cDNA-AFLP analysis of Psidium guajava L. cultivars under water stress and mechanical injury: methodological implications

Abstract

Studies involving amplified fragment length polymorphism (cDNA-AFLP) have often used polyacrylamide gels with radiolabeled primers in order to establish best primer combinations, to analyze, and to recover transcript-derived fragments. Use of automatic sequencer to establish best primer combinations is convenient, because it saves time, reduces costs and risks of contamination with radioactive material and acrylamide, and allows objective band-matching and more precise evaluation of transcript-derived fragments intensities. This study aimed at examining the gene expression of commercial cultivars of P. guajava subjected to water and mechanical injury stresses, combining analyses by automatic sequencer and fluorescent kits for polyacrylamide gel electrophoresis. Firstly, 64 combinations of EcoRI and MseI primers were tested. Ten combinations with higher number of polymorphic fragments were then selected for transcript-derived fragments recovering and cluster analysis, involving 45 saplings of P. guajava. Two groups were obtained, one composed by the control samplings, and another formed by samplings undergoing stress, with no clear distinction between stress treatments. The results revealed the convenience of using a combination of automatic sequencer and fluorescent kits for polyacrylamide gel electrophoreses to examine gene expression profiles. The Unweighted Pair Group Method with Arithmetic Mean analysis using Euclidean distances points out a similar induced response mechanism of P. guajava undergoing water stress and mechanical injury.

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

“…The CTAB-based methodology has also been used successfully by Jaakola et al (2001) for the extraction of high-quality RNA from Vaccinium myrtillus [41], and by Zeng & Yang (2002) in Cinnamomum tenuipilum [29], both [43,44]. In the study involving P. guajava, Furlan et al (2012) isolated RNA using the guanidine thiocyanate buffer evaluated in this study [37]. However, in the comparative analysis, this method did not show the best results.…”

“…The third method is based on the association of lysis buffer with phenol-chloroform, the TRIzol reagent. The last protocol in this group is the guanidine thiocyanate method and it is the only one already described for the isolation of P. guajava RNA [37]. The goal of this study was to identify and/or perfect an efficient RNA isolation method for various P. guajava tissues to be used in plant molecular biology studies.…”

Efficient Method for Isolation of High-Quality RNA from Psidium Guajava L. Tissues

“…More recently, researchers of species from the Myrtaceae family have isolated RNA by the CTAB method and performed analyses of transcriptome and gene expression, such as Guzman et al (2014) in Eugenia uniflora [41], and Vining et al (2015) and Favreau et al (2019) in Eucalyptus grandis [42,43]. In the study involving P. guajava, Furlan et al (2012) isolated RNA using the guanidine thiocyanate buffer evaluated in this study [37]. However, in the comparative analysis, this method did not show the best results.…”

“…The third method is based on the association of lysis buffer with phenol-chloroform, the TRIzol ® reagent. The last protocol in this group is the guanidine thiocyanate method, the only already described for the isolation of P. guajava RNA [37]. Our goal was to identify an efficient RNA isolation method for various P. guajava tissues that can be used in plant molecular biology studies.…”

“…The pellet was dried at room temperature and the RNA was solubilized in 30 μL of RNase-free ultrapure water. The RNA was stored in an ULT freezer at -80°C.Guanidine Thiocyanate MethodExtraction buffer: 4 M guanidine thiocyanate, 100 mM Tris-HCl (pH 8.0), 25 mM sodium citrate pH 8.0, 0.5% N-laurylsarcosine, 1% PVP (mol wt 30,000), 2% β-mercaptoethanol (added just before use).This method was used byFurlan et al (2012) for RNA extraction from P. guajava tissue, but was adapted by Salzman et al(1999). Here, we proposed a small adjustment to the initial buffer volume and amount of tissue.…”