Genetic diversity in the highbush blueberry evaluated with microsatellite markers

Sixty-nine accessions representing wild and domesticated highbush blueberry (Vaccinium corymbosum L.) germplasm were genotyped using 28 simple sequence repeats (SSRs). A total of 627 alleles was detected and unique fi ngerprints were generated for all accessions. Suspected duplicate accessions of 'Coville' and 'Ivanhoe' had DNA fi ngerprints that were identical to 'Coville' and 'Ivanhoe', respectively. Genetic similarity measures placed wild and cultivated blueberries in separate groups. Northern highbush blueberries grouped among ancestral clones that were used extensively in blueberry breeding such as 'Rubel' and 'Stanley'. Southern highbush blueberries formed a separate group from northern highbush blueberries. The microsatellite markers used here show excellent promise for further use in germplasm identifi cation, in genetic studies of wild Vaccinium L. populations, and for constructing linkage maps. The highbush blueberry, Vaccinium corymbosum, is a peren- nial shrub cultivated for its edible fruit. Highbush blueberry is a high-value crop produced in the United States, Canada, Europe, Australia, New Zealand, Chile, and Argentina (Galletta and Ballington, 1996). The wild highbush blueberry is indigenous to subtropical and temperate climates in the eastern third of North America. Highbush is also the major cultivated blueberry type grown in temperate North America and the world. Vaccinium corymbosum is believed to occur at three ploidy levels, 2n = 2X = 24, 4X = 48, and 6X = 72 (Vander Kloet, 1988) but its taxonomy remains unsettled. The cultivated hexaploid was considered as a form of V. corymbosum by Vander Kloet. How- ever, it is considered ecologically and morphologically distinct by some breeders (Qu et al., 1998; Ritzinger and Lyrene, 1998) and is referred to in the literature as V. ashei Reade or V. virgatum Aiton. In this article we refer to it as V. virgatum. The highbush blueberry was domesticated in the 20th century. The fi rst breeding program for the predominant type, known as "northern highbush," was initiated by F. Coville of the U.S. Department of Agriculture (USDA) (Coville, 1937). Ehlenfeldt (1994) summarized the contributions of wild germplasm to the highbush blueberry genepool. Founder selections 'Brooks', 'Sooy', and 'Rubel' have contributed the majority of genes to the highbush blueberry genepool at overall levels of 23.5%, 12.5%, and 28.6%, respectively, for the period from 1908 to 1994. The lowbush blueberry, V. angustifolium Aiton, also contributed to the initial genepool through 'Russell' and 'North Sedgewick'. There has also been a consistent increase in the average inbreeding coeffi cients of new highbush blueberry cultivars. Southern highbush blueberry cultivars were recently developed from northern highbush cultivars through the introgression of genes from V. darrowii Camp, and to a lesser extent, V. virgatum and V. tenellum Aiton. Southern highbush blueberries are better adapted to warmer climates than northern highbush blueberries. Among southern highbush blueberry cultivars, V. darrowii is the largest contributor of genetic material after V. corymbosum. Despite concerns that inbreeding depression may be a serious problem in future blueberry breeding efforts (Haghigi and Hancock, 1992; Hancock and Siefker, 1982), some of the most successful cultivars have high inbreeding coeffi cients (Ehlen- feldt, 1994). Traditional methods of characterizing and identifying cultivars in fruit crops are based on phenotypic observations, a slow ap- proach that is subject to environmental infl uences. DNA-based markers reveal a high degree of polymorphism and can accelerate genotype fi ngerprinting and studies of genetic relationships among cultivars. Randomly amplifi ed polymorphic DNA (RAPD) mark- ers were used to distinguish 26 genotypes of lowbush blueberry (V. angustifolium Aiton) (Burgher et al., 2002) and for cultivar identifi cation of rabbiteye blueberry (V. virgatum) and highbush blueberry (Aruna et al., 1993, 1995; Levi and Rowland, 1997) as well as closely related cranberry (V. macrocarpon Aiton) (Novy and Vorsa, 1995, 1996a, 1996b; Novy et al., 1994). Three inter-simple sequence repeat (ISSR) and 15 RAPD primer pairs differentiated 15 highbush blueberry cultivars and two rabbiteye blueberry cultivars (Levi and Rowland, 1997). ISSR techniques are similar to RAPD techniques in their dominant nature and use of random primers. However, ISSR primer sequences are designed from microsatellite regions and the annealing tem- peratures used are higher than those used for RAPD markers. Clustering of V. corymbosum accessions based on molecular data did not agree with pedigree information (Levi and Rowland, 1997). Furthermore, RAPD markers are dominant and limited by lack of reproducibility between labs due to slight differences in thermocycling conditions or buffer composition (Powell et al., 1996). Therefore, more reproducible marker systems are needed in blueberry. Rowland et al. (2003) recently developed expressed sequence Tag-PCR (EST-PCR) and cleaved amplifi ed polymorphism (CAPS) markers for blueberry (Rowland et al., 2003), which present a signifi cant improvement over random