Alternative Splicing in the Gbssi Gene Involved in Amylose-Free Cassava

Introduction: Cassava is an important source of starch in tropics. Cassava starch derivatives are traded in the international starch market making it a potential cash crop for small holder farmers. Diversity of starch sources in cassava was unknown until 2007 when a source of amylose-free starch (waxy) was discovered at the International Center for Tropical Agriculture (CIAT). The GBSSI gene is responsible for this trait.

Results: Phylogenetic analysis of waxy and non-waxy genotypes showed a clear evolutionary and functional division with a waxy and a non-waxy cluster indicating a complex gene structure for this trait. Sequence analysis identified between two and eight allelic variants of the GBSSI gene indicating that the waxy trait may be controlled by more than one form of the GBSSI allele. We found evidence of alternative splicing in the GBSSI gene. This finding was further validated by transcript reconstruction by cDNA cloning and RNAseq approach. Transcript reconstruction gave evidence of alternative splicing, mainly driven by exon skipping or intron retention and the presence of multiple open-reading frames. These variations at transcript level contribute to the protein diversity in GBSSI.

Conclusions: We confirm multiple allelism through polymorphisms between the overlapping fragments after cloning and sequencing approach, suggesting the internal variability within M. esculenta for GBSSI gene. Taken together, our results indicate the existence of new isoforms for GBSSI gene and constitutes the first report of alternative splicing phenomenon in cassava.