Abstract Medicago murex Willd. is an annual species (2n = 14) widespread in the wild and of remarkable in- terest for pastures in regions with a mediterranean cli- mate. It is considered closely related to Medicago le- sinsii E. Small (2n = 16) but, up to now, there is no evi- dence demonstrating their genetic affinity. This research was undertaken to investigate the genomic relationships between M. murex and M. lesinsii by using genomic in situ hybridization (GISH). In this study GISH experi- ments were performed using both species as sources of chromosomes and genomic probes. To better evaluate the results of the hybridization, the labelled DNA of each species was hybridized to chromosomes of the same species and to chromosomes of the diploid Medi- cago littoralis (2n = 16). Strong hybridization signals were found on chromosomes of M. murex and M. lesinsii after GISH. Differences in the hybridization strength were not observed when slides from interspecific hybrid- ization were compared with the control preparations. These results suggest that consistent divergences of the DNA sequences did not occur after the separation of the two species. Instead very reduced cross hybridization was found on chromosome spreads of M. littoralis hy- bridized with the DNA of M. lesinsii or M. murex. The distribution of the ribosomal genes (rDNA) investigated by fluorescent in situ hybridization (FISH) appeared similar in both M. murex and M. lesinsii. The GISH tech- nique may be a valuable approach to obtain information on evolution of the 2n = 14 species and on the origin of the polyploids Medicago rugosa (2n = 30) and Medicago scutellata (2n = 30). The first attempt to investigate the genomic composition of M. scutellata using a genomic probe is reported in this paper. Keywords Medicago murex · Medicago lesinsii · Genomic in situ hybridization · Fluorescent in situ hybridization · rDNA Introduction Annual species constitute the most numerous component of the genus Medicago. They originated in the Mediter- ranean Basin from which they spread to the temperate regions of the world. The chromosome number 2n = 16 is the most frequently occurring in the group of diploid annual Medicago species, but there are a few species with 2n = 14 (Lesins and Lesins 1979). Contrary to the perennials which frequently exhibit polyploidy, the an- nuals are prevalently diploid with the exception of Medicago scutellata and Medicago rugosa that are poly- ploid with a chromosome number of 2n = 30. Medicago murex Willd. is among the 2n = 14 species. It is spread extensively in the wild and is of considerable interest for pastures in some countries of the Mediterra- nean area and in Australia (Francis and Gillespie 1981; Gillespie 1988). For a long time the chromosome number of M. murex was given as 2n = 16 (Heyn 1956; Clement 1962) but, after the discovery of populations with 2n = 14 (Lesins et al. 1970), both chromosome numbers have been used to describe this species. Soon afterwards it was dem- onstrated that the 2n = 14 and 2n = 16 chromosome taxa can be identified morphologically. Hence, it appeared more appropriate to classify them as separate species (Small and Brookes 1985). The 2n = 14 form maintained the name M. murex Willd., while the 2n = 16 variant was described as a new species: Medicago lesinsii E. Small. Although the two taxa are considered closely related, up to now, conclusive evidence has not been provided demonstrating their genetic relationships. Communicated by F. Salamini This research was funded by C.N.R. strategic project “Biodiversity”. The authors wish to thank Dr. Mary Traynor for checking the English form of the manuscript E. Falistocco ( ✉ ) · R. Torricelli · M. Falcinelli Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Universitá degli Studi di Perugia, Borgo XX Giugno, 06121 Perugia, Italy e-mail: egiziaf@unipg.it Tel.: +39-75-5856209, Fax: +39-75-5856224 Theor Appl Genet (2002) 105:829–833 DOI 10.1007/s00122-002-1055-5 E. Falistocco · R. Torricelli · M. Falcinelli Genomic relationships between Medicago murex Willd. and Medicago lesinsii E. Small. investigated by in situ hybridization Received: 2 July 2001 / Accepted: 4 February 2002 / Published online: 19 September 2002 © Springer-Verlag 2002