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Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).
Cuttings from Rubus ursinus Cham. & Schlecht, the trailing blackberry, were collected in Oregon, Washington, and British Columbia from 21 sites. The cuttings were rooted and placed in pots in the greenhouse. After the plants began to grow, leaves were harvested for ELISA testing using standard procedures. Each sample represented three clones from a site. Plants from 18 sites were represented by five samples and two sites were represented by three samples. None of the samples tested positive for the presence of raspberry bushy dwarf virus or tomato ringspot virus. Forty-four percent of the samples tested positive for tobacco streak virus. Only 33% of the sites on the Pacific coast tested positive for tobacco streak, whereas, 100% of the Cascade Mountain sites and 88% of the sites in the coastal range type environment tested positive. The only site in the Willamette Valley had no positive tests. With one exception, all of the sites that tested negative for the virus were also low elevation sites 0-90 m.
The relative susceptibility of 44 genotypes of wild Fragaria L. and commercial cultivars of strawberry Fragaria ×ananassa Duch. to Meloidogyne hapla Chitwood and Pratylenchus penetrans (Cobb) Filipjev & Shuurmans Stekhoven was evaluated in the greenhouse. Eleven genotypes were highly resistant to populations of M. hapla from Washington State and Oregon, with Rf values (initial nematode density/final population density) less than 0.5. However, root growth of most genotypes, including resistant genotypes, was reduced by M. hapla. Thirteen genotypes were ranked more resistant to P. penetrans than F. ×ananassa `Totem', a susceptible cultivar. Root growth of most genotypes was not affected by P. penetrans under these experimental conditions. We conclude that commercial cultivars and wild Fragaria genotypes can provide a readily exploitable source of resistance to M. hapla. Conversely, sources of resistance to P. penetrans were uncommon in the germplasm evaluated. The F. ×ananassa cultivars, which already have commercially important characteristics, appear to be a better source of resistance for both nematode species than the wild, unimproved germplasm.