Development of SCAR Markers for DNA Fingerprinting and Germplasm Analysis of American Cranberry

DNA fingerprinting has been useful for genotypic classification of American cranberry (Vaccinium macrocarpon Ait.). Polymerase chain reaction (PCR) based methodologies including randomly amplified polymorphic DNA (RAPD) markers are relatively easy to use, and inexpensive as compared to other methods. However, RAPD markers have some limitations including seamless interlaboratory transferability and susceptibility to certain types of error. An alternative method, sequence characterized amplified regions (SCARs), was developed for cranberry germplasm analysis. Nine primer sets were designed from RAPD-identified polymorphic markers for use in two multiplex PCR reactions. These primer sets generated 38 markers across a cranberry germplasm collection. Estimates of genetic relatedness deduced from employment of the RAPD and SCAR methods were compared among 27 randomly chosen cranberry germplasm accessions. Although both methods produced comparable results above 0.90 coefficient of similarity, branches below this level exhibited variation in clustering. SCAR and RAPD markers can be employed for identifying closely related genotypes. However, the inferences of more distant genetic relationships are less certain. SCAR marker reactions provided more polymorphic markers on a per reaction basis than RAPD marker reactions and as such more readily separated closely related progeny. When SCAR primers were fluorescent dye-labeled for computerized detection and data collection, reduced marker intensity relative to unlabeled reactions was one problem encountered. Accurate computer automation would facilitate scoring, cataloging, and comparisons of cranberry DNA fingerprinting data. However, some of the shortcomings of RAPD markers make the implementation of computer-based analysis problematic. In this case, reproducibility must be increased, markers must be unambiguous, and monomorphic markers must be minimized. Sequence characterized amplified regions (SCARs) which are derived from RAPD markers, have the advantages of RAPD markers and the additional benefits of increased specificity and reproducibility (Paran and Michelmore, 1993). SCAR primer sets can be strategically multiplexed in a PCR reaction to improve efficiency (i.e., increase the number of polymorphic markers produced per reaction). Thus, it is conceivable that the number of reactions required to fingerprint one sample could be reduced to one. The most significant drawback of SCAR markers is development time and expense. Once established, however, the cost of individual SCAR PCR reactions are similar to those of RAPD markers, and a reduction in the number of required reactions can decrease overall expenses. Additionally, SCAR markers lend themselves readily to computer automation (i.e. scoring, database cataloging, and comparative analysis). The advantages of SCAR markers over RAPD markers for large-scale and ongoing DNA fingerprinting of cranberry samples could easily justify the added development effort. Therefore, the objectives of this research were to 1) develop a fast, inexpensive and reliable method for DNA fingerprinting of American cranberry, 2) evaluate the efficiency of the SCAR marker and RAPD marker methods for discriminatory analysis, 3) compare estimates of genetic relatedness among cranberry germplasm based on RAPD and SCAR marker derived data, and 4) assess application of the SCAR methodology for computer automation using fluorescent dye-labeled primers and laser detection of products. Materials and Methods PLANT MATERIAL, DNA EXTRACTION, AND QUANTIFICATION. Cultivars used were deduced previously to be true-to-type by examinaReceived for publication 21 Aug. 2001. Accepted for publication 8 Apr. 2002. Assistant research director and corresponding author. Professor. Cultivated beds of American cranberry (Vaccinium macrocarpon) are generally started using pressed-in vines or stem cuttings harvested from established beds. Newly planted beds typically take 2 to 3 years to reach full production potential and can remain productive for many years. Most cultivars are clonal, but some older cultivars appear to have been mixtures of genotypes when originally selected from the wild (Polashock and Vorsa, unpublished). Evidence suggests that over time, encroachment by wild vines or seedlings and unproductive volunteer seedlings from selfor crosspollinations become established in cultivated beds, lowering yield potential (Novy et al., 1996). Since cranberry lacks qualitative morphological characters to allow for visual classification, an alternative method of genotype identification is necessary. Since beds of most cultivars are supposed to be pure clones but are often contaminated as noted above, a genotyping method would be useful in determining if a problem such as poor yield has a genetic (i.e., due to the establishment of a contaminating genotype) or environmental basis. For breeding purposes, confirmation of genetic identity and estimating genetic similarity would be valuable. Since large sample numbers must be processed for any of the purposes listed above, a rapid and reliable method is required. The method of choice should provide for good genome coverage, be relatively simple to use, and be cost effective. Random amplified polymorphic DNA (RAPD) markers meet these general requirements, but are prone to certain types of error that affect reproducibility (Davin-Regli et al., 1995; Hansen et al., 1998; Perez et al., 1998; Staub et al., 1996). These reproducibility problems limit interlaboratory method transferability, and make routine scoring and comparison to an existing database difficult. In addition, most bands (markers) generated using RAPD by any one primer are not polymorphic. This necessitates execution of multiple polymerase chain reactions (PCR) with different primers for adequate fingerprinting of each sample.