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

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.

Genetic characterization of Iranian safflower (Carthamus tinctorius) using inter simple sequence repeats (ISSR) markers

“…Amplified fragment length polymorphism (AFLP), isozymes, simple sequence repeats (SSR), random amplified microsatellite polymorphisms (RAMP), random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) have all been used to determine genetic diversity in plant populations (Wang et al 1994;Godwin et al 1997;Hollngsworth et al 1998;Blair et al 1999;Amsellem et al 2000;Mengoni et al 2000). Microsatellites or simple sequence repeats are tandemly repeated mono-, di-, tri-, tetra-or pentanucleotide units (Sehgal et al 2009).…”

Genetic structuring in the Pyramid Elimia, Elimia potosiensis (Gastropoda, Pleuroceridae), with implications for pleurocerid conservation

“…First, choice of our primer sequences was probably not ideal, employing dinucleotide repeats and/ or degenerate 5'-bases ahead of the repeated portion. Results from the literature suggest that trinucleotide repeats and 3'-degenerate anchors after the repeats increase resolution and repeatability (Godwin et al 1997). Second, we visualized our bands on agarose instead of polyacrylamide.…”

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

“…The advantages of ISSR markers are well documented in agronomic species (Godwin, Aitken & Smith, 1997). Genome analysis in woody perennials using ISSR has also been shown to be possible in Douglas fir and Sugi (Tsumura et al, 1996), Citrus Fang et al, , 1998, Cocoa (Charters & Wilkinson, 2000), Larix (Arcade et al, 2000) and Morus (Awasthi et al, 2004).…”