APPLICATION OF INTER SIMPLE SEQUENCE REPEAT (ISSR) MARKERS TO PLANT GENETICS

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eu karyotic genomes. Single-locus SSR markers have been developed for a n umber of species, although there is a major bottleneck in developing S SR markers whereby flanking sequences must be known to design 5'-ancho rs for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) finge rprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a mult ilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reac tion yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and re port here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis us ually detects a higher level of polymorphism than that detected with r estriction fragment length polymorphism (RFLP) or random amplified pol ymorphic DNA (RAPD) analyses. Our data indicate that this is not a res ult of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.