Chromosomal DNA content of sweet pepper determined by association of cytogenetic and cytometric tools

Abstract

The nuclear DNA content of sweet pepper (Capsicum annuum L. var. annuum, 2n = 24) has been measured by flow and image cytometries but the DNA content of each chromosome of this species has not yet been regarded. DNA content of individual chromosomes has been quantified by the flow karyotyping technique, which requires a great quantity of intact metaphasic chromosomes and methods that allow the characterization of individual chromosomes; however, the obtainment of adequate number of metaphases can be difficult in some species like C. annuum. In order to estimate the DNA content of each C. annuum var. annuum cv. "New Mexican" chromosome, flow and image cytometries were associated with the cytogenetic methodology. First, the DNA amount (2C = 6.90 pg) was established by flow cytometry. Integrated optical density (IOD) values were calculated by image cytometry for each Feulgen stained metaphasic chromosome. Then, by distributing the correspondent metaphasic value (4C = 13.80 pg) proportionally to average IOD values, the following chromosomal DNA contents were obtained in pg: 0.74 (chromosome 1), 0.67 (2), 0.61 (3, 4), 0.60 (5), 0.59 (6, 7), 0.58 (8), 0.57 (9), 0.56 (10) and 0.39 (11, 12). This study reports an alternative and reproducible technique that makes quantifying the chromosomal DNA content possible.

Nuclear DNA content of three Eucalyptus species estimated by flow and image cytometry

“…In this method, the nuclear preparations are fixed in advance (Doležel et al 1998). The root tips were fixed with methanol : acetic acid solution (Vilhar et al 2001;Abreu et al 2008) and, subsequently, with 95% alcohol, as suggested by Greilhuber and Ebert (1994). In accordance with Clarindo and Carvalho (2009), this fixative contributed to maintaining the integrity of the nuclei, avoiding any bias by providing high chromatin stability.…”

“…Seeds of E. grandis, E. urophylla, E. globulus and internal standards (R. sativus and S. lycopersicum) were germinated in Petri dishes with distilled water, at 30 C, and the root tips were fixed with a methanol : acetic acid solution (3 : 1, v/v) (Merck, KGaA) for 24 h (Vilhar et al 2001;Abreu et al 2008). The solution was then replaced with 95% ethanol, as suggested by Greilhuber and Ebert (1994).…”

DNA Amount of X and B Chromosomes in the Grasshoppers <i>Eyprepocnemis plorans</i> and <i>Locusta migratoria</i>

“…But it may also be applied to any other chromosome within the genome, or even to all chromosomes [Rosado et al, 2005;de Abreu et al, 2008].…”

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

“…An adequate protocol is a key step for supplying adequate nuclei and chromosome ICM (Vilhar et al 2001;Clarindo and Carvalho 2009). Derenzini (1999), Greilhuber (2005), and Abreu et al (2008) related that hydrolysis is the most critical step in ICM, requiring meticulous determination of the appropriate concentration of HCl, incubation time, and optimal temperature to ensure the stoichiometric DNA staining with Schiff's reagent. During the hydrolysis step, residual cytoplasmic background noises were removed, increasing the precision of the nuclei and chromosome digital segmentation by image analysis.…”

“…The Feulgen reaction was performed according to Haroske et al (2001), Abreu et al (2008), and Clarindo and Carvalho (2009), with modifications. The slides were postfixed in 4% formaldehyde solution (Merck) for 60 min, washed in distilled water, air dried, and hydrolyzed in 5 M HCl (Merck) for 10-60 min at 25C.…”