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

VIVIANA ECHENIQUE; FELITTI, S.; PANIEGO, N.; CERVIGNI, G.; SELVA, J.P.; PESSINO, S.; SPANGENBERG, G.

San Diego, California

Conferencia; XIV Plant and Animal Genome Conference; 2006

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 Tanganyika (2n=4x=40), a dihaploid sexual strain derived from it (2n=2x=20) and a tetraploid sexual strain (2n=4x=40) obtained by colchicine 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), apomictic tetraploid flowers (02EC), apomictic tetraploid leaves (03EC) and sexual tetraploid flowers (04EC). Total RNA was extracted from flowers or leaves using the RNeasy® total RNA isolation kit (Promega).The cDNAs were obtained using a the SMART® PCR synthesis kit, cloned into the pGEM®-T easy vector and used for transformation of XL10-Gold® Ultracompetent E. coli cells. 2400 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.