Estimation of the nuclear DNA content of gossypium species

Abstract

Background and aims: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices.

Methods: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates.

Key results: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes.

Conclusions: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.

Fig. 1.

Example of standard curve used to calculate Gossypium nuclear DNA content values (n = 8 for all species). In all instances, the relationship between PI fluorescence and DNA content was better defined by a polynomial (e.g. this curve y = −1·9131x² + 86·157x − 10·917, r² = 0·995). The arrow highlights a set of fluorescence measurements for G. hirsutum ‘DP491’ applied to the regression equation. Cotton nuclei fluorescence is lower than that of corn (2C = 5·47 pg) and barley (2C = 11·12 pg) but higher than that of rice (2C = 1·01 pg), indicative of tetraploid cotton's genome size (2C = 4·93).

Fig. 2.

Compounds from H. vulgare ‘Sultan’ enhance the PI fluorescence G. hirsutum ‘DP491’. Peak A: fluorescence peak of cotton processed alone. Peak B and C: fluorescence peaks of co-processed cotton and barley, respectively. Co-processed sample was mixed with the independently processed cotton sample after separate measurements to illustrate the effect.

Fig. 3.

Ancestral genome sizes (±s.e.) predicted with the PGLS method based on phylogeny developed by Cronn et al. (2002). Evidence of bidirectional genome size change is evident within Gossypium. Note that the K genome, sister to the C/G genome clade, was not included in Wendel et al. (2002), hence had to be omitted from the calculation.

Fig. 4.

Comparison of 2C DNA content values for 20 Gossypium species determined with Feulgen microspectrophotometry (Kadir, 1976) to values reported in this study. Bennett et al. (1982) converted the arbitrary fluorescence units reported by Kadir (1976) into picograms using H. vulgare ‘Sultan’ as an internal standard. The revised values shown in this figure were derived from arbitrary fluorescence units converted into picograms using the G. arboreum DNA content reported in this study (2C = 3·5 pg). y = 0·4462367 + 0·8759436x (r² = 0·961).