ORIGINAL PAPER C. Tamborindeguy Æ C. Ben Æ T. Liboz Æ L. Gentzbittel Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower Received: 3 August 2003 / Accepted: 3 February 2004 / Published online: 9 March 2004 Ó Springer-Verlag 2004 Abstract Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs rep- resented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previ- ously reported for sunflower. Several potentially inter- esting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The li- braries described here represent an original and valuable resource for the discovery of yet unknown genes puta- tively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos. Keywords Sunflower Æ Embryogenesis Æ In vitro protoplast culture Æ Seed development Æ Expressed Sequence Tags (ESTs) Introduction The structural establishment of the dicotyledonous (di- cot) embryo occurs under the influence of genes that control complex networks of molecular interactions. Although several genes implicated in embryo develop- ment have been identified in recent years (Ju¨rgens et al. 1997; Souter and Lindsey 2000; Chaudhury et al. 2001; Jurgens 2001), the puzzle is still far from being fully assembled. Arabidopsis thaliana mutants that display abnormal embryo development have greatly facilitated the identification of genes expressed in the developing embryo (Torres-Ruiz and Ju¨rgens 1994; Busch et al. 1996; Hardtke and Berleth 1998; Chen et al. 2001). However, although the morphological course of pattern development in Arabidopsis is well known (West and Harada 1993; Goldberg et al. 1994; Ju¨rgens 1995, 2001), analysis of the early stages of embryogenesis at the molecular level is difficult, because the zygote is tightly surrounded by maternal tissues. In angiosperms, embryogenesis begins with a double fertilization event: pollen nuclei fertilize the egg cell and the central cell to form the zygote and the endosperm, respectively. The zygote then divides asymmetrically to give rise to a large basal cell and a small apical cell. The basal cell forms the suspensor, an embryonic structure that senesces during early embryogenesis, leaving only the uppermost cell, which becomes part of the embryonic root. The apical cell develops into the embryo proper. Embryo develop- ment proceeds along two main axes: apical-basal and radial. Since the young embryo develops deep within the maternal tissues, making it very difficult to investigate using conventional methods (West and Harada 1993; Goldberg et al. 1994; Ju¨rgens 2001), alternative ap- proaches, such as the study of somatic embryogenesis or the use of Fucus (an alga) as a model (Zimmerman 1993; Belanger and Quatrano 2000), have sometimes been adopted. In recent years, Helianthus annuus, a member of the Compositae originating from North America but Communicated by R. Hagemann C. Tamborindeguy Æ C. Ben Æ T. Liboz Æ L. Gentzbittel (&) Laboratoire de Biotechnologie etAme´lioration des Plantes, Poˆ le de Biotechnologie Ve´ge´tale, IFR40, Institut Nationale Polytechnique de Toulouse-Ecole Nationale Supe´rieure de Toulouse, 18 Chemin de Borde Rouge, Auzeville, 31326 Castanet Tolosan, France E-mail: gentz@ensat.fr Tel.: +33-562-193596 Fax: +33-562-193587 Mol Gen Genomics (2004) 271: 367–375 DOI 10.1007/s00438-004-0989-5