Indica rice genome assembly, annotation and mining of blast disease resistance genes

Sequence data size for rice cultivars. (XLSX 15 kb

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae

<p>Abstract</p> <p>Background</p> <p>Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited.</p> <p>Results</p> <p>Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, <it>Magnaporthe oryzae</it>. Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively.</p> <p>Conclusions</p> <p>Our findings suggest <it>M. oryzae </it>small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.</p

Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods

BACKGROUND: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. RESULTS: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. CONCLUSION: The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants

Robust analysis of 5′-transcript ends (5′-RATE): a novel technique for transcriptome analysis and genome annotation

Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5′-transcript ends (5′-RATE) for rapid and cost-effective isolation of long 5′ transcript ends (∼80 bp). It consists of three major steps including 5′-oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5′-RATE library revealed complex alternative transcription start sites and a 5′ poly(A) tail in maize transcripts. Our results demonstrate that 5′-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes

Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.

Background: Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Methods: Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. Results: We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.  Conclusions: We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause

Additional file 22 of Genomic and transcriptomic analysis of sacred fig (Ficus religiosa)

Additional file 22: Table S9.1. The total differentially expressed genes from F. religiosa leaf tissue sample of day and night period