Chromosomes of Bromus auleticus Trin . ex Nees ( Poaceae )

The chromosome number of 14 accessions of Bromus auleticus Trin. ex Nees, native of Rio Grande do Sul, was 2n = 6x = 42, same ploidy level found in other South-American Bromus species. Its chromosomes were metacentric or submetacentric, ranging from ca. 4 μm to ca. 8 μm in length. Up to two satellite-bearing chromosome pairs were sometimes observed. However, as already reported for other species, the high symmetry and homogeneity of the karyotypes made it difficult to detect possible intraspecific differences.


Introduction
The genus Bromus comprises more than 100 annual and perennial species (Mabberley, 1997) distributed around the world, typically cool-season grasses, varying greatly in adaptation and use, and including some important forage and range species, such as the perennials B. inermis, B. anomalus, B. pumpellianus, B. catharticus, and the annuals B. mollis and B. rigidus, among others (Carlson and Newell, 1985).
In Rio Grande do Sul, Southern Brazil, the genus is represented by the native B. auleticus, B. brachyanthera, B. catharticus, and the exotic B. commutatus and B. mollis species (Longhi, 1977).Among these, the perennial allogamous B. auleticus, popularly known as "cevadilha" or "cevadilha vacariana" (Araújo, 1971;Mohrdieck, 1973), occupies an outstanding position as a forage in the native pastures of that State.This species has called the attention of plant breeders and agronomists (Mohrdieck, 1993;Soares, 1999, among others), and some populations have already been characterized by isozyme and RAPD analysis (Yanaka, 2002).Cytological data on this species, however, is scarce.Only isolated chromosome counts in a few plants (2n = 42) have been performed (Elliott, 1948and 1949, in Federov, 1969;Bowden and Senn, 1962) and, to our knowledge, no other cytogenetic information on the species has been published.This paper reports data on chromosome numbers and morphology of 14 natural populations of B. auleticus from the State of Rio Grande do Sul.

Material and Methods
Seeds were collected at several locations of Rio Grande do Sul, Southern Brazil (ca.33°to 28°S) (Table 1).Each sample from each collection site was considered as an accession.Seeds from 11 of these accessions were kindly provided by the germplasm bank of EMBRAPA-CPPSul (Empresa Brasileira de Pesquisa Agropecuária -Centro de Pesquisa de Pecuária dos Campos Sul-Brasileiros).
For somatic chromosome counts, seeds were germinated in Petri dishes with moistened filter paper, and the 1-1.5 mm long roots were pre-treated with a saturated solution of paradichlorobenzene (PDB) for 18-20 h at 4 °C, fixed in 3:1 ethanol-acetic acid for 24 h, and stored in 70% ethanol below 0 °C until required.For slide preparation, root tips were hydrolyzed with HCl 1 N for 10 to 15 min at 60 °C, stained with Feulgen for ca. 2 h, and squashed in a drop of acetic carmine.Semi-permanent slides were examined by light microscopy.Only intact and well-spread metaphases were analyzed.Each root tip was considered as one individual.
Up to two satellite-bearing chromosome pairs could be seen in a few cells of some accessions, but in most cells the satellites were very difficult to distinguish.Armstrong (1982) already pointed out the difficulty in detecting satellites when they are small or when the chromosomes are very condensed.Naranjo (1985), reviewing data from the literature, commented that there might be actual differences in the number of satellite-bearing chromosomes (or in the number of these chromosomes expressed) among different Bromus individuals or species, but that the high symmetry and homogeneity of the karyotypes could also make it difficult to detect possible differences.
A pilot FISH (fluorescent in situ hybridization) test performed in one B. auleticus accession (data not shown) suggested the presence of up to fourteen 45 S rDNA sites.Bromus chromosomes are a good material for cytogenetic analysis, regarding for example their size, however the little success obtained so far in detecting variability with conventional staining and C-banding techniques suggests that more advanced techniques should be employed.

Figure 1 -
Figure 1 -Metaphase plate of Bromus auleticus (2n = 42) (one chromosome on the lower left side of the cell is folded).Scale bar equal to 10 µm.

Table 1 -
List of the accessions of Bromus auleticus examined, number of individuals and cells analyzed per accession, and somatic chromosome numbers (2n).
a accession from the germplasm bank of EMBRAPA-CPP Sul.