Inferring Genetic Variability between Two Crayfish Species and Two Fish Species Using RAPD-PCR




Christopher L. Romano

Introduction

Random amplified polymorphic DNA PCR (RAPD-PCR) is an effective technique for studies of population and conservation genetics, taxonomy, genetic variability and insights into geographic origins and invasion routes of colonizing species. The principle behind RAPD-PCR is the detection of polymorphisms through the PCR amplification of DNA fragments with primers of arbitrary nucleotide sequence. Polymorphisms are detected as either the presence or absence of bands, caused by nucleotide sequence differences between individuals.

Primers used for RAPD-PCR are usually 10 nucleotides in length and between 50-70% G + C content. Each RAPD-PCR reaction volume contains a single primer which functions as both the forward and reverse primer for amplification. DNA amplification products are generated from any region where primers bind in the proper orientation and within ~5 kbp of each other. Following the PCR, amplification products are visualized on sizing gels for analysis.

RAPD-PCR has significant advantages over techniques such as microsatellites and RFLPs. For example, RAPD-PCR is relatively fast, simple and inexpensive to conduct. It requires only small quantities of template DNA and potentially provides a large number of polymorphic loci. Also, no prior knowledge of the target DNA sequence is needed.

The aim of this study was to employ RAPD-PCR to infer genetic variability between two species of crayfish (Freckled Crayfish: Cambarus maculatus and Noble Crayfish: Astacus astacus) and two species of fish (Goldfish: Carassius auratus and Molly: Poecilia latipinna). An additional goal of this study was to determine the reproducibility of RAPD-PCR results, in light of claims that this is a weakness of the technique.

Figures


Figure 1-Reproducibility of RAPD-PCR markers. From left to right, duplicate RAPD patterns obtained with primers RAPD 1, RAPD 2, RAPD 3, RAPD 4 and RAPD 5 with P. latipinna genomic DNA.


Figure 2-RAPD banding profile of primers RAPD 1, RAPD 2, RAPD 3, RAPD 4 and RAPD 5. From left to right, duplicates of noble crayfish Astacus astacus (C1), freckled crayfish Cambarus maculates (C2), goldfish Carassius auratus (F1) and molly Poecilia latipinna (F2). Arrows indicate bands shared by both C. auratus and P. latipinna.


Summary
The ten decamer oligonucleotide primers used were arbitrarily designed with 50 and 60% G + C content. These primers were tested in PCR amplification on C. auratus and A. astacus DNA for their capability of producing clear bands. Five primers were subsequently chosen from these ten for use with all four species. Reproducibility of RAPD-PCR results was successfully achieved (Figure 1) by holding variables known to yield divergent results constant, such as DNA concentration. The five primers generated 90 bands for the crayfish DNA, 100% of which were polymorphic. The five primers generated 131 bands for the fish DNA, 94.7% of which were polymorphic. A high level of polymorphism was observed at the interspecific level,suggesting a high degree of genetic variability between the pairs of species compared. For future studies, the use of more primers would likely reveal a greater degree of similarity between the species evaluated and decrease the level of polymorphism observed. Also, the incorporation of additional species into the analysis would allow degrees of relatedness to be determined.

Full Paper

Acknowledgments

I would like to thank Jinsong Qiu and Lisa Sarran for their continuous help and enduring patience. I would also like to thank Dr. Berish Rubin for his helpful assistance and for the use of his laboratory. In addition, I thank Dr. James Fetzner at the Carnegie Museum of Natural History in Pittsburgh for his generous contribution of crayfish genomic DNA. Last but not least, I thank my classmates for providing good cheer and encouragement.


This document was last modified 05/17/2006.
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