Functional categorisation of expressed sequence tags from flowers of Eragrostis curvula genotypes showing different ploidy levels and reproductive modes

VIVIANA ECHENIQUE; SILVINA FELITTI; NORMA PANIEGO; GERARDO CERVIGNI; JUAN PABLO SELVA; SILVINA PESSINO; GERMAN SPANGENBERG

San Diego, California

Conferencia; Plant and Animal Genome XIV; 2006

Scherago International Inc.

The aim of this work was the discovery and functional classification of expressed sequence tags (ESTs) from immature inflorescences of the apomictic E. curvula cultivar Tanganyka (2n=4x=40) obtained by colchicines duplication of the dihaploid, in order to identify candidate genes involved in diplospory and/or ploidy level gene regulation. Four cDNA libraries were constructed from sexual dihaploid flowers (01EC), apomicitc tetraploids flowers (02EC), apomicitic tetraploid leaves (03EC) and sexual tetraploids flowers (04EC). Total RNA was extracted from flowers or leaves using the RNeasy® total RNA isolation kit (Promega). The cDNAs were obtained using the SMART® PCR synthesis kit, cloned into pGEM®-T easy vector and used for transformation of XL10-Gold® Ultracompetent E. coli cells. 2,400 randomly selected cDNA clones from each library were sequenced using a MegaBACETM DNA sequence analyzer. Raw EST sequence traces were processed and annotated after the analysis using the XGI pipeline (http://www.ncgr.org/xgi) for automated EST clustering. Processed ESTs were clustered into consensus sequences that were compared using BLASTX. Functional categories were assigned by means of gene ontology (GO) annotations. A total of 2,218 unique sequences (719 for EC01, 564 for EC02, 589 for EC03 and 346 for EC04) were identified. BLASTX analysis revealed no significant similarities at an E-value greater than 10-6 for 54% of the non-redundant sequences. Functional categories were assigned to 402 out of 1198 annotated sequences. The EST collections showed a distinctive distribution among different functional categories.