Molecular analysis of Arabidopsis thaliana transparent testa (tt) genes in Brassica napus Tina Lotz 1 , Rod Snowdon 1 , Renate Horn 2 , Gieta Dewal 3 , Bernd Weisshaar 3 , Wolfgang Friedt 1 , Harry Belcram 4 , Michel Caboche 4 and Boulos Chalhoub 4 1 Institute of Crop Science and Plant Breeding I,Justus-Liebig-University, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, e-mail: tina.lotz@agrar.uni-giessen.de 2 Department of Genetics & Biochemistry, University of Clemson, 100 Long Hall, Clemson, SC 29634-0324, USA 3 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Köln, Germany 4 Unité de Recherches en Génomique Végétale - Institut National de Recherche Agronomique, 2, rue Gaston Crémieux, CP 5708, 91057 Évry Cedex, France ABSTRACT The yellow seed colour trait, which is controlled by flavonoid biosynthesis, is of particular interest for rapeseed breeding because of the associated improvements in feed grain quality after oil extraction. Numerous genes involved in flavonoid biosynthesis have been identified in Arabidopsis thaliana that cause when mutated the transparent testa (tt) phenotype. We are applying the accumulated knowledge from A. thaliana to oilseed rape (Brassica napus), the most important European oilseed crop. Our goal is to identify and characterise homologous genes implicated in yellow seed colour of oilseed rape. Oligonucleotide primers derived from A. thaliana sequence data for different tt loci were used to identify corresponding sequences in oilseed rape by PCR screening of a Brassica napus BAC library. Positive BAC clones are being analysed in detail to determine gene structure in a polyploid context. Key words: seed colour – transparent testa – tt loci – Arabidopsis – candidate genes INTRODUCTION The flavonoid biosynthetic pathway has been studied in detail at the biochemical and genetic level in a number of plant species, particularly in Zea mays, Petunia hybrida, Antirrhinum majus and the model flowering plant Arabidopsis thaliana (Winkel-Shirley 2001). With regard to the latter, numerous loci that play a role in the synthesis of flavonoids have been studied in detail (Shirley et al. 1995; Graham 1998) and insertional mutagenesis experiments indicate that several additional loci are also involved (Wisman et al. 1998). Gene loci that affect the synthesis of brown pigments (condensed tannins) in the A. thaliana seed coat are collectively named transparent testa (tt) loci. The locus TT1 is involved in development of the seed endothelium, in which brown tannin pigments accumulate (Sagasser et al. 2002). Several further loci (TT3, TT4, TT5, TT6 and TTG) are also required for the accumulation of purple anthocyanins in leaves and stems, and TTG also plays additional roles in trichome and root hair development (Koornneef 1994). Numerous single-copy A. thaliana flavonoid biosynthetic genes have been identified and correlated with specific loci: chalcone synthase (CHS) with TT4 (Feinbaum and Ausubel 1988), chalcone isomerase (CHI/CFI) with TT5 (Shirley et al. 1992), dihydroflavonol 4-reductase (DFR) with TT3 (Shirley et al. 1992), flavanone 3- hydroxylase (F3H) with TT6 (Wisman et al. 1998) and flavanone 3’-hydroxylase (F3’H) with TT7 (Schoenbohm et al. 2000), respectively. The different tt homologs identified in B. napus will be sequenced and compared both to each other and to orthologues identified in other species. Genetic distances between the different gene loci will be established and it is hoped that the homoeologous loci can ultimately be mapped in B. napus and compared to seed colour QTL identified in a project funded by the German genome programme “GABI” (see Badani et al. 2003), giving important information about the genome organisation of yellow seed colour genes in Brassica napus.