt tolerance was validated in this study. Our findings represent potential targets of genetic manipulation to understand and improve salinity tolerance in wheat.

Marker-trait association for grain weight of spring barley in well-watered and drought environments.

Climate change will increase the effect of drought stress which is one of major constrains for barley production and productivity in Egypt. Identification and development new cultivars having a high drought tolerance combined with a high yield are urgently needed. In this study, a set of 60 highly homozygous and diverse barley genotypes was evaluated in well-watered (N) and dry (D) environments for two successive seasons. Five yield traits were scored; plant height, spike length, days to flowering, grain yield per spike (GYPS), and thousand kernel weight (TKW). High genetic variation was found among genotypes in all studied traits under N and D. High heritability for all traits was observed in both seasons. The drought susceptibility index (DSI) for GYPS and TKW was estimated to determine the tolerant and susceptible genotypes in both seasons. As a result, four spring barley genotypes were considered drought tolerant for TKW and GYPS in both seasons. A set of ten single sequence repeats primers, developed from wheat genome, were tested in the 60 genotypes. All SSR primers had a high polymorphism among the genotypes producing 82 marker alleles. Single marker analysis was performed for DSI, TKW, and GYPS in both seasons. Twenty QTLs were found to be associated with low DSI and high GYPS and TKW in N and D. The marker alleles associated with the 20 QTL were screened in the four tolerant genotypes. PNBYT15 included only one marker allele associated with one QTL, while, SCYT-28 included six marker alleles controlling nine QTL. The high genetic variation and heritability for the studied traits indicated that these traits could be used for selection for high yielding and drought tolerance. The four drought tolerant genotypes can be used for a further breeding program to improve drought tolerance in barley.

Genome-wide association mapping for root traits associated with frost tolerance in faba beans using KASP-SNP markers.

Frost is an abiotic stress factor that threatens plant development and crop productivity not only in cold regions but also in temperate zones. Roots play an important role in plant growth during frost stress. Therefore, variation in root characteristics could be studied to improve frost tolerance in winter faba bean. The present study aimed to identify the genomic regions that control frost tolerance in a winter faba bean population by focusing on root-related traits. A set of 185 genotypes were tested for frost tolerance under artificial frost growth conditions at -16°C, -18°C, and -19°C in a growth chamber. Frost stress reduced the root-related parameters in all genotypes, with a wide variation among genotypes. A genome-wide association study identified nine novel single-nucleotide polymorphisms that are associated with the root-related traits. The most frost-tolerant genotypes were identified; two genotypes, S_028 and S_220, exhibited remarkable performance under frost stress. Moreover, they harbored all four of the alleles favorable for frost tolerance. Remarkably, two markers showed genetic pleiotropic effects with positive allele effects on root fresh matter and root dry matter. Thus, both genotypes can be implemented in a breeding program to provide the alleles for healthier roots under frost conditions to develop more frost-tolerant varieties, and the two markers can be used to screen large collections to select for frost tolerance. These results may provide novel insights for improving frost tolerance in faba beans and in other legume crops.

Association mapping and candidate genes for physiological non-destructive traits: Chlorophyll content, canopy temperature, and specific leaf area under normal and saline conditions in wheat.

Wheat plants experience substantial physiological adaptation when exposed to salt stress. Identifying such physiological mechanisms and their genetic control is especially important to improve its salt tolerance. In this study, leaf chlorophyll content (CC), leaf canopy temperature (CT), and specific leaf area (SLA) were scored in a set of 153 (103 having the best genotypic data were used for GWAS analysis) highly diverse wheat genotypes under control and salt stress. On average, CC and SLA decreased under salt stress, while the CT average was higher under salt stress compared to the control. CT was negatively and significantly correlated with CC under both conditions, while no correlation was found between SLA and CC and CT together. High genetic variation and broad-sense-heritability estimates were found among genotypes for all traits. The genome wide association study revealed important QTLs for CC under both conditions (10) and SLA under salt stress (four). These QTLs were located on chromosomes 1B, 2B, 2D, 3A, 3B, 5A, 5B, and 7B. All QTLs detected in this study had major effects with R2 extending from 20.20% to 30.90%. The analysis of gene annotation revealed three important candidate genes (TraesCS5A02G355900, TraesCS1B02G479100, and TraesCS2D02G509500). These genes are found to be involved in the response to salt stress in wheat with high expression levels under salt stress compared to control based on mining in data bases.

Genetic factors controlling nTiO2 nanoparticles stress tolerance in barley (Hordeum vulgare) during seed germination and seedling development.

Titanium dioxide nanoparticle (nTiO2) is one of the most produced nanoparticles worldwide. Its mechanism on crop development and performance is unclear as it is hard to predict their toxicity or benefit. Therefore, understanding the genetics of crop development under nTiO2 is a prerequisite for their applications in agriculture and crop improvement. Here, we aimed to examine the influnce of 300ppm nTiO2 on seed germination, seedling morphology, root-related traits in 121 worldwide spring barley (Hordeum vulgare L.) accessions. Results show that nTiO2 significantley affected all traits scored in this study. Response to nTiO2 treatment, clear wide natural variation among accesions was detected. Remarkably, 10 genotypes showed increased root length under nTiO2 at the seedling stage indicating that nTiO2 enhanced the root elongation. Genome-wide association scan (GWAS) was applied using 9K single nucleotide polymorphism (SNPs) in a mixed-linear model that revealed 86 significant marker-trait associations with all traits scored in this study. Many significant SNPs were physically located near candidate genes, of which 191 genes were detected within the linkage disequilibrium and distributed over all barley chromosomes. Mostly, the genes harboured by chromosome 2H, specially calcium-binding genes family, regulate the variation of seedling length-related traits. Candidate genes on 7H encode zinc finger protein that controls the rate of germination. Therefore, these genomic regions at 2H and 7H can be targeted to select for improved seedling development and seed germination under nTiO2 stress in soils. These results improve understanding the genetic control of seed germination and seedling development under high levels of nTiO2 that can support plant breeding and crop improvement programmes.

Natural Variation Uncovers Candidate Genes for Barley Spikelet Number and Grain Yield under Drought Stress.

Drought stress can occur at any growth stage and can affect crop productivity, which can result in large yield losses all over the world. In this respect, understanding the genetic architecture of agronomic traits under drought stress is essential for increasing crop yield potential and harvest. Barley is considered the most abiotic stress-tolerant cereal, particularly with respect to drought. In the present study, worldwide spring barley accessions were exposed to drought stress beginning from the early reproductive stage with 35% field capacity under field conditions. Drought stress had significantly reduced the agronomic and yield-related traits such as spike length, awn length, spikelet per spike, grains per spike and thousand kernel weight. To unravel the genetic factors underlying drought tolerance at the early reproductive stage, genome-wide association scan (GWAS) was performed using 121 spring barley accessions and a 9K single nucleotide polymorphisms (SNPs) chip. A total number of 101 significant SNPs, distributed over all seven barley chromosomes, were found to be highly associated with the studied traits, of which five genomic regions were associated with candidate genes at chromosomes 2 and 3. On chromosome 2H, the region between 6469300693-647258342 bp includes two candidate drought-specific genes (HORVU2Hr1G091030 and HORVU2Hr1G091170), which are highly associated with spikelet and final grain number per spike under drought stress conditions. Interestingly, the gene expression profile shows that the candidate genes were highly expressed in spikelet, grain, spike and leaf organs, demonstrating their pivotal role in drought tolerance. To the best of our knowledge, we reported the first detailed study that used GWAS with bioinformatic analyses to define the causative alleles and putative candidate genes underlying grain yield-related traits under field drought conditions in diverse barley germplasm. The identified alleles and candidate genes represent valuable resources for future functional characterization towards the enhancement of barley cultivars for drought tolerance.

Detailed Genetic Analysis for Identifying QTLs Associated with Drought Tolerance at Seed Germination and Seedling Stages in Barley.

Drought induces several challenges for plant development, growth, and production. These challenges become more severe, in particular, in arid and semiarid countries like Egypt. In terms of production, barley ranks fourth after wheat, maize, and rice. Seed germination and seedling stages are critical stages for plant establishment and growth. In the current study, 60 diverse barley genotypes were tested for drought tolerance using two different treatments: control (0-PEG) and drought (20%-PEG). Twenty-two traits were estimated for seed germination and seedling parameters. All traits were reduced under drought stress, and a significant variation was found among genotypes under control and stress conditions. The broad-sense heritability estimates were very high under both control and drought for all traits. It ranged from 0.63 to 0.97 under the control condition and from 0.89 to 0.97 under drought, respectively. These high heritabilities suggested that genetic improvement of drought tolerance in barley at both stages is feasible. The principal component analysis revealed that root-related parameters account for the largest portion of phenotypic variation in this collection. The single-marker analysis (SMA) resulted in 71 quantitative trait loci (QTLs) distributed across the seven chromosomes of barley. Thirty-three QTLs were detected for root-length-related traits. Many hotspots of QTLs were detected for various traits. Interestingly, some markers controlled many traits in a pleiotropic manner; thus, they can be used to control multiple traits at a time. Some QTLs were constitutive, i.e., they are mapped under control and drought, and targeting these QTLs makes the selection for drought tolerance a single-step process. The results of gene annotation analysis revealed very potential candidate genes that can be targeted to select for drought tolerance.

Genetic basis of drought tolerance during seed germination in barley.

Drought is one of the harshest abiotic stresses hindering seed germination, plant growth, and crop productivity. A high rate and uniformity of germination under stressful conditions are vital for crop establishment and growth; thus, for productivity. A better understanding of the genetic architecture of seed germination under drought stress is a prerequisite for further increasing yield potential. Barley is considered one of the most abiotic stresses-tolerant cereals. Elucidating the drought tolerance of barley during seed germination would indeed pave the way towards improving the performance of all cereals. However, we still know relatively little about the genetic control of drought tolerance during the seed germination phase. In our study, 218 worldwide spring barley accessions were subjected to PEG-induced drought during seed germination. Induced drought stress "20% PEG" significantly reduced the seed germination parameters and seedling related traits. A genome-wide association scan (GWAS) was used to identify genomic regions associated with our trait of interest. In total, 338 single nucleotide polymorphisms (SNPs) were found to be associated with several traits distributed across seven barley chromosomes, of which 26 genomic regions were associated with candidate genes. The current study found some of the quantitative trait loci (QTL) that have previously been reported to be linked to seed germination-related traits under drought conditions, as well as some new associations. Noteworthy, the identified QTL colocalized with a number of genes (within interval ±0.5 Mbp) that are exclusively distributed on chromosomes 1H, 2H, and 5H. The annotation of these genes in barley shows their roles in drought tolerance through encoding different transcription factors. The function of the identified genes during seed germination was also confirmed by the annotation of their counterparts in Arabidopsis. The current analyses show the power of the GWAS both for identifying putative candidate genes and for improving plant adaptive traits in barley.