Computational protocol: Fine mapping and epistatic interactions of the vernalization gene VRN-D4 in hexaploid wheat

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Protocol publication

[…] To delimit the region on B. distachyon that is colinear with the VRN-D4 candidate region in wheat, sequences of the wheat Expressed Sequence Tags (ESTs) flanking VRN-D4 were used as queries to search the B. distachyon genome (Brachy1.0 database, http://www.brachypodium.org/) using the BLASTN algorithm. The B. distachyon genes were then used to find the rice orthologs at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and Department of Energy’s Joint Genome Institute and the Center for Integrative Genomics website (http://www.phytozome.net). Since rice does not have a vernalization response and is evolutionarily less related to wheat compared to B. distachyon, we focused on the B. distachyon colinear region to search for potential VRN-D4 candidate genes.Conserved domains present in the B. distachyon proteins encoded by the genes present in the colinear candidate region were analyzed using the Conserved Domain Search at NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and InterPro Scan at EMBL-EBI (http://www.ebi.ac.uk/Tools/pfa/iprscan/).Based on this information and on the annotation of these genes in other species, we identified transcription factors and genes known to be involved in development. We searched for the closest wheat homologs in wheat NCBI databases, genomic sequences from flow-sorted wheat chromosomes arms developed by the International Wheat Genome Sequencing consortium (IWGSC, http://urgi.versailles.inra.fr), and recent wheat transcriptomes (Krasileva et al. (), http://maswheat.ucdavis.edu/Transcriptome/). We then did a reverse BLASTP of the best wheat candidate to the B. distachyon proteome to confirm that the correct homolog was found. […]

Pipeline specifications

Software tools BLASTN, InterPro, BLASTP
Databases Phytozome
Applications Phylogenetics, Protein sequence analysis
Organisms Triticum aestivum, Brachypodium distachyon, Arabidopsis thaliana