Development and Application of Novel SSR Markers to Assess the Genetic Diversity and Population Structure of Phacelia secunda Along an Altitudinal Gradient in the Central Chile Andes

<i>Phacelia secunda</i> J.F. Gmel. (Boraginaceae) is a widely distributed insect-pollinated perennial herb. In central Chile (33° S), it occurs from the sea level up to 3600 m in the Andes, exhibiting broad morphological variation. In this study, we developed and characterized novel polymorphic microsatellites for this species, using an Illimina MiSeq sequencing platform. Nineteen polymorphic loci were obtained, with alleles numbers ranging from 3 to 13 per locus (mean = 5.84). Observed (<i>H</i>_O) and expected heterozygosities (<i>H</i>_E) ranged from 0.050 to 0.900 and from 0.049 to 0.825, respectively. These markers were applied to assess the genetic diversity and population structure along an altitudinal spanning from 1600 to 3600 m. The highest elevation population exhibited significantly lower within-population genetic diversity compared to lower-elevation populations. Significant population differentiation was observed along the gradient. Gene flow estimates support a stepping-stone like mode of migration, with greater exchange between adjacent elevations. These new microsatellites provide a valuable tool for elucidating the influence of altitude on genetic diversity and structure, and for evaluating the roles of local adaptation and phenotypic plasticity in shaping population variation.