Computational protocol: The developmental and genetic bases of apetaly in Bocconia frutescens (Chelidonieae: Papaveraceae)

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[…] Fresh inflorescences carrying floral buds and flowers during organ initiation (Fig. ), as well as young flowers in S0–S4 (see above in “Plant material”) and flowers in anthesis from three different cultivated plants, were mixed together and were ground using liquid nitrogen. Further total RNA extraction was carried out using TRIzol reagent (Invitrogen). The RNAseq experiment was conducted using the truseq mRNA library construction kit (Illumina) and sequenced in a HiSeq 2000 instrument reading 100 bases paired end reads. A total of 25,185,945 raw read pairs were obtained. The transcriptome was assembled de novo. Read cleaning was performed with prinseq-lite with a quality threshold of Q35, and contig assembly was computed using Trinity package following default settings. Contig metrics are as follows: total assembled bases: 149,710,500; total number of contigs (>101 bases): 211,821; average contig length: 706 b; largest contig: 17,004 b; contig N50: 1877 bp; contig GC%: 40.09; number of Ns: 0. Orthologous gene search was performed using BLASTN [] with the reference sequences downloaded from GenBank (see below). Protein domain searches were carried out using HMMER and the PFAM database downloaded from the Danger Institute FTP server. Top hits for each sequence were individually analyzed and confirmed by protein domain searches, prior to phylogenetic analysis. [...] Sequences in the transcriptome were added to each dataset consisting of sequences available from NCBI, as well as genes available in Phytozome, specifically in the Aquilegia coerulea genome (http://www.phytozome.net/). Other homologs for all MADS-box gene lineages studied here were isolated from the 1kp transcriptome database (http://218.188.108.77/Blast4OneKP/home.php), Phytometasyn (http://www.phytometasyn.ca) and Plantrans DB (http://lifecenter.sgst.cn/plantransdb) after a search in the available transcriptomes of members of the Ranunculales (Additional file : Table S1). A matrix for each gene lineage was generated. All sequences were then compiled using Bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html), where they were cleaned to keep exclusively the open reading frame. Nucleotide sequences were then aligned using the online version of MAFFT (http://mafft.cbrc.jp/alignment/server/) [], with a gap open penalty of 3.0, an offset value of 0.8 and all other default settings. The alignment was then refined by hand using Bioedit taking into account the protein domains and amino acid motifs that have been reported as conserved for the five gene lineages. Maximum likelihood (ML) phylogenetic analyses using the nucleotide sequences were performed in RaxML-HPC2 BlackBox [] on the CIPRES Science Gateway []. The best performing evolutionary model was obtained by the Akaike information criterion, using ModelTest incorporated in MEGA6 []. Bootstrapping was performed according to the default criteria in RAxML where bootstrapping stopped after 200–600 replicates when the criteria were met. Trees were observed and edited using FigTree v1.4.0. […]

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