Microsatellite Analysis Reveals a Distinct Genetic Cluster of Galician and Northern Portuguese Rye Used in Traditional Rye Breads

Galician and Northern Portuguese rye varieties constitute the basis for traditional bread in the northwestern Iberian Peninsula. This study evaluated the genetic diversity and structure of Galician and Portuguese ecotypes, primarily conserved at the Agricultural Research Centre of Mabegondo (CIAM) and the Universidade de Trás-os-Montes e Alto Douro (UTAD), in order to determine their origins and to differentiate them from commercial cultivars for traceability in flours and breads. A total of 411 samples, including 378 rye samples, 14 flours, and 19 breads, were evaluated using 17 simple sequence repeat (SSR) markers. Fifteen Galician ecotypes (163 samples), 22 Portuguese ecotypes (109 samples), and 9 commercial cultivars (106 samples) were analyzed. The analysis identified 310 diploid genotypes and 68 putative trisomics, which, according to the literature, are associated with meiotic disturbances and environmental adaptation. The SSR loci revealed 147 alleles, with 139 detected in diploids, 6 in putative trisomics, and 2 unique to Galician flours. Galician and northern Portuguese ryes constitute a unique and distinct genetic group, moderately differentiated from commercial cultivars (Fst = 0.089). A total of 72 alleles were unique to ecotypes, Galician flours, or bread samples. Moreover, the number of alleles was significantly higher in diploid ecotypes compared to commercial cultivars (134 vs. 67, respectively). This trend remained consistent when putative trisomics were included in the analysis (142 vs. 75, respectively). Portuguese ryes showed a higher number of putative trisomics (37) than Galician ones (25), whereas Galician ecotypes showed a slightly higher number of alleles (127 vs. 118). Neighbor-joining and principal component analyses (PCA) support the separation between Galician and Portuguese ecotypes and commercial cultivars, highlighting the singularity of these Galician and Portuguese ryes. Flour and bread derivatives produced from Galician ecotypes and cultivars were clearly differentiated from commercial cultivars, even when blends were included in the analyses. These findings underscore the potential for genetic traceability in quality bread production and emphasize the importance of this distinct genetic pool for breeding programs.