Uncovering the transcriptional responses of tobacco (Nicotiana tabacum L.) roots to Ralstonia solanacearum infection: a comparative study of resistant and susceptible cultivars.

BACKGROUND: Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious soil-borne disease of tobacco that significantly reduces crop yield. However, the limited availability of resistance in tobacco hinders breeding efforts for this disease. RESULTS: In this study, we conducted hydroponic experiments for the root expression profiles of D101 (resistant) and Honghuadajinyuan (susceptible) cultivars in response to BW infection at 0 h, 6 h, 1 d, 3 d, and 7d to explore the defense mechanisms of BW resistance in tobacco. As a result, 20,711 and 16,663 (total: 23,568) differentially expressed genes (DEGs) were identified in the resistant and susceptible cultivars, respectively. In brief, at 6 h, 1 d, 3 d, and 7 d, the resistant cultivar showed upregulation of 1553, 1124, 2583, and 7512 genes, while the susceptible cultivar showed downregulation of 1213, 1295, 813, and 7735 genes. Similarly, across these time points, the resistant cultivar had downregulation of 1034, 749, 1686, and 11,086 genes, whereas the susceptible cultivar had upregulation of 1953, 1790, 2334, and 6380 genes. The resistant cultivar had more up-regulated genes at 3 d and 7 d than the susceptible cultivar, indicating that the resistant cultivar has a more robust defense response against the pathogen. The GO and KEGG enrichment analysis showed that these genes are involved in responses to oxidative stress, plant-pathogen interactions, cell walls, glutathione and phenylalanine metabolism, and plant hormone signal transduction. Among the DEGs, 239 potential candidate genes were detected, including 49 phenylpropane/flavonoids pathway-associated, 45 glutathione metabolic pathway-associated, 47 WRKY, 48 ERFs, eight ARFs, 26 pathogenesis-related genes (PRs), and 14 short-chain dehydrogenase/reductase genes. In addition, two highly expressed novel genes (MSTRG.61386-R1B-17 and MSTRG.61568) encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins were identified in both cultivars at 7 d. CONCLUSIONS: This study revealed significant enrichment of DEGs in GO and KEGG terms linked to glutathione, flavonoids, and phenylpropane pathways, indicating the potential role of glutathione and flavonoids in early BW resistance in tobacco roots. These findings offer fundamental insight for further exploration of the genetic architecture and molecular mechanisms of BW resistance in tobacco and solanaceous plants at the molecular level.

Ferroptosis of Endothelial Cells Triggered by Erythrophagocytosis Contributes to Thrombogenesis in Uremia.

BACKGROUND: Although thrombosis events are the leading complication of uremia, their mechanism is largely unknown. The interaction between endothelial cells (ECs) and red blood cells (RBCs) in uremic solutes and its prothrombotic role need to be investigated. METHODS AND RESULTS: Here, we established an in vitro co-incubation model of uremic RBC and EC as well as a uremic rat model induced by adenine. Using flow cytometry, confocal microscopy, and electron microscopy, we found increased erythrophagocytosis by EC accompanied by increased reactive oxygen species, lipid peroxidation, and impairment of mitochondria, indicating that ECs undergo ferroptosis. Further investigations showed increased proteins' expression of heme oxygenase-1 and ferritin and labile iron pool accumulation in EC, which could be suppressed by deferoxamine (DFO). The ferroptosis-negative regulators glutathione peroxidase 4 and SLC7A11 were decreased in our erythrophagocytosis model and could be enhanced by ferrostatin-1 or DFO. In vivo, we observed that vascular EC phagocytosed RBC and underwent ferroptosis in the kidney of the uremic rat, which could be inhibited by blocking the phagocytic pathway or inhibiting ferroptosis. Next, we found that the high tendency of thrombus formation was accompanied by erythrophagocytosis-induced ferroptosis in vitro and in vivo. Importantly, we further revealed that upregulated TMEM16F expression mediated phosphatidylserine externalization on ferroptotic EC, which contributed to a uremia-associated hypercoagulable state. CONCLUSION: Our results indicate that erythrophagocytosis-triggered ferroptosis followed by phosphatidylserine exposure of EC may play a key role in uremic thrombotic complications, which may be a promising target to prevent thrombogenesis of uremia.

Comprehensive transcriptome analysis reveals heat-responsive genes in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis) using RNA sequencing.

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee, 2n=20, AA) is a vegetable species in southern parts of China that faces high temperatures in the summer and winter seasons. While heat stress adversely impacts plant productivity and survival, the underlying molecular and biochemical causes are poorly understood. This study investigated the gene expression profiles of heat-sensitive (HS) '3T-6' and heat-tolerant (HT) 'Youlu-501' varieties of flowering Chinese cabbage in response to heat stress using RNA sequencing. Among the 37,958 genes expressed in leaves, 20,680 were differentially expressed genes (DEGs) at 1, 6, and 12 h, with 1,078 simultaneously expressed at all time points in both varieties. Hierarchical clustering analysis identified three clusters comprising 1,958, 556, and 591 down-regulated, up-regulated, and up- and/or down-regulated DEGs (3205 DEGs; 8.44%), which were significantly enriched in MAPK signaling, plant-pathogen interactions, plant hormone signal transduction, and brassinosteroid biosynthesis pathways and involved in stimulus, stress, growth, reproductive, and defense responses. Transcription factors, including MYB (12), NAC (13), WRKY (11), ERF (31), HSF (17), bHLH (16), and regulatory proteins such as PAL, CYP450, and photosystem II, played an essential role as effectors of homeostasis, kinases/phosphatases, and photosynthesis. Among 3205 DEGs, many previously reported genes underlying heat stress were also identified, e.g., BraWRKY25, BraHSP70, BraHSPB27, BraCYP71A23, BraPYL9, and BraA05g032350.3C. The genome-wide comparison of HS and HT provides a solid foundation for understanding the molecular mechanisms of heat tolerance in flowering Chinese cabbage.

Genome-wide identification and development of InDel markers in tobacco (Nicotiana tabacum L.) using RAD-seq.

Insertions and deletions (InDels) can be used as molecular markers in genetic studies and marker-assisted selection breeding. However, genetic improvement in tobacco has been hindered by limited genetic diversity information and relatedness within available germplasm. A Chinese tobacco variety, Yueyan-98, was resequenced using restriction-site associated DNA (RAD-seq) approach to develop InDel markers. In total, 32,884 InDel loci were detected between Yueyan-98 and the K326 reference sequence [18,598 (56.55%) deletions and 14,288 (43.45%) insertions], ranging from 1 to 62 bp in length. Of the 6,733 InDels (> 4 bp) that were suitable for polyacrylamide gel electrophoresis, 150 were randomly selected. These 150 InDels were unevenly distributed on 23 chromosomes, and the highest numbers of InDels were observed on chromosomes Nt05, Nt13, and Nt23. The average density of adjacent InDels was 19.36 Mb. Thirty-seven InDels were located in genic regions. Polymerase chain reaction (PCR)-based markers were developed to validate polymorphism; 113 (79.80%) of the 150 InDel markers showed polymorphism and were further used for genetic diversity analysis of 50 tobacco accessions (13 from China, 1 from Mexico, and 36 from the USA). The average expected heterozygosity (He) and polymorphism information content (PIC) values were 0.28 ± 0.16 and 0.38 ± 0.10, respectively. The average Shannon diversity index (I) was 0.34 ± 0.18, with genetic diversity ranging from 0.13-0.57. The 50 accessions were classified into two groups with a genetic similarity coefficient of 0.68. Principal coordinate analysis (PCoA) and population structure analysis showed similar results and divided the population into two groups unrelated to their geographical origins. AMOVA showed 4% variance among the population and the remaining 96% within the population, suggesting low genetic differentiation between two subpopulations. Furthermore, 10 InDels (19 alleles) were significantly identified for tobacco plant height using GLM+Q model at P < 0.005. Among these, three markers (Nt-I-26, Nt-I-41, and Nt-I-44) were detected in at least two environments, with phenotypic variance explained (PVE) ranging from 14.03 to 32.68%. The polymorphic InDel markers developed can be used for hybrid identification, genetic diversity, genetic linkage map construction, gene mapping, and MAS breeding programs of tobacco. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01187-3.

TMEM16F mediated phosphatidylserine exposure and microparticle release on erythrocyte contribute to hypercoagulable state in hyperuricemia.

The link between hyperuricemia (HUA) and the risk of venous thromboembolism (VTE) has been well established. However, the mechanisms of thrombus generation and the effect of HUA on procoagulant activity (PCA) of erythrocytes remain unclear no matter in uremia or hyperuricemia. Here, phosphatidylserine (PS) exposure, microparticles (MPs) release, cytosolic Ca2+, TMEM16F expression, reactive oxygen species (ROS) and lipid peroxidation of erythrocyte were detected by flow cytometer. PCA was assessed by coagulation time, purified coagulation complex and fibrin production assays. The fibrin formation was observed by scanning electron microscopy (SEM). We found that PS exposure, MPs generation, TMEM16F expression and consequent PCA of erythrocyte in HUA patients significantly increased compared to those in healthy volunteers. Furthermore, high UA induced PS exposure, and MPs release of erythrocyte in concentration and time-dependent manners in vitro, which enhanced the PCA of erythrocyte and was inhibited by lactadherin, a PS inhibitor. Additionally, using SEM, we also observed compact fibrin clots with highly-branched networks and thin fibers supported by red blood cells (RBCs) and RBC-derived MPs (RMPs). Importantly, we demonstrated UA enhanced the production of ROS and lipid peroxidation and reduced the generation of glutathione (GSH) of erythrocyte, which enhanced TMEM16F activity and followed PS externalization and RMPs formation. Collectively, these results suggest that Ca2+-dependent TMEM16F activation may be responsible for UA-induced PS exposure and MPs release of RBC, which thereby contribute to the prothrombotic risk in HUA.

[Effects of exhaustive exercise on coagulation state in rats].

Objective: To investigate the effects of one-time exhaustive exercise on coagulation state in rats and its mechanism. Methods: Forty-eight SD rats were randomly divided into control group and exhaustive exercise group, 24 rats in each group. Rats in exhaustive exercise group were trained with treadmill training for 25～50 min at a time on non-slope treadmill and the initial speed of 5 m/min was uniformly accelerated to 25 m/min until the rats exhausted. Thrombelastography (TEG) was used to monitor the coagulation function of rats after training. The ligation model of inferior vena cava (IVC) was established to evaluate thrombosis. The phosphatidylserine (PS) exposure and Ca2+ concentration were detected by flow cytometry. The production of FXa and thrombin was detected by microplate reader. The clotting time was measured by using coagulometer. Results: Compared with the control group, the blood of rats in the exhaustive exercise group exhibited hypercoagulable state. The probability of thrombus formation, weight, length and ratio in the exhaustive exercise group were significantly higher than those in the control group (P＜0.01). The levels of PS exposure and intracellular Ca2+ concentration of red blood cells (RBCs) and platelets in the exhaustive exercise group were increased significantly (P＜0.01). The blood clotting time of RBCs and platelets was shortened (P＜0.01), and the production of FXa and thrombin was increased significantly (P＜0.01) in the exhausted exercise group, and both were inhibited by lactadherin (Lact, P＜0.01). Conclusion: The blood of exhaustive exercise rats is in a hypercoagulable state and the risk of thrombosis is increased. The increased PS exposure of RBCs and platelets caused by exhaustive exercise may be an important mechanism of thrombosis.

Identification of Novel Quantitative Trait Nucleotides and Candidate Genes for Bacterial Wilt Resistance in Tobacco (Nicotiana tabacum L.) Using Genotyping-by-Sequencing and Multi-Locus Genome-Wide Association Studies.

Tobacco bacterial wilt (TBW) is a devastating soil-borne disease threatening the yield and quality of tobacco. However, its genetic foundations are not fully understood. In this study, we identified 126,602 high-quality single-nucleotide polymorphisms (SNPs) in 94 tobacco accessions using genotyping-by-sequencing (GBS) and a 94.56 KB linkage disequilibrium (LD) decay rate for candidate gene selection. The population structure analysis revealed two subpopulations with 37 and 57 tobacco accessions. Four multi-locus genome-wide association study (ML-GWAS) approaches identified 142 quantitative trait nucleotides (QTNs) in E1-E4 and the best linear unbiased prediction (BLUP), explaining 0.49-22.52% phenotypic variance. Of these, 38 novel stable QTNs were identified across at least two environments/methods, and their alleles showed significant TBW-DI differences. The number of superior alleles associated with TBW resistance for each accession ranged from 4 to 24; eight accessions had more than 18 superior alleles. Based on TBW-resistant alleles, the five best cross combinations were predicted, including MC133 × Ruyuan No. 1 and CO258 × ROX28. We identified 52 candidate genes around 38 QTNs related to TBW resistance based on homologous functional annotation and KEGG enrichment analysis, e.g., CYCD3;2, BSK1, Nitab4.5_0000641g0050, Nitab4.5_0000929g0030. To the best of our knowledge, this is the first comprehensive study to identify QTNs, superior alleles, and their candidate genes for breeding TBW-resistant tobacco varieties. The results provide further insight into the genetic architecture, marker-assisted selection, and functional genomics of TBW resistance, improving future breeding efforts to increase crop productivity.

Identification and Analysis of Small Interfering RNAs Associated With Heat Stress in Flowering Chinese Cabbage Using High-Throughput Sequencing.

Endogenous small interfering RNAs (siRNAs) are substantial gene regulators in eukaryotes and play key functions in plant development and stress tolerance. Among environmental factors, heat is serious abiotic stress that severely influences the productivity and quality of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). However, how siRNAs are involved in regulating gene expression during heat stress is not fully understood in flowering Chinese cabbage. Combining bioinformatical and next-generation sequencing approaches, we identified heat-responsive siRNAs in four small RNA libraries of flowering Chinese cabbage using leaves collected at 0, 1, 6, and 12 h after a 38°C heat-stress treatment; 536, 816, and 829 siRNAs exhibited substantial differential expression at 1, 6, and 12 h, respectively. Seventy-five upregulated and 69 downregulated differentially expressed siRNAs (DE-siRNAs) were common for the three time points of heat stress. We identified 795 target genes of DE-siRNAs, including serine/threonine-protein kinase SRK2I, CTR1-like, disease resistance protein RML1A-like, and RPP1, which may play a role in regulating heat tolerance. Gene ontology showed that predictive targets of DE-siRNAs may have key roles in the positive regulation of biological processes, organismal processes, responses to temperature stimulus, signaling, and growth and development. These novel results contribute to further understanding how siRNAs modulate the expression of their target genes to control heat tolerance in flowering Chinese cabbage.

Comparative Analysis of miRNA Expression Profiles Between Heat-Tolerant and Heat-Sensitive Genotypes of Flowering Chinese Cabbage Under Heat Stress Using High-Throughput Sequencing.

Heat stress disturbs cellular homeostasis, thus usually impairs yield of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). MicroRNAs (miRNAs) play a significant role in plant responses to different stresses by modulating gene expression at the post-transcriptional level. However, the roles that miRNAs and their target genes may play in heat tolerance of flowering Chinese cabbage remain poorly characterized. The current study sequenced six small RNA libraries generated from leaf tissues of flowering Chinese cabbage collected at 0, 6, and 12 h after 38 °C heat treatment, and identified 49 putative novel miRNAs and 43 known miRNAs that differentially expressed between heat-tolerant and heat-sensitive flowering Chinese cabbage. Among them, 14 novel and nine known miRNAs differentially expressed only in the heat-tolerant genotype under heat-stress, therefore, their target genes including disease resistance protein TAO1-like, RPS6, reticuline oxidase-like protein, etc. might play important roles in enhancing heat-tolerance. Gene Ontology (GO) analysis revealed that targets of these differentially expressed miRNAs may play key roles in responses to temperature stimulus, cell part, cellular process, cell, membrane, biological regulation, binding, and catalytic activities. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified their important functions in signal transduction, environmental adaptation, global and overview maps, as well as in stress adaptation and in MAPK signaling pathways such as cell death. These findings provide insight into the functions of the miRNAs in heat stress tolerance of flowering Chinese cabbage.

Non-coding RNAs: Functional roles in the regulation of stress response in Brassica crops.

Brassica crops face a combination of different abiotic and biotic stresses in the field that can reduce plant growth and development by affecting biochemical and morpho-physiological processes. Emerging evidence suggests that non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long ncRNAs (lncRNAs), play a significant role in the modulation of gene expression in response to plant stresses. Recent advances in computational and experimental approaches are of great interest for identifying and functionally characterizing ncRNAs. While progress in this field is limited, numerous ncRNAs involved in the regulation of gene expression in response to stress have been reported in Brassica. In this review, we summarize the modes of action and functions of stress-related miRNAs and lncRNAs in Brassica as well as the approaches used to identify ncRNAs.

Identification of conserved and novel miRNAs responsive to heat stress in flowering Chinese cabbage using high-throughput sequencing.

Plant microRNAs (miRNAs) are noncoding and endogenous key regulators that play significant functions in regulating plant responses to stress, and plant growth and development. Heat stress is a critical abiotic stress that reduces the yield and quality of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). However, limited information is available on whether miRNAs are involved in the regulation of heat stress in B. campestris. A high-throughput sequencing approach was used to identify novel and conserved heat-responsive miRNAs in four small RNA libraries of flowering Chinese cabbage using leaves collected at 0 h, 1 h, 6 h and 12 h after a 38 °C heat-stress treatment. The analysis identified 41 conserved miRNAs (belonging to 19 MIR families), of which MIR156, MIR159, MIR168, MIR171 and MIR1885 had the most abundant molecules. Prediction and evaluation of novel miRNAs using the unannotated reads resulted in 18 candidate miRNAs. Differential expression analysis showed that most of the identified miRNAs were downregulated in heat-treated groups. To better understand functional importance, bioinformatic analysis predicted 432 unique putative target miRNAs involved in cells, cell parts, catalytic activity, cellular processes and abiotic stress responses. Furthermore, the Kyoto Encyclopedia of Genes and Genomes maps of flowering Chinese cabbage identified the significant role of miRNAs in stress adaptation and stress tolerance, and in several mitogen-activated protein kinases signaling pathways including cell death. This work presents a comprehensive study of the miRNAs for understanding the regulatory mechanisms and their participation in the heat stress of flowering Chinese cabbage.

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Development of EST-SSR markers in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) based on de novo transcriptomic assemblies.

Flowering Chinese cabbage is one of the most important vegetable crops in southern China. Genetic improvement of various agronomic traits in this crop is underway to meet high market demand in the region, but the progress is hampered by limited number of molecular markers available in this crop. This study aimed to develop EST-SSR markers from transcriptome sequences generated by next-generation sequencing. RNA-seq of eight cabbage samples identified 48,975 unigenes. Of these unigenes, 23,267 were annotated in 56 gene ontology (GO) categories, 6,033 were mapped to 131 KEGG pathways, and 7,825 were assigned to clusters of orthologous groups (COGs). From the unigenes, 8,165 EST-SSR loci were identified and 98.57% of them were 1-3 nucleotide repeats with 14.32%, 41.08% and 43.17% of mono-, di- and tri-nucleotide repeats, respectively. Fifty-eight types of motifs were identified with A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT the most abundant. The lengths of repeated nucleotide sequences in all SSR loci ranged from 12 to 60 bp, with most (88.51%) under 20 bp. Among 170 primer pairs were randomly selected from a total of 4,912 SSR primers we designed, 48 yielded unambiguously polymorphic bands with high reproducibility. Cluster analysis using 48 SSRs classified 34 flowering Chinese cabbage cultivars into three groups. A large number of EST-SSR markers identified in this study will facilitate marker-assisted selection in the breeding programs of flowering Chinese cabbage.

Genetic variations of HvP5CS1 and their association with drought tolerance related traits in barley (Hordeum vulgare L.).

Delta-1-pyrroline-5-carboxylate synthase gene1 (P5CS1) is the key gene involved in the biosynthesis of proline and is significantly induced by drought stress. The exploration of genetic variation in HvP5CS1 may facilitate a better understanding of the mechanism of drought adaptation in barley. In the current study, 41 polymorphisms including 16 single nucleotide polymorphisms (SNPs) and 25 insertions/deletions (indels) were detected in HvP5CS1 among 287 barley (Hordeum vulgare L.) accessions collected worldwide, with 13 distinct haplotypes identified in the barley collection. Five polymorphisms in HvP5CS1 were significantly (P < 0.001) associated with drought tolerance related traits in barley. The phenotypic variation of a given trait explained by each associated polymorphism ranged from 4.43% to 9.81%. Two sequence variations that were significantly (P < 0.0001) associated with grain yield had marginally significant positive Tajima's D values in the sliding window, so they might have been selected for environmental adaptation. Meanwhile, two haplotypes HvP5CS1_H1 and HvP5CS1_H4, which contained desired alleles of the two variations mentioned above, were significantly (P < 0.001) associated with drought tolerance related traits, and explained 5.00~11.89% of the phenotypic variations. These variations associated with drought tolerance related traits can be used as potential markers for improving drought tolerance in barley.

Development of a simple and effective silver staining protocol for detection of DNA fragments.

Silver staining is one of the widely used methods for DNA fragment detection in biological research. Silver staining protocols have been steadily optimized to improve detection efficiency. This research reports a continuous effort to simplify the existing silver staining protocols, lower experiment cost, and improve DNA detection sensitivity and image clarity. The new method only requires three reagents (silver nitrate, sodium hydroxide, and formaldehyde) and 6-7 min with high detection sensitivity to visualize as low as 14.6 pg (3.3 pg/mm2 ) of DNA in a non-denaturing polyacrylamide gel. In comparison to previous reported protocols, the new one has the highest resolution, is the easiest to operate, takes the shortest time, and uses the fewest chemical reagents. Therefore, the new method can be used for quick generation of high quality molecular marker data in genetic analysis.

Single nucleotide polymorphisms in HSP17.8 and their association with agronomic traits in barley.

Small heat shock protein 17.8 (HSP17.8) is produced abundantly in plant cells under heat and other stress conditions and may play an important role in plant tolerance to stress environments. However, HSP17.8 may be differentially expressed in different accessions of a crop species exposed to identical stress conditions. The ability of different genotypes to adapt to various stress conditions resides in their genetic diversity. Allelic variations are the most common forms of genetic variation in natural populations. In this study, single nucleotide polymorphisms (SNPs) of the HSP17.8 gene were investigated across 210 barley accessions collected from 30 countries using EcoTILLING technology. Eleven SNPs including 10 from the coding region of HSP17.8 were detected, which form nine distinguishable haplotypes in the barley collection. Among the 10 SNPs in the coding region, six are missense mutations and four are synonymous nucleotide changes. Five of the six missense changes are predicted to be deleterious to HSP17.8 function. The accessions from Middle East Asia showed the higher nucleotide diversity of HSP17.8 than those from other regions and wild barley (H. spontaneum) accessions exhibited greater diversity than the cultivated barley (H. vulgare) accessions. Four SNPs in HSP17.8 were found associated with at least one of the agronomic traits evaluated except for spike length, namely number of grains per spike, thousand kernel weight, plant height, flag leaf area and leaf color. The association between SNP and these agronomic traits may provide new insight for study of the gene's potential contribution to drought tolerance of barley.

Allelic variations of a light harvesting chlorophyll a/b-binding protein gene (Lhcb1) associated with agronomic traits in barley.

Light-harvesting chlorophyll a/b-binding protein (LHCP) is one of the most abundant chloroplast proteins in plants. Its main function is to collect and transfer light energy to photosynthetic reaction centers. However, the roles of different LHCPs in light-harvesting antenna systems remain obscure. Exploration of nucleotide variation in the genes encoding LHCP can facilitate a better understanding of the functions of LHCP. In this study, nucleotide variations in Lhcb1, a LHCP gene in barley, were investigated across 292 barley accessions collected from 35 different countries using EcoTILLING technology, a variation of the Targeting Induced Local Lesions In Genomes (TILLING). A total of 23 nucleotide variations were detected including three insert/deletions (indels) and 20 single nucleotide polymorphisms (SNPs). Among them, 17 SNPs were in the coding region with nine missense changes. Two SNPs with missense changes are predicted to be deleterious to protein function. Seventeen SNP formed 31 distinguishable haplotypes in the barley collection. The levels of nucleotide diversity in the Lhcb1 locus differed markedly with geographic origins and species of accessions. The accessions from Middle East Asia exhibited the highest nucleotide and haplotype diversity. H. spontaneum showed greater nucleotide diversity than H. vulgare. Five SNPs in Lhcb1 were significantly associated with at least one of the six agronomic traits evaluated, namely plant height, spike length, number of grains per spike, thousand grain weight, flag leaf area and leaf color, and these SNPs may be used as potential markers for improvement of these barley traits.