Research Article |
Corresponding author: Ekaterina D. Badaeva ( katerinabadaeva@gmail.com ) Academic editor: Manoj Kumar Dhar
© 2019 Ekaterina D. Badaeva, Sergei A. Surzhikov, Alexander V. Agafonov.
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:
Badaeva ED, Surzhikov SA, Agafonov AV (2019) Molecular-cytogenetic analysis of diploid wheatgrass Thinopyrum bessarabicum (Savul. and Rayss) A. Löve. Comparative Cytogenetics 13(4): 389-402. https://doi.org/10.3897/CompCytogen.v13i4.36879
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Thinopyrum bessarabicum (T. Săvulescu & T. Rayss, 1923) A. Löve, 1980 is diploid (2n=2x=14, JJ or EbEb), perennial self-fertilizing rhizomatous maritime beach grass, which is phylogenetically close to another diploid wheatgrass species, Agropyron elongatum (N. Host, 1797) P. de Beauvois, 1812. The detailed karyotype of Th. bessarabicum was constructed based on FISH with six DNA probes representing 5S and 45S rRNA gene families and four tandem repeats. We found that the combination of pAesp_SAT86 (= pTa-713) probe with pSc119.2 or pAs1/ pTa-535 allows the precise identification of all J-genome chromosomes. Comparison of our data with the results of other authors showed that karyotypically Th. bessarabicum is distinct from A. elongatum. On the other hand, differences between the J-genome chromosomes of Th. bessarabicum and the chromosomes of hexaploid Th. intermedium (N. Host, 1797) M. Barkworth & D.R. Dewey, 1985 and decaploid Th. ponticum (J. Podpěra, 1902) Z.–W. Liu & R.–C. Wang, 1993 in the distribution of rDNA loci and hybridization patterns of pSc119.2 and pAs1 probes could be an indicative of (1) this diploid species was probably not involved in the origin of these polyploids or (2) it could has contributed the J-genome to Th. intermedium and Th. ponticum, but it was substantially modified over the course of speciation
Chromosome, evolution, FISH-karyotyping, J genome, rRNA gene distribution, Thinopyrum bessarabicum
Thinopyrum bessarabicum (T. Săvulescu & T. Rayss, 1923) A. Löve 1980 (syn. Agropyron bessarabicum T. Săvulescu & T. Rayss, 1923 or A. junceum (K. Linnaeus, 1753) P. de Beauvois, 1812) is a diploid (2n = 2x = 14, JJ or EbEb), perennial self-fertilizing rhizomatous maritime beach grass (
The natural distribution range of Th. bessarabicum spans along Black sea shore from southeastern and eastern Europe to Turkey (
Th. bessarabicum is characterized by symmetric karyotype consisting of metacentric and submetacentric chromosomes. Four chromosomes carry satellites (SAT) on their short arms. Due to similarity of size and morphological parameters of the J-genome chromosomes, additional methods are necessary for their identification.
The C-banding technique, which was broadly used at the end of XXth for chromosome identification in wheat and related species, was also employed for the analysis of Th. bessarabicum chromosomes (
Fluorescence in situ hybridization or FISH provides a broad prospective for plant chromosome analysis. This approach has already been applied for Th. bessarabicum, and a standard set of probes – 45S rDNA, pSc119.2, or pAs1 was used for chromosome identification (
In a current study we mapped six “classical” DNA probes, including 45S and 5S rDNAs (
Thinopyrum accessions used in analyses, their origin and genome constitution are given in Table
Fixation of the material, slide preparation and fluorescence in situ hybridization (FISH) were carried out as described earlier (
No | Species | Accession # | 2n | Ploidy level | Genome composition (per 1n)* | Origin | Donor name |
---|---|---|---|---|---|---|---|
1 | Thinopyrum bessarabicum | W6 10232 | 14 | 2× | J or Eb | Russia, Crimea | USDA-ARS (U.S.A.) |
2 | Th. bessarabicum | PI 531711 | 14 | 2× | J or Eb | Russia, Crimea | USDA-ARS (U.S.A.) |
3 | Th. intermedium | – | 42 | 6× | EstEstSt or EtEst(V-J-R) | Russia, unknown | obtained from collection of Moscow Scientific-Research Agricultural Institute of Nonchernozem Zone “Nemchinovka” |
4 | Th. ponticum | – | 70 | 10× | EEEEstEst or EEEStSt | Russia, on a sea shore of the island Sergeevskyi, White sea | collected by Dr. A.A. Pomortsev, Vavilov Institute of General Genetics RAS, Moscow, Russia |
FISH with pTa71 probe revealed four prominent 45S rDNA signals in the regions of secondary constrictions of two pairs of Th. bessarabicum chromosomes (Fig.
Hybridization pattern of oligo-pAs1 and oligo-pSc119.2 probes obtained in a current study (Fig.
Distribution of rDNA probes and different tandem repeats on metaphase chromosomes of perennial grass species: Th. bessarabicum W6 10232 (a–c) and PI 531711 (d), Th. intermedium (e, f); and Th. ponticum (g, h). Probe combination in a, e, g pTa71, red + pTa794, green b, h pSc119.2, green + pAs1, red c pSc119.2, green + pTa-535, red d pSc119.2, green + pAesp_SAT86, red f pAs1, red. The letters from A to K designate pairs of homologous chromosomes identified in Th. intermedium (e) or Th. ponticum (g) mitotic cells based on characteristic patterns of 5S and/or 45S rDNA probes. Yellow arrows (b–d) show position of secondary constrictions on Th. bessarabicum chromosomes. 5S rDNA sites on Th. intermedium (e) or Th. ponticum (g) chromosomes are indicated with small arrows. White arrows (h) show homologous Th. ponticum chromosomes with contrasting pSc119.2 patterns. Scale bar: 10 µm.
Hybridization with pAs1 probe resulted in fuzzy labelling of distal chromosome halves; signal intensities varied from medium to relatively high depending on a chromosome and fluorochrome used (signals generated by Fluorescein-labelled pAs1 probe (Fig.
Hybridization with the pAesp_SAT86 probe produced sharp, large diagnostic signals on four (1J, 4J, 5J, and 6J) out of seven pairs of Th. bessarabicum chromosomes (Figs
Relatively faint pAesp_SAT86 signals were detected on chromosomes 2J and 7J, which both carried sharp pSc119.2 sites in their short arms (Fig.
Distribution of different tandem repeats on Th. bessarabicum chromosomes; their idiograms are given on the right. The probe combinations are shown on the top, probe color corresponds to signal color. 1 – 7 – genetic groups. The pAs1 probe on lanes B, and G was labelled with Cy-3/TAMRA, while on lanes D and E with fluorescein resulting in lower pAs-1 signal intensities.
FISH with pTa71 and pTa794 probes on hexaploid Th. intermedium revealed twelve 5S rDNA signals (Fig.
Eighteen chromosomes of decaploid Th. ponticum possessed 5S rDNA clusters of variable sizes (Fig.
Diploid Th. bessarabicum is considered as one of genome donors to Th. intermedium (
The distribution of rDNA loci is often used in phylogenetic studies of plants. In the Triticinae, major NORs can be located on group 1, 5 and 6 chromosomes (
Earlier
Both Th. bessarabicum and A. elongatum contain a pair of 5S rDNA loci on group 1 chromosomes. Major clusters of 45S rDNA probe are located on group 1 and 5 chromosomes of Th. bessarabicum (
Interestingly, polyploid Thinopyrum possess higher number of 5S rDNA loci per 1x compared to diploids species. Thus, we detected twelve pTa794 sites (two per 1x) in hexaploid Th. intermedium (Fig.
We found similar pattern in decaploid Th. ponticum (Fig.
The karyotype of Th. bessarabicum shared many common features with karyotypes of other diploid grasses. These are distinct pSc119.2 sites in subtelomeric chromosome regions and high amount of pAs1 repeat, which is accumulated predominantly in the distal chromosome halves (
As was shown in a current study, the sequence pAesp_SAT86 (= pTa-713) hybridizes specifically to six out of seven Th. bessarabicum chromosomes. Probe distribution is species-specific, because it differs from the pTa-713 labeling patterns of wheat (
A detailed karyotype of Th. bessarabicum was constructed using FISH with six DNA probes representing 5S and 45S rDNAs and four tandem repeats belonging to different families. A combination of pAesp_SAT86 (= pTa-713) probe with either pSc119.2 or pAs1/ pTa-535 was found to be most effective for the identification of J-genome chromosomes. Comparison of our results with data available from literature showed that the J-genome of Th. bessarabicum is distinct from genomes of other diploid wheatgass species. Differences between chromosomes of Th. bessarabitum, on one hand, and Th. intermedium and Th. ponticum, on the other hand, indicate that probably Th. bessarabitum did not contribute genome to these polyploid species. Alternatively, the J-genome could be present in polyploid wheatgrasses, but in significantly rearranged form.
All authors declare that there is no conflict of interests exists. All of the authors have contributed substantially to the manuscript and approved the submission.
We thank Dr. A.A. Pomortsev (Vavilov Institute of General Genetics Russian Academy of Sciences) and Dr. Dave Stout (USDA-ARS, WRPIS Washington State University, U.S.A.) for providing the material. This work was supported by State Budgetary Project No 0112-2019-0002 and by the grant from Russian Stated Foundation for Basic Research 17-04-00087.