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Sample GSM1658565

Query DataSets for GSM1658565

Status

Public on Sep 29, 2017

Title

Medicago / Rhizophagus replicate 3

Sample type

SRA

Source name

M. truncatula roots associated with R. irregularis

Organism

Medicago truncatula

Characteristics

fungal strain: DAOM197198
plant specie: Medicago truncatula A17
tissue: Roots

Treatment protocol

no treatment was applied

Growth protocol

To increase colonization of M. truncatula and B. distachyon, a nursery system was used: Leek seeds (Allium porum cv. Elboeuf) were germinated on vermiculite for 2 weeks at room temperature, and then transferred to 4L pots with sterile charred clay (Oil-dri, Klasmann, France) as substrate, mixed with 400sp/L of R. irregularis. Leeks were grown for 3 months at 25/22°C under a long day photoperiod (16h/8h) with 80% humidity. Seeds of M. truncatula A17 were surface sterilized with 3.2% sodium hypochlorite for 10 min, 95% ethanol for 2 min and washed four times with sterile distiled water. Seeds were germinated on water agar 1% for 5 days at 4°C in the dark. Seeds of B. distachyon were surface sterilized with Ethanol 70% for 30 seconds, rinsed with water and treated with 10% sodium hypochlorite added with a drop of tween 20 for 20 minutes under agitation. Seeds were rinsed 3 times for 5 minutes with sterile water, and soaked in water for 1 hour. They were finaly rinsed 3 times and germinated for 3 days on water agar 0,7% at 22°C under 16h photoperiod. Seedlings were than transferred to pots containing mycorrhized Leeks to increase fungal colonization. Plantlets were removed after 3 weeks, they were transferred in 250mL pots with sterile charred clay as substrate and grown for 2 weeks. Root colonization reached 67% and 42% for M. truncatula and B. distachyon respectively. Lunulari cruciata (liverwort) subcultures were cultivated in vitro on solid M medium (4% phytagel) + sucrose, in association with R. irregularis under a long day photoperiod (16h/8h). After 6 weeks, thalli were harvested and distal parts were cut with a scalpel and discarded, as these zone are not colonized by the fungus. To generate extra radical mycelium, R. irregularis was cultivated in association with Daucus carota in split plate on solid M medium (4g/L phytagel) at 22°C in the dark. Once the fungal compartment was colonized, the gel was removed with a scalpel and replaced by liquid M medium without sucrose. After two weeks of growth in the same previous conditions, ERM were collected with forceps and frozen with liquid nitrogen.

Extracted molecule

total RNA

Extraction protocol

Plant roots and extra radical mycelium were collected and 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 (Toulouse, France). 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 Ri Mt vs ERM
33_ATGTCA_L004

Data processing

Reads from R. irregularis symbiotic samples and extraradical mycelium (ERM) samples were mapped back to the Gloin1 genre repertoire to calculate gene expression as RPKM, using RNA-seq functionality in CLC software 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 R. irregularis gene in symbiotic samples were compared to their respective ERM using proportion-based test statistics with a False Discovery Rate (FDR) correction for multiple testing. R. irregularis genes were considered as significantly upregulated when meeting the requirements of RPKM fold change>5 and FDR corrected p value<0.05.
Genome_build: GCA_000439145.2

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