| Overall design |
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 and 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. 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). Plates were opened only once, at harvest time, to ensure the same growth conditions for all seedlings. Sampling occurred mid-afternoon. Ten segments (1cm) of the root tip 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. Four replicates of each of the 77 genotypes were grown. Between 7 and 14 root tips, 3 to 4 days post-germination, were collected and 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. 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.
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