The draft genome of Tibetan hulless barley reveals adaptive patterns to the high stressful Tibetan Plateau.
ABSTRACT: The Tibetan hulless barley (Hordeum vulgare L. var. nudum), also called "Qingke" in Chinese and "Ne" in Tibetan, is the staple food for Tibetans and an important livestock feed in the Tibetan Plateau. The diploid nature and adaptation to diverse environments of the highland give it unique resources for genetic research and crop improvement. Here we produced a 3.89-Gb draft assembly of Tibetan hulless barley with 36,151 predicted protein-coding genes. Comparative analyses revealed the divergence times and synteny between barley and other representative Poaceae genomes. The expansion of the gene family related to stress responses was found in Tibetan hulless barley. Resequencing of 10 barley accessions uncovered high levels of genetic variation in Tibetan wild barley and genetic divergence between Tibetan and non-Tibetan barley genomes. Selective sweep analyses demonstrate adaptive correlations of genes under selection with extensive environmental variables. Our results not only construct a genomic framework for crop improvement but also provide evolutionary insights of highland adaptation of Tibetan hulless barley.
Project description:Powdery mildew is a fungal disease that represents a ubiquitous threat to crop plants. Transcriptomic and metabolomic analyses were used to identify molecular and physiological changes in Tibetan hulless barley in response to powdery mildew. There were 3418 genes and 405 metabolites differentially expressed between the complete resistance cultivar G7 and the sensitive cultivar Z13. Weighted gene coexpression network analysis was carried out, and the differentially expressed genes were enriched in five and four major network modules in G7 and Z13, respectively. Further analyses showed that phytohormones, photosynthesis, phenylpropanoid biosynthesis, and flavonoid biosynthesis pathways were altered during Qingke-Blumeria graminis (DC.) f.sp. hordei (Bgh) interaction. Comparative analyses showed a correspondence between gene expression and metabolite profiles, and the activated defenses resulted in changes of metabolites involved in plant defense response, such as phytohormones, lipids, flavone and flavonoids, phenolamides, and phenylpropanoids. This study enabled the identification of Bgh responsive genes and provided new insights into the dynamic physiological changes that occur in Qingke during response to powdery mildew. These findings greatly improve our understanding of the mechanisms of induced defense response in Qingke and will provide new clues for the development of resistant Tibetan hulless barley varieties.
Project description:Hulless barley (Hordeum vulgare L. var. nudum) is a barley variety that has loose husk cover of the caryopses. Because of the ease in processing and edibility, hulless barley has been locally cultivated and used as human food. For example, in Tibetan Plateau, hulless barley is the staple food for human and essential livestock feed. Although the draft genome of hulless barley has been sequenced, the assembly remains fragmented. Here, we reported an improved high-quality assembly and annotation of the Tibetan hulless barley genome using more than 67X PacBio long-reads. The N50 contig length of the new assembly is at least more than 19 times larger than other available barley assemblies. The new genome assembly also showed high gene completeness and high collinearity of genome synteny with the previously reported barley genome. The new genome assembly and annotation will not only remove major hurdles in genetic analysis and breeding of hulless barley, but will also serve as a key resource for studying barley genomics and genetics.
Project description:Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUB? (Alpha-tubulin), TUB?6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1? (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUB?6, E2, TUB?, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUB? and HSP90 showed peak stability under salt stress, TUB? and EF-1? were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.
Project description:The harsh environment on the Qinghai-Tibetan Plateau gives Tibetan hulless barley (Hordeum vulgare var. nudum) great ability to resist adversities such as drought, salinity, and low temperature, and makes it a good subject for the analysis of drought tolerance mechanism. To elucidate the specific gene networks and pathways that contribute to its drought tolerance, and for identifying new candidate genes for breeding purposes, we performed a transcriptomic analysis using two accessions of Tibetan hulless barley, namely Z772 (drought-tolerant) and Z013 (drought-sensitive).There were more up-regulated genes of Z772 than Z013 under both mild (5439-VS-2604) and severe (7203-VS-3359) dehydration treatments. Under mild dehydration stress, the pathways exclusively enriched in drought-tolerance genotype Z772 included Protein processing in endoplasmic reticulum, tricarboxylic acid (TCA) cycle, Wax biosynthesis, and Spliceosome. Under severe dehydration stress, the pathways that were mainly enriched in Z772 included Carbon fixation in photosynthetic organisms, Pyruvate metabolism, Porphyrin and chlorophyll metabolism. The main differentially expressed genes (DEGs) in response to dehydration stress and genes whose expression was different between tolerant and sensitive genotypes were presented in this study, respectively. The candidate genes for drought tolerance were selected based on their expression patterns.The RNA-Seq data obtained in this study provided an initial overview on global gene expression patterns and networks that related to dehydration shock in Tibetan hulless barley. Furthermore, these data provided pathways and a targeted set of candidate genes that might be essential for deep analyzing the molecular mechanisms of plant tolerance to drought stress.
Project description:Salinity stress represents one of the most harmful abiotic stresses for agricultural productivity. Tibetan hulless barley is an important economic crop widely grown in highly stressful conditions in the Qinghai-Tibet Plateau and is often challenged by salinity stress. To investigate the temporal metabolic responses to salinity stress in hulless barley, we performed a widely targeted metabolomic analysis of 72 leaf samples from two contrasting cultivars. We identified 642 compounds 57 % of which were affected by salt stress in the two cultivars, principally amino acids and derivatives, organic acids, nucleotides, and derivatives and flavonoids. A total of 13 stress-related metabolites including piperidine, L-tryptophan, L-glutamic acid, L-saccharopine, L-phenylalanine, 6-methylcoumarin, cinnamic acid, inosine 5'-monophosphate, aminomalonic acid, 6-aminocaproic acid, putrescine, tyramine and abscisic acid (ABA) represent the core metabolome responsive to salinity stress in hulless barley regardless of the tolerance level. In particular, we found that the ABA signalling pathway is essential to salt stress response in hulless barley. The high tolerance of the cultivar 0119 is due to a metabolic reprogramming at key stress times. During the early salt stress stages (0-24 h), 0119 tended to save energy through reduced glycolysis, nucleotide metabolism and amino acid synthesis, while increased antioxidant compounds such as flavonoids. Under prolonged stress (48-72 h), 0119 significantly enhanced energy production and amino acid synthesis. In addition, some important compatible solutes were strongly accumulated. By comparing the two cultivars, nine salt-tolerance biomarkers, mostly unreported salt-tolerance compounds in plants, were uncovered. Our study indicated that the salt tolerant hulless barley cultivar invokes a tolerance strategy which is conserved in other plant species. Overall, we provide for the first time some extensive metabolic data and some important salt-tolerance biomarkers which may assist in efforts to improve hulless barley tolerance to salinity stress.
Project description:Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third-generation sequencer (PacBio), we de novo assembled a 4.84-Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59-Gb sequence to seven chromosomes. Of the 46,787 annotated 'high-confidence' genes, 31 564 were validated by RNA-sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low-diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.
Project description:BACKGROUND: Hulless barley is attracting increasing attention due to its unique nutritional value and potential health benefits. However, the molecular biology of the barley grain development and nutrient storage are not well understood. Furthermore, the genetic potential of hulless barley has not been fully tapped for breeding. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we investigated the transcriptome features during hulless barley grain development. Using Illumina paired-end RNA-Sequencing, we generated two data sets of the developing grain transcriptomes from two hulless barley landraces. A total of 13.1 and 12.9 million paired-end reads with lengths of 90 bp were generated from the two varieties and were assembled to 48,863 and 45,788 unigenes, respectively. A combined dataset of 46,485 All-Unigenes were generated from two transcriptomes with an average length of 542 bp, and 36,278 among were annotated with gene descriptions, conserved protein domains or gene ontology terms. Furthermore, sequences and expression levels of genes related to the biosynthesis of storage reserve compounds (starch, protein, and ?-glucan) were analyzed, and their temporal and spatial patterns were deduced from the transcriptome data of cultivated barley Morex. CONCLUSIONS/SIGNIFICANCE: We established a sequences and functional annotation integrated database and examined the expression profiles of the developing grains of Tibetan hulless barley. The characterization of genes encoding storage proteins and enzymes of starch synthesis and (1-3;1-4)-?-D-glucan synthesis provided an overview of changes in gene expression associated with grain nutrition and health properties. Furthermore, the characterization of these genes provides a gene reservoir, which helps in quality improvement of hulless barley.
Project description:Nitrogen (N) deprivation or excess can lead to dramatic phenotype change, disrupt important biological processes, and ultimately limit plant productivity. To explore genes in Tibetan hulless barley responsive to varied N stress, we utilized a comparative transcriptomics method to investigate gene expression patterns under three nitrate treatments. The transcriptome of the control (optimal-nitrate, ON) sample was compared with that of free-nitrate (FN), low-nitrate (LN), and high-nitrate (HN) treatment samples, identifying 2428, 1274, and 1861 genes, respectively, that exhibited significant differences in transcript abundance. Among these, 9 genes encoding ribulose bisphosphate carboxylases exhibited up-regulated expression under varied N stress. We further compared FN vs. ON and LN vs. ON to investigate the impact of stress degree on gene expression. With the aggravation of stress, more genes were differentially expressed and thus possibly involved in the response to nitrogen deficiency. Cluster and functional enrichment analysis indicated that the differentially expressed genes (DEGs) in FN were highly enriched in response to stress, defense response, and gene expression regulation. Comprehensive comparison analysis further suggested that Tibetan hulless barley could respond to varied N stress by regulating multiple common biological processes and pathways such as nitrogen metabolism, carbon metabolism, and photosynthesis. A large number of specific DEGs involved in diverse biological processes were also detected, implying differences in the potential regulatory patterns of low- and high-N stress response. Notably, we also identified some NIN-like proteins and other transcription factors significantly modulated by these stresses, suggesting the involvement of these transcription factors in N stress response. To our knowledge, this study is the first investigation of the Tibetan hulless barley transcriptome under N stress. The identified N-stress-related genes may provide resources for genetic improvement and promote N use efficiency.
Project description:Barley (Hordeum vulgare L.) is one of the most important cereal crops worldwide. In the Qinghai-Tibet Plateau, six-rowed hulless (or naked) barley, called "qingke" in Chinese or "nas" in Tibetan, is produced mainly in Tibet. The complexity of the environment in the Qinghai-Tibet Plateau has provided unique opportunities for research on the breeding and adaptability of qingke barley. However, the genetic architecture of many important agronomic traits for qingke barley remains elusive. Heading date (HD), plant height (PH), and spike length (SL) are three prominent agronomic traits in barley. Here, we used genome-wide association (GWAS) mapping and GWAS with eigenvector decomposition (EigenGWAS) to detect quantitative trait loci (QTL) and selective signatures for HD, PH, and SL in a collection of 308 qingke barley accessions. The accessions were genotyped using a newly-developed, proprietary genotyping-by-sequencing (tGBS) technology, that yielded 14,970 high quality single nucleotide polymorphisms (SNPs). We found that the number of SNPs was higher in the varieties than in the landraces, which suggested that Tibetan varieties and varieties in the Tibetan area may have originated from different landraces in different areas. We have identified 62 QTLs associated with three important traits, and the observed phenotypic variation is well-explained by the identified QTLs. We mapped 114 known genes that include, but are not limited to, vernalization, and photoperiod genes. We found that 83.87% of the identified QTLs are located in the non-coding regulatory regions of annotated barley genes. Forty-eight of the QTLs are first reported here, 28 QTLs have pleotropic effects, and three QTL are located in the regions of the well-characterized genes HvVRN1, HvVRN3, and PpD-H2. EigenGWAS analysis revealed that multiple heading-date-related loci bear signatures of selection. Our results confirm that the barley panel used in this study is highly diverse, and showed a great promise for identifying the genetic basis of adaptive traits. This study should increase our understanding of complex traits in qingke barley, and should facilitate genome-assisted breeding for qingke barley improvement.
Project description:Tibetan hulless barley is widely grown in the extreme environmental conditions of the Qinghai-Tibet Plateau which is characterized by cold, high salinity, and drought. Osmotic stress always occurs simultaneously with drought and its tolerance is a vital part of drought tolerance. The diversity of metabolites leading to osmotic stress tolerance was characterized using widely-targeted metabolomics in tolerant (XL) and sensitive (D) accessions submitted to polyethylene glycol. XL regulated a more diverse set of metabolites than D, which may promote the establishment of a robust system to cope with the stress in XL. Compounds belonging to the group of flavonoids, amino acids, and glycerophospholipids constitute the core metabolome responsive to the stress, despite the tolerance levels. Moreover, 8 h appeared to be a critical time point for stress endurance involving a high accumulation of key metabolites from the class of nucleotide and its derivative which provide the ultimate energy source for the synthesis of functional carbohydrates, lipids, peptides, and secondary metabolites in XL. This intrinsic metabolic adjustment helped XL to efficiently alleviate the stress at the later stages. A total of 22 diverse compounds were constantly and exclusively regulated in XL, representing novel stress tolerance biomarkers which may help improving stress tolerance, especially drought, in hulless barley.