Mutations in the Alpha-D-Galactosidase Gene Suggest Molecular Basis of the Mutant "Makapuno" Coconut (Cocos nucifera L.) Phenomenon

Alpha-D-galactosidases modulate the post -depositional degree of galactose substitution in galactomannans in the cell walls during seed development. The natural mutant makapuno is reported to have a deficiency 'of the alpha-D-galactosidase enzyme. To elucidate the possible explanation of this deficiency, the genomic sequences of the alpha-D-galactosidase gene of normal and makapuno coconut (Cocos nucifera L.) were isolated and characterized. Genomic DNA was extracted from young leaflets of normal and makapuno coconut using the cetyltrimethylammonium bromide (CTAB) DNA extraction method. Primers for the gene designed using Primer Basic Local Alignment System Tool (BLAST) and NetPrimer softwares were used for polymerase chain reaction (PCR) amplification of genomic DNA. The PCR products were gel extracted using Vivantis GF-1 Ambiclean kit and sequenced using ABI3730XL (Macrogen, Inc., Korea). Results revealed that the obtained sequence is the alpha-D-galactosidase. The nucleotide sequences were 1,149 bp in length for both normal and makapuno designated as ngal and mgal, respectively. BLASTn analysis revealed 93% identity to alpha-D-galactosidases of coconut. In silico translation revealed that the nucleotide sequences coded for 383 amino acids for the normal and makapuno coconut. Upon protein BLAST analysis, the identity of the sequence was confirmed to be the Cocos nucifera alpha galactosidase with the presence of conserved domains of glycoside hydrolase super family (IPR017853) and aldolase-type triose-phosphate isomerase (TIM) barrel (IPR013785). Alignment of DNA sequences of normal and makapuno revealed deletions, insertions and substitutions in makapuno.These mutations of the alpha-D-galactosidase gene could be one of the major causes of the makapuno phenomenon.