Optimization of the Analysis of Almond DNA Simple Sequence Repeats (SSRs) Through Submarine Electrophoresis Using Different Agaroses and Staining Protocols

Authors

1 Department of Landscape Engineering, Faculty of Agriculture, Malayer University, Malayer, Iran

2 Department of Horticulture, Agriculture and Natural Resources Research Center, Shahrekord, Iran

3 Department of Biology, Faculty of Sciences, Malayer University, Malayer, Iran

4 Department of Plant Breeding, CEBAS-CSIC, P.O. Box 164, 30100 Campus Universitario, Espinardo, Murcia, Spain

Abstract

Simple sequence repeat (SSR markers or microsatellites), based on the specific PCR amplification of DNA sequences, are becoming the markers of choice for molecular characterization of a wide range of plants because of their high polymorphism, abundance, and codominant inheritance. Different methods have been used for the analysis of the SSR amplified fragments being submarine agarose electrophoresis the more suitable method for the routine application. In this work we have performed a comparative study of the utilization of four different types of low melting (Metaphor®, Sea Kem®, and MS-8®) and regular (LD-2®) agaroses and two different staining protocols using Ethidium Bromide and Gel Red Nucleic Acid Gel Sating®. Almond cultivars assayed included the Spanish cultivars ‘Antoñeta’, ‘Marta’, ‘Penta’, ‘Tardona’ ‘Desmayo’ and ‘Guara’, the French cultivars ‘Ferragnés’ and ‘R1000’, the USA cultivar ‘Mission’, the Tunisian cultivar ‘Achaak’, the Italian cultivar ‘Tuono’ and the Australian cultivar ‘Chellaston’. SSR detection using Metaphor® agarose gel electrophoresis was the most efficient with higher resolution and would be able to resolve most of allelic variation in comparison with the other three agaroses assayed. In addition, gel staining using Ethidium Bromide showed similar results than the GelRedTM Nucleic Acid Gel Stain® although it is much more toxic. The use of MetaPhor® agarose and GelRedTM Nucleic Acid Gel Stain® appears good indicated for molecular characterization of mapping of population due to its good resolution in comparison with the rest of agaroses, less toxicity in comparison with the use of Ethidium Bromide, and lower cost and easier routine application in comparison with the automatic capillary sequencing.

Keywords