Project description:BACKGROUND: Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro. RESULTS: In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1? were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants. CONCLUSIONS: geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1? had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.
Project description:BACKGROUND: Transcriptome sequencing can be used to determine gene sequences and transcript abundance in non-model species, and the advent of next-generation sequencing (NGS) technologies has greatly decreased the cost and time required for this process. Transcriptome data are especially desirable in bamboo species, as certain members constitute an economically and culturally important group of mostly semelparous plants with remarkable flowering features, yet little bamboo genomic research has been performed. Here we present, for the first time, extensive sequence and transcript abundance data for the floral transcriptome of a key bamboo species, Dendrocalamus latiflorus, obtained using the Illumina GAII sequencing platform. Our further goal was to identify patterns of gene expression during bamboo flower development. RESULTS: Approximately 96 million sequencing reads were generated and assembled de novo, yielding 146,395 high quality unigenes with an average length of 461 bp. Of these, 80,418 were identified as putative homologs of annotated sequences in the public protein databases, of which 290 were associated with the floral transition and 47 were related to flower development. Digital abundance analysis identified 26,529 transcripts differentially enriched between two developmental stages, young flower buds and older developing flowers. Unigenes found at each stage were categorized according to their putative functional categories. These sequence and putative function data comprise a resource for future investigation of the floral transition and flower development in bamboo species. CONCLUSIONS: Our results present the first broad survey of a bamboo floral transcriptome. Although it will be necessary to validate the functions carried out by these genes, these results represent a starting point for future functional research on D. latiflorus and related species.
Project description:MicroRNAs (miRNAs), a class of non-coding small endogenous RNAs of approximately 22 nucleotides, regulate gene expression at the post-transcriptional levels by targeting mRNAs for degradation or by inhibiting protein translation. Thousands of miRNAs have been identified in many species. However, there is no information available concerning miRNAs in ma bamboo (Dendrocalamus latiflorus), one of the most important non-timber forest products, which has essential ecological roles in forests. To identify miRNAs in D. latiflorus, a small RNA library was constructed from leaf tissues. Using next generation high-throughput sequencing technology and bioinformatics analysis, we obtained 11,513,607 raw sequence reads and identified 84 conserved miRNAs (54 mature miRNAs and 30 star miRNAs) belonging to 17 families, and 81 novel miRNAs (76 mature miRNAs and five star miRNAs) in D. latiflorus. One hundred and sixty-two potential targets were identified for the 81 novel bamboo miRNAs. Several targets for the novel miRNAs are transcription factors that play important roles in plant development. Among the novel miRNAs, 30 were selected and their expression profiles in response to different light conditions were validated by qRT-PCR. This study provides the first large-scale cloning and characterization of miRNAs in D. latiflorus. Eighty-four conserved and 81 novel miRNAs were identified in D. latiflorus. Our results present a broad survey of bamboo miRNAs based on experimental and bioinformatics analysis. Although it will be necessary to validate the functions of miRNAs by further experimental research, these results represent a starting point for future research on D. latiflorus and related species.
Project description:BACKGROUND: Bamboo occupies an important phylogenetic node in the grass family with remarkable sizes, woodiness and a striking life history. However, limited genetic research has focused on bamboo partially because of the lack of genomic resources. The advent of high-throughput sequencing technologies enables generation of genomic resources in a short time and at a minimal cost, and therefore provides a turning point for bamboo research. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset for the Ma bamboo (Dendrocalamus latiflorus Munro). RESULTS: The Ma bamboo transcriptome was sequenced using the Illumina paired-end sequencing technology. We produced 15,138,726 reads and assembled them into 103,354 scaffolds. A total of 68,229 unigenes were identified, among which 46,087 were annotated in the NCBI non-redundant protein database and 28,165 were annotated in the Swiss-Prot database. Of these annotated unigenes, 11,921 and 10,147 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 45,649 unigenes onto 292 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. The annotated unigenes were compared against Moso bamboo, rice and millet. Unigenes that did not match any of those three sequence datasets are considered to be Ma bamboo unique. We predicted 105 unigenes encoding eight key enzymes involved in lignin biosynthesis. In addition, 621 simple sequence repeats (SSRs) were detected. CONCLUSION: Our data provide the most comprehensive transcriptomic resource currently available for D. latiflorus Munro. Candidate genes potentially involved in growth and development were identified, and those predicted to be unique to Ma bamboo are expected to give a better insight on Ma bamboo gene diversity. Numerous SSRs characterized contributed to marker development. These data constitute a new valuable resource for genomic studies on D. latiflorus Munro and, more generally, bamboo.
Project description:The mechanism of floral transition in bamboo remains unclear. Dendrocalamus latiflorus (Bambusease, Bambusoideae, Poaceae) is an economically and ecologically important clumping bamboo in tropical and subtropical areas. We evaluated morphological characteristics and gene expression profiling to study floral induction and early flower development in D. latiflorus. The detailed morphological studies on vegetative buds and floral organography were completed using paraffin sectioning and scanning electron microscopy. The 3 mm floral buds commence the development of stamen primordia and pistil primordium. Furthermore, homologs of floral transition-related genes, including AP1, TFL1, RFL, PpMADS1, PpMADS2, SPL9, FT, ID1, FCA, and EMF2, were detected and quantified by reverse transcriptase PCR and real-time PCR in vegetative and floral buds, respectively. Distinct expression profiles of ten putative floral initiation homologues that corresponded to the developmental stages defined by bud length were obtained and genes were characterized. Six of the genes (including DlTFL1, DlRFL, DlMADS2, DlID1, DlFCA, DlEMF2) showed statistically significant changes in expression during floral transition. DlAP1 demonstrated a sustained downward trend and could serve as a good molecular marker during floral transition in D. latiflorus. The combined analysis provided key candidate markers to track the transition from the vegetative to reproductive phase.
Project description:Elucidating the differences in gene expression profiles of plants with different ploidy levels and how they affect phenotypic traits is vital to allow genetic improvement of plants such as Ma bamboo (Dendrocalamus latiflorus Munro). We previously obtained triploid (2n = 3X = 36), hexaploid (2n = 6X = 72), and dodecaploid (2n = 12X = 144) Ma bamboo plants from embryogenic callus by anther culturing. Phenotypic differences between these plants appeared to be correlated with differences in ploidy. Here, we performed transcriptome profiling and sequencing of anther-regenerated plants and F1 seedlings of different ploidy levels using RNA-Seq technology. Pair-wise comparisons of the four resulting libraries revealed 8,396 differentially expressed genes. These differentially expressed genes were annotated, functionally classified, and partially validated. We found that the chromosome doubling led to substantially up- or down-regulation of genes that were involved in cell growth and differentiation; the polyploidy levels altered the anatomical, physiological and growth characteristics, such as leaf thickness, fusoid cell and stomatal size, shoot number, photosynthesis and respiration rate and so on. Additionally, two candidate genes, EXPB3 and TCP with potenitial regulatory roles in cell division and differentiation, were identified through gene coexpresseion network analysis. These results highlight the significance of potential applications of polyploidy, and provide valuable information for the genetic breeding of bamboo species.
Project description:Although bamboo is one of the most important woody crops in Asia, information on its genome is still very limited. To investigate the relationship among Poaceae members and to understand the mechanism of albino mutant generation in vitro, the complete chloroplast genome of two economically important bamboo species, Dendrocalamus latiflorus Munro and Bambusa oldhamii Munro, was determined employing a strategy that involved polymerase chain reaction (PCR) amplification using 443 novel primers designed to amplify the chloroplast genome of these two species. The lengths of the B. oldhamii and D. latiflorus chloroplast genomes are 139,350 and 139,365 bp, respectively. The organization structure and the gene order of these two bamboos are identical to other members of Poaceae. Highly conserved chloroplast genomes of Poaceae facilitated sequencing by the PCR method. Phylogenetic analysis using both chloroplast genomes confirmed the results obtained from studies on chromosome number and reproductive organ morphology. There are 23 gaps, insertions/deletions > 100 bp, in the chloroplast genomes of 10 genera of Poaceae compared in this study. The phylogenetic distribution of these gaps corresponds to their taxonomic placement. The sequences of these two chloroplast genomes provide useful information for studying bamboo evolution, ecology and biotechnology.