Development and validation of genome-wide simple sequence repeat markers for large scale genotyping applications in barnyard millet (Echinochloa spp.)

Genomic resources for domesticated Echinochloa species are inadequately characterized, while research on extensive diversity panels and trait mapping remains in its nascent stages. This study aimed to develop and validate genome-wide simple sequence repeat (SSR) markers from the E. crus-galli genome and assess their utility in the genetic analysis of cultivated barnyard millet species. A total of 25,108 perfect and 271,509 imperfect SSRs were identified from a sequence size of 132,420,621 bp in barnyard millet, resulting in a relative density of 3,249.39 bp/Mb. Trinucleotide repeats represented the principal category of microsatellites, accounting for 89.81%, and were predominantly enriched with GC-rich motifs. A total of 21,655 primer pairs were developed, from which 228 hyper-variable SSR loci were chosen based on repeat length and motif attributes. A chromosome-level analysis revealed uneven distribution of SSRs and distinct subgenome-specific variations, with the CH subgenome exhibiting the highest density of SSRs. Of 100 randomly selected SSR markers evaluated across 73 barnyard millet genotypes, 58 demonstrated efficient amplification, whereas 42 were polymorphic.The population structure, neighbor-joining clustering and principal coordinate analysis reliably classified the genotypes into four principal subpopulations with negligible admixture. This study presents the inaugural thorough genome-wide development and validation of SSR markers in cultivated barnyard millet. The newly developed chromosome-specific SSR markers demonstrated moderate to high polymorphism and successfully elucidated genetic relationships among diverse Echinochloa genotypes.