Molecular Ecology Notes (2001) 1, 205–208 © 2001 Blackwell Science Ltd Blackwell Science, Ltd TECHNICAL NOTE Intersimple sequence repeat (ISSR) polymorphisms as a genetic marker system in cotton BAO LIU and JONATHAN F. WENDEL Department of Botany, Bessey Hall, Iowa State University, Ames IA 50011, USA Abstract We studied the applicability of intersimple sequence repeat (ISSR) polymorphism in cotton. We found that: (i) the resolving power of agarose gels is poor relative to that provided by sequencing gels; (ii) fluorescent labelling of ISSR amplification primers produced numerous scorable bands; (iii) primer mixing (double priming) generated more bands than the sum of fragments resulting from two single primers, although an unexplained dis- appearance of several larger fragments also reproducibly occurred; (iv) ISSR fingerprinting patterns are highly heritable; and (v) double priming ISSR is an easy and informative genetic marker system in cotton for revealing both inter- and intraspecific variations. Keywords: cotton, fluorescent-labelling, Gossypium, intersimple sequence repeat (ISSR) polymorphism, primer mixing Received 31 January 2001; revision accepted 22 March 2001 Microsatellites or simple sequence repeats (SSR) are short DNA sequence stretches consisting of motifs of one to six bases that are tandemly repeated. Owing to their ubiquity, hypervariability, abundance and genome-wide distribution, SSR loci represent a new generation of powerful genetic markers for eukaryotes. Use of this marker system, however, is hampered by the requirement for sequence information from flanking regions, from which primers are designed for polymerase chain reaction (PCR) amplification. Discovery and characterization of a large number of SSRs is therefore time-consuming and expensive for many taxa. A recently developed modification of SSR-based marker systems, i.e. ISSR (intersimple sequence repeat) analysis, circumvents this requirement for flanking sequence informa- tion, and thus has found wide applicability in a variety of plants. Popularized largely by A. Wolfe and colleagues (Wolfe et al . 1998), ISSR analysis involves PCR amplifica- tion of genomic DNA using a single primer that targets the repeat per se , with 1– 3 bases that anchor the primer at the 3 ′ or 5 ′ end. In addition to freedom from the necessity of obtaining flanking genomic sequence information, ISSR analysis is technically simpler than many other marker systems. The method provides highly reproducible results and generates abundant polymorphisms in many systems. Most applications to date have used either agarose gel electrophoresis with ethidium bromide detection (Nagaoka & Ogihara 1997; Joshi et al . 2000) or polyacrylamide gel electrophoresis (PAGE) separation combined with either silver staining or isotopic detection (Blair et al . 1999). Cotton ( Gossypium ) provides the world’s most important textile fibre. Genetic diversity in cultivated cotton has been studied using several approaches (Wendel et al . 1992; Brubaker & Wendel 1994; Pillay & Myers 1999; Liu et al . 2000). A consensus reached by these studies is that genetic variation among cotton cultivars is very low. Thus, more efficient marker systems are needed for both breeding applications and basic evolutionary/ecological studies. A recent modification of ISSR analysis utilizes fluorescein- labelling of amplification primers and automated detection on sequencing gels (Huang & Sun 2000). Here we extend these modifications and in addition demonstrate the flexibility of ISSR analysis to incorporate data on genome size and relative SSR frequency into experimental design. To assess the efficiency of ISSR markers in revealing polymorphisms in cotton, nine accessions of Gossypium hirsutum and two accessions of G. barbadense were selected (Fig. 1c,d). These same accessions were used in earlier studies of allozyme and restriction fragment length poly- morphism (RFLP) variation, permitting a comparison of techniques. To test whether ISSR markers were stably Correspondence: Jonathan F. Wendel. Fax: 515 294 1337; E-mail: jfw@iastate.edu