ABSTRACT
Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.
Subject(s)
DNA, Plant/isolation & purification , Liquid-Liquid Extraction/methods , RNA, Plant/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Arabidopsis/chemistry , Arabidopsis/genetics , Avicennia/chemistry , Avicennia/genetics , Cacao/chemistry , Cacao/genetics , Chloroform/chemistry , DNA, Plant/chemistry , DNA, Plant/genetics , Epigenesis, Genetic , Fruit/chemistry , Fruit/genetics , Lithium Chloride/chemistry , Paspalum/chemistry , Paspalum/genetics , Phenol/chemistry , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Roots/chemistry , Plant Roots/genetics , Plant Stems/chemistry , Plant Stems/genetics , RNA, Plant/chemistry , RNA, Plant/genetics , Seedlings/chemistry , Seedlings/genetics , Sorghum/chemistry , Sorghum/geneticsABSTRACT
Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA(+) bands only in the satellites, but no DAPI(+) bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.
Subject(s)
Cytogenetic Analysis/methods , Passiflora/genetics , Analysis of Variance , Chromosome Banding , Chromosomes, Plant , Haploidy , In Situ Hybridization, Fluorescence , Indoles/metabolism , Karyotyping , Metaphase , Passiflora/cytology , Species SpecificityABSTRACT
We tested four C-banding protocols to obtain heterochromatic bands in the passion fruit species Passiflora edulis and P. cacaoensis (Passifloraceae). Three of these protocols had been previously described. The three published protocols were not adequate to obtain C-bands in these species. An adapted protocol demonstrated heterochromatin distribution in metaphasic chromosomes of species of Passiflora for the first time. The differentiated coloration for C-bands was obtained with immersion of the slides in 99% ethanol, 45% acetic acid (additional step), 0.2 N hydrochloric acid, hydroxide of barium, 45% acetic acid, and 2X standard saline citrate at four different temperatures. The C-bands were observed in the satellites and in the telomere and centromere regions of all chromosomes, both in P. edulis and in P. cacaoensis.
