Genetic relationships among Stylosanthes species as revealed by sequence-tagged site markers

Nineteen sequence-tagged site (STS) primer pairs were designed on coding an d non-coding regions in nine published Stylosanthes genes, which were mostl y derived from cDNA. Direct sequencing of PCR products derived from genomic DNA allowed us to identify introns and to design specific primers flanking these introns. The use of 23 STS primer pairs for the detection of intra- and inter-specific variation in Stylosanthes based on size differences was tested on a core set of Stylosanthes species. Based on these results, 20 ST S markers were selected to determine genetic relationships among 63 genotyp es representing 24 Stylosanthes species. A total of 148 alleles were amplif ied and analyzed, resulting in a genetic similarity value ranging from 0.62 to 0.98 among the species. Based on duster analysis, three main groups and three subgroups were determined, and most of the species were classified u nambiguously. Alloploid species were recognized by the occurrence of more t han one allele per STS marker, indicating fixed heterozygosity. Sixteen STS markers were useful for the identification of genotypes within a species. Inter-species relationships, as revealed by STS, were in general agreement with previous morphological and molecular relationship studies. These STS m arkers are useful as an additional tool for the identification of species, subspecies and genotypes in Stylosanthes, with a view to plant conservation and breeding.