Research Article |
Corresponding author: Ilya V. Kirov ( kirovez@gmail.com ) Academic editor: Andrzej Joachimiak
© 2016 Ilya V. Kirov, Katrijn Van Laere, Nadine Van Roy, Ludmila I. Khrustaleva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kirov IV, Van Laere K, Van Roy N, Khrustaleva LI (2016) Towards a FISH-based karyotype of Rosa L. (Rosaceae). Comparative Cytogenetics 10(4): 543-554. https://doi.org/10.3897/CompCytogen.v10i4.9536
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The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding activities are going on supported by molecular studies. However, the cytogenetic studies of rose species are scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping. Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore, in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crépin, 1888), a rose species with several benefits for advanced molecular cytogenetic studies of genus Rosa (
Cytogenetic markers, fluorescence in situ hybridization, interstitial telomeric repeat (ITR), 5S rDNA, 45S rDNA, Rosa wichurana
Rosa Linnaeus, 1753 is an economically important ornamental genus belonging to the Rosaceae. Of the approximately 200 described Rosa species (
Rosa wichurana (Crépin, 1888) is a valuable model species for molecular cytogenetic studies in Rosa genus (
Because the chromosomes are difficult to distinguish, further progress in cytogenetic mapping depends on the development of cytogenetic markers useful for chromosome identification. The conservative tandemly organized repetitive sequences 5S and 45S rRNA genes are valuable sources of cytogenetic markers, and have been used for chromosome identification in many plant species including Rosa species (
Development of an effective cytogenetic marker system is an important step in answering many biological questions (
This study aims to explore the opportunities of ITRs, 5S and 45S rDNA as cytogenetic markers allowing to distinguish individual chromosomes of Rosa. FISH with 5S rDNA, 45S rDNA and the Arabidopsis-type telomeric repeat was performed. These FISH results were combined with chromosome morphology measurements (
Rosa wichurana plants were grown in the field. For chromosome slide preparations, cuttings were made. Rooted cuttings were transferred to terracotta stone pots and grown in the greenhouse (moderate climatic conditions, East Flanders, Belgium). To prepare mitotic chromosome slides, young meristems were harvested. For meiotic (pachytene) chromosome slides, flowers buds with a hypanthium size of 3 mm were harvested.
Plasmids containing 5S rRNA genes of rye (pSCT7,
Pachytene and mitotic chromosomes were prepared according to the “SteamDrop” protocol (
For FISH we used the protocol described in
For sequential FISH experiments, the slides were washed in the series of ethanol (70%, 90% and 100%) after the first round of FISH and then the above-mentioned FISH procedure was applied.
Images were acquired using a Zeiss AxioImager M2 fluorescence microscope (400× and 1000× magnification) equipped with an AxioCam MRm camera and Zen software (Zeiss, Belgium). Final image adjustments were performed using Photoshop (Adobe Inc., USA). Measurements of chromosome lengths and karyotyping was done in MicroMeasure version 3.2 (
FISH using the common hybridisation temperature of 37°C with 45S rDNA revealed a signal on chromosome 7, while the Arabidopsis type telomere-based probe hybridized on chromosome 5 (Fig.
FISH on the chromosomes of R. wichurana. AFISH with Arabidopsis-type telomere probe (red) and 45S (green) under hybridization at 37°C BFISH with Arabidopsis-type telomere probe under the low hybridization stringency condition (23-25°C). Arrows indicate the major ITRs on chromosome 5 and arrowheads show the ITRs which are visible under the low hybridization stringency condition C The same metaphase as in 1B rehybridized with 5S rDNA under the common hybridization stringency (37°C). Arrows indicate the 5S rDNA signals. Sacale bar: 5 µm.
To further evaluate the value of the telomeric repeat (TR) as a cytogenetic marker, FISH was carried out at room temperature (the low hybridization temperature). We observed the Arabidopsis-type TR signals on all chromosome ends (Fig.
Sequential FISH at the low hybridization temperature with the Arabidopsis-type telomere-based probe and 5S rDNA showed co-localization of these signals on chromosome 7. We also performed double-color FISH with the Arabidopsis-type telomere repeat-based probe and the 45S rDNA probe under the low temperature of hybridization (Fig.
A summary of the karyotypic features and distribution of FISH probes is given in Fig.
Double-color FISH under the low hybridization conditions using the Arabidopsis-type telomere repeat-based (red) and 45S rDNA (green) probes to R. wichurana mitotic chromosomes. Scales bar: 10 µm.
Distribution of the repetitive sequences on the mitotic R. wichurana chromosomes. 1 – ITR1: signals that are visible under hybridization at 37°C as well as at low temperature (23–25°C). 2 – ITR2: signals that are visible only under hybridization at low temperature (23–25°C).
All the other chromosomes can only be distinguished at this time based on their morphological parameters. Differentiation between chromosome 1 and 2 is possible by their centromeric indices which are 46.00 ±1.2% and 40.30 ±1.3%, respectively (
FISH experiments with 5S rDNA, 45S rDNA, and the Arabidopsis-type TR on rose pachytene chromosomes provide a much higher resolution of the mapped sequences. 5S rDNA-FISH on pachytene chromosomes did not reveal any reliable signals, while FISH with the 45S rDNA probe resulted in a clear signal at the subtelomeric region of the NOR-bearing chromosome (Fig.
High resolution physical mapping of ITR on R. wichurana pachytene chromosomes. FISH with the Arabidopsis-type telomere repeat probe (red) and 45S (green). Merged (A) and the DAPI gray scale (B) pictures are shown. FISH was performed under the low hybridization stringency condition. Dotted lines show the regions that were digitally enlarged (A’ and B’). Scales bar: 5 µm.
Rosa mitotic and meiotic chromosomes are difficult to distinguish by common karyotype analysis (Kirov et al. 2014,
In this study, FISH with the 45S rDNA and the Arabidopsis-type telomere probe, reliably identified 2 (chromosome 5 and 7) of the 7 pachytene bivalents of R. wichurana. These markers will accelerate the ongoing physical mapping of pachytene chromosomes of R. wichurana as their identification by morphological parameters or specific heterochromatin patterns is impossible (
ITRs can be used to trace ancient chromosomes rearrangements such as chromosome fusions, Robertsonian translocations and duplications resulting in dysploidy (
Interestingly, FISH under the low hybridization temperature – and thus low stringency – revealed more chromosomes possessing the telomeric repeat compared to FISH performed under the common hybridization temperature. This result suggest that these chromosomes (1, 2 and 7) may contain truncated or diverged telomere motifs. As a consequence for our experiments, the telomeric probe may be much more informative as cytogenetic marker when hybridized at a lower temperature than at 37°C (
High-resolution FISH on pachytene chromosomes with the telomere probe resulted in a signal in the centromere of chromosome 5, indicating that the telomere-like motifs may be the components of the R. wichurana functional centromere as it has been shown for potato (
This is the first report describing valuable cytogenetic markers for four mitotic chromosomes and two pachytene bivalents of R. wichurana. Moreover, by combining our FISH results with the chromosome morphology measurements (
The authors would like to thank Oleg S. Alexandrov for providing the telomere probe.