Graduate Thesis Or Dissertation
 

Determining ploidy level and nuclear DNA content in Rubus by flow cytometry

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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 that included: increasing the stain concentration, adding the stain after the RN ase treatment instead of adding it to the chopping buffer, reducing the tissue sample size, and using trout red blood cells (TRBC) as an internal standard. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose ploidy had been determined by chromosome counting, indicated that fluorescence increased concurrently with an increase in chromosome number. Ploidy level accounted for ninety-nine percent of the variation in fluorescence intensity (r = 99%) and variation among the ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus, which is widely represented in the USDA-ARS breeding program and has been reported to have 6x, 8x, 9x, lOx, 1 lx and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x and 1 lx forms were found as well. 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 subgenus Rubus, 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. Jdaeobatus 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 (0.47pg). Ploidy level of 84 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 due to the function of non-reduced gametes. Flow cytometry was effective in differentiating chromosome numbers differing by lx but was not able to differentiate aneuploids.
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