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Corresponding author: Marta Molnar-Lang ( molnar.marta@agrar.mta.hu ) Academic editor: Luiz Gustavo Souza
© 2016 Klaudia Kruppa, Marta Molnar-Lang.
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:
Kruppa K, Molnár-Láng M (2016) Simultaneous visualization of different genomes (J, JSt and St) in a Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid (Poaceae) and in its parental species by multicolour genomic in situ hybridization (mcGISH). Comparative Cytogenetics 10(2): 283-293. https://doi.org/10.3897/CompCytogen.v10i2.7305
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Multicolour genomic in situ hybridization (mcGISH) using total genomic DNA probes from Thinopyrum bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (genome Jb or Eb, 2n = 14), and Pseudoroegneria spicata (Pursh, 1814) Á. Löve, 1980 (genome St, 2n = 14) was used to characterize the mitotic metaphase chromosomes of a synthetic hybrid of Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 and Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu et R.-C.Wang, 1993 named „Agropyron glael” and produced by N.V. Tsitsin in the former Soviet Union. The mcGISH pattern of this synthetic hybrid was compared to its parental wheatgrass species. Hexaploid Th. intermedium contained 19 J, 9 JSt and 14 St chromosomes. The three analysed Th. ponticum accessions had different chromosome compositions: 43 J + 27 JSt (PI531737), 40 J + 30 JSt (VIR-44486) and 38 J + 32 JSt (D-3494). The synthetic hybrid carried 18 J, 28 JSt and 8 St chromosomes, including one pair of J-St translocation and/or decreased fluorescent intensity, resulting in unique hybridization patterns. Wheat line Mv9kr1 was crossed with the Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid in Hungary in order to transfer its advantageous agronomic traits (leaf rust and yellow rust resistance) into wheat. The chromosome composition of a wheat/A.glael F1 hybrid was 21 wheat + 28 wheatgrass (11 J + 14 JSt+ 3 S). In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the type of Thinopyrum chromosomes in a Th. intermedium/Th. ponticum synthetic hybrid called A. glael.
multicolour GISH, Thinopyrum intermedium , Thinopyrum ponticum , Agropyron glael, J, Jst, St genomes
N.V. Tsitsin produced a synthetic hybrid in the former Soviet Union by crossing Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 (=Agropyron glaucum Roemer & Schultes, 1817, 2n=6x=42) with Thinopyrum ponticum (Podpěra, 1902) Z.-W.Liu & R.-C.Wang, 1993 (=Agropyron elongatum Host ex P. Beauvois, 1812, 2n=10x=70) (Tsitsin 1954). The hybrid plants were named “Agropyron glael” (A. glael, 2n=8x=56,
Both wheatgrass species are long been known to have superior resistance to various diseases (
Polyploid Thinopyrum (Á. Löve, 1980) species contain genomes similar to the J (Eb, Jb) genome of the diploid Th. bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (2n=2x=14) (
Genomic in situ hybridization (GISH) or multicolour genomic in situ hybridization (mcGISH) offered new opportunities for testing genome relationships in plants (Bennett et al. 1991), for describing hybrid character (
Multicolour genomic in situ hybridization was used in the present study for the simultaneous visualization of the J and St genomic DNA of A. glael and their parental wheatgrass species (Th. intermedium, Th. ponticum) and to describe the chromosome composition of these materials. As previously published papers had different findings and the authors proposed different genome formulas in Th. intermedium, difficulties in identification of the different genomes were expected in our study. As Th. ponticum chromosomes belonged to two different genomes (J and JSt), precise detection and identification of them was probable despite of the high chromosome number. There were no former molecular cytogenetic data about the A. glael, but the presence of all the three different chromosome types (J, JSt, St) of the two parental wheatgrass species was hoped-for.
Thinopyrum intermedium, Th. ponticum, their synthetic hybrid A.glael, and the wheat/A. glael F1 hybrid were analysed cytogenetically (Table
Genotype | Accession number | Genebank | Geographic origin |
---|---|---|---|
Thinopyrum intermedium | PI565004 | USDA ARS GRIN | Russia |
Thinopyrum ponticum | PI 636523 | USDA ARS GRIN | Argentina |
Th. ponticum | PI531737 | USDA ARS GRIN | Argentina |
Th. ponticum | PI 547313 | USDA ARS GRIN | Russia |
Th. intermedium × Th. ponticum synthetic hybrid: Agropyron glael | glael-8/2008 | Martonvásár Cereal Genebank | Russia |
Mv9kr1 × A. glael F1 hybrid | 112705 | Martonvásár Cereal Genebank | Hungary |
McGISH, performed using J and St genomic DNA probes, simultaneously discriminated three different genomes in the segmental autoallohexaploid Th. intermedium (Fig.
Results of multicolour genomic in situ hybridization on Thinopyrum intermedium. a Karyotype of a complete cell using Thinopyrum bessarabicum (J, green) and Pseudoroegneria spicata (St, red) genomic DNA as probes. Chromosome with satellite is indicated with arrow b Karyogram of Thinopyrum intermedium chromosomes. Top row: J chromosomes; middle row: JSt chromosomes with the St pericentromeric region; bottom row: St chromosomes. Bar = 10 μm.
The analysed Th. ponticum contained 70 chromosomes and two groups could be distinguished based on their mcGISH pattern (Fig.
Multicolour genomic in situ hybridization on Thinopyrum ponticum. a Karyotype of Th. ponticum (accession VIR-44486) carrying 40 J and 30 JStchromosomes, using Thinopyrum bessarabicum (J, green) and Pseudoroegneria spicata (St, red) genomic DNA as probes b 38 J and 32 JStchromosomes identified in Th. ponticum (accession D-3494) c JS chromosomes with different lengths of St DNA in the centromeric region. JSt chromosomes were marked with asterisks. Bar = 10 μm.
McGISH made it possible to discriminate three different groups of A. glael chromosomes (Fig.
Multicolor genomic in situ hybridization pattern of Agropyron glael and the wheat (Mv9kr1 genotype)/A. glael F1 hybrid. a Karyotype of a partial cell of A. glael using Thinopyrum bessarabicum (J, green) and Pseudoroegneria spicata (St, red) DNA probes. Translocation between J and St chromosomes were marked with arrows b Karyotype of a complete cell of wheat (Mv9kr1 genotype)/A. glael F1 hybrid using Th. bessarabicum (J, green) and Ps. spicata (St, red) genomic DNA simultaneously as probes and wheat genomic DNA as block simultaneously c Karyogram of A.glael chromosomes present in the wheat/A.glael F1 hybrid. Nine A. glael chromosomes with hybridization patterns different to their parental species are marked with asterisks. Bar = 10 μm.
Chromosome counting detected 49 chromosomes in the wheat/A. glael F1 hybrid (21 wheat + 28 wheatgrass), 28 of which hybridized with the J and/or St genomes during mcGISH, discriminating the wheatgrass chromosomes from the unlabelled wheat (Fig.
GISH or mcGISH, a modification of fluorescence in situ hybrization, has been used to characterize genomes and chromosomes in polyploid Thinopyrum species (
As the number of J and JSt chromosomes was usually odd [19 J + 9 JSt in Th. intermedium and 43 J + 27 JSt (PI531737) in Th. ponticum], it is possible that J-JSt chromosome pairing can occur in meiosis, as reported by
Several types of genome composition and chromosome numbers have been reported for Th. intermedium (
Nucleolar dominance, an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid or during allopolyploidization, first reported in the 1930s (
As A. glael contains chromosomes from the two most valuable Thinopyrum species, changes in its genome could result in new invaluable genetic material, especially for wheat breeding.
In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the genome composition and the type of Thinopyrum chromosomes in a Th. intermedium/Th. ponticum synthetic hybrid called A. glael.
This work was funded by the Hungarian National Scientific Research Fund (OTKA K 104382 and K 108555). Special thanks to Dezső Szalay, and to the Moscow Research Institute of Agriculture -“Nemchinovka”, who kindly provided the A. glael plants. The authors gratefully acknowledge the excellent technical assistance of F. Tóth. Thanks are due to Barbara Hooper for revising the manuscript linguistically.