Rapid identification of Amaranthus caudatus and Amaranthus hypochondriacus by sequencing and PCR-RFLP analysis of two starch synthase genes.

The objective of this study was to develop a PCR-RFLP method to identity the cultivated species of grain amaranth based on variations in the sequences of their starch synthase genes. We sequenced the SSSI and GBSSI loci in 126 accessions of cultivated grain amaranth collected from diverse locations around the world. We aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. Our analyses indicated that EcoRI would recognize the sequence 5'-GAATT/C-3' in the SSSI gene from Amaranthus caudatus L., and TaqI would recognize the sequence 5'-T/CGA-3' in the GBSSI gene from Amaranthus hypochondriacus L. The PCR products obtained using gene-specific primers were 423 bp (SSSI) and 627 or 635 bp (GBSSI) in length. These products were cut with different restriction enzymes resulting in species-specific RFLP patterns that could be used to distinguish among the cultivated grain amaranths. The results clearly showed that A. caudatus and A. hypochondriacus were easily differentiated at the species level using this method. Therefore, the PCR-RFLP method targeting amaranth starch synthase genes is simple and rapid, and it will be a useful tool for the identification of cultivated species of grain amaranth.