Phylogenetic Approaches to Ortholog Identification in Sunflower (Helianthus annuus, Compositae)

FUSARI C.; HEINZ R.; PANIEGO N.; LIA V.V.

Punta del Este, Uruguay

Congreso; Darwin 200. A South American celebration.; 2009

Determination of gene orthology is a prerequisite for mining and utilizing the rapidly increasing amount of sequence data for genome-scale phylogenetics and comparative genomic studies. Commonly, most researchers use pairwise distance comparisons algorithms, such as BLAST, COG, RBH, RSD and INPARANOID, to determine gene orthology (Chiu et al., 2006). In contrast, orthology determination within a character-based phylogenetic framework has been only partially exploited due to the lack of efficiency and automation, and because is restricted to model species (Chiu et al., 2006). Here we present a protocol of Ortholog Identification in a phylogenetic context using cladistic and probabilistic reconstruction methods. The routine starts with the optimization of parameters and settings in searching and sequence recovery limits for the different BLAST algorithms (BLASTx, tBLASTx) (Altschul et al., 1990), using as query the transcript of the model species which ortholog is about to identify in the species of interest. The goal is to obtain, besides the sequence of the species of interest, a group of related sequences representative of different plant families. Subsequently, the group of sequences selected are traduced into the correct open reading frame and then aligned together using the different routines implemented in MAFFT software (Katoh et al., 2002). The phylogenetic relationships are inferred by 2 strategies: (a) Maximum Parsimony: the traditional trees searching by heuristic methods are complemented by new technology searches proposed by Goloboff (1999); and (b) Maximun Likelihood: the model of election is chosen using ProtTest software (Abascal et al., 2005) and the searches are run through PhyML (Guindon et al., 2003). Finally, the ortholog and paralog sequences of the species of interest are determined based on the previous reconstruction results obtained. This routine was applied in the search of candidate genes related to resistance in Sunflower Head Rot disease, caused by the fungus Sclerotinia sclerotiorum. Arabidopsis’ sequences were used as query since they are the only information available about gene expression profiles associated with fungus infection (Zhao et al., 2007). As starting point of experimental validation, the aligned orthologs identified by this routine were used as template for the design of degenerated primers using iCODEHOP software (Boyce et al., 2009).