Application of inter simple sequence repeat (ISSR) markers to plant genetics.
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.
Godwin
I D
ID
Department of Agriculture, University of Queensland, Brisbane, Australia. i.godwin@mailbox.uq.edu.au
Aitken
E A
EA
Smith
L W
LW
D001345:Autoradiography; D016172:DNA Fingerprinting; D018744:DNA, Plant / Q000032:analysis; D002523:Edible Grain / Q000235:genetics; D005638:Fruit / Q000737:chemistry; D017343:Genes, Plant; D005819:Genetic Markers; D018598:Minisatellite Repeats; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D018744:DNA, Plant; D005819:Genetic Markers