Karyotype and C-Banding Patterns of Mitotic Chromosomes in Diploid Bromegrass (Bromus riparius Rehm)

nomic relationships among species (Vosa, 1975; Cai and Chinnappa, 1987; Fominaya et al., 1988; Gill and Sears, Previous cytogenetic studies of the genus Bromus L. were limited 1988; Tayyar et al., 1994; Falistocco et al., 1995). The to chromosome counts and construction of karyotypes on the basis of Feulgen staining. Since the chromosomes of Bromus are similar in C-banding procedure has not been explored fully for morphology, these karyotypes are of limited use for chromosome chromosome identification and genome analysis in identification and genome analysis. The objectives of this study were Bromus. to develop and evaluate a Giemsa C-banding procedure to use in Bromus inermis L. (2n 5 56), or smooth bromegrass, identification of individual bromegrass chromosomes and to develop is one of the most widely used forage species in agricula karyotype for diploid Bromus riparius Rehm. (2n 5 14; PI 440215). ture. Progenitor species of this complex polyploid have All chromosomes had one or more C-bands which were located mainly not been fully delineated (Vogel et al., 1996). In this at telomeric regions. A group (I) of four pairs of chromosomes had report, we describe an effective C-banding procedure telomeric bands on only one arm and could be differentiated. In this for bromegrasses that was used to identify chromosomes group, one pair had an interstitial C-band along with a telomeric band, and construct a karyotype of a possible progenitor, the one pair had a nucleolus organizer region (NOR) at a subtelomeric location on the short arm, and the other two pair could be distinguished diploid species (B. riparius Rehm.), of the B. inermis by centromere location. The other group (II) of three pairs of chromocomplex. The karyotype is based on chromosome somes had telomeric bands on both arms. The unequivocal identificalength, arm ratio, and C-banding patterns. tion of specific chromosomes of Group II was not possible in all cells The B. riparius accession used in this study, PI 440215 because of their similarity and differential condensation of chromo(National Plant Germplasm System, 1999; Armstrong, somes. Chromosomes of both groups were either metacentric or sub1987), was collected from Chimkent in Kazakhstan in metacentric. The total length of individual chromosomes ranged from 1977. Armstrong (1987) determined its ploidy level as 5.58 to 6.87 mm and the arm ratios ranged from 1.02 to 1.5. The 2n 5 14 and suggested that it could be a progenitor of homologous chromosomes were paired and assigned numbers I to the Bromus inermis complex since its morphology reVII in decreasing length. A karyotype was constructed by means of the sembles that of the tetraploid forms of B. inermis colC-bands, mean chromosome lengths, and arm ratios. The C-banding procedure used in this study could be used to developed karyotypes for lected from the same region. It is the only diploid B. the other species of the genus Bromus and these C-banded karyotypes riparius in the USDA Plant Germplasm System that we could be used to compare genomes within the genus. have identified to date. MATERIALS AND METHOD T genus bromus contains more than 100 species distributed over all continents (Gould and Shaw, Seeds of diploid Bromus riparius Rehm. (PI 440215) were 1983). The ploidy level within the genus varies from obtained from the USDA Regional Plant Introduction Station, diploid to decaploid (Armstrong, 1991). Feulgen stainPullman, WA. Seeds were used to grow plants in the greening based karyotypes have been constructed for some house and to produce seedlings in germination boxes. Twenty plants were grown in pots filled a mixture of soil, Perlite, and species of the genus Bromus (Rychlewski, 1970; Armpeat moss (2:1:1 v/v/v). Plants were exposed to 16-h photopestrong, 1977; Stebbins, 1981). It has been difficult to riod during the period when root tips were collected. They identify all of the chromosomes because the chromowere maintained in a vegetative stage by clipping. Actively somes are similar in morphology. Genetic relationships growing root tips were collected from potted plants 3 to 4 wk among a few species have been studied by means of after each clipping. Seeds were germinated in germination crossability and chromosome pairing data (Stebbins, boxes containing germination paper saturated with distilled 1981; Armstrong, 1991). Giemsa C-banding technique, water. Imbibed seeds were kept at room temperature for 1 d which stains constitutive heterochromatin, is a techbefore they were transferred to a refrigerator at 0 to 48C for nique that has been used successfully in many species one to a few days (until the majority of seeds appeared to be to identify individual chromosomes and to establish gegerminating). Boxes were then placed in the dark at room temperature and fast growing root tips were collected when they reached 1 to 1.5 cm in length. Harvested root tips from M. Tuna, Dep. of Agronomy, Tekirdag Agriculture Faculty, Univ. of potted plants or germinated seeds were placed in vials conTrakya, Tekirdag, Turkey; K.S. Gill, Dep. of Agronomy, Univ. of taining 0.05% colchicine (w/v). The colchicine solution was Nebraska, Lincoln, NE 68583-0915; K.P. Vogel, USDA-ARS, Wheat, drained from the vials after 1 to 1.5 h and replaced with a Sorghum, and Forage Res. Unit, 344 Keim Hall, Univ. of Nebraska, fixative of ethanol: glacial acetic acid (3:1, v/v) for 2 wk to a P.O. Box 830937, Lincoln, NE 68507-0937. Contribution from Nebraska Agric. Res. Div., Journal Series No. 12 973. Reported research few months. is from a dissertation submitted by the senior author (Metin Tuna) The C-banding method used was that described by Giraldez in partial fulfillment of the requirements for the Ph.D. degree at et al. (1979) and slightly modified as follows. Root tips were the University of Nebraska. Received 3 April 2000. *Corresponding author ([email protected]). Abbreviations: Mb, megabases; NOR, nucleolus organizer region; Chr, chromosome; Sat, satellite. Published in Crop Sci. 41:831–834 (2001).