Theor Appl Genet (1993) 85:882-888 9 Springer-Verlag 1993 Genetic variation in agronomically important species of Stylosanthes determined using random amplified polymorphic DNA markers K. Kazan, J. M. Manners, and D. F. Cameron CSIRO, DivisionofTropical Crops and Pastures,Cunningham Laboratory, 306 Carmody Road,St. Lucia,QLD,4067,Australia Received April 14, 1992; Accepted June 3, 1992 Communicated by M. Koorneef Summary. Random amplified polymorphic DNA (RAPD) markers were generated from 20 cultivars and accessions representing four agronomically important species of Stylosanthes, S. scabra, S. hamata, S. 9uia- nensis, and S. humilis. Approximately 200 fragments generated by 22 primers of arbitrary sequence were used to assess the level of DNA variation. Relatively low levels of polymorphism (0-16% of total bands in pairwise comparisons) were found within each species, while polymorphisms between the species were much higher (up to 46%). Very few polymorphisms (0-2%) were detected between the individuals of the same cultivar or accession. A phenogram of relationships among the species was constructed based on band sharing. Four main clusters corresponding to each species were readily distinguished on this phenogram. The allotetraploid species S. hamata and its putative diploid progenitor, S. humilis, were more similar to each other than to S. seabra and S. 9uianensis. No variation in RAPD markers was found between the two commercial S. hamata cvs 'Verano' and 'Amiga'. Cultivar 'Oxley' in S. 9uianensis was considerably different from the other cultivars and accessions of this species. The phylogenetic distinctions obtained with RAPDs were in agreement with other studies from morphology, cytology, and enzyme electro- phoresis. The low level of polymorphisms observed within each species suggested that interspecific crosses may be a better vehicle for the construction of RAPD linkage maps in Stylosanthes. Key words: Polymerase chain reaction-Random amplified polymorphic DNA- Stylosanthes spp. Correspondence to: J. M. Manners Introduction The genus Stylosanthes (Leguminosae) has been estimated to consist of about 40 species that are classified into two sections (Stylosanthes and Stypo- santhes) on the basis of morphological characteristics (Kirkbride and de Kirkbride 1985). The section Stylosanthes contains all of the known diploid species (2n=2x=20), whereas the section Styposanthes includes both diploid and polyploid species (Mannetje 1984). Four species of the genus Stylosanthes, namely S. scabra, S. hamata, S. guianensis and S. humilis, have been widely cultivated as tropical forage legumes. Among these, S. scabra and S. hamata are allotet- raploids (2n--2x=40) and appear to be highly diploidized with regular bivalent formation at meiosis and disomic inheritance (Stace and Cameron 1984); the other 2 species, S. 9uianensis and S. humilis, are diploid (2n = 2x = 20). These 4 cultivated species are predominantly self-pollinating with a low but variable degree of outcrossing (Miles 1985; Stace 1982). The limited studies that have been carried out on interspecific hybrids have reported complete sterility (Cameron 1974), although normal bivalent formation and moderate seed set have been observed in the synthetic S. humilis x S. hamata allotetraploid hybrid (Cameron 1968). Breeding for disease resistance and other characters would benefit from the development of molecular marker technology for this genus of legumes. Variation at the DNA level as measured with molecular techniques offers a number of advantages over mor- phological data with respect to examination of the basic taxonomic relationships of the species. Variation detected by the molecular analysis of DNA can be quantified, provides many characters, and is not