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Sample GSM1657858

Query DataSets for GSM1657858

Status

Public on Mar 07, 2016

Title

Medicago / Gigaspora replicate 1

Sample type

SRA

Source name

M. truncatula roots associated with R. rosea

Organism

Medicago truncatula

Characteristics

fungus strain: DAOM 194757
plant species: Medicago truncatula A17
tissue: Roots
growth time: 4 weeks

Treatment protocol

no treatment was applied

Growth protocol

Colonized B. distachyon was achieved by directly inoculating the seedlings of 3 days with sterile spores (50-60 spores per plant), before planting into 1L pot containing sterile charred clay (1 plant per pot). After 6 weeks of growth under a long day photoperiod (16h light/8h dark at 22°C/20°C) with 40% humidity, the finer and yellowish root fragments that tend to be well mycorrhized (Hong et al., 2012) were selected for RNA extraction. A nursery system strategy was adopted to generate the highly mycorrhized M. truncatula, using 4-week of B. distachyon pre-colonized by G.rosea as the nursery plant. After 4 weeks of co-cultivation with B. distachyon under a long day photoperiod (16h light/8h dark at 22°C/20°C) with 40% humidity, only the M. truncatula roots to which there were abundant hyphae attached were enriched under stereomicroscope for RNA extraction. The Colonization level of both hosts was over 80%. Extraradical mycelium of G.rosea was directly extracted from the microbox purchased from Agronutrition S.A. (Labège, France). After dissolving the gel using citrate buffer (Doner & Bécard, 1991), mycelium pellets free of any root material were collected and rinsed in sterile water for further RNA extraction.

Extracted molecule

total RNA

Extraction protocol

Biological samples were crushed with a motar and a pestle in liquid nitrogen and mRNA were extracted using RNeasy plant kit (Qiagen), according to the manufacturer instructions.
Samples were sequenced at the Genome and Transriptome platform in Toulouse, France (GeT). mRNA were isolated with polyT beads before processing, libraries were constructed according to the manufacturer's protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RAW Gr Mt vs ERM
55_AGTCAA_L001

Data processing

G. rosea raw reads trimming and filtration: Short pair-end sequencing reads generated on Illumina platforms (2x100 bp from Hiseq2000 and 2x150 bp from Miseq1000) were trimmed based on the quality scores (limit: 0.05), end ambiguity (maximum number of ambiguities: 2) and adaptor sequences using the commercial software CLC Genomic Workbench (v6.0, CLC Bio, Denmark). The reads less than 50bp after trimming were discarded. As the ERM corresponds to axenic cultures, the trimmed reads from non-symbiotic samples were used for de novo assembly directly. Whereas, in the symbiotic samples (SYM+BD, SYM+MT) which were obtained from plants cultivated in pot, the reads from other organisms including the host plant and potential contaminants need to be filtered away. The plant–originated reads were eliminated when they mapped on the genomes of Brachypodium distachyon (Bd21) v1.0 and Medicago truncatula (A17) v198 (ftp://ftp.jgipsf.org/pub/compgen/phytozome/v9.0/ Mtruncatula/) respectively, using CLC mapping tool. In order to eliminate the plant-originated reads sequences specifically induced in the symbiotic condition but not included in the present genome annotations, the large gap mapping (length fraction: 0.9 and similarity: 0.95) and transcript discovery (with default settings) strategies in CLC software were chosen. After removing the symbiotic reads mapped (length fraction: 0.9 and similarity: 0.9) on the plant genomes (with updated annotations based on the large-gap mapping and transcript discovery mentioned above), the remaining symbiotic reads were also further mapped onto the transcriptomes of non-mycorrhized B. distachyon and M. truncatula roots which were also generated in this study, to detect potential contaminant reads which might be introduced in the greenhouse condition. The symbiotic reads retained after all these filtrations were used for the following de novo assembly.
Gigaspora transcript assembly: in house de novo assembly using 230 084 705 cleaned reads (21 630 065 trimmed and filtered symbiotic reads plus 208 454 640 trimmed nonsymbiotic reads) was performed with CLC workbench. A total of 97 463 contigs were obtained, with an average length of 611bp. To discard possible contaminant sequence relics, we compared these 97463 contigs with NCBI nr/nt DNA database and removed 11131 sequences showing high similarity (blastn, e value cut-off at E-50) to those from other organisms than G. rosea. The remaining 86 332 contigs, constituting the first transcriptome of G. rosea (GiroV1), were considered as the nonredundant virtual transcripts (NRVTs) that will be used for the further annotation and gene expression analysis. The G. rosea transcriptome GiroV1 is approximately 55.5M in size, with an average trancript length of 643 bp and 33% of GC content. The accuracy of this refined assembly was demonstrated by the blastn result that 332 of 428 published G. rosea ESTs (downloaded from NCBI EST database using gigaspora rosea as the key word) could be found in this pool of NRVTs (e value cutoff at e-20, identity>85%). Nearly all (235 of 248) Core Eukaryotic Genes (CEGs) were found to be present in the current transcriptome (tblastn, e value cut-off at E-10), suggesting it covers most of the real transcriptome of G. rosea.
Selection of libraries: Pairwise Pearson correlations were calculated among each triplicates. Libraires with Pearson correlation lower than 0.9 were excluded: Library H4_GTCCGC (Gigaspora ERM replicate 2) and Library 55_AGTCAA (Medicago/Gigaspora replicate 1).
Read mapping: trimmed reads from all triplicates of G. rosea symbiotic samples and extraradical mycelium sample, were mapped back to the G. rosea transcriptome GiroV1 genre repertoire. To calculate gene expression, RNA-seq functionality in CLC software was used, with the settings of minimum mapped read length fraction at 0.95 and minimum similarity at 0.98. To find the NRVTs significantly upregulated during symbiosis, the RPKM of G. rosea NRVT in symbiotic samples was compared with that in their respective extraradical mycelium (ERM) using proportion-based test statistics with a False Discovery Rate (FDR) correction for multiple testing. G. rosea NRVTs were considered as significantly upregulated when meeting the requirements of RPKM fold change>5 and FDR corrected p value<0.05.

Submission date

Apr 15, 2015

Last update date

May 15, 2019

Contact name

Christophe Roux

E-mail(s)

[email protected]

Organization name

University of Toulouse/CNRS

Lab

Plant Science Laboratory - Joint Unit University of Toulouse/CNRS 5546