determined for each DNA content measurement, based on FCM assessments of standards and samples. Fig. 1. Representative cytograms of fluorescence intensity by flow cytometry of G0/G1 nuclei from some C. arabica cultivars and standards. Representative
Josue Ortega-Ortega, Francisco Arturo Ramírez-Ortega, Roberto Ruiz-Medrano and Beatriz Xoconostle-Cázares
Mohammed Elsayed El-Mahrouk, Mossad K. Maamoun, Antar Nasr EL-Banna, Soliman A. Omran, Yaser Hassan Dewir and Salah El-Hendawy
repeats ( Muranty et al., 2002 ), and flow cytometry ( Kiełkowska and Adamus, 2010 ; Lotfi et al., 2003 ). The first use of DNA flow cytometry in plants was reported by Galbraith et al. (1983) . The nuclei are classified according to their relative
Ying Wang, Cale A. Bigelow and Yiwei Jiang
morphological characteristics. This method, however, has not proven always accurate or sufficient. Flow cytometry is the newest technique to determine ploidy level, which appears to become a powerful method. This technique combines the advantages of microscopy
Milica Ćalović, Qibin Yu, Vladimir Orbović, Frederick G. Gmitter Jr, Jude W. Grosser and Chunxian Chen
determination. Somatic regenerants were screened by flow cytometry to determine their ploidy level while they were still growing in vitro. The relative amount of nuclear DNA content was measured in leaf samples of regenerated plants using a tabletop ploidy
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.
Xia Xu, Jiang Lu and Zhongbo Ren
Ploidy level in grapevines varies, especially since in vitro techniques are employed in the breeding process and after plants are treated with either chemicals or radiation. Detection of ploidy level in grapevines by microscopic chromosome counting is complicated by their high number and the small size of chromosomes. Flow cytometry provides an accurate and rapid method in determining the ploidy level in plant tissue by measuring the nuclear DNA content in living cells and thus is a very useful tool in plant breeding or genetic studies. The objective of this research was to analyze the ploidy level of a selected group of muscadine vines that were different from normal diploid vines in morphology. These grapes were derived from either chemical treatment of known varieties or from controlled/open pollinations. Among the 26 grapevines investigated, 8 were found to be diploids, 11 were tetraploids, and 7 were chimeric aneuploids. Results of this study indicate that flow cytometry is a quick, reliable tool for determining ploidy levels of grapevines.
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.
Mary Ann Start, James Luby, Robert Guthrie and Debby Filler
The hardy Actinidia species represent a source of genetic diversity for improving A. deliciosa (kiwifruit) as well as for creating new economically important cultivars through intra- and interspecific crosses. Attempts at breeding in Actinidia have been complicated by the existence of intraspecific as well as interspecific variation in ploidy. The haploid chromosome number in Actinidia is 29 and diploid (2n=2x=58), tetraploid (2n=4x=116), and hexaploid (2n=6x=174) levels have been identified. Because of the problems encountered when crossing parents differing in ploidy level, it is desirable to know the ploidy levels of plants to be used in breeding. We determined the ploidy levels of 61 Actinidia accessions currently available in the U.S., including primarily accessions of relatively winter-hardy species. The 61 accessions, representing eight species and three interspecific hybrids, were screened for ploidy using flow cytometry. Mitotic root tip cells from one plant from each putative ploidy level were examined microscopically to confirm the ploidy level derived from flow cytometry. There were 17 diploids, 40 tetraploids, and 4 hexaploids. Intraspecific variation was not found among accessions of the species arguta, callosa, deliciosa, kolomikta, melanandra, polygama, or purpurea. All kolomikta and polygama accessions were diploid. All arguta, callosa, melanandra, and purpurea accessions were tetraploid. Actinidia deliciosa was hexaploid. One chinensis accession was tetraploid. Two accessions (NGPR 0021.14 and 0021.3), acquired as chinensis, were hexaploid and may, in fact, be A. deliciosa based on their morphology. `Issai' (arguta × polygama) was hexaploid and `Ken's Red' and `Red Princess' (both melanandra × arguta) were tetraploid.
W.E. Jones, A.R. Kuehnle and K. Arumuganathan
Flow cytometry (FC) has proven to be an efficient and reliable method to estimate nuclear DNA content (genome size) in quantifiable units useful for genetic and molecular biology studies. This method also makes possible determination of the variation in nuclear DNA content between related taxa, which gives insights into the process of speciation. In this study, DNA content was determined in nuclei isolated from leaves of 21 Dendrobium species representing each of the major taxonomic groups used in the Univ. of Hawaii breeding program. Nuclei were mechanically isolated, stained with the nucleic acid-specific fluorochrom propidium iodide, and DNA content determined using a Coulter Epics 753 laser flow cytometer. Chicken erythrocyte nuclei (2C = 2.33 pg DNA) were used as an internal standard for direct comparative measurement. The mean diploid genome (2C) values for Dendrobium species ranged from 3.36 to 5.06 pg. Genome sizes were evaluated for possible use as discrete characters for taxonomic group assignment and compared to previous data on breeding compatibility and evolutionary relationship between species.