Optimizing reproducibility evaluation for random amplified polymorphic DNA markers

Abstract

ABSTRACT. The random amplified polymorphic DNA (RAPD) technique is often criticized because it usually shows low levels of repeatability; thus it can generate spurious bands. These problems can be partially overcome by rigid laboratory protocols and by performing repeatability tests. However, because it is expensive and time-consuming to obtain genetic data twice for all individuals, a few randomly chosen individuals are usually selected for a priori repeatability analysis, introducing a potential bias in genetic parameter estimates. We developed a procedure to optimize repeatability analysis based on RAPD data, which was applied to evaluate genetic variability in three local populations of Tibochina papyrus, an endemic Cerrado plant found in elevated rocky fields in Brazil. We used a simulated annealing procedure to select the smallest number of individuals that contain all bands and repeated the analyses only Optimizing reproducibility evaluation for RAPD markers for those bands that were reproduced in these individuals. We compared genetic parameter estimates using HICKORY and POPGENE softwares on an unreduced data set and on data sets in which we eliminated bands based on repeatability of individuals selected by simulated annealing and based on three randomly selected individuals. Genetic parameter estimates were very similar when we used the optimization procedure to reduce the number of bands analyzed, but as expected, selecting only three individuals to evaluate the repeatability of bands produced very different estimates. We conclude that the problems of repeatability attributed to RAPD markers could be due to bias in the selection of loci and primers and not necessarily to the RAPD technique per se.

Genetic analysis of five sedentary fish species in middle Laranjinha River (upper Paraná River basin): A case study

“…Despite the limitations of the technique, prudent application of these markers can provide equivalent results and interpretations to those derived from codominant marker analysis (Ramos et al, 2008(Ramos et al, , 2012Phong et al, 2011). Moreover, the results produced by RAPD markers are reproducible (Pascual et al, 2006).…”

“…Furthermore, such information can be very helpful in understanding the effects of anthropogenic activities, such as dams, on the fish populations throughout the river basin. In the last decade, use of PCR-RAPD (random amplified polymorphic DNA) markers has been widely adopted to study genetic diversity and structure in neotropical fish populations in South America (Piorski et al, 2008), producing results that are comparable with those from other markers (Ramos et al, 2008;Muneer et al, 2009;Phong et al, 2011).…”

The narrow endemic Norwegian peat moss Sphagnum troendelagicum originated before the last glacial maximum

“…It has been shown that RAPD markers may have low reproducibility (Pérez et al, 1998;Rabouam et al, 1999, but see Ramos et al (2008)), and previous results concerning levels of variability and inferred recombination in S. troendelagicum are therefore difficult to interpret with confidence. The aim of this study is to learn more about the evolutionary history of this species, more specifically to (1) determine which of the parental taxa are maternal and paternal progenitors, respectively; (2) quantify patterns of genetic variability and structuring in natural populations of S. troendelagicum and the parental species using variable markers with high reproducibility; (3) test the hypothesis that the species originated multiple times; (4) quantify historical parameters, most importantly time since speciation event(s), to determine whether the species likely originated in central Norway or whether it immigrated after the last ice age; and finally, (5) make suggestions concerning the continued preservation of S. troendelagicum.…”

Molecular characterization of cajá, Spondias mombin (Anacardiaceae), by RAPD markers

“…In the present study, repeatability and reproducibility tests revealed that the amplification reactions were carried out under adequate and optimized conditions that generated consistent RAPD bands. High levels of repeatability in RAPD loci have also been reported for the frog Physalaemus cuvieri (85-99%; Telles, 2005) and for a Cerrado plant Tibouchina papyrus (85%; Ramos et al, 2008). Some studies have shown that different brands of Taq DNA polymerase can produce dissimilar results in PCRs (Evangelopoulos et al, 2001;Verweij et al, 2004;Al-Soud et al, 2005), and in several cases enhancers such as bovine serum albumin, casein, formamide, and polyvinylpolypyrrolidone are required to facilitate amplification.…”