HORTSCIENCE, V OL. 38(7), DECEMBER 2003 1428 BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES Utility of Blueberry-derived EST-PCR Primers in Related Ericaceae Species Lisa J. Rowland 1 and Anik L. Dhanaraj U.S. Department of Agriculture, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Fruit Laboratory, Bldg. 010A, 10300 Baltimore Avenue, Beltsville, MD 20705 James J. Polashock U.S. Department of Agriculture, Agricultural Research Service, Blueberry and Cranberry Research Center, 125A Lake Oswego Road, Chatsworth, NJ 08019 Rajeev Arora Iowa State University, Department of Horticulture, 139 Horticulture Hall, Ames, IA 50011-1100 Additional index words. cranberry, expressed sequence tags, molecular markers, rhododendron, STS markers, Vaccinium Abstract. Expressed sequence tag-polymerase chain reaction (EST-PCR) markers for DNA fingerprinting and mapping in blueberry (Vaccinium sp.) had previously been developed from expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants. Because EST-PCR markers are derived from gene coding regions, they are more likely to be conserved across popula- tions and species than markers derived from random regions of DNA, such as randomly amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers. In this study, we tested whether many of the EST-PCR primer pairs developed for blueberry are capable of amplifying DNA fragments in other members of the family Ericaceae. In addition, we cloned and sequenced a selection of 13 EST-PCR fragments to determine if they showed homology to the original blueberry cDNA clones from which the EST-PCR primer pairs were derived. Closely related cranberry genotypes (two wild selections of V. oxycoccus L. and two cultivars of V. macrocarpon Aiton, ‘Early Black and ‘Stevens ) and more distantly related rhododendron genotypes (one wild selection each of Rhododendron arboreum Marsh, R. maximum L., and R. ponticum L. and three complex species hybrids, ‘Sonata , ‘Grumpy Yellow , and ‘Roseum elegans ) were used. Of 26 primer pairs tested in cranberry, 23 (89%) resulted in successful amplification and eight of those (35%) amplified polymorphic fragments among the cranberry genotypes. Of 39 primer pairs tested in rhododendron, 29 (74%) resulted in successful amplifica- tion and 21 of those (72%) amplified polymorphic fragments among the rhododendron genotypes. Approximately 50% of the 13 sequenced EST-PCR fragments were found to be homologous to the original blueberry cDNA clones. These markers should be useful for DNA fingerprinting, mapping, and assessing genetic diversity within cranberry and rhododendron species. The markers which are shown to be homologous to the blueberry cDNA clones by DNA sequencing should also be useful for comparative mapping and genetic diversity studies between some genera of the family Ericaceae. Polymerase chain reaction (PCR)-based molecular markers, such as randomly amplified polymorphic DNAs (RAPDs), amplified frag- ment length polymorphisms (AFLPs), simple sequence repeat (SSR) markers, and sequence- tagged-site (STS) markers, have been widely used in recent years for DNA fingerprinting, genome mapping, and population genetic studies. Their popularity is due, in part, to the technical simplicity of setting up PCRs and the small amount of genomic DNA that is required in the reactions. The many expressed sequence tag (EST) databases available today are provid- ing sequences for developing a subcategory of STS markers, termed expressed sequence tag-polymerase chain reaction (EST-PCR) markers. There are a number of advantages to using EST-PCR markers or other cDNA- based markers over random-primed markers for genetic studies. First, they target expressed genes; thus, they should be particularly useful for quantitative trait loci (QTL) mapping. If an EST-PCR marker is found linked to a QTL, it is possible that the gene itself, from which the EST-PCR marker was derived, controls the trait in question. Second, because they are derived from gene coding regions, which are more likely to be conserved across populations and species than noncoding regions, EST-PCR markers should be useful for comparative map- ping studies. Third, EST-PCR markers have the potential for being codominantly inherited, which allows the identification of two different alleles at heterozygous loci in diploid organ- isms. Schubert et al. (2001) reported that six out of seven polymorphic EST-PCR markers developed for Norway spruce [Picea abies (L.) Karst.] were inherited in a codominant fashion. In an effort to obtain markers with these advantages for use in blueberry (Vaccinium L., section Cyanococcus A. Gray, family Eri- caceae), we have been producing ESTs using a cDNA library derived from floral buds of cold acclimated blueberry plants (Rowland et al., 2003a), and have developed a set of EST-based primer pairs with general utility for DNA fingerprinting and mapping (Rowland et al., 2003b). Recently, EST-PCR markers have been developed for many woody plant spe- cies, including loblolly pine (Pinus taeda L.) (Temesgen et al., 2001), black spruce [Picea mariana (Mill.) B.S.P.] (Perry and Bousquet, 1998), Norway spruce (Schubert et al., 2001), and sugi (Cryptomeria japonica D. Don) (Tsumura et al., 1997). Generally, amplification using EST-PCR primers must be followed by digestion with restriction enzymes to gener- ate cleaved amplified polymorphic sequences (CAPS) markers, heteroduplex analysis, or single-stranded conformational polymorphism (SSCP) analysis to detect polymorphisms (Cato et al., 2001). In the case of blueberry, half (15 out of 30) of the tested EST-PCR primer pairs resulted in amplification of polymorphic frag- ments that were detectable directly after ethid- ium bromide staining of agarose gels (Rowland et al., 2003b). The fact that blueberry species are primarily outcrossing and exhibit low to moderate levels of self-fertility (Galletta and Ballington, 1996) may explain the high percentage of polymorphic EST-PCR mark- ers observed without any need for additional manipulation or analysis. Although there are many advantages to using EST-PCR markers, considerable cost is involved in terms of time, money, and other resources in developing this type of marker. RNA must be extracted from sometimes small amounts of the appropriate tissue, a cDNA li- brary must be constructed, hundreds of clones must be picked and plasmid DNA isolated from them, the clones must be sequenced, and primers must be designed, synthesized, and tested. If the EST-PCR primers designed for one species can be used in related species, then the cost involved in developing markers for DNA fingerprinting, genetic relationship studies, mapping, etc. for those other species is significantly reduced. In addition, if the markers are shown to be homologous, they can be used for comparative mapping studies between the species. The ability to transfer mapped STS markers within the Poaceae fam- ily has been demonstrated, both between cereal species (Erpelding et al., 1996) and between cereal and forage grass species (Taylor et al., 2001). STS primers developed from black spruce cDNA sequences have been shown to direct amplification in several different conifer species. Nearly all (95% to 97%) of the black spruce STS primers worked in congeneric tri- als, while some (21% to 33%) worked in other Pinaceae genera as well (Perry and Bousquet, 1998). Here, we have investigated whether HORTSCIENCE 38(7):1428–1432. 2003. Received for publication 31 Dec. 2002. Accepted for publication 22 May 2003. 1 To whom reprint requests should be addressed. E-mail: rowlandj@ba.ars.usda.gov