Transcriptome Analysis of Brassica juncea

Two lines of Brassica juncea, namely Varuna (Indian gene pool) and Heera (east European gene pool) were maintained in the field by selfing for more than ten generations. Seedling tissue and plant parts were collected from field grown plants. The following three tissue amalgams were taken and processed for RNA isolation: i.

Five days old seedlings grown in a growth chamber (10/14 h day/night at 20/18°C).

ii.

Young inflorescence, comprising unopened flower buds and small leaves from field grown plants.

iii.

Post fertilization, 10, 20, 30, and 40 day old pods, all taken together as one sample.

RNA was isolated from each of the three samples types using a Total RNA Spectrum kit (Sigma-Aldrich, St Louis, MO, USA) and subsequently treated with DNase A (Ambion, TX, USA). Equal amounts of RNA, 6 μg from each sample, were pooled and used for cDNA library preparation following the method described for RNA-seq of B. rapa lines [22 (link)].
Libraries prepared for Varuna and Heera were sequenced as 2×101 nt paired end reads on a Genome Analyzer IIx instrument (Illumina Inc, San Diego, USA). Sequence data were obtained from four lanes of the flowcell for Heera, and from five lanes for Varuna. Quality checking and de novo assembly was carried out using the Fastx-toolkit [54 ] and Velvet de novo assembly programs respectively, following the parameters described by Paritosh et al. [22 (link)].