Fluorescence chromosome banding and FISH mapping in perennial ryegrass, Lolium perenne L

Abstract

Background: The unambiguous identification of individual chromosomes is a key part of the genomic characterization of any species. In this respect, the development and application of chromosome banding techniques has revolutionised mammalian and especially, human genomics. However, partly because of the traditional use of chromosome squash preparations, consistent fluorescence banding has rarely been achieved in plants. Here, successful fluorescence chromosome banding has been achieved for the first time in perennial ryegrass (Lolium perenne), a forage and turf grass with a large genome and a symmetrical karyotype with chromosomes that are difficult to distinguish.

Results: Based on flame-dried chromosome preparations instead of squashes, a simple fluorescence Q-banding technique using quinacrine mustard, unambiguously identified each chromosome and enabled the development of a banded karyotype and ideogram of the species. This Q-banding technique was also shown to be compatible with sequential FISH mapping enabling labelled genes and molecular markers to be precisely assigned to specific cytogenetic bands. A technique for DAPI-banding, which gave a similar pattern to Q-banding, was also introduced. This was compatible with FISH mapping and was used to anchor a single copy gene from an earlier mapped linkage group of L. perenne, thus providing a step towards integration of the genetic and cytogenetic maps.

Conclusions: By enabling the allocation of genes mapped by other methods to physically identified chromosome positions, this work will contribute to a better understanding of genomic structures and functions in grasses.

Keywords: Banded ideogram; Chromosome Q-banding; Chromosome identification; FISH mapping; Lolium perenne; Perennial ryegrass; Standardized banded karyotype.

Fig. 1

Q-banding and sequential FISH mapping in L. perenne. Early metaphase cells of diploid (a, b) and haploid (c, d) L. perenne after Q-banding (a, c) and sequential FISH mapping (b, d) with 5S and 18S rDNA sequences. Dotted lines in (a) and (c) denote decondensed NORs. Bar represents 5 μm

Fig. 2

Q-band karyotype standardization of L. perenne with sequential FISH mapping of cytogenetic markers, Representative Q-banded karyotype (grayscale) and sequential FISH mapping of 5S (red) and 18S (green) rDNA sequences. Diagrammatic representation of Q-banding on left of each chromosome. Lines across chromosomes represent centromeric positions

Fig. 3

Q-banded ideogram of L. perenne

Fig. 4

DAPI-banding and FISH mapping of LpGI gene in L. perenne. Metaphase cell after (a) DAPI staining and (b) FISH mapping with LpGI (red) and 18S rDNA (green) sequences. In (c), the left inset shows an example of Q-banded chromosome 2 (grayscale) with sequential hybridization of 18S rDNA. The right inset shows DAPI-banded chromosome 2 in grayscale along with LpGI (red) and 18S rDNA (green) hybridizations from two cells at different condensation levels. At the bottom right, LpGI is positioned on the banded ideogram of chromosome 2. Red arrows in (b) and (c) indicate LpGI FISH signals. Lines across chromosomes in (c) represent centromeric positions. Bar represents 5 μm