Computational protocol: Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa

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Protocol publication

[…] The sequences of the B. rapa AsA D-Man/L-Gal pathway genes were retrieved from the Brassica database (BRAD), according to previous reports (). The genome data set of B. oleracea was downloaded from Bolbase (), and that of B. napus was downloaded from the Brassica napus Genome Browser (). The gene sequences from Vitis vinifera, Carica papaya, Populus trichocarpa, and Amborella trichopoda were downloaded from Phytozome v9.1 (). The sequences of the B. oleracea and B. napus homologs to these AsA-related genes in B. rapa were identified through a BLASTp search (E-value 1e ≤ 20, identity ≥ 40%) (). The GGP homologs in V. vinifera, C. papaya, P. trichocarpa, and A. trichopoda genomes were identified in the same way. Then, we verified these sequences in the NCBI database. [...] The position of each AsA D-Man/L-Gal pathway gene on the syntenic blocks was verified by searching for homologs among A. thaliana and the LF, MF1 and MF2 subgenomes of B. rapa, B. oleracea, and B. napus using BRAD (last accessed January 8, 2015) (). Conservation of chromosomal synteny around the GGPs in A. thaliana, A. trichopoda, C. papaya, P. trichocarpa, V. vinifera, and B. rapa was evaluated with CoGe. An in-house Perl program was used to draw the syntenic diagram.The potential duplicated genes in the B. rapa, B. oleracea and B. napus genomes were identified using MCScanX (). The resulting blast hits were incorporated, along with the chromosome coordinates of all protein-coding genes, as an input for MCScanX and classified into segmental, tandem, proximal and dispersed duplications under the default criteria.The set of core eukaryotic genes and a set of randomly selected genes from the microsyntenic regions corresponding to the AsA D-Man/L-Gal pathway genes and a set of genes flanking the A. thaliana AsA D-Man/L-Gal pathway genes (10 on either side) were established according our previous study (). [...] The amino acid sequence alignments of the full-length protein sequences of the AsA-related genes were aligned with the MUSCLE program using the default parameters (; ). The phylogenetic trees were then constructed with the ML method in each analysis by using MEGA 5.2 (). The confidence level of the monophyletic clade was estimated using a bootstrap analysis of 1,000 replicates.The putative AsA-related protein sequences used for the phylogenetic analysis were detected by MEME program version 4.9.0 to analyze the possible conserved motifs by using default parameters (), except for the following parameters: the maximum number of motifs was set to 10 and the optimum motif width was set to ≥10 and ≤100. […]

Pipeline specifications

Software tools BLASTP, MCScanX, MUSCLE, MEGA, MEME
Applications Genome annotation, Phylogenetics, Nucleotide sequence alignment, Genome data visualization
Organisms Brassica rapa, Brassica oleracea, Brassica napus, Homo sapiens
Chemicals Ascorbic Acid, Aspirin, Sodium Chloride