Genetic Relationships and Characterization of Persian Walnut (Juglans regia L.) Cultivars Using Restriction Fragment Length Polymorphisms (RFLPs)

RFLP markers were used to investigate genetic diversity among California walnut (Juglans regia) cultivars and germplasm collected worldwide. Sixteen of 21 RFLP markers were polymorphic in the 48 walnut accessions tested. RFLP markers were useful for identifying walnut cultivars. All genotypes were heterozygous at ≈20% of the loci for both California and worldwide germplasm. California walnut germplasm contained 60% of the worldwide allelic diversity. Cluster analysis of genetic distance between accessions and principal component analysis of allelic genotypes showed two major groups of walnut domestication. California germplasm was associated with germplasm from France, central Europe, and Iran and had less genotypic similarity with germplasm from Nepal, China, Korea, and Japan. successfully used to study genetic diversity in cross-pollinated and self-pollinated crop species (Brummer et al., 1991; Chase et al., 1991; Kesseli et al., 1991; Miller and Tanksley, 1990). In the current study, 22 walnut RFLP loci were used to determine the genetic diversity and relationships of 48 walnut accessions of diverse origins. Materials and Methods Germplasm. Walnut germplasm used for this study consisted of cultivars and selections from the Univ. of California, Davis, walnut breeding program; chance seedling cultivars; and plant introductions (Table 1). Trees were maintained at the Wolfskill Experimental Farm in Winters, Calif., at evaluation orchards in Davis, and as seedlings at the National Clonal Germplasm Repository (NCGR) greenhouses in Davis. Germplasm was classified into 12 groupings (Table 1) for combined cluster analysis. Carpathian germplasm is comprised of cold-hardy cultivars grown in the United States considered to have originated from eastern Europe. The germplasm classified as French germplasm are selections made in California from French lines. DNA isolation. Leaves were collected from 48 J. regia accessions (Table 1). Leaves were ground in liquid N and stored frozen at –70C before DNA was isolated by a modification of the method of Doyle and Doyle (1987). Five grams of frozen leaves was added to 20 ml of 60C preheated 2x CTAB buffer (2% CTAB, 1% PVP, 1% b-mercaptoethanol, 0.1% sodium bisulfite, 1.4 M NaCl, 50 mM tris, 20 mM sodium EDTA, pH 8.0), and incubated at 60C for 30 min. The aqueous solution was extracted with 20 ml 24 chloroform : 1 isoamyl alcohol and centrifuged for 10 min at 1800 rpm in a bench-top centrifuge at 25C, and the aqueous layer was retained. Fifteen milliliters of isopropanol was added to precipitate the nucleic acids. The precipitate was spooled and washed in 76% ethanol with 10 mM ammonium acetate. The nucleic acid precipitate was air-dried overnight, rehydrated in 1 ml TE buffer (10 mM tris, 1 mM EDTA, pH 8.0), digested with 10 μg RNase at 37C for 1 h, and precipitated with ethanol. The DNA precipitate was washed in 75% ethanol and dried overnight before rehydration in 200 μl TE, pH 8.0. The DNA was quantified spectrophotometrically at 260 nm and visually in a 0.8% agarose gel stained with ethidium bromide containing lambda phage standards, with ≈25 μg·ml DNA = 1.0 Å at 260 nm. RFLP detection. Six micrograms of walnut DNA was digested Received for publication 23 Aug. 1993. Accepted for publication 27 Dec. 1993. This research was funded in part by the California Agricultural Experiment Station, the California Genetic Resources Program, and the U.S. Dept. of Agriculture– Agricultural Research Service. We thank Don Edwards, Univ. of California, Davis, Dept. of Pomology, for graphics production. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Present address: U.S. Dept. of Agriculture–Agricultural Research Service, National Forage Seed Production Research Center, Corvallis, OR 97331. Juglans regia, the Persian or English walnut, is an important commercial crop in California where production has greatly benefited from the release of cultivars developed by the Univ. of California walnut breeding program. This breeding program was founded on crosses between late-season French selections (‘Franquette’ and ‘Mayette’) and laterally fruitful selections (‘Chandler’ and ‘Vina’), which combined both traits (Forde, 1975; McGranahan and Forde, 1985). Lateral fruitfulness was derived from ‘Payne’, but was also found in ‘Sharkey’. Because western Chinese walnut germplasm frequently exhibits lateral fruitfulness and French cultivars do not, it was suggested that ‘Payne’ and ‘Sharkey’ may have been derived from Chinese germplasm (Tulecke and McGranahan, 1994). Central Asian germplasm has also been used. ‘Payne’ was used in crosses with PI159568 from Afghanistan to produce ‘Sunland’ and ‘Tulare’. Since a limited number of parents has been used to develop new California cultivars, an assessment of genetic variability in J. regia cultivars was needed to verify the availability of genetic diversity, which is required for continued crop improvement. Little genetic information is available for this crop species despite its widespread commercial use, making genetic variability measurements difficult. Morphological variability, although readily recorded, can be an unreliable measure of genetic diversity because of the confounding effects of environment, pleiotropy, and the unknown genetic basis of most morphological attributes. Isozymes and RFLPs have advantages as genetic markers for appraising variability because 1) their codominant nature allows a plant genotype to be assayed directly and 2) they are relatively free from environmental effects. Arulsekar et al. (1985, 1986) identified isozyme loci using four enzyme systems (glucose phosphate isomerase, aspartate amino transferase, phosphoglucomutase, and esterase) in walnuts. To circumvent the potentially limiting number of isozyme loci available in walnuts, RFLP markers were developed to measure walnut genetic diversity. RFLPs have been