Species identification in seven small millet species using polymerase chain reaction-restriction fragment length polymorphism of trnS-psbC gene region

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 access ions from seven small millet species were amplified by polymerase chain rea ction (PCR) and digested with eight restriction enzymes individually as wel l as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymor phism between only some species. However, all the species could be distingu ished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding pa tterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhy ncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed int raspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.