Characterization of 14 Microsatellite Markers for Genetic Analysis and Cultivar Identifi cation of Walnut

One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm x J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profi les; the accessions with identical profi les appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identifi cation, verifi cation of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections. The persian walnut cultivars currently grown in California are the product of more than 200 years of cultivation in the state. The University of California (UC)–Davis Walnut Breeding Program, begun in 1948, produced many cultivars that contribute to the prominence of the California industry in the world market (Tulecke and McGranahan, 1994). The materials used to develop these cultivars originated in various parts of the world, including Europe, South America (where they were fi rst brought from Europe by Spanish settlers), and Asia. Several techniques have been used to examine genetic diversity and relationships among cultivars of persian walnut, including isozymes (Arulsekar et al., 1985; Solar et al., 1993, 1994), restriction fragment-length polymorphism (RFLP) (Fjellstrom et al., 1994), randomly amplifi ed polymorphic DNA (RAPD) markers (Nicese et al., 1998), and, most recently, inter-simple sequence repeat (ISSR) markers (Potter et al., 2002a). These studies refl ect the need for accurate cultivar identifi cation (genetic fi ngerprints), and for verifi cation of paternity and genealogy. Microsatellite (SSR) markers are well suited to meet these needs. In addition, SSR markers informative in J. regia may also be polymorphic in other Juglans L. species (Aldrich et al., 2003) and have utility for breeding hybrid rootstocks. Paradox, an important rootstock in the California walnut industry, is generally produced by open-pollination of J. hindsii by J. regia. SSR markers will be useful for determining the specifi c J. regia cultivar or other species involved in the production of Paradox hybrids, a level of precision not heretofore possible (Potter et al., 2002b). SSR-based paternity analysis can also contribute to an understanding of pollen fl ow within and among walnut orchards: a critical issue as pollen is the vector for blackline disease (Mircetich et al., 1985) and because surplus pollen from pollinizers can result in yield loss from pistillate fl ower abscission (McGranahan et al., 1994). Simple sequence repeat markers also have proven useful in the repository setting (Mitchell et al., 1997) to examine potential redundancies and propagation errors within collections (Dangl et al., 2001; Phippen et al., 1997). Thus, a short-term goal of this research was to identify SSRs for characterizing the UC–Davis and U.S. Dept. of Agriculture (USDA) walnut germplasm collections based on SSR loci identifi ed in J. nigra (Woeste et al., 2002). Received for publication 8 July 2004. Accepted for publication 29 Aug. 2004. We gratefully acknowledge fi nancial support from the Walnut Marketing Board, the California Dept. of Food and Agriculture, and the Genetic Resources Conservation Program at Univ. of California–Davis. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable. 1Current address: Chuck Simon, USDA, ARS PGRU, Cornell University, Collier Dr., Geneva, NY 14456-0462. 2To whom reprint requests should be addressed. E-mail address: ghmcgranahan @ucdavis.edu; phone 530-752-0113; fax 530-752-8502. Book 1.indb 348 4/8/05 12:54:31 PM 349 J. AMER. SOC. HORT. SCI. 130(3):348–354. 2005. Materials and Methods PLANT MATERIAL AND DNA EXTRACTIONS. Forty-seven accessions of J. regia, including many cultivars and selections from the UC–Davis Walnut Breeding Program, and one J. hindsii x J. regia hybrid rootstock, were selected for this study (Table 1). The accessions represent more than 90% of the currently cultivated cultivars. Two trees of each cultivar were tested (one tree from Wolfskill Experimental Orchard, Winters, Calif., and one from New Stuke Block on the UC–Davis campus). Only one tree of the plant introduction (PI) 159568, located at Wolfskill, was available. Young leaves were collected on two occasions from each tree. The fi rst samples were ground in liquid nitrogen and DNA extracted using the method of Doyle and Doyle (1987) with an additional chloroform extraction step. A second set of samples was rapidly dried using Drierite (W.A. Hammond Drierite Co., Xenia, Ohio). Approximately 20 mg of dried young leaf tissue were ground in liquid nitrogen and the DNA extracted using a DNeasy Plant Mini Kit (QIAGEN, Valencia, Calif.). DNA quality and quantity were determined using 2% agarose gels (SeaKem, Cambrex Bio Science, East Rutherford, N.J.) stained with ethidium bromide and visualized using ultraviolet (UV) light. As both DNA samples from both trees of a given cultivar generated an identical genotype based on SSR profi les, this genotype was treated as one result for further analysis. PCR AMPLIFICATION. Genomic DNA was diluted to approximately 25 ng·μL–1 in water. Polymerase chain reactions were conducted in a total volume of 15 μL containing 5 ng·μL–1 DNA, 1X Gold Buffer [Applied Biosystems Inc., Foster City, Calif. (ABI)], 2 mM MgCl2, 0.2 mM each dNTP, 0.2 pmol·μL–1 each primer and 0.024 units/μL AmpliTaq Gold DNA polymerase (ABI). The thermal cycle profi le for all primers was 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 1 min at 58 °C, and 40 s at 72 °C, concluding with 1 cycle of 2 min at 72 °C. FRAGMENT SEPARATION AND SIZING. Amplifi ed fragments were separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer using a 47 cm × 5 μm capillary with POP4 as the matrix (ABI). Run parameters were: travel length 30 cm, run time 24 min, electrophoresis power 15 kV, and temperature 60 °C. Forward primers were labeled with one of the three dyes of Virtual dye Set D (ABI); the fourth dye was the tag for the internal size standard (400HD-ROX; ABI). During the initial screening of primer pairs, color multiplexing allowed analysis of three primer pairs per injection. In later phases of screening, size multiplexing (combining markers with non-overlapping fragment size ranges) was used in combination with color multiplexing. Depending on the concentration of amplifi ed product, injection samples were prepared by mixing 0.6–0.8 μL of amplifi ed product from separate reactions with 0.74 μL of size standard and 9.26 μL of formamide. Fragment size in absolute base pairs was determined using GeneScan Analysis Software 3.1 (ABI) using local southern option as the size calling method. Peak binning and label editing were performed using GenoTyper version 2.5 (ABI). SCREENING OF PRIMER PAIRS. Primer pairs designed to amplify SSRs in J. nigra (Woeste et al., 2002) were initially screened against 12 J. regia accessions (Table 1). Priority was given to the most polymorphic markers with peaks that could be scored easily. The secondary primer screen consisted of the accessions in the primary screen as well as 12 other accessions (Table 1). DATA ANALYSIS. Observed heterozygosity (HO) and null allele frequency estimates were calculated for each locus using the Table 1. Juglans regia cultivars, selections, and hybrid investigated in this study. Accession UCD accession no. Nation of origin UC56-224y UC56-224 USA UC59-124 UC59-124 USA UC61-25 UC61-25 USA UC67-13x UC67-13 USA UC86-011 UC86-011 Germany Alsoszentivani-117x UC85-042 Hungary Amigoy UC56-226 USA Ashley UC4 USA Badajoz UC87-002 Spain Carmello UC29 USA Cascade UC87-18 USA Chandlery UC64-172 USA Chicoy UC56-206 USA Ciscoy UC66-178 USA Conway Mayettey UC31 USA Early Ehrhardt UC6 USA Eurekay UC7 USA Franquettex UC96-002 France Graves Franquette UC32 USA Gustine UC52-061 USA Hartley UC2 USA Howardy UC64-182 USA Lara UC86-016 France Lompocx UC52-046 USA Lozeronne UC92-002 France Manregian (PI18256)x UC49 China Marchettiy UC41 USA Meylany UC42 France Midland UC49-047 USA Paradox Burbankw UC45 USA Paynex UC1 USA Pedroy UC53-113 USA PI159568 DJUG 99v Afghanistan Pioneer UC51-170 USA Placentia UC51 UNK Poe UC91-136 USA Scharsch Franquette UC3 USA Serrx UC59-129 USA Sharkeyy UC53 USA Sinensis-5x UC54 Japan Sunlandx UC66-004 USA Tehamax UC58-011 USA Tularex UC67-011 USA Vina UC49-049 USA Waterloox UC56 USA