Functional characterization of fifteen hundred transcripts from Ziarat juniper (Juniperus excelsa M.Bieb)

Background: Ziarat juniper (Juniperus excelsa M.Bieb) is an evergreen and dominant species of Balochistan juniper forests. This forest is providing many benefits to regional ecosystems and surrounding populations. No functional genomics study is reported for this important juniper plant. This research is aimed to characterize the Ziarat juniper functional genome based on the analyses of 1500 transcripts.  Methods: Total RNA from shoot of Juniperus excelsa was extracted and subjected for transcriptome sequencing using Illumina HiSeq 2000 with the service from Macrogen, Inc., South Korea. The Illumina sequenced data was subjected to bioinformatics analysis. Quality assessment and data filtration was performed for the removal of low-quality reads, ambiguous reads and adaptor sequences. The high-quality clean reads data was deposited in the Sequence Read Archive (SRA) at NCBI, and used for downstream processes. Fifteen hundred transcripts were randomly chosen and used for functional characterization.Results: As a result of homology search 80.3% transcripts showed significant similarities and were placed  in significant similarities category, 19.3% transcripts showed low similarities and assigned to the ‘‘unclassified’’ category while 0.4% transcripts are defined as no hits. The functional characterization results showed that most (18%) of the transcripts are involved in metabolism, followed by 11.7% in transcription and 11.5% as structural protein. 8.8% transcripts are engaged in stress response, whereas the transcripts involved in growth and development constituted 6.7%. Transcripts involved in signal transduction represented 5.6%, while 3.5% facilitating transport and 34.1% are involved in hypothetical functions.Conclusion: The functional annotation data produced in this study will be very useful for future functional genome analysis of Juniperus excelsa.

IDENTIFICATION OF MICRORNAS IN 12 PLANT SPECIES OF FABACEAE

MicroRNAs (miRNAs) are tiny, non-coding and regulatory RNAs approximately 21 nucleotides in length.They are reported in various plants but still needs discovery in important plant species. The 12 plant species ofFabaceae were subjected this time to identify their miRNAs. The comparative genomics approaches withcombination of various bioinformatics’ tools were applied to find the novel miRNAs. This research leads to thefinding of 29 miRNAs belonging to 13 miRNA families. From the 29 miRNAs, nine belongs to Arachis duranensis,four to Lotus japonicas, three to each Pisum sativum and Arachis hypogaea, two to each Arachis ipaensis andPhaseolus vulgaris and one to each Cicer arietinum, Phaseolus acutifolius, Lupinus luteus, Glycyrrhiza uralensis,Robinia pseudoacacia and Lathyrus odoratus. These findings will be useful in the future to design and developdesirable traits in the 12 plant species of Fabaceae

GO-biological processes.

<p>Based on agriGo, more complicated enriched biological processes was built and wheat miRNAs are involved in many different biological processes, mainly in reproduction, multi-organism process, cell communication, cell recognition, respiration and biosynthesis processes.</p

GO-cellular processes.

<p>Based on agriGo, more complicated enriched cellular component processes was built and wheat miRNAs are involved in many different cellular components, mainly in membrane and membrane-linked, mitochondrion and small ribosomal subunit.</p

Identification and annotation of newly conserved microRNAs and their targets in wheat (<i>Triticum aestivum</i> L.)

<div><p>MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs produce by cell endogenously. They are 18–26 nucleotides in length and play important roles at the post-transcriptional stage of gene regulation. Evolutionarily, miRNAs are conserved and their conservation plays an important role in the prediction of new miRNAs in different plants. Wheat (<i>Triticum aestivum</i> L.) is an important diet and consumed as second major crop in the world. This significant cereal crop was focused here through comparative genomics-based approach to identify new conserved miRNAs and their targeted genes. This resulted into a total of 212 new conserved precursor miRNAs (pre-miRNAs) belonging to 185 miRNA families. These newly profiled wheat’s miRNAs are also annotated for stem-loop secondary structures, length distribution, organ of expression, sense/antisense orientation and characterization from their expressed sequence tags (ESTs). Moreover, fifteen miRNAs along with housekeeping gene were randomly selected and subjected to RT-PCR expressional validation. A total of 32927 targets are also predicted and annotated for these newly profiled wheat miRNAs. These targets are found to involve in 50 gene ontology (GO) enrichment terms and significant processes. Some of the significant targets are RNA-dependent DNA replication (GO:0006278), RNA binding (GO:0003723), nucleic acid binding (GO:0003676), DNA-directed RNA polymerase activity (GO:0003899), magnesium ion transmembrane transporter activity (GO:0015095), antiporter activity (GO:0015297), solute:hydrogen antiporter activity (GO:0015299), protein kinase activity (GO:0004672), ATP binding (GO:0005524), regulation of Rab GTPase activity (GO:0032313) Rab GTPase activator activity (GO:0005097), regulation of signal transduction (GO:0009966) and phosphoprotein phosphatase inhibitor activity (GO:0004864). These findings will be helpful to manage this economically important grain plant for desirable traits through miRNAs regulation.</p></div

Wheat miRNAs RT-PCR expressional validation.

<p>One housekeeping gene and nine wheat miRNAs; 1 (Housekeeping gene Ta54227- AAA-superfam. ATPases), 2 (tae-miR5040), 3 (tae-miR6220), 4 (tae-miR169), 5 (tae-miR172d), 6 (tae-miR827), 7 (tae-miR5523), 8 (tae-miR530b), 9 (tae-miR530a) and 10 (tae-miR1522), were selected and subjected to RT-PCR expression analysis for the experimental validation. The product of each sample was separated on a 1.5% (w/v) agarose gel with 100 base pair DNA leader.</p

The newly identified wheat miRNAs’ secondary structures.

<p>The wheat pre-miRNAs’ secondary structures were developed through the Mfold algorithm. These structures clearly show the mature miRNAs in green, in the stem portion of the stem-loop structures.</p

Putative wheat targets enrichment analysis in GO-terms.

<p>Where, BP = Biological Process, MF = Molecular Function, CC = Cellular Component, and FDR = False Discovery Rates.</p

GO-molecular functions.

<p>Based on agriGo, more complicated enriched molecular functional processes was built and wheat miRNAs are involved in many different molecular functions, mainly in bindings, nutrient reservoir activity, hydrolase activity and in RNA polymerase activity.</p