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

Abstract

BackgroundThe 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.ResultsBased 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.ConclusionsBy 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.

Distribution patterns of rDNA loci in the Schedonorus-Lolium complex (Poaceae)

“…Three of these, M. tuberosa , L. multiflorum MRCN and F. simensis , were previously unmapped. The results for L. perenne and N European L. multiflorum agree with previous studies ( Thomas et al 1996 ; Ansari et al 2016 ). The new results are discussed first and then rDNA chromosomal patterns across the complex are reviewed.…”

“…Seeds were germinated and grown in a glasshouse. Somatic chromosome preparations were obtained from the meristematic tissue of actively growing root tips according to the flame-drying technique described earlier ( Ansari et al 1999 , 2016 ). Good quality cytological preparations were selected after screening using phase contrast optics.…”

“…Therefore, conventional karyological information is of little value for evaluating evolutionary changes ( Malik and Thomas 1966 ; Namaganda et al 2006 ; Kopecký et al 2010 ). Molecular cytogenetic mapping of 5S and 35S rDNA has detected variations in the distributional patterns of the two rDNA markers among diploids and polyploids in this complex ( Thomas et al 1996 , 1997 ; Ksia¸zczyk et al 2010 ; Inda and Wolny 2013 ; Ansari et al 2016 ; Ezquerro-López et al 2017 ; Shafiee et al 2020 ). Based on their report, Ezquerro-López et al (2017) made a preliminary attempt to decipher the evolutionary relationships among Festuca species belonging to this complex.…”

A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

“…Typically, to deduce the position of FISH signals in relation to banding, fluorescent differential staining and FISH are done separately on different slides or infrequently—sequentially on the same slide. In the latter case, after subjecting to a fluorescent differential staining, the preparation is then destained and treated according to the FISH protocol (e.g., Golczyk et al 2010 ; Ansari et al 2016 ). The disadvantage of the sequential procedures is however that previously identified and photographed chromosomes/nuclei, when further processed for FISH (detaching of the coverslip, removing the mounting medium from the slide, destaining and in situ hybridization procedure), are difficult to find, fragmented, lost, and/or changing their position on the slide, which renders such a highly troublesome and tricky mapping extremely difficult.…”

Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid

“…Somatic chromosome preparations were obtained from actively growing root tips using a flame-drying technique after enzyme maceration as described earlier [ 25 ] with minor modifications [ 49 ]. Meiotic chromosome preparations were obtained by squashing PMCs from young floral buds after enzymatic maceration.…”