-spread metaphase stage. Table 2. Ploidy level, chromosome numbers, and karyotypic characteristics of the 11 studied Lonicera cultivars. Table 3. DNA ploidy level, relative DNA content, z and nuclear DNA content in Lonicera cultivars as estimated by flow
Juan Chen, Nianhe Xia, Xiaoming Wang, Richard C. Beeson Jr., and Jianjun Chen
Ying Wang, Cale A. Bigelow, and Yiwei Jiang
and biochemical analysis to measure quantitative traits such as DNA and RNA content and total cellular protein content with high precision and rapid throughput ( Eaton et al., 2004 ; Muirhead et al., 1985 ). Flow cytometry has been used to determine
Jessica Gaus Barb, Dennis J. Werner, and Shyamalrau P. Tallury
Skyrocket’; and 3) determine the absolute nuclear 2C DNA content of diploid and tetraploid plants using flow cytometry. Materials and Methods Plant material. Diploid accessions of Stokesia (‘Alba’, ‘Colorwheel’, ‘Honeysong Purple’, ‘Mary
Hamidou F. Sakhanokho and Nurul Islam-Faridi
about the genetics of C. obcordata . For this reason, we decided to determine the chromosome number using a modern protoplast technique to spread root tip chromosomes, nuclear DNA content and base composition using flow cytometry, and the location of
Peggy Ozias-Akins and Robert L. Jarret
The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei. Ploidy level and DNA content were significantly correlated within the genus, but more highly so within species that contained multiple cytotypes. DNA content of cultivated Z. batatas (L.) Lam. (4.8 to 5.3 pg/2C nucleus) and feral tetraploid I. batatas (3.0 to 3.5 pg/2C nucleus) was estimated from the known DNA content of chicken erythrocytes (2.33 pg), which were used as an internal standard. Tetraploid forms of Z. cordato-triloba Dennstedt also were identified. Ploidy analysis using flow cytometry is rapid and suitable for large-scale experiments such as studying the genetic structure of populations of Z. batatas and related species. Chemical name used: 4′,6-diamidino-2-phenylindole (DAPI).
Rodomiro Ortiz, D.E. Costich, T.P. Meagher, and N. Vorsa
DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.
Peter H. Velguth and Harold Pellett
We evaluated flow cytometric measurement of nuclear DNA content to determine ploidy level in azalea. If ploidy level correlates with DNA content, ploidy level could be determined more readily than by direct chromosome counts and assist in planning crosses and evaluating progeny. Tested plants included azalea cultivars, materials from the azalea breeding project at the Univ. of Minnesota, and species from the Rhododendron Species Botanic Garden and the North Carolina Arboretum. Data compiled from DNA assays of practically all material analyzed fell into distinct groups consistent with their being either diploid, triploid, or tetraploid. Additionally, a known diploid plant of each of four diploid species, together with a natural or derived tetraploid plant of each of these species was obtained. Results showed that the four diploids had a similar DNA content compared to one another. DNA content of the tetraploids was also similar, and the tetraploid's DNA content was approximately twice that of the diploids, as expected. Unfortunately, success with direct chromosome counts in other material has proven elusive, currently precluding direct correlation of DNA amount with ploidy level across other species and cultivars. Although many cases exist in the literature where DNA content has a direct relationship to ploidy level, this does not always hold. Although the majority of plants tested fell into a diploid, triploid, or tetraploid grouping based on DNA content, further study is required to determine the exact relationship between ploidy level and DNA content in azalea.
W. Vance Baird, Agnes S. Estager, and John K. Wells
Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera [Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)], two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60±0.03 pg (or 5.8×108 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (× = 8).
Rengong Meng and Chad Finn
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).
Rengong Meng, Chad E. Finn, and Robert P. Doss
Knowledge of the chromosome number in Rubus would be valuable when planning crosses and identifying plants, etc., however, preparation of tissue for microscopic evaluation and chromosome counting is difficult and time-consuming. Flow cytometry offers a more-efficient approach to this task. DNA flow cytometry was used to determine the nuclear DNA content in 22 Rubus genotypes. The genotypes represented a range of reported chromosome numbers from 2x to 12x. Six of the genotypes were representatives of Rubus ursinus, which is reported to have both 8x and 12x forms. Samples of nuclei were prepared from leaf discs of newly emerged and mature leaves following published protocols with some modifications. The DNA content was estimated by comparison of the fluorescence of Rubus nuclei with an internal DNA standard. There was an increase in nuclear DNA content concurrent with the increase in chromosome number. In these studies DNA flow cytometry could differentiate genotypes that differed by 2x, such as 6x and 8x, but could not reliably distinguish genotypes that differed by 1x, such as 7x vs. 8x or 6x. Aneuploids cannot be differentiated at this time.