Theor Appl Genet (2004) 109:361–369 DOI 10.1007/s00122-004-1635-7 ORIGINAL PAPER Cherdsak Liewlaksaneeyanawin · Carol E. Ritland · Yousry A. El-Kassaby · Kermit Ritland Single-copy, species-transferable microsatellite markers developed from loblolly pine ESTs Received: 14 July 2003 / Accepted: 9 February 2004 / Published online: 26 March 2004  Springer-Verlag 2004 Abstract Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the “markers of choice” in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers devel- oped for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species trans- ferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of mi- crosatellites, confirming that EST-SSRs are more con- served than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine. Introduction In the past several years, the advent of molecular markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and microsatellites [or simple sequence repeats (SSRs)] have helped greatly in population genetic studies in areas such as gene diversity, mating systems, and gene mapping. Microsatellites are often regarded as the “marker of choice”, being codominant and showing high variability but requiring significant investment to develop, as primer pairs specific to the microsatellite locus must be designed. Microsatellite markers can be developed in several ways: via genomic libraries, enriched genomic libraries, BAC/ YAC libraries, and cDNA libraries (Scott 2001). How- ever, reliable microsatellites are difficult to develop for conifer species due to their large genome size and the extensive repetitive nature of their DNA (Kinlaw and Neale 1997). Often, complex banding patterns (multiple loci) are obtained because of duplications, and null alleles are more frequent due to variation at primer binding sites. Several approaches have been applied to eliminate highly repetitive DNA in conifer libraries. Elsik and Williams (2001) developed microsatellites in loblolly pine using a low-copy-enrichment method. In their study, they suggested that low-copy microsatellites provided more polymorphic and informative markers than total genomic microsatellites. Zhou et al. (2002) also reported an alter- native method of microsatellite development in loblolly pine. Using a methylation-sensitive restriction enzyme (McrBC), they developed microsatellites from a library of undermethylated (UM) DNA. Although this method only eliminated some of the highly repetitive DNA and pro- vided a single-locus inheritance microsatellite, the level of polymorphisms for UM microsatellites was lower than that of low-copy microsatellites. Scotti et al. (2000) developed six microsatellite loci by screening a cDNA library in Picea abies for repeats. They reported that four of the six expressed sequence tag mi- crosatellites (EST-SSRs) that they found provided clear banding patterns and a high level of polymorphism. This Communicated by O. Savolainen C. Liewlaksaneeyanawin ( ) ) · C. E. Ritland · Y. A. El-Kassaby · K. Ritland Department of Forest Sciences, University of British Columbia, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada e-mail: cliewlak@interchange.ubc.ca Tel.: +1-604-8225609 Fax: +1-604-8229102