Application of inter simple sequence repeat (ISSR) markers to plant genetics

Electrophoresis. 1997 Aug;18(9):1524-8. doi: 10.1002/elps.1150180906.

Abstract

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting 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)8RG or (AGC)6TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with 32P or 33P via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoradiography
  • DNA Fingerprinting
  • DNA, Plant / analysis
  • Edible Grain / genetics
  • Fruit / chemistry
  • Genes, Plant*
  • Genetic Markers*
  • Minisatellite Repeats
  • Polymerase Chain Reaction
  • Polymorphism, Genetic
  • Repetitive Sequences, Nucleic Acid*

Substances

  • DNA, Plant
  • Genetic Markers