Variation in Granule Bound Starch Synthase I (GBSSI) loci amongst Australian wild cereal relatives (Poaceae) F.M. Shapter * , P. Eggler, L.S. Lee 1 , R.J. Henry Grain Foods CRC, Centre for Plant Conservation Genetics, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia article info Article history: Received 30 July 2007 Received in revised form 12 November 2007 Accepted 20 June 2008 Keywords: Australian native grasses Starch Crop wild relatives Cereal Phylogenetic abstract A complex cascade of enzymes is responsible for the development of starch granules in grain endosperm. Granule Bound Starch Synthase I (GBSSI), encoded by the Waxy gene, is a key enzyme of starch synthesis and determines the accumulation of amylose in the starch granules. The complete genomic GBSSI sequence was ascertained for eight Australian cereal wild relatives (CWR) to determine diversity within the gene. A phylogeny derived from the coding sequence of the entire Waxy gene was compared to established phylogenetic relationships. Starch granule morphology observed in conjunction with this phylogeny suggests that small polygonal starch granules arranged as compound granules are the ancestral state, evolving subsequently to bimodal starch granules and to larger simple granules. Genomic sequence length varied within the species from 2800 to 3572 bp. Most variation occurred within the intron sequences, the largest insertion showing strong homology to a retrotransposon. One wild species was determined to have a deletion in the 3 0 -end of exon 1 resulting in a putatively non-functional allele. Alignment of the amino acid sequence showed strong homology throughout the central fragments of the gene but broad variation in the transit peptides. All putative functional alleles maintained the reported active sites for glycogen synthesis, though with variations in other highly conserved areas of the gene. These variations within the wild relatives of cultivated cereals may provide novel sources of genetic diversity for future cereal improvement programs. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Granule Bound Starch Synthase I (GBSSI), encoded by the Waxy gene, is responsible for the accumulation of amylose during the development of starch granules in cereal endosperm. GBSSI has been reported with changing enzyme nomenclature over the last two decades. The most recent, EC 2.4.1.242, clearly delineates this enzyme from previously recorded enzymes EC 2.4.1.11 (Taira et al., 1995) and EC 2.4.1.21 (Baldwin, 2001; Demeke et al., 1999), as being able to use both UDP-glucose and ADP-glucose as a substrate for starch synthesis. Although GBSSI and II share the same enzyme nomenclature and approximately 69% sequence identity, suggest- ing that they are homologous, the genes encoding them are situated at independent loci. The gene encoding GBSSI is predom- inantly expressed in endosperm and pollen whereas GBSSII is expressed in the leaf, culm and pericarp (Vrinten and Nakamura, 2000). Equally well reported is that cereal species with a null allele at all Waxy loci will produce an amylose free endosperm. The amylose status of cereal starches affects their functional properties and therefore their end uses (Domon et al., 2002; Gaines et al., 2000; Kim et al., 2003) and a simple, rapid iodine test for the presence/absence of amylose in endosperm has been recently optimised (Pedersen et al., 2004). Within cultivated cereals Waxy mutants are common from induced (Nakamura et al., 1995) and naturally occurring origins (Domon et al., 2002), with mutations remaining conserved in the genome over generations. The concentration of Waxy mutants occurring in cereal wild relatives (CWR) is as yet undetermined. Of the waxy and low-amylose cereal species sequenced to date, it appears that there is no specific polymorphism across all the species which results in the waxy phenotype. Rather, any disrup- tion to the production or regulation of this enzyme is critical in cereal species (Mangalika et al., 2003; Rahman et al., 2000; Shure et al., 1983; Yan et al., 2000). Within any one cereal species, multiple alleles of the GBSSI coding gene are common and are denoted by a subscript or chromosomal reference which is species specific (Mangalika et al., 2003; Pedersen et al., 2007). In the case of polyploid species such as wheat, the number of functional GBSSI coding alleles is correlated with the concentration of amylose in the grain (Mangalika et al., 2003). The phylogenetic utility of the GBSSI encoding gene has been repeatedly reported (Evans and Campbell, 2002; Mason-Gamer * Corresponding author. Tel.: þ61 2 6620 3466; fax: þ61 2 6622 2080. E-mail address: frances.shapter@scu.edu.au (F.M. Shapter). 1 Present address: Centre for Tropical Crops and Biocommodities, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs 0733-5210/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2008.06.013 Journal of Cereal Science 49 (2009) 4–11