Optimization of SCoT and iPBS-Retrotransposon Marker Systems for PCR-Based Genetic Analysis in Alfalfa (Medicago sativa L.)

Abstract

The correct selection and appropriate optimization of molecular markers and primers used in genetic diversity studies are crucial for the unveiling of true polymorphism among genotypes and to make sure that the data obtained can be used effectively in breeding programs. Two important molecular marker systems that are widely used in genetic diversity analyses are SCoT and iPBS-retrotransposon markers due to their PCR-based structures and high discriminatory ability. Optimization of these markers is of high importance in alfalfa, which has a complex, polyploid, cross-pollinated genome structure. Alfalfa is not only among the most important forage crops of the world but also a strategic agricultural crop that needs continuous development because of its economic value and environmental advantages. Thus, genotype identification, population structure characterization, and determination of genetic relationships between populations constitute the basic research areas of the sustainable breeding efforts. In the present study, six alfalfa genotypes collected from distinct geographical regions of Turkey and representing the gene pool, together with two commercial varieties, were used as plant materials. Different DNA volumes were tested to find the optimal template DNA amount in the PCR amplification of the SCoT and iPBS-retrotransposon marker systems. The conditions determined after optimization were validated by screening with different SCoT and iPBS primers. The obtained results provide a reliable and reproducible methodological framework for studies on genetic diversity, population structure, and breeding in alfalfa.