Project description:Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta.
Project description:Background:Ammopiptanthus nanus is a rare broad-leaved shrub that is found in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here, we report a draft genome assembly using the Pacific Biosciences (PacBio) platform and gene annotation for A. nanus. Findings:A total of 64.72 Gb of raw PacBio sequel reads were generated from four 20-kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59× coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated using Benchmarking Universal Single-Copy Orthologs; 1,327 of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements and 53.44% is composed of long terminal repeat elements. We predicted ?37,188 protein-coding genes, of which 96.53% were functionally annotated. Conclusions:The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.
Project description:<i>Ammopiptanthus mongolicus</i> is a tertiary relict evergreen broad-leaf shrub in family Fabaceae with remarkable tolerance to desiccation and low temperature. In this study, we report the complete mitochondrial genome of <i>A. mongolicus.</i> The total genome length was 475,396?bp and contained a total of 127 genes, including 79 protein-coding genes (28 novel genes, 45 known functional genes, and six known orf genes), three rRNA genes, and 45 tRNA genes. Most of the genes were single-copy genes, only six were duplicated and two were multi-copy. The mitochondrial genome also contained 'promiscuous' sequences from the chloroplast, 16 intact tRNAs of mitochondrial origin, and 29 intact and potentially functional chloroplast-derived tRNAs. The overall GC content of the mitochondrial DNA was 42.75%. A neighbour-joining phylogenomic analysis showed that <i>A. mongolicus</i> was closely related to <i>Medicago truncatula</i>, which also belongs to family Leguminosae.
Project description:BACKGROUND: Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f., an evergreen broadleaf legume shrub, is distributed in Mid-Asia where the temperature can be as low as -30°C during the winter. Although A. mongolicus is an ideal model to study the plant response to cold stress, insufficient genomic resources for this species are available in public databases. To identify genes involved in cold acclimation (a phenomenon experienced by plants after low temperature stress), a high-throughput sequencing technology was applied. RESULTS: We sequenced cold-treated and control (untreated) samples of A. mongolicus, and obtained 65,075,656 and 67,287,120 high quality reads, respectively. After de novo assembly and quantitative assessment, 82795 all-unigenes were finally generated with an average length of 816 bp. We then obtained functional annotations by aligning all-unigenes with public protein databases including NR, SwissProt, KEGG and COG. Differentially expressed genes (DEGs) were investigated using the RPKM method. Overall, 9309 up-regulated genes and 23419 down-regulated genes were identified. To increase our understanding of these DEGs, we performed GO enrichment and metabolic pathway enrichment analyses. Based on these results, a series of candidate genes involved in cold responsive pathways were selected and discussed. Moreover, we analyzed transcription factors, and found 720 of them are differentially expressed. Finally, 20 of the candidate genes that were up-regulated and known to be associated with cold stress were examined using qRT-PCR. CONCLUSIONS: In this study, we identified a large set of cDNA unigenes from A. mongolicus. This is the first transcriptome sequencing of this non-model species under cold-acclimation using Illumina/Solexa, a next-generation sequencing technology. We sequenced cold-treated and control (untreated) samples of A. mongolicus and obtained large numbers of unigenes annotated to public databases. Studies of differentially expressed genes involved in cold-related metabolic pathways and transcription factors facilitate the discovery of cold-resistance genes.
Project description:Ammopiptanthus mongolicus is an ecologically important desert plant. miRNA are endogenous 20-22 nt small RNAs that play important roles in regulating gene expression in plants. To identify to the target genes of miRNA in Ammopiptanthus mongolicus, degradome sequencing was performed and a batch of transcripts were found to be targeted by miRNA. The result is helpful in understanding the roles on miRNA in plant development and environmental stress response. Overall design: A degradome sequencing library was constructed using pooled mRNA isolated from leaves and roots of A. mongolicus seedlings. The library was sequenced using Illumina Hiseq 2500 platform.
Project description:BACKGROUND: De novo assembly of transcript sequences produced by next-generation sequencing technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. Ammopiptanthus mongolicus, a super-xerophytic broadleaf evergreen wood, is an ecologically important foundation species in desert ecosystems and exhibits substantial drought tolerance in Mid-Asia desert. Root plays an important role in water absorption of plant. There are insufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the drought tolerance of A. mongolicus. Thus, high throughput transcriptome sequencing from A. mongolicus root is helpful to generate a large amount of transcript sequences for gene discovery and molecular marker development. RESULTS: A total of 672,002 sequencing reads were obtained from a 454 GS XLR70 Titanium pyrosequencer with a mean length of 279 bp. These reads were assembled into 29,056 unique sequences including 15,173 contigs and 13,883 singlets. In our assembled sequences, 1,827 potential simple sequence repeats (SSR) molecular markers were discovered. Based on sequence similarity with known plant proteins, the assembled sequences represent approximately 9,771 proteins in PlantGDB. Based on the Gene ontology (GO) analysis, hundreds of drought stress-related genes were found. We further analyzed the gene expression profiles of 27 putative genes involved in drought tolerance using quantitative real-time PCR (qRT-PCR) assay. CONCLUSIONS: Our sequence collection represents a major transcriptomic resource for A. mongolicus, and the large number of genetic markers predicted should contribute to future research in Ammopiptanthus genus. The potential drought stress related transcripts identified in this study provide a good start for further investigation into the drought adaptation in Ammopiptanthus.
Project description:BACKGROUND:Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., an endangered ancient legume species, endemic to the Gobi desert in north-western China. As the only evergreen broadleaf shrub in this area, A. mongolicus plays an important role in the region's ecological-environmental stability. Despite the strong potential of A. mongolicus in providing new insights on drought tolerance, sequence information on the species in public databases remains scarce. To both learn about the role of gene expression in drought stress tolerance in A. mongolicus and to expand genomic resources for the species, transcriptome sequencing of stress-treated A. mongolicus plants was performed. RESULTS:Using 454 pyrosequencing technology, 8,480 and 7,474 contigs were generated after de novo assembly of RNA sequences from leaves of untreated and drought-treated plants, respectively. After clustering using TGICL and CAP3 programs, a combined assembly of all reads produced a total of 11,357 putative unique transcripts (PUTs). Functional annotation and classification of the transcripts were conducted by aligning the 11,357 PUTs against the public protein databases and nucleotide database (Nt). Between control and drought-treated plants, 1,620 differentially expressed genes (DEGs) were identified, of which 1,106 were up-regulated and 514 were down-regulated. The differential expression of twenty candidate genes in metabolic pathways and transcription factors families related to stress-response were confirmed by quantitative real-time PCR. Representatives of several large gene families, such as WRKY and P5CS, were identified and verified in A. mongolicus for the first time. CONCLUSIONS:The additional transcriptome resources, gene expression profiles, functional annotations, and candidate genes provide a more comprehensive understanding of the stress response pathways in xeric-adapted plant species such as A. mongolicus.
Project description:MicroRNAs (miRNAs) regulate target gene expression to modulate plant growth, development, and biotic and abiotic stress response at the post-transcriptional level. Ammopiptanthus mongolicus, an ecologically important desert plant, is increasingly used as a model for studying stress tolerance in plants. The miRNA-mediated gene regulatory network might remarkably contribute to the high stress tolerance of A. mongolicus. However, a genome-wide identification of miRNAs and their targets is still lacking in A. mongolicus. In this study, 170 conserved and 156 non-conserved miRNAs were identified in A. mongolicus. We experimentally identified 298 miRNA-target pairs from the degradome data. Quantitative real-time polymerase chain reaction analyses identified 28 drought-responsive miRNAs in leaves and 15 in roots. Some characteristics of the miRNA-mediated regulatory network were found in A. mongolicus. Multiple miRNAs, including 2 newly identified non-conserved miRNAs, miR-P11 and miR-P14, generated from the precursors of miR169, were found to be involved in drought stress response. Further, miR2118 and miR858 participated in drought stress response by up-regulating OZF1 gene and certain MYB genes that were involved in the regulation of flavonol biosynthesis in A. mongolicus. The findings of this study might provide new insights for understanding the functions of miRNA in stress response in plants.