The genetic diversity of alfalfa (Medicago sativa) significantly impacts forage and seed quality, making the authentication of its populations and varieties crucial for farmers and seed suppliers. This study employed genome-wide bioinformatics approaches to discover and validate SSR (simple sequence repeat) markers, with a focus on long-core SSR motifs, for distinguishing alfalfa populations and ensuring seed authenticity. Genomic data were obtained from the NCBI database (Project: PRJNA۶۸۵۲۷۷) and the Chinese Academy of Genomic Sciences (GWH: GWHBECI۰۰۰۰۰۰۰۰), with the tetraploid alfalfa genome estimated at ۳.۱ Gb. Using Geneious R۹ and Phobos software, repetitive sequences were identified, and over one million microsatellite loci were screened. Key criteria for marker selection included
long-core SSR motifs (۳–۶ nucleotides), uniform genome-wide distribution (at least one locus per chromosome), absence of repetitive flanking sequences (within ۲۰۰ bp), and loci shorter than ۳۰۰ bp. Special emphasis was placed on selecting loci within ۱۰ Mb of chromosome ends, where recombination rates are higher, enhancing their utility for genetic studies. A total of ۴۶۴ loci met these criteria, with
long-core SSR motifs prioritized for their higher polymorphism and stability. From these, ۲۳ primer pairs were synthesized after rigorous screening for multiplexing compatibility, secondary structure stability (ΔG > -۹ kcal/mol), and absence of off-target binding. PCR optimization and validation were performed using DNA from ۳۰ seeds of each alfalfa population. Among the ۲۳ primer pairs, ۱۲ demonstrated high reproducibility, polymorphism, and the ability to differentiate alfalfa populations effectively. These markers, enriched with long-core SSR motifs, were distributed across all chromosomes, with all allele sizes ranging from ۱۷۰ to ۳۴۰ bp, ensuring broad applicability for genetic diversity studies. This study highlights the power of bioinformatics-driven approaches in developing SSR markers for alfalfa breeding and seed authentication. The selected markers provide a robust toolset for genetic diversity analysis, population differentiation, and quality control in alfalfa cultivation. Future applications include marker-assisted breeding and the development of high-yield, stress-resistant alfalfa varieties.
