Computational protocol: High resolution mapping of traits related to whole-plant transpiration under increasing evaporative demand in wheat

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Protocol publication

[…] SNPs underlying the target QTL were assigned to contigs of the wheat chromosome survey sequences (CSS) based on the information provided by . To obtain gene expression information for genes located in the QTL region we used the PopSeq map for bread wheat together with the high confidence (HC) gene predictions described in and the publicly available RNA-Seq data for cv. Chinese Spring (http://wheat-urgi.versailles.inra.fr/Seq-Repository/RNA-Seq). This dataset covers five different organs (root, leaf, stem, spike, grain) at three developmental stages, each in two replicates. We used the HC gene predictions, version 2.1 (ftp://ftpmips.helmholtz-muenchen.de/plants/wheat/IWGSC/genePrediction_v2.1/) as reference for mapping the RNA-Seq reads. The reference was prepared by extracting the genomic sequence for each of the predicted genes with up to 2kb upstream and downstream bases whenever available from the corresponding IWGSC-CSS contig. The coordinates in the corresponding GTF/GFF transcript annotations file were adjusted accordingly. RNA-Seq reads were quality-, adapter- and length-trimmed using Trimmomatic (), version 0.30, with a custom list of adapter sequences and the following settings: ‘ILLUMINACLIP:adapters.fa:1:6:6 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:6 MINLEN:60’. After indexing the reference using Bowtie2 () version 2.2.1, trimmed reads were aligned to the reference using TopHat () version 2.2.1, not allowing any mismatches or indels. Paired reads were required to map concordantly (--no-discordant setting) to the same (--no-mixed setting) reference sequence. BAM files for the biological replicates were merged, apart from a single tissue/stage sample (spike_Z39) where only one sample was available. Expression was quantified by Cufflinks () version 2.1.1 utilizing (through the --GTF option) the adjusted version of the reference transcript annotations provided with the HC gene predictions. The remaining settings were left at their defaults, except for: --max-multiread-fraction 1, --frag-len-std-dev 50 --max-intron-length 5 000. FPKMs (fragments per kilobase of exon per million fragments mapped) per gene (rather than per isoform) were extracted and aggregated in tabular form. Functional annotation was obtained by comparison of the HC gene predictions to MSU Rice Genome, Release 7, and Arabidopsis TAIR using BlastX (expect-value <10−10). […]

Pipeline specifications

Software tools Trimmomatic, Bowtie2, TopHat, Cufflinks, BLASTX
Databases TAIR
Application RNA-seq analysis
Organisms Triticum aestivum