Genomic organization and phylogenetic relationships in the genus Dasypyrum analysed by Southern and in situ hybridization of total genomic and cloned DNA probes I. Galasso 1,3 , A. Blanco 2 , A. Katsiotis 3 , D. Pignone 1 , J. S. Heslop-Harrison 3 1 C.N.R., Istituto del Germoplasma, Via G. Amendola 165/A, I-70126 Bari, Italy 2 Istituto di Miglioramento Genetico delle Piante Agrarie, Università di Bari, Via G. Amendola 165/A, I-70126 Bari, Italy 3 Karyobiology Group, Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK &misc:Received: 29 March 1996; in revised form: 28 December 1996 / Accepted: 2 February 1997 &p.1:Abstract. Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the two spe- cies as probes for in situ hybridization to chromosomes, we found that the pericentromeric regions of the chromo- some arms of both species are similar, while distal re- gions show substantial differences. Two dispersed repeti- tive DNA sequences were isolated: pDbKB45 is distrib- uted along the chromosomes but amplified in the subtelo- meric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size-separated restriction enzyme digests of DNA showed many repetitive fragments, but few in com- mon between the two species. After probing Southern transfers with D. breviaristatum genomic DNA, all lanes showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum lanes. In contrast, probing with D. villosum DNA showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an inter- specific hybrid showed seven bivalents of D. breviaris- tatum origin and seven univalents from D. villosum. We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Our data support an autotetraploid origin for D. breviaristatum, but its genome and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum. Introduction The genus Dasypyrum (Cosson et Durieu) T. Durand (tribe Triticeae, family Poaceae), comprises two allo- gamous species: D. villosum (L.) Candargy [an annual diploid, 2n=2x=14, genome designated by Sears (1953) as VV], distributed in the Mediterranean region and south-western Asia, and D. breviaristatum (Lindb. f.) Frederiksen [a perennial tetraploid, 2n=4x=28, synonym D. hordeaceum (Cosson et Durieu) Candargy], mainly distributed in North Africa (Algeria, Morocco), with iso- lated populations present in Greece (Frederiksen 1991). The taxonomy and relationships of the two species have long been controversial; the species were first placed in Secale Linnaeus and later in Haynaldia Schur; the latter is still occasionally used. According to Löve (1984), D. breviaristatum contains the same genome as D. villosum, and is an autotetraploid (whose genomic formula would be VVVV). The autoploid origin is supported by Sarkar (1957), Sakamoto (1986), Bothmer and Claesson (1990), and Frederiksen (1991) after observation of up to six quadrivalents in D. breviaristatum meiotic metaphases. However, meiotic analysis of D. breviaristatum×D. villo- sum hybrids (Sakamoto 1986; Frederiksen 1991), and comparison of C-banding karyotypes of the two species (Linde-Laursen and Frederiksen 1991) does not support the autoploid origin of D. breviaristatum from D. villo- sum. The genus Dasypyrum includes useful characters that might be introduced into the wheat genome to improve powdery mildew resistance, seed protein content and quality (De Pace et al. 1990; Murray et al. 1994), as well as resistance to abiotic stress. In evolutionary and plant breeding studies there is a need to trace the origin of species and their genomic relationships with closely re- lated ones, to understand better how genomes function and evolve, and to provide vital information able to boost the introduction of alien genes (Law 1981; Jauhar 1996). Genomic in situ hybridization, using total DNA as a probe, has been shown to be a valuable technique for distinguishing autopolyploid from allopolyploid species (Bennett et al. 1992), for discriminating between closely related genomes (Anamthawat-Jònsson et al. 1990), for establishing the parental genomes of natural amphidiplo- Edited by: W. Hennig Correspondence to: I. Galasso (e-mail: germtg13@area.ba.cnr.it)&/fn-block: Chromosoma (1997) 106:53–61 © Springer-Verlag 1997