
Kim
In many studies, FISH technique plays a vital role in clarifying genome structures, chromosome counts, physical locations of repeats and genes, and revealing genome rearrangements like chromosomal segment inversions, duplications and translocations (Koo
The 5S rDNA repeat unit consists of ∼120 bp coding region gene and a comparatively short intergenic spacer (IGS) with 100 to 1,000 bp long (Galián
In this study, FISH karyotype analyses of
Seeds of
Following the method used by Waminal
The 5S rDNA with 120 bp long was attained through PCR labeling using a PCR DIG Probe Synthesis Kit (Roche, Germany) and genomic DNA as a template (Koo
The procedures carefully followed those of Waminal
The FISH karyotype was analyzed with mitotic metaphase chromosome spread and paired according to chromosome length, centromeric position and ribosomal DNA signals. The chromosomes were arranged in decreasing order based on length.
We properly inspected metaphase chromosomes using seed germination. Fresh roots germinated from the seeds of the two
The results of karyotypic analysis based on chromosome lengths, type, and rDNA signals are shown in Table 1 and 2. The total chromosome length of ‘Jangbaek’ was in the range of 2.34 ± 0.13-3.99 ± 0.197 μm; its short arm, 1.15 ± 0.08-1.74 ± 0.11 μm and long arm, 1.19 ± 0.08-2.25 ± 0.10 μm. In case of the tetraploid cultivar ‘Etteumbaek’, those were estimated as 2.37 ± 0.08-4.20 ± 0.16 μm, 1.12 ± 0.07-1.52 ± 0.11 μm, and 1.25 ± 0.08-2.68 ± 0.13 μm, respectively. Considering the chromosomal length variation according to the mitotic stage, the chromosomal length difference was not considered as significant between the two cultivars.
Table 1 . FISH karyotype analysis of the diploid
Chr. no. | Chromosome length (μm) | Arm ratio | Chr. type | FISH signal no. (n) | |||
---|---|---|---|---|---|---|---|
Short arm (S) | Long arm (L) | Total (S+L) | 5S | 45S | |||
1 | 1.74 ± 0.11 | 2.25 ± 0.10 | 3.99 ± 0.197 | 1.30 ± 0.06y | m | - | - |
2 | 1.50 ± 0.09 | 1.99 ± 0.10 | 3.50 ± 0.17 | 1.33 ± 0.07 | m | - | - |
3 | 1.54 ± 0.07 | 1.93 ± 0.12 | 3.46 ± 0.15 | 1.25 ± 0.08 | m | 2 | - |
4 | 1.39 ± 0.09 | 1.76 ± 0.07 | 3.15 ± 0.07 | 1.27 ± 0.12 | m | - | - |
5 | 1.28 ± 0.16 | 1.72 ± 0.15 | 2.99 ± 0.10 | 1.37 ± 0.28 | m | - | - |
6* | 1.06 ± 0.06 | 1.84 ± 0.03 | 2.90 ± 0.07 | 1.75 ± 0.09 | sm | 2 | 2 |
7 | 1.20 ± 0.08 | 1.44 ± 0.12 | 2.64 ± 0.17 | 1.20 ± 0.09 | m | - | - |
8 | 1.09 ± 0.05 | 1.37 ± 0.02 | 2.46 ± 0.05 | 1.25 ± 0.08 | m | - | - |
9 | 1.15 ± 0.08 | 1.19 ± 0.08 | 2.34 ± 0.13 | 1.04 ± 0.09 | m | - | - |
Total: 16m + 2sm | 4 | 2 |
m, metacentric; sm, submetacentric; *, satellite chromosome; y, mean ± standard deviation.
Table 2 . FISH karyotype analysis of the colchicine-induced tetraploid
Chr. no. | Chromosome length (μm) | Arm ratio | Chr. type | FISH signal no. (n) | |||
---|---|---|---|---|---|---|---|
Short arm (S) | Long arm (L) | Total (S+L) | 5S | 45S | |||
1 | 1.52 ± 0.11 | 2.68 ± 0.13 | 4.20 ± 0.16 | 1.77 ± 0.17y | sm | 1 | - |
2 | 1.32 ± 0.06 | 2.28 ± 0.12 | 3.61 ± 0.12 | 1.73 ± 0.11 | sm | - | - |
3 | 1.52 ± 0.15 | 2.00 ± 0.17 | 3.52 ± 0.21 | 1.33 ± 0.18 | m | 2 | - |
4 | 1.43 ± 0.15 | 1.68 ± 0.11 | 3.11 ± 0.21 | 1.19 ± 0.12 | m | - | - |
5 | 1.22 ± 0.10 | 1.55 ± 0.08 | 2.77 ± 0.11 | 1.29 ± 0.15 | m | 1 | - |
6* | 0.81 ± 0.11 | 1.68 ± 0.17 | 2.49 ± 0.19 | 2.12 ± 0.39 | sm | 4 | 4 |
7 | 1.18 ± 0.08 | 1.27 ± 0.15 | 2.45 ± 0.16 | 1.08 ± 0.15 | m | - | - |
8 | 1.10 ± 0.11 | 1.31 ± 0.10 | 2.41 ± 0.12 | 1.22 ± 0.18 | m | - | - |
9 | 1.12 ± 0.07 | 1.25 ± 0.08 | 2.37 ± 0.08 | 1.13 ± 0.13 | m | - | - |
Total: 24m + 12sm | 8 | 4 |
sm, submetacentric; m, metacentric; *, satellite chromosome; y, mean ± standard deviation.
In chromosomal type based on the arm ratio as described by (Levan
The somatic metaphase chromosome spreads of both cultivars displayed clear 5S and 45S rDNA tandem repeats signals (Fig. 1). In the diploid cultivar ‘Jangbaek,’ a pair of 5S rDNA signals were detected and noticed in the interstitial or pericentromeric region of chromosome 3 and the colocalization of 5S and 45S rDNA signals on chromosome 6 (Fig. 2A). Whereas in the tetraploid cultivar ‘Etteumbaek,’ only a pair of intense 5S rDNA signal was detected on chromosome 2 and dispersed minor signals on the single chromosome of chromosomes 1 and 5. The location of 5S rDNA varied in some regions of the chromosomes. For instance, the vague signal of 5S rDNA in one of the chromosomes 1 was found to be in the subtelomeric region and the two chromosomes in chromosome 3 were found in the interstitial or pericentromeric region alike with the signal in one chromosome of chromosome 5 (Fig. 2B). Both cultivars showed colocalization of 5S and 45S rDNA in the NOR regions of chromosomes 6 (Fig. 2). The co-localization of 5S and 45S rDNA probes displayed a yellow signal on the chromosomes.
Polyploid plants were established in agriculture and horticulture as they usually possess desirable agronomic traits over their diploid counterparts (Boo
In this present study, FISH karyotype between two
To investigate chromosome characterization, cytogenetic markers such as ribosomal DNA (rDNA) tandem repeats could provide information about genome organization and allow the identification of homologous chromosome pairs (Heslop-Harrison 2000). In this study, we used the highly conserved coding region of both the non-NOR-forming 5S rDNA and the nucleolar organizing region (NOR) of 45S rDNA to characterize chromosome composition (Pellerin
In conclusion, FISH karyotype analysis using 5S and 45S rDNA probes revealed the occurrence of the doubling of chromosomes in the tetraploid cultivars of
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