A new method for the design of degenerate primers and its use to identify homologues of apomixis - associated genes in brachiaria
Apomixis is a reproductive phenomenon that occurs in flowering plants. It allows a plant to produce asexual seeds, with its same genetic constitution. The existence of a genetic basis for apomixis is crushing, but the molecular mechanisms are unclear. The search for the “master apomixis gene” had led to the isolation of various candidate transcripts, but neither of them could be confirmed in different plant species. Here we tried to isolate homologues to all those transcripts in one unique plant, Brachiaria. In order to achieve this, a new method for degenerate primer design was employed, since classical methods have proven to be unsuccessful. We used multiple local alignments, instead of global, with the Multiple Expectation - Maximization for Motif Elicitation (MEME) algorithm, to find conserved blocks and motifs. These alignments were followed by ePCR simulation and standard primer pair design programs. The method demonstrated to be useful to amplify fragments homologous to genes poorly molecular and biologically characterized, with which multiple global alignments showed no conserved regions. The obtained amplicons showed differential expression according to tissue in some cases. This technique can be used to design degenerate primers in cases where one sequence exhibits poor global similarity and has low biological characterization, and it is useful to amplify orthologous genes in an organism weakly described at the molecular level.
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