74,146.OrTP2

Carrot root tissue was collected from three yellow (yyY₂Y₂) and three orange (yyy₂y₂) pigmented biological replicates, plants from the progenitor F₂ population of population 74,146, at 40 (time point one) and 80 (time point two) days after planting (DAP). Two time points were sampled to detect potential variation in expression across development. Time point one corresponds to the onset of visual detection of carotenoid accumulation in the storage root, and time point two corresponds to the onset of the plateau in carotenoid accumulation. Total RNA was extracted from storage root tissue using the TRIzol Plus RNA Purification Kit (Life Technologies, Carlsbad, CA) in accordance with the manufacturer’s protocol. Contaminating DNA was removed with the TurboDNA-free kit (Life Technologies, Carlsbad, CA). RNA quantity and integrity was confirmed with an Experion RNA StdSens Analysis kit (Bio-Rad, Hercules, CA). All samples had RQI values >8.0.

For each sample, a 133-nt insert size paired-end library was prepared at the Biotechnology Center, UW-Madison. Libraries were sequenced on Illumina HiSeq2000 lanes using 2 × 100 nt reads. Reads were filtered with Trimmomatic version 0.32 with adapter trimming and using a sliding window of length ≥50 and quality ≥28, i.e., “ILLUMINACLIP:adapterfna:2:40:15 LEADING:28 TRAILING:28 MINLEN:50 SLIDINGWINDOW:10:28.”

Short reads from each replicate were independently mapped against the carrot genome sequence using Bowtie2 (Langmead and Salzberg 2012) and Tophat2 (Kim et al. 2013) (Table S4 in 10.1534/g3.117.043067 Supplementary File S1.xlsx).

Differential expression was analyzed with Cufflinks (Trapnell et al. 2012). The three technical replicates for each sample were merged during analysis. Expression data from this analysis is derived from the cufflinks "cds_exp.diff" file.