Characterization and expression analysis of Isoamylasei in cassava and its wild relatives
Characterization of starch genes in Manihot esculenta is limited. The cDNA (764 bp) of Isoamylase1 was isolated from the cassava storage roots; its copy number, spatial and temporal expression patterns were studied. Moreover, comparative single nucleotide polymorphisms (SNPs) of two sugar metabolizing genes (Isoamylase1 and N- glyceraldehyde 3-phosphate dehydrogenase) were studied between cultivated cassava and its wild relatives (M. esculenta subsp. flabellifolia). The BLAST similarity search showed Isoamylase1 has the highest nucleotide (90 %) and protein (92 %) identity with Recinus communis Isoamylase1. The spatial and temporal expression studies showed that Meisa1 is highly expressed in fibrous roots and in early stages of storage roots development. Isoamylase1 exist in at least two copies in the genome. 49 SNPs, 31 (1,317 bp) in cultivated and 18 (1,271 bp) in wild, and one InDel (G3pdh of wild) were identified. The estimated SNPs frequencies were 1/42 bp in cultivated and 1/70 bp in wild. Nucleotide polymorphisms (θw) suggested that two randomly chosen wild or cultivated cassava sequences vary 1 in ~250 or ~111 nucleotides and 1 in ~90 or ~250 nucleotides for Meisa1 and G3pdh, respectively. This suggests that Meisa1 is more diverse in cultivated cassava than in subsp. flabellifolia whereas G3pdh is more diverse in the wild. Tajima’s D showed that Meisa1 has been evolving under different selection pressures, diversifying in cultivated and purifying in wild. The abundance of Meisa1 in fibrous roots and early stage of tuber development suggests that Meisa1 is involved in differentiation of fibrous roots into storage roots. The highest genetic diversity of Meisa1 observed in cultivated than the wild could suggest a role of Isoamylase1 in improved quality starch trait in cultivated than wild. The results showed that G3pdh and Meisa1could are good markers for phylogeny and intra- and inter-cultivar diversity studies, respectively.