A New System for Strawberry Cultivar Identification Developed at Foundation Plant Services ( FPS ) , University of California , Davis , Using Simple Sequence Repeat ( SSR ) Primers

Early and correct cultivar identification of strawberry (Fragaria ×ananassa Duch.), a clonally propagated crop with an octaploid genome, is critical to the industry. Sixteen previously published primer pairs that flanked simple sequence repeats (SSRs) in the strawberry cultivar Earliglow were examined for their ability to distinguish commonly grown strawberry cultivars. Six primer pairs both amplified consistently and produced variable fragment sizes. One primer pair, ARSFL-09, generated unique profiles for 29 of 45 accessions. The remaining 16 fell into eight profile groups of two cultivars each, which were easily resolved with any one of three other primer pairs. Although the octaploid nature of strawberry complicates interpretation, this system can reliably identify the most commonly grown strawberry cultivars at an early developmental stage. Strawberries are California’s seventh most valuable crop, with direct agricultural sales of more than $1.2 billion in 2004 (CDFA, 2006). More than 60% of the state’s strawberry acreage is planted with cultivars developed by the strawberry breeding program at the Department of Plant Sciences, UC Davis (CSC, 2006). Foundation Plant Services (FPS) is the UC service department which produces and distributes virus-tested propagation stock plants of UC-patented strawberry cultivars licensed by the UC Office of Technology Transfer. Cultivated strawberry (Fragaria ×ananassa Duch.) plants are vegetatively propagated. Maintaining correct cultivar identity during this process is critical, a task made difficult because propagation stock is distributed prior to development of distinguishing morphological characteristics. Many biochemical and molecular marker systems have been used for strawberry cultivar identification, including isozymes (Bell and Simpson, 1994), Randomly amplified polymorphic DNA (RAPD) markers (Degani et al., 1998; Garcia et al., 2002; Hancock et al., 1994), Amplified Fragment Length Polymorphism (AFLP) markers (Miroslaw et al., 2002), and Inter Simple Sequence Repeat (ISSR) markers (Arnau et al., 2002). Each of these systems has its own technical limitations; all have known problems with reproducibility of data among labs. Simple sequence repeats (SSRs) are abundant and scattered throughout the eukaryotic genome (Hamada et al., 1982). They have been widely used for cultivar identification in grape, walnut, and other clonally propagated crops (Dangl et al., 2001, 2005), but SSR markers take considerable time and expertise to develop (Lewers et al., 2005). Application of this technology to strawberry is particularly difficult because strawberries are octaploid, with eight copies of each gene, and because inbreeding can lead to cultivars with multiple copies of the same allele at a single locus. Since SSR technology can detect only the presence or absence of an allele, but not copy number, there are often several genetic combinations that could lead to a particular profile. Here we describe an SSR marker system that can distinguish closely related, UC-patented strawberry cultivars that are widely planted in California. This system uses published markers to reduce development time, and scores the amplified fragments as dominant markers. Because primer binding is highly specific, this system is both highly accurate and reproducible by other laboratories. Materials and Methods PLANT MATERIAL AND DNA EXTRACTIONS. Initial young leaves were collected and stored frozen from a set of 23 accessions of cultivated strawberry; these included cultivars and selections from the UC Davis breeding program and one diverse cultivar (Table 1). The frozen leaves were ground and DNA extracted using the DNeasy 96 Plant Kit (Qiagen Inc., Valencia, CA) according to the manufacture’s protocol. Leaves from a second set of accessions (Table 1) were dried rapidly using Drierite (W.A. Hammond Drierite Co., Xenia, OH) prior to DNA extraction. Subsequently, DNA has been successfully extracted from various other tissues: root, stem, calyx, and fruit, using several standard protocols. A total of 45 accessions were genotyped with six selected primer pairs using the optimized PCR protocol. Multiple plants were tested for most accessions. AMPLIFICATION CONDITIONS. Sixteen primer pairs (Table 2) for PCR amplification of SSR containing loci cloned from the strawberry cultivar ‘Earliglow’ (Lewers et al., 2005; Nourse et al., 2002) were screened with the initial set of 23 strawberry accessions using a range of PCR reaction mixes and thermal cycle profiles. The optimal PCR reaction mix was approximately 6.25 ng·μL–1 genomic DNA, 2 mM MgCl2, 0.8 mM of each dNTP [dNTP mix, Applied Biosystems Inc. (ABI), Foster City, CA], 0.025 units/μL AmpliTaq Gold (ABI), 1× Gold Buffer (ABI), and 0.25 pmol·μL–1 of each primer in a total volume of 20 uL. One primer of each pair was labeled with a fluorescent dye. For the