Microsatellite Markers Distinguish Hawaiian Ohelo from Other Vaccinium L. Section Myrtillus Species

The US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, collection of Vaccinium L. contains >1700 accessions representing 66 species from 33 countries. About 377 accessions belong to 10 species of Vaccinium section Myrtillus. This section is restricted to the Northern Hemisphere where its primary center of diversity occurs around the Pacific Rim in high elevations from Alaska to Guatemala and in mountains of Japan, Kurile, Sakhalin, and Hawaiian Islands, and Russian Siberia. The first objective of this study was to use microsatellite or simple sequence repeat markers (SSR) previously isolated from domestic blueberry, to uniquely identify three clones of Ohelo berry, V. reticulatum, selected for their ornamental qualities. The second objective was to evaluate the SSR markers for cross-transference to other species within Section Myrtillus including: two genotypes from another endemic Hawaiian species, V. calycinum; 17 representatives from seven North American species (V. caespitosum, V. deliciosum, V. membranaceum, V. myrtillus, V. ovalifolium, V. parvifolium, and V. scoparium), and in two accessions of V. praestans from Sakahlin Island, Russian Federation and one genotype from Hokaido, Japan. As many as eighteen microsatellite markers cross-amplified and appeared polymorphic in most of the genotypes evaluated. The three V. reticulatum genotypes were uniquely identified with eight SSR primer pairs. UPGMA clustering based on nine microsatellite primer pairs clearly separated the Hawaiian taxa and grouped V. praestans together. Two accessions of V. myrtillus (V myrt 1684-1, V myrt 1684-3) collected from within the Kalmiopsis Wilderness of southern Oregon had identical fingerprints indicating a clonal relationship. Conserved primers that amplify what appear to be single loci SSRs are recommended for population genetic analyses in diploid Vaccinium section Myrtillus taxa including V. calycinum, V. reticulatum, V. scoparium and V. praestans. SSR-based analysis indicated high diversity in other taxa of section Myrtillus and the need for a larger amount of accessions from each species, additional markers or sequence-based markers, for taxonomic resolution. INTRODUCTION The genus Vaccinium L. contains more than 400 species of terrestrial or epiphytic shrubs or small trees (Van der Kloet, 1988) and is traditionally divided into 33 sections (Sleumer, 1941). In their morphologic treatment of the taxonomy of section Myrtillus based on phenetic analyses, Van der Kloet and Dickinson (1999) described five clusters that contain seven species (V. caespitosum Michaux, V. deliciosum Piper, V. membranaceum Douglas ex Torrey, V. myrtillus L., V. ovalifolium Smith, V. parvifolium J.E. Smith, and V. scoparium Leiberg). These species share a number of morphological features including, most distinctively, flowers that are borne singly in the axils of the lowermost leaves, and a pedicel that is continuous with the calyx tubes. Phylogenetic analysis based on molecular data from the nuclear ribosomal internal transcribed spacer a [email protected] Proc. IS on Molecular Markers in Horticulture Eds.: N.V. Bassil and R. Martin Acta Hort. 859, ISHS 2010 82 (nrITS) and the chloroplast genes matK and ndhF resulted in the identification of three Hawaian species (V. dentatum Smith, V. calycinum Smith and V. reticulatum Smith) previously placed in Macropelma as a monophyletic group that is derived from within section Myrtillus (Powell and Kron, 2002). Furthermore, the Myrtillus clade was sister to a clade that includes sections Oxycoccoides, Praestantia (V. praestans Lambert) and the two Asian representatives (V. hirtum Thunb. and V. smallii A. Gray) of section Hemimyrtillus. Species from these sections share the character of perennating bud scales with two prophylls and appear to form a monophyletic Pacific Rim clade (Powel and Kron, 2002). V. praestans is considered in section Myrtillus sensu lato in this study. The center of diversity of species in section Myrtillus is found along the Pacific Rim from Japan, arching through Alaska, southerly to Guatemala (Van der Kloet and Dickinson, 1999). The mountains of Honshu, Japan, form a secondary center of diversity. Fruits of these wild plants are highly aromatic and harvested mostly for home use as fresh or processed products. The fruit pulp in addition to the skin is intensely colored in Myrtillus, as opposed to green or white in blueberries (section Cyanococcus). Differences in anthocyanin content or in the aglycone profiles identify fruits of V. parvifolium, V. deliciosum, V. membranaceum and V. ovalifolium of the Pacific Northwest (Ballington et al., 1988). Interspecific hybridization between cultivated V. corymbosum species from section Cyanococcus and V. membranaceum species were attempted to introgress the aroma of fruits from these species into the cultivated blueberry but did not result in any releases (Chad Finn, pers. commun.). The USDA-ARS Pacific Basin Agricultural Research Center (PBARC) identified and selected three clones of ohelo (Vaccinium reticulatum Smith) for their highly attractive foliage and berries. On the Islands of Hawaii and Maui, residents gather berries in summer from the wild in disturbed, open sites at 640 to 3700 m elevation for use in jam, jelly and pie filings. A long term goal of this project consists of developing sustainable ohelo berry production in Hawaii for culinary and value added product use; and to determine nutraceutical components and value-added products for consumers. The USDA-ARS National Clonal Germplasm Repository in Corvallis, Oregon, preserves a collection of >1700 Vaccinium accessions representing 66 species from 33 countries. Ten species in section Myrtillus are represented. The first objective of this study was to use microsatellite markers previously developed in blueberry, Vaccinium corymbosum L., to identify the three Hawaiian ohelo, V. reticulatum, selections. The second objective was to evaluate these SSR primer pairs for cross-transference to other species within section Myrtillus including: two genotypes of the another Hawaiian endemic, V. calycinum; 17 representatives from seven North American species [V. caespitosum (dwarf bilberry), V. deliciosum, V. membranaceum, V. myrtillus (North American and Eurasian: bilberry, whinberry, whortleberry, European blueberry), V. ovalifolium (Alaska blueberry), V. parvifolium (red huckleberry), and V. scoparium; and in three accessions of V. praestans from Sakhalin Russian Federation and Hokaido, Japan. MATERIALS AND METHODS DNA was extracted from actively-growing leaves of thirty-two accessions (Table 1) of Vaccinium section Myrtillus plants growing in screenhouses at the National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon. Tissue samples were homogenized with an MM 301 Mixer Mill (Retsch International, Haan, Germany) and DNA was extracted using a modified Puregene (Gentra Systems Inc. Minneapolis, Minn) protocol used routinely in the NCGR lab. Twenty-three SSR primer pairs previously developed in blueberry were reported to amplify in V. parvifolium (Boches et al., 2005). These primer pairs were evaluated for amplification of a product in 24 accessions (Table 1) representing ten Vaccinium section Myrtillus species by 2% agarose gel electrophoresis. PCRs were performed in 10 μl volume containing 1× reaction buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.3 μM fluorescent WellRed forward primer, 0.3 μM reverse primer, 0.25 U of Biolase Taq DNA polymerase (Bioline), and 2.5 ng genomic DNA. The