Sequences reported here have been submitted to Genbank as Accession numbers AF 076679, AF 076680 Communicated by P. Langridge S. Rahman ( ) · S. Abrahams · R. Appels · M. K. Morell CSIRO Plant Industry, P.O. Box 1600, ACT 2601, Australia Fax:# 61-6-246-5000 E-mail: s.rahman@pican.pi.csiro.au Z. Li · D. Abbott Cooperative Research Centre for Plant Science, P.O. Box 1600, ACT 2601, Australia Theor Appl Genet (1999) 98 : 156—163  Springer-Verlag 1998 S. Rahman · Z. Li · S. Abrahams · D. Abbott R. Appels · M. K. Morell Characterisation of a gene encoding wheat endosperm starch branching enzyme - I Received: 14 January 1998 / Accepted: 14 July 1998 Abstract A genomic DNA fragment from ¹riticum tauschii, the donor of the wheat D genome, contains a starch branching enzyme-I (SBE-I) gene spread over 6.5 kb. This gene (designated wSBE I-D4) encodes an amino acid sequence identical to that determined for the N-terminus of SBE-I from the hexaploid wheat (¹. aestivum) endosperm. Cognate cDNA sequences for wSBE I-D4 were isolated from hexaploid wheat by hybridisation screening from an endosperm library and also by PCR. A contiguous sequence (D4 cDNA) was assembled from the sequence of five overlapping partial cDNAs which spanned wSBE I-D4. D4 cDNA encodes a mature polypeptide of 87 kDa that shows 90% ident- ity to SBE-I amino acid sequences from rice and maize and contains all the residues considered essential for activity. D4 mRNA has been detected only in the en- dosperm and is at a maximum concentration mid-way through grain development. The wSBE I-D4 gene con- sists of 14 exons, similar to the structure for the equiva- lent gene in rice; the rice gene has a strikingly longer intron 2. The 3 end of wSBE I-D4 was used to show that the gene is located on group 7 chromosomes. The sequence upstream of wSBE I-D4 was analysed with respect to conserved motifs. Key words ¹riticum tauschii · Starch branching enzyme genes · Wheat · Endosperm Introduction Starch is an important constituent of the wheat grain, accounting for approximately 65% of the weight of the grain at maturity. It is produced in the amyloplast of the endosperm by the concerted action of a number of enzymes that include ADPglucose pyrophosphorylase (EC 2.7.7.27), granule-bound and soluble starch syn- thases (EC 2.4.1.21), branching enzymes (EC 2.4.1.18) and debranching enzymes (EC 3.2.1.41 and EC 3.2.1.68) (Martin and Smith 1995; Morell et al. 1995). Some of the proteins involved in the synthesis of starch can be recovered from the starch granule (Denyer et al. 1995; Rahman et al. 1995). Branching enzymes catalyse a transglycosylation reaction in which the reducing terminus produced by the hydrolysis of an 1,4 glucan is linked to the C6 hydroxyl of another 1,4 linked glucosyl residue. This reaction is the only known mechanism for the introduc- tion of 1,6 linkages in starch. There are two types of branching enzymes in plants, starch branching enzyme- I (SBE I) and starch branching enzyme-II (SBE-II), and both are about 85—90 kDa in mass. At the nucleic acid level there is about 65% sequence identity between types I and II in the central portion of the molecules; the sequence identity between SBE I from different cereals is about 80% overall (Burton et al. 1995; Morell et al. 1995). While SBE-I and SBE-II catalyse identical reactions, evidence from mutational and gene sup- pression experiments demonstrate that the enzymes differ in their roles, and biochemical evidence suggests that they differ in their patterns of action (Guan et al. 1997). In maize (Boyer and Preiss 1981), rice (Mizuno et al. 1993) and pea (Smith 1988), null mutations in SBE-II reduce starch branching and lead to a high amylose phenotype (amylose extender in maize). In con- trast, the partial suppression by antisense of SBE-I activity in the potato tuber leads to subtle alterations in starch physico-chemical properties but not to