Microsatellite-Based Fingerprinting of Western Blackberries from Plants, IQF Berries and Puree

The blackberry industry needs a reliable method to ensure trueness-to-type of blackberry products. Microsatellite markers or simple sequence repeats (SSRs) are ideal for cultivar fingerprinting, paternity testing and identity certification. Fingerprinting is valuable for variety identification, quality control and as a legal method to protect against infringement by competitors. The objectives of this study were to develop a DNA extraction protocol and SSR-based identification for individually quick-frozen (IQF) ‘Marion’ and ‘Kotata’ whole berries and concentrate and to generate genetic fingerprints for 16 important western blackberry cultivars. IQF berries and frozen concentrate of ‘Marion’ and ‘Kotata’ were generously provided by reliable commercial sources. The FAST ID Kit worked better than two other DNA extraction methods for isolating DNA from IQF berries and from frozen and thawed concentrate. Out of twenty-nine SSRs tested, ten polymorphic SSRs differentiated between ‘Marion’ and ‘Kotata’ leaves and were chosen for subsequent analyses. SSR-based fingerprinting of individual IQF berries (using the receptacle for DNA extraction) revealed a mixture of ‘Kotata’ and ‘Marion’ berries in the commercial ‘Marion’ bag while fingerprinting of frozen ‘Marion’ concentrate identified ‘Kotata’ in the small frozen puree sample evaluated and possible contamination from seed DNA. The ten SSRs differentiated between each of the 16 western cultivars included in this study. In fact, one SSR marker, Rubus 275a was sufficient to distinguish these 16 cultivars. In summary, blackberry can be reliably identified with SSR markers, using leaves and frozen berries as sources of DNA. Fingerprinting from concentrate does not appear reliable for identity certification due to possible contamination from seed DNA. INTRODUCTION Blackberry, an aggregate fruit of Rubus species subgenus Rubus in the Rosaceae family, has long been a favorite wild fruit. Rubus species are native to several countries where they are picked for personal or commercial use. The US is the leading producer of cultivated blackberries in the world. Oregon dominates the US production and the Corvallis-based breeding program is the oldest continuously active blackberry breeding program in the world (Clark et al., 2007). Most of the cultivars released from that program and grown for the processing industry are of the trailing type. They are largely derived from the western dewberry (Rubus ursinus Cham. & Schlt.), but have several other species in their background. Over 95% of the blackberries in Oregon are harvested for processing, most commonly as individually quick-frozen (IQF) or pureed product. In 2008, Oregon produced 39.2 million pounds of blackberries valued at 3.8 million dollars as fresh fruit and 19 million dollars in processed products (USDANASS, 2009). Over 4000 acres of this production is in the cultivar Marion, believed to a [email protected] Proc. IS on Molecular Markers in Horticulture Eds.: N.V. Bassil and R. Martin Acta Hort. 859, ISHS 2010 74 have ideal processing characteristics such as: high soluble solids, high titratable acidity, excellent flavor, low perception of seediness, and good color and size (Yorgey and Finn, 2005). The blackberry industry is in critical need of a reliable method for ensuring trueness-to-type of their fruit products. Molecular markers and, in particular, microsatellite or simple sequence repeat (SSR) markers are ideal for cultivar fingerprinting, paternity testing and identity certification. When this study was initiated, the number of SSR markers in blackberry was limited. Eight SSRs from ‘Marion’ blackberry and four markers from ‘Meeker’ raspberry were developed in our lab and used to develop genetic fingerprints for 48 blackberry cultivars (Castillo et al., 2009). Additional microsatellite markers were developed mostly from R. idaeus (Graham et al., 2002, 2004, 2006). A small number of SSRs were isolated from other Rubus species: eight from R. alceifolius Poir. (Amsellem et al., 2001) and 15 from R. hochstetterorum Seub. (Lopes et al., 2006). A minimum of 14 R. idaeus and one R. alceifolius SSRs were polymorphic in parents of a mapping blackberry population (Stafne et al., 2005) and 13 of 15 microsatellite markers cross-amplified in R. fruticosus auct. aggr. (Lopes et al., 2006). The objectives of this study were to evaluate existing SSRs for cultivar identification in blackberry. Our goals were to develop a microsatellite-based identification protocol for IQF ‘Marion’ and ‘Kotata’ berries and puree using polymorphic SSR markers; and to fingerprint 16 of the most important western cultivars. MATERIALS AND METHODS Fresh leaves and berries of ‘Marion’ and ‘Kotata’ were collected from the USDA-ARS blackberry breeding field in Corvallis, Oregon. Actively growing leaves of the remaining fourteen cultivars were obtained in the spring from the NCGR screenhouse (Table 1). IQF ‘Marion’ and ‘Kotata’ berries and frozen concentrate were obtained from reputable commercial sources. Tissue samples were homogenized with an MM 301 Mixer Mill (Retsch International, Haan, Germany) and DNA was extracted using three different methods including: a modified Puregene (Gentra Systems Inc., Minneapolis, MN) protocol used routinely in the NCGR lab (Proteinase K and RNase A digestion steps were included in the extraction and protein precipitation was repeated twice); a Qiagen DNeasy mini kit (Qiagen Inc., Valencia, CA); and a Fast ID kit (Genetic ID NA, Inc., Fairfield, IA). The samples used to identify ‘Marion’ and ‘Kotata’ included leaves, receptacles from fresh berries, drupelets from fresh berries, receptacles from IQF berries, drupelets from IQF berries and frozen concentrates. For fingerprinting the 16 western cultivars, DNA was extracted from actively-growing leaves. Thirty-two primer pairs were tested for polymorphism between ‘Marion’ and ‘Kotata’ by visual inspection of ethidium bromide-stained agarose (3%) gel electrophoresis of the PCR products. The primers included: six SSRs from ‘Marion’ (RhM001, RhM011, RhM021, RhM023, RhM031, and RhM043) (Castillo et al., 2009); three primers pairs from the red raspberry ‘Meeker’ (RiM017, RiM019, and RiM036) (Castillo et al., 2009); thirteen R. idaeus SSR markers (Graham et al., 2004) that cross-amplified in the parents of a blackberry mapping population (Rubus 105b, Rubus 107a, Rubus 117b, Rubus 119a, Rubus 145a, Rubus 194h, Rubus 270a, Rubus 275a, Rubus 262b, RubfruitC1 and RubfruitE4) (Stafne et al., 2005); and ten microsatellite primer pairs developed in Rubus hochstetterorum that generated two or more alleles in Rubus fruticosus (ssrRhCBA5, ssrRhCBA6, ssrRhCBA12, ssrRhCBA15, ssrRhCBA16, ssrRhCBA18, ssrRhCBA21, ssrRhCBA23, ssrRhCBA27, and ssrRhCBA28) (Lopes et al., 2006). Ten SSRs clearly differentiated between ‘Marion’ and ‘Kotata’ and were subsequently used (Table 2). PCRs were performed in 10 μl volume containing 1× reaction buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.3 μM of each primer, 0.25 U of Biolase Taq DNA polymerase (Bioline), and 2.5 ng genomic DNA. The PCR protocol consisted of initial