Research Article |
Corresponding author: Pavel Yu. Kroupin ( pavelkroupin1985@gmail.com ) Academic editor: Xutong Wang
© 2019 Pavel Yu. Kroupin, Victoria M. Kuznetsova, Ekaterina A. Nikitina, Yury Ts. Martirosyan, Gennady I. Karlov, Mikhail G. Divashuk.
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:
Kroupin PYu, Kuznetsova VM, Nikitina EA, Martirosyan YuTs, Karlov GI, Divashuk MG (2019) Development of new cytogenetic markers for Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang. Comparative Cytogenetics 13(3): 231-243. https://doi.org/10.3897/CompCytogen.v13i3.36112
|
Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu & R.-C.Wang, 1993 is an important polyploid wild perennial Triticeae species that is widely used as a source of valuable genes for wheat but its genomic constitution has long been debated. For its chromosome identification, only a limited set of FISH probes has been used. The development of new cytogenetic markers for Th. ponticum chromosomes is of great importance both for cytogenetic characterization of wheat-wheatgrass hybrids and for fundamental comparative studies of phylogenetic relationships between species. Here, we report on the development of five cytogenetic markers for Th. ponticum based on repetitive satellite DNA of which sequences were selected from the whole genome sequence of Aegilops tauschii Cosson, 1849. Using real-time quantitative PCR we estimated the abundance of the found repeats: P720 and P427 had the highest abundance and P132, P332 and P170 had lower quantity in Th. ponticum genome. Using fluorescence in situ hybridization (FISH) we localized five repeats to different regions of the chromosomes of Th. ponticum. Using reprobing multicolor FISH we colocalized the probes between each other. The distribution of these found repeats in the Triticeae genomes and its usability as cytogenetic markers for chromosomes of Th. ponticum are discussed.
Thinopyrum ponticum, cytogenetic markers, repetitive DNA, fluorescence in situ hybridization, quantitative PCR, copy number
Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu & R.-C.Wang, 1993 (=Agropyron elongatum Host ex P. Beauvois, 1812, 2n =10×=70) is an allodecaploid perennial grass used as a valuable pasture crop because of its heavy fibrous root system and good regrowth (
The polyploid nature of Th. ponticum is still debatable, and the question about the possible origin of its subgenomes remains open to discussion.
Repetitive DNA is the most promising for the development of chromosomal markers (
We have developed an algorithm to develop cytogenetic markers based on satellite DNA and identified five of the most copy satellite repeats in Aegilops tauschii Cosson, 1849 genome, which were successfully applied to A, B and D subgenomes of wheat and R of rye (
Plants of Th. ponticum (accession PI636523, Germplasm Research International Network, USA) were grown under optimum temperature and soil water conditions in a greenhouse.
Genomic DNA was extracted from plants according to the protocol in
Designed primers for tandem repeats and oligonucleotide sequence used as probes (for P332 and P132).
Repeat | Primers / oligonucleotide probe |
---|---|
P720 | F: 5’-AGCCACGTCATCAACTTTCA-3’ R: 5’-TGTCCAGTTTGTACGCGAAG-3’ |
P170 | F: 5’-TCCTTGGAAGAATCTAGTCGTCA-3’ R: 5’-TCGGTTTTGCGCAGTGTTAA-3’ |
P427 | F:5’-CGCCTCGACTCGCGTTACCC-3’ R:5’-GCCGAGACGAGCACGTGACA-3’ |
P332 | F: 5’-GCTCTTCACTCGGTAGGATTT-3’ R: 5’-TCCCGTACTCGCCTAAGT-3’ BIO-5’-CGAGTGAGAGGATTGCTCTTCACTCGGTAGATTTTT-3’ |
P132 | F:5’-TTTTACACTAGAGTTGAACTTGCTC-3’ R:5’-TGTAAAATTATTTGAACTAGGCTAT-3’ 6-FAM-5’-TTTTACACTAGAGTTGAACTTGCTCTATAGGCTAGTAC-3’ |
A chromosome spread preparation was made according to
The detection was performed using FITC conjugated to antidigoxigenin for P170 and P427 and Cy3 conjugated to streptavidin (Roche) for P720 and P332; the chromosomes were counterstained with DAPI. Signals in all variants were visualized using an AxioZeiss Imager V1 (Carl-Zeiss, Oberkochen, Germany) fluorescence microscope with Cy3 or FITC filter. The results were recorded with an AxioCam MRm Zeiss camera (Carl-Zeiss, Oberkochen, Germany) and contrasted using AxioVision. Reprobing of the slides was performed according to
In order to develop cytogenetic markers for Th. ponticum, we chose five of the largest copy number repeats identified in the genome-wide sequence of A. tauschii: P720, P170, P427, P332, P132 (
The decimal logarithm of the quantity of tandem repeats relative to the reference gene (VRN1) in Th. ponticum as revealed using qPCR. Error bar=standard deviation.
As a result of the FISH procedure, five studied repeats were localized to the chromosomes of Th. ponticum (Figure
Fluorescence in situ hybridization in Th. ponticum using the following probes a P720 (red, asterisks show chromosomes with pericentromeric localization of P720) b P427 (green, asterisks show chromosomes with strong signal of P427, a and b show chromosomes with interstitial localization of P427) c P132 (green) d P332 (red) e P170 (green). P720, P427, and P132 are PCR products labeled with biotin (P720) and digoxigenin (P427 and P132), P332 and P170 are oligonucleotide probes labeled with biotin (P332) and 6-carboxyfluorescein (P170). Scale bar: 10µm.
P720 was localized to all chromosomes and the signal was of varying intensity (Figure
P427 produced strong reproducible signals at 16 chromosomes that were localized partially to the pericentromeric regions and partially to the terminal regions (indicated by asterisks in Figure
P132 was localized to two chromosomes in the pericentromeric region (Figure
Fluorescence in situ hybridization in Th. ponticum using the reprobing multicolor FISH technique a P720 (red), P427 (green) b P332 (red), P132 (white, pseudocolor), P170 (green). P720, P427, and P132 are PCR products labeled with biotin (P720) and digoxigenin (P427 and P132), P332 and P170 are oligonucleotide probes labeled with biotin (P332) and 6-carboxyfluorescein (P170). The designation of the asterisks, arrowheads and letters (a-f) is given in the text. Scale bar: 10µm.
As a result of reprobing, we revealed the following groups of chromosomes: 9 chromosomes with both subtelomeric P427 and P720 signals at the same arm (Figure
The development of molecular cytogenetic markers is a long and routine process. Previously, we developed an algorithm allowing to select the most likely candidates for the role of chromosomal markers based on the genome-wide sequence using qPCR (
According to the results of qPCR, P720 and P427 had the highest abundance in the genome of Th. ponticum, which we localized to all or most of the chromosomes as a result of FISH. P720 showed a comparable copy number in bread and durum wheat and rye, which may indicate its prevalence among Triticeae (
P720 and P427 were localized both to the terminal and pericentromeric regions of all or most of the chromosomes of Th. ponticum, which may indicate the conservative nature of these repeats. We also localized P720 to both the pericentromeric and terminal regions on the chromosomes of A, B and D subgenomes of wheat and R of rye (
P720 produced a brighter signal on the D subgenome chromosomes on the chromosomes of bread wheat (
In our work, we have shown that P170 is localized to four chromosomes of Th. ponticum, while the repeat itself is mostly characteristic for D subgenome of bread wheat and is absent on chromosomes of A, B and R subgenomes (
P332, P170, and P132 were hybridized on several (from two to four) chromosomes of Th. ponticum. P170 was hybridized specifically on chromosomes of the D subgenome, and P332 was not identified in the R subgenome of rye (
We colocalized the developed probes using reprobing FISH and revealed that the chromosome set of Th. ponticum is imbalanced: chromosomes a-f (Figure
In conclusion, using our algorithm, we selected five repeats, two highly abundant repeats with localization on all or most of the chromosomes, and three repeats with a lower copy number with localization on several individual chromosomes. The repeats we selected can be used as a tool for the comparative cytogenetics of Triticeae to study phylogenetics and evolutionary relationships between species.
The work is supported by Russian Science Foundation grant № 18-76-00035.
Figures S1–S5
Data type: Adobe Acrobat Document (.pdf)
Explanation note: Supplementary Figure 1. Fluorescence in situ hybridization in Th. ponticum using P720 as a probe (red, PCR product labeled with biotin). Supplementary Figure 2. Fluorescence in situ hybridization in Th. ponticum using P427 as a probe (green, PCR product labeled with digoxigenin). Supplementary Figure 3. Fluorescence in situ hybridization in Th. ponticum using P132 as a probe (white pseudocolor, PCR product labeled with digoxigenin). Supplementary Figure 4. Fluorescence in situ hybridization in Th. ponticum using P332 as a probe (red, oligonucleotide labeled with biotin).