Abstract
BackgroundMicrosatellites, also called Simple Sequence Repeats (SSRs), repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis. A total of five different primer pairs were optimized for polymerase chain reaction (PCR) to amplify microsatellite loci in total genomic DNA of bunchgrass lizards (Sceloporus slevini) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains.FindingsThe primers used for current investigation were originally designed for the Eastern Fence Lizard (Sceloporus undulatus). Five primer pairs were selected based on annealing temperatures for optimizing the PCR conditions to amplify with bunchgrass lizards. Different concentrations of DNA and annealing temperature were optimized. While keeping other reagents constant, a DNA concentration, 37.5 ng in the final reaction volume and PCR conditions of an initial denaturation of 94°C for five minutes, an annealing temperature of 55°C and final extension of 72°C for four minutes gave the best amplification for all the primer pairs.ConclusionsModifying the standard protocol for annealing temperatures and final extension time increases the success of cross amplification of specific microsatellite loci in the bunchgrass lizard. A loading volume of 5 ul DNA at a concentration of 10 ng/ul and a 2% agarose for gel electrophoresis were observed the best for cross amplification of selected five primer pairs on bunch grass lizard.Trial RegistrationThe research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.
Highlights
Microsatellites, called Simple Sequence Repeats (SSRs), repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis
Modifying the standard protocol for annealing temperatures and final extension time increases the success of cross amplification of specific microsatellite loci in the bunchgrass lizard
The polymerase chain reaction (PCR) conditions standardized for microsatellite amplification in this study were an initial denaturation at 94°C
Summary
Microsatellites, called Simple Sequence Repeats (SSRs), repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis. To amplify microsatellite loci in total genomic DNA of bunchgrass lizards (Sceloporus slevini) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains. Slevin’s bunchgrass lizard, Sceloporus slevini [4] inhabits both disjunct mountain grasslands in southeastern Arizona, southwestern New Mexico, the Sierra Madre Occidental of Mexico [5,6,7] and lower elevation grasslands in the Sonoita plain and Canelo grasslands in Arizona [7,8]. Species-specific microsatellite markers [9] can be used to elucidate gene flow patterns and genetic diversity studies [10]. The primer sequences used for the current project were originally developed for the Eastern
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