Computational protocol: Understanding the basis of a novel fruit type in Brassicaceae: conservation and deviation in expression patterns of six genes

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Protocol publication

[…] RNA was extracted from developing flowers and fruits using Concert Plant RNA Reagent kit (Invitrogen, Carlsbad, CA, USA), DNAse1 treated to remove DNA (Fermentas, Honover, MD, USA) and then cleaned with RNAeasy MiniKit (Qiagen, MD, USA). mRNA was purified from total RNA with the Dynabeads mRNA Purification Kit (Invitrogen, Karlsruhe, Germany). cDNA was then synthesized from the mRNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St Leon-Rot, Germany) or the SuperScriptII reverse transcriptase (Invitrogen, Carlsbad, CA, USA) by priming with polyT primer []. ALC-, FUL-, IND-, RPL-, and SHP1/2-like genes were amplified via PCR from Erucaria and Cakile cDNA. In order to establish copy number, we used a set of broadly degenerate primers that picked up other related loci and used multiple primer combinations (see Additional file : Table S1, for primer information and sequences). Primers were designed using mostly a nested primer design and by comparison to known sequences, particularly within Arabidopsis thaliana. PCR products were cloned into TOPO-TA (Invitrogen, Carlsbad, CA, USA). An average of 42 independent clones were sequenced per identified locus with a minimum number of 10 sequenced. Restriction analysis was also employed to screen colonies for potentially different copies.Sequences were manually edited in MacVector v. 11.1 (MacVector Inc., Cary, NC, USA) and putative gene identity was initially established with BLASTn searches. To confirm gene homology, phylogenetic analyses were then conducted. Amino acid sequences were aligned with GenBank sequences (Additional file : Table S2) in either MacVector or BioEdit v.7.0.9.0 []. Neighbor-joining (NJ) trees were generated in MEGA v.5 []. As we are primarily interested in identifying homologs, not reconstructing phylogenies to infer relationships amongst genes, the NJ approach was sufficient to assess homology. […]

Pipeline specifications

Software tools MacVector, BLASTN, BioEdit, MEGA-V
Applications Phylogenetics, GWAS
Organisms Arabidopsis thaliana