Genome-wide transcriptomic and functional analyses provide new insights into the response of spring barley to drought stress.

Drought is a major abiotic stress that impairs the physiology and development of plants, ultimately leading to crop yield losses. Drought tolerance is a complex quantitative trait influenced by multiple genes and metabolic pathways. However, molecular intricacies and subsequent morphological and physiological changes in response to drought stress remain elusive. Herein, we combined morpho-physiological and comparative RNA-sequencing analyses to identify core drought-induced marker genes and regulatory networks in the barley cultivar 'Giza134'. Based on field trials, drought-induced declines occurred in crop growth rate, relative water content, leaf area duration, flag leaf area, concentration of chlorophyll (Chl) a, b and a + b, net photosynthesis, and yield components. In contrast, the Chl a/b ratio, stoma resistance, and proline concentration increased significantly. RNA-sequence analysis identified a total of 2462 differentially expressed genes (DEGs), of which 1555 were up-regulated and 907 were down-regulated in response to water-deficit stress (WD). Comparative transcriptomics analysis highlighted three unique metabolic pathways (carbohydrate metabolism, iron ion binding, and oxidoreductase activity) as containing genes differentially expressed that could mitigate water stress. Our results identified several drought-induced marker genes belonging to diverse physiochemical functions like chlorophyll concentration, photosynthesis, light harvesting, gibberellin biosynthetic, iron homeostasis as well as Cis-regulatory elements. These candidate genes can be utilized to identify gene-associated markers to develop drought-resilient barley cultivars over a short period of time. Our results provide new insights into the understanding of water stress response mechanisms in barley.

Correction to: Genetic diversity and genetic variation in morpho-physiological traits to improve heat tolerance in Spring barley.

The correct spelling of the third author's surname is Elakhdar and his current address is Agri-Bio Research Laboratory, Kyushu University, Motooka 744, Japan. The correct address for the fourth author is Agri-Bio Research Laboratory, Kyushu University, Motooka 744, Japan.

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 analysis provides insights into the genetic basis of photosynthetic responses to low-temperature stress in spring barley.

Low-temperature stress (LTS) is among the major abiotic stresses affecting the geographical distribution and productivity of the most important crops. Understanding the genetic basis of photosynthetic variation under cold stress is necessary for developing more climate-resilient barley cultivars. To that end, we investigated the ability of chlorophyll fluorescence parameters (FVFM, and FVF0) to respond to changes in the maximum quantum yield of Photosystem II photochemistry as an indicator of photosynthetic energy. A panel of 96 barley spring cultivars from different breeding zones of Canada was evaluated for chlorophyll fluorescence-related traits under cold acclimation and freeze shock stresses at different times. Genome-wide association studies (GWAS) were performed using a mixed linear model (MLM). We identified three major and putative genomic regions harboring 52 significant quantitative trait nucleotides (QTNs) on chromosomes 1H, 3H, and 6H for low-temperature tolerance. Functional annotation indicated several QTNs were either within the known or close to genes that play important roles in the photosynthetic metabolites such as abscisic acid (ABA) signaling, hydrolase activity, protein kinase, and transduction of environmental signal transduction at the posttranslational modification levels. These outcomes revealed that barley plants modified their gene expression profile in response to decreasing temperatures resulting in physiological and biochemical modifications. Cold tolerance could influence a long-term adaption of barley in many parts of the world. Since the degree and frequency of LTS vary considerably among production sites. Hence, these results could shed light on potential approaches for improving barley productivity under low-temperature stress.

Genetic diversity and population structure assessment of Western Canadian barley cooperative trials.

Studying the population structure and genetic diversity of historical datasets is a proposed use for association analysis. This is particularly important when the dataset contains traits that are time-consuming or costly to measure. A set of 96 elite barley genotypes, developed from eight breeding programs of the Western Canadian Cooperative Trials were used in the current study. Genetic diversity, allelic variation, and linkage disequilibrium (LD) were investigated using 5063 high-quality SNP markers via the Illumina 9K Barley Infinium iSelect SNP assay. The distribution of SNPs markers across the barley genome ranged from 449 markers on chromosome 1H to 1111 markers on chromosome 5H. The average polymorphism information content (PIC) per locus was 0.275 and ranged from 0.094 to 0.375. Bayesian clustering in STRUCTURE and principal coordinate analysis revealed that the populations are differentiated primarily due to the different breeding program origins and ear-row type into five subpopulations. Analysis of molecular variance based on PhiPT values suggested that high values of genetic diversity were observed within populations and accounted for 90% of the total variance. Subpopulation 5 exhibited the most diversity with the highest values of the diversity indices, which represent the breeding program gene pool of AFC, AAFRD, AU, and BARI. With increasing genetic distance, the LD values, expressed as r2, declined to below the critical r2 = 0.18 after 3.91 cM, and the same pattern was observed on each chromosome. Our results identified an important pattern of genetic diversity among the Canadian barley panel that was proposed to be representative of target breeding programs and may have important implications for association mapping in the future. This highlight, that efforts to identify novel variability underlying this diversity may present practical breeding opportunities to develop new barley genotypes.

Agrobacterium-mediated Transformation of Japonica Rice Using Mature Embryos and Regenerated Transgenic Plants.

Identification of novel genes and their functions in rice is a critical step to improve economic traits. Agrobacterium tumefaciens-mediated transformation is a proven method in many laboratories and widely adopted for genetic engineering in rice. However, the efficiency of gene transfer by Agrobacterium in rice is low, particularly among japonica and indica varieties. In this protocol, we elucidate a rapid and highly efficient protocol to transform and regenerate transgenic rice plants through important key features of Agrobacterium transformation and standard regeneration media, especially enhancing culture conditions, timing, and growth hormones. With this protocol, transformed plantlets from the embryogenetic callus of the japonica cultivar 'Taichung 65' may be obtained within 90 days. This protocol may be used with other japonica rice varieties.

Serine hydroxymethyltransferase participates in the synthesis of cysteine-rich storage proteins in rice seed.

The low level of cysteine-rich proteins (lcrp) mutation indicates a decrease in cysteine-rich (CysR) prolamines, α-globulin, and glutelin. To identify the causing factor of lcrp mutation, to elucidate its function, and to elucidate the role of CysR proteins in the formation of protein bodies (PBs), lcrp mutant was analyzed. A linkage map of the LCRP gene was constructed and genomic DNA sequencing of a predicted gene within the mapped region demonstrated that LCRP encodes a serine hydroxymethyltransferase, which participates in glycine-serine interconversion of one-carbon metabolism in the sulfur assimilation pathway. The levels of l-Ser, Gly, and Met in the sulfur assimilation pathway in the lcrp seeds increased significantly compared to that in the wildtype (WT). As the lcrp mutation influences the growth of shoot and root, the effects of the addition to the medium of amino acids and other compounds on the sulfur assimilation pathway were studied. Electron-lucent PBs surrounded by ribosome-attached membranes were observed accumulating cysteine-poor prolamines in the lcrp seeds. Additionally, glutelin-containing PBs were smaller and distorted in the lcrp seeds compared to those in the WT. These analyses of PBs in the lcrp seeds suggest that cysteine-rich proteins play an important role in the formation of PBs in rice.

QTL Mapping and Phenotypic Variation for Seedling Vigour Traits in Barley (Hordeum vulgare L.).

Seed vigour is considered a critical stage for barley production, and cultivars with early seedling vigour (ESV) facilitate rapid canopy formation. In this study, QTLs for 12 ESV-related traits were mapped using 185 RILs derived from a Xena x H94061120 evaluated across six independent environments. DArT markers were used to develop a genetic map (1075.1 cM; centimorgans) with an average adjacent-marker distance of 3.28 cM. In total, 46 significant QTLs for ESV-related traits were detected. Fourteen QTLs for biomass yield were found on all chromosomes, two of them co-localized with QTLs on 1H for grain yield. The related traits: length of the first and second leaves and dry weight of the second leaf, biomass yield and grain yield, had high heritability (>30%). Meanwhile, a significant correlation was observed between grain yield and biomass yield, which provided a clear image of these traits in the selection process. Our results demonstrate that a pleiotropic QTL related to the specific leaf area of the second leaf, biomass yield, and grain yield was linked to the DArT markers bPb-9280 and bPb-9108 on 1H, which could be used to significantly improve seed vigour by marker-assisted selection and facilitate future map-based cloning efforts.

Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm.

Prolamines are alcohol-soluble proteins classified as either cysteine-poor (CysP) or cysteine-rich (CysR) based on whether they can be alcohol-extracted without or with reducing agents, respectively. In rice esp1 mutants, various CysP prolamines exhibit both reduced and normal amounts of isoelectric focusing bands, indicating that the mutation affects only certain prolamine classes. To examine the genetic regulation of CysP prolamine synthesis and accumulation, we constructed a high-resolution genetic linkage map of ESP1. The ESP1 gene was mapped to within a 20 kb region on rice chromosome 7. Sequencing analysis of annotated genes in this region revealed a single-nucleotide polymorphism within eukaryotic peptide chain release factor (eRF1), which participates in stop-codon recognition and nascent-polypeptide release from ribosomes during translation. A subsequent complementation test revealed that ESP1 encodes eRF1. We also identified UAA as the stop codon of CysP prolamines with reduced concentration in esp1 mutants. Recognition assays and microarray analysis confirmed that ESP1/eRF1 recognizes UAA/UAG, but not UGA. Our results provide convincing evidence that ESP1/eRF1 participates in the translation termination of CysP prolamines during seed development.

Population structure and marker-trait association of salt tolerance in barley (Hordeum vulgare L.).

Association mapping is becoming an important tool for identifying alleles and loci responsible for dissecting highly complex traits in barley. This study describes the population structure and marker-trait association using general linear model (GLM) analysis on a site of 60 barley genotypes, evaluated in six salinity environments. Ninety-eight SSR and SNP alleles were employed for the construction of a framework genetic map. The genetic structure analysis of the collection turned out to consist of two major sub-populations, mainly comprising hulled and naked types. LD significantly varied among the barley chromosomes, suggesting that this factor may affect the resolution of association mapping for QTL located on different chromosomes. Numerous significant marker traits were associated in different regions of the barley genome controlling salt tolerance and related traits; among them, 46 QTLs were detected on 14 associated traits over the two years, with a major QTL controlling salt tolerance on 1H, 2H, 4H and 7H, which are important factors in improving barley's salt tolerance.