Optimization of PCR conditions to amplify microsatellite loci in the bunchgrass lizard (Sceloporus slevini) genomic DNA

<p>Abstract</p> <p>Background</p> <p>Microsatellites, 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 (<it>Sceloporus slevini</it>) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains.</p> <p>Findings</p> <p>The primers used for current investigation were originally designed for the Eastern Fence Lizard (<it>Sceloporus undulatus</it>). 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.</p> <p>Conclusions</p> <p>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. 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.</p> <p>Trial Registration</p> <p>The research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.</p

Discriminating durum wheat cultivars using highly polymorphic simple sequence repeat DNA markers

The winter type durum wheat varieties of Anatolia used in this study were differentiated for the first time by using simple sequence repeat (SSR) DNA markers or microsatellites. Seven microsatellite markers were used to distinguish four well-adapted landrace selections. five cultivars and seven recently obtained advancing lines. The loci of seven microsatellites were all homozygous, but the WMS6 locus occurred with two alleles in all the genotypes. The genotypes were all distinguished from each other, with the number of alleles ranging from live to 13. The lowest and highest polymorphism information content (PIC) values were observed to be 0.609 and 0.872, respectively. Three markers alone, WMS6, WMS30 and WMS120. can distinguish all 16 genotypes. UPGMA dendogram, based on a similarity matrix by a simple matching coefficient algorithm, is in accordance with the available pedigree information

SSR markers in the genetic relationship studies of Turkish durum wheat varieties

DNA markers are the essential tools for differentiating genotypic characters of varieties and cultivars. Microsatellite or Simple Sequence Repeat (SSR) markers are PCR-based molecular markers. SSRs are the type of markers that can be used most efficiently for DNA fingerprinting due to their reproducible and high level of polymorphic features. The variations in the number of nucleotide repeats, SSR length polymorphism, were detected by separating (alpha P-32)-dATP labeled PCR products obtained with locus specific primer pairs on the DNA denaturing gels. The durum wheat (Triticum durum L.) varieties of Anatolia used in this study were differentiated using microsatellite markers. The genotypes were all distinguished from each other, with the average PIC value of 0.756 +/-0.082. In addition, an enriched library in terms of a dinucleotide repeat type SSR is in progress for the development of new SSR markers

Molecular and Elemental Characterization of Selected Turkish Durum Wheat Varieties

Combination of elemental and protein studies along with molecular data using microsatellite markers may lead CO the better and realistic determination of relatedness between the varieties and their populations. In this study, the extent of diversity among five Turkish durum wheat cultivars and their populations has been assessed using seven microsatellite markers and the elemental analysis together with the differences in their protein content. In molecular analysis, total 23 alleles have been obtained among all the genotypes with middling of 4.6 per primer. On employing UPGMA Dendrogram, Principle Coordinate Analysis (PCoA) and Winboot analyses, both inter and inn-a varietal polymorphic studies had shown similar clustering with minor differences. As a result of AMOVA performed, the extent of diversity was found to be higher among the genotypes (76%) in comparison to the variability within the genotypes (24%). In elemental analyses, 'Selcuklu-97' was found to be the most efficient variety with high content of several elements. Also, strong and positive correlation has been observed between magnesium-phosphorus, magnesium-sulphur and sulphur-sodium, while noteworthy negative correlation has been observed between sodium and zinc. The protein content of the genotypes was found in the range of 15.17-16.90%. The diversity revealed in durum genotypes can be employed in genetic expansion of the crop. These involved varieties may aid to avoid genetic attrition coming up from the landraces. The information provided can be utilized by breeders for appropriate selection of both, generically and nutritionally efficient durum wheat varieties