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Sample GSM2323615

Query DataSets for GSM2323615

Status

Public on Jan 01, 2018

Title

IL6-2 rep4

Sample type

SRA

Source name

Root

Organism

Solanum lycopersicum x Solanum pennellii

Characteristics

solanum pennellii introgression line: IL6-2
tissue: Root
solanum lycopersicum cultivar: M82

Treatment protocol

The experiments were carried out in a growth chamber with a 16:8 light:dark cycle at 22ºC and 50-75% humidity with a light intensity of 100μE.

Growth protocol

Seedlings of S. lycopersicum (cv. M82), S. pennellii (LA0716), and 75 introgression lines between those parents were grown according to (Ron et al., 2013). Plates were placed vertically in a rack in a randomized block design.

Extracted molecule

total RNA

Extraction protocol

The germination date was monitored for each seed. Immediately prior to harvesting root tissue for RNA-seq, each replicate of each genotype had between 2 and 5 plates, all of which were scanned with a Epson Perfection V700 photo flatbed scanner into 24-bit RGB TIF image files at 300 dpi. Images were analyzed with the imageJ program (Rasband 1997-2014) and custom macros. Based on germination date and scan date, seedling age in days after germination was determined, and primary roots of seedlings that were 3 or 4 days post-germination were traced with the imageJ polyline tool, following the root centerline from the top of the root (at start of hairs and change from green to white) to the tip. The coordinates of the polylines were recorded by the macros in data tables saved as files, along with metadata including genotype, plate ID, plating date, germination day of each seedling, plate harvest date, measurer, and two flags for each root: “collide” was true if the root contacted or crossed another root, and “along” was true if the root contacted another root and grew along it for any distance. The polylines in the vetted data were analyzed to measure root length and angle using multiple traits (Figure 1A). Additional data was calculated from the metadata: root age (days from germination to harvest), “germination age” (days from plating to germination), “plating age” (days from plating to harvest), and biological replicate number (based on plating date; replicates were named REP2 through REP5, and later replicates for fixing genotype errors were named REPN2 through REPN5 and REPX1 through REPX6). Four replicates of each of the 77 genotypes were grown. Plates were opened only once, at harvest time, to ensure the same growth conditions for all seedlings. Sampling occurred mid-afternoon. Between 7 and 14 root tips (1 cm), 3 to 4 days post-germination, from each genotype replicate were cut and immediately placed into a labeled 2ml tube containing silica beads and immersed in liquid nitrogen. Samples were stored at −80ºC until library preparation.
Libraries were created using a modified Kumar et. al. protocol. Library enrichment was done using 12 cycles. Each library was barcoded, using a unique bar code for each IL, but the same barcode for all replicates of an IL. The libraries of each replicate were pooled, and the four pools were purified with Ampure beads. The library pooling was tested using a Bioanalyzer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Each pool was sequenced in 50 bp single-end format on a total of 5 lanes of a HiSeq 2000 platform (Illumina Inc. San Diego, CA, USA). Two replicates were lost during library creation, S. pennellii #3 and IL7-5 #3.
Demultiplexed data and filtered and trimmed bad base calls. Note that FASTQ files provided to GEO have been demultiplexed but not trimmed.
A chimeric reference was created for each IL by combining parental matched references (SlCDS_ITAG2.3_pseudoGMAP5V80passNoDif.fa and SpCDS_ITAG2.3_pseudoGMAP5V80passNoDif.fa) using the latter pennellii reference in the introgression region obtained from (Chitwood et al. 2013) or if not there then using introgression coordinates obtained by using the sequence data to locate the introgression.
Reads were mapped to chimeric references using BWA with parameters “aln -t 4 -k 1 -l 25 -n 0.1 -e 15 -i 10” and mapped read counts extracted with samtools.
Custom R code was used to combine count files, normalized using R edgeR package function calcNormFactors() method TMM, differentially expressed genes (DEG) called using edgeR. Custom R code tested DEG for GO and other biological category enrichments and special gene set enrichments and looked for common enrichments in ILs exhibiting QTLs for various root traits. Gene-gene and gene-trait correlations were done. Co-expression networks were built using partial correlation R package SPACE and using a probabilistic graphical model with bootstrap aggregation with R package DAGBAG.
Genome_build: Matched references from (Koenig et al. 2013) and chimeric references described above, based on International Tomato Annotation Group SL2.40 ITAG 2.3 genome at SolGenomics.net.

Submission date

Sep 21, 2016

Last update date

May 15, 2019

Contact name

Siobhan M Brady

E-mail(s)

sbrady@ucdavis.edu

Organization name

UC Davis

Department

Plant Biology