Regular Article
Diversity in CMA- and DAPI-Banded Regions in Ten Germplasms of Crotalaria from Bangladesh
2022 Volume 87 Issue 3 Pages 209-213
Details
2022 Volume 87 Issue 3 Pages 209-213
Ten germplasms of three Crotalaria species viz. C. pallida (Acc. No. 4250, 4803, 4805, 4806, and 4807), C. incana (Acc. No. 4790, 4801, 4804, and 4809), and C. juncea (Local) were investigated cytogenetically through Chromomycin A3 (CMA) and 4′-6 diamidino-2-phenylindole (DAPI) fluorescent bandings. After CMA-banding, 10 Crotalaria germplasms showed 31 centromeric and 34 terminal bands which indicated a tendency of accumulating GC-rich sequences at centromeric regions or chromosomal ends. The number of DAPI bands was less than that of the CMA bands found in different germplasms of Crotalaria. Maximum terminal DAPI-bands indicated a tendency of accumulating AT-rich sequences at the chromosomal ends. Few chromosomes of Acc. No. 4803 of C. pallida and 4804 of C. incana could be used as a marker of respective germplasms due to their unique DAPI-banding pattern. Fluorescent banding revealed the occurrence of genomic alteration within these germplasms. In Acc. No. 4790 and 4804 of C. incana, 2n=17 chromosomes were observed indicating its the aneuploid origin that correlates with their phenotypic features like seedless pod formation. The diversity in CMA- and DAPI-banded regions in ten germplasms of Crotalaria from Bangladesh can help in further breeding programs.
The genus Crotalaria (Sun hemp) is a heterogenous member of Fabaceae. In Bangladesh 25 Crotalaria species were recorded of which only C. pallida, C. incana and C. juncea are available in the gene bank of Bangladesh Jute Research Institute (BJRI), Dhaka (Khan et al. 1996, Ahmed et al. 2009). This multi-purpose crop has better quality fiber than Corchorus because it showed good luster, higher tensile strength, and better resistance to environmental exposure thus it could be used as an alternative to Corchorus in Bangladesh (Chaudhury 1950, Mukherjee 1953). In Bangladesh, the main fiber-yielding crop is Corchorus. Besides this different species of Crotalaria would be an alternative to Corchorus and be considered as a possible industrial crop of Bangladesh. On the other hand, the demand for fiber is increasing day by day in the sectors of industry. So, it is very essential to increase the production of fiber to satisfy the ever-increasing demands for fiber in Bangladesh. In such a situation it is very important to develop special fiber-yielding crops and to draw attention to the farmers in cultivating this crop. A good number Crotalaria species were collected randomly from different regions and stored in the gene bank of BJRI. Authentic characterization and genomic information of these germplasms would be a wealth for the breeding program. But these were only characterized based on their phenotypic features. The phenotypic features are not always reliable. For these reasons this type of characterization sometimes creates the problem. For this purpose, authentic characterization and conservation of each germplasm are very essential for a successful breeding program.
Different researchers characterized Crotalaria germplasm with traditional karyotype analysis (Senn 1938, Datta and Biswas 1963, Bir and Kumari 1977, Gupta and Gupta 1978, Mangotra and Koul 1979, Raina and Verma 1979, Verma and Raina 1983, Warasy et al. 2021). Most of the earlier cytological investigations on Crotalaria were confined to 2n chromosome number and karyotype. It is well known that fluorescent banding with CMA and DAPI is satisfactory for the determination of quantity and location of GC- and AT-rich sequences in chromosomes, respectively (Bonna et al. 2017, Dash et al. 2017). But there was no detailed report on the fluorescent banding pattern of Crotalaria germplasm available in Bangladesh. Therefore, in the present study, the fluorescent banding method will be used for an authentic characterization and to find out the chromosomal variation of Crotalaria germplasms.
Ten germplasms of Crotalaria species viz. C. pallida (Acc. No. 4250, 4803, 4805, 4806, and 4807), C. incana (Acc. No. 4790, 4801, 4804, and 4809), and C. juncea (Local) were studied in this work (Table 1). Seeds collected from the gene bank of BJRI were maintained in the Botanical Garden, Department of Botany, the University of Dhaka for more than five years. Seeds were collected from the three mature plants of each germplasm and then the seeds were sown on soil from which healthy root tips were collected for cytogenetical studies. The maximum number of dividing cells (about 90%) were found in the root tip cells studied from May to November. The young healthy roots were cut 0.5 cm away from the tip by a blade and pretreated with 2 mM 8-hydroxyquinoline for 1.15 h at room temperature (28–30°C) followed by 15 min fixation in 45% acetic acid at 4°C. These were then hydrolyzed for 10 s at 60°C in a mixture of 1 M HCl and 45% acetic acid (2 : 1). For CMA- and DAPI bandings, Alam and Kondo’s (1995) method was used with slight modification. These methods are described below.
| Species | Acc. No. | Original number | Country of origin |
|---|---|---|---|
| C. pallida | 4250 | NY/0231C | Tanzania |
| 4803 | NY/105CR | Kenya | |
| 4805 | NY/143CR | Kenya | |
| 4806 | NY/160CR | Kenya | |
| 4807 | NY/209CR | Kenya | |
| C. incana | 4790 | NY/045CR | Kenya |
| 4801 | NY/102CR | Kenya | |
| 4804 | NY/126CR | Kenya | |
| 4809 | NY/261CR | Kenya | |
| C. juncea | Local | — | Bangladesh |
After hydrolyzing and dissecting, the materials were tapped and squashed with 45% acetic acid and kept in −80°C freezes for 3 min. The cover glass was removed quickly and dried in air for at least 24 h before the study. After 24 h of air drying for CMA-staining, the slide was first pre-incubated in McIlvaine buffer (pH 7.0) for 30 min. At once one drop of 0.1 mg mL−1 distamycin-A was added to the materials of slides and a cover glass was placed on it. The slide was kept in a humid chamber for 10 min. Then the slide was washed with distilled water in such a way that the cover glasses were removed. The slide was rinsed mildly in McIlvaine buffer supplemented with MgSO4 for 15 min. Then one drop of 0.1 mg mL−1 CMA was added to the materials of a slide and a clean cover glass was placed on it. The slide was kept in a humid chamber for 12 min. The slide was washed with distilled water in such a way that the cover glasses were removed. The slides were treated again for 10 min in McIlvaine buffer with Mg2+and 10 min McIlvaine buffer without Mg2+. Slides were mounted in 50% glycerol and kept at 4°C overnight before observation. These were observed under a Nikon (Eclipse 50i) fluorescence microscope with a blue-violet filter cassette. After 24 h of air drying for DAPI-staining, the slide was first pre-incubated in McIlvaine buffer (pH 7.0) for 25 min. The slide was treated in 0.25 mg mL−1 actinomycin D for 10 min in a humid chamber. After antibiotic treatment, the slide was washed with distilled water in such a way that the cover glass was removed. The slide was immersed again in McIlvaine buffer (pH 7.0) for 15 min followed by treatment in 0.1 mg mL−1 DAPI solution for 12 min. After rinsing in McIlvaine buffer (pH 7.0) for 10 min, the slide was mounted with 50% glycerol and kept at 4°C. These were observed under a Nikon (Eclipse 50i) fluorescence microscope with an ultraviolet filter cassette. At least ten mitotic metaphase cells from 10 different individuals of each germplasm of three Crotalaria species were observed for each staining.
With CMA, 31 centromeric and 34 terminal bands were observed in 10 Crotalaria germplasms which indicated that centromere and terminal portions of these chromosomes were GC-rich. In addition, entirely fluoresced 12 chromosomes were present where GC-rich sequences were distributed along the chromosomes. The possible reason for these entirely fluoresced chromosomes might be the tandem duplication of GC-rich sequences (Bonna et al. 2017, Dash et al. 2017).
The number of CMA bands varied from three (Acc. No. 4804 and 4809 of C. incana) to 16 (C. juncea, and Acc. No. 4803 of C. pallida) (Figs. 1–10, 21–30, Table 2). The total length of the CMA-positive banded region ranges from 0.60±0.05 µm (Acc. No. 4809 of C. incana) to 7.21±0.05 µm (Acc. No. 4807 of C. pallida) (Figs. 1–10, 21–30, Table 2). The percentage of GC-rich regions was lowest (2.53%) in the Acc. No. 4809 of C. incana and highest (31.57%) in the Acc. No. 4807 of C. pallida (Table 2).
| Species | Acc. No. | No. of CMA-bands | The total length of CMA-banded region (x̅±SD µm) | % of CMA-banded region | CMA-banded karyotype formulae |
|---|---|---|---|---|---|
| C. pallida | 4250 | 12 | 4.05±0.05 | 18.26 | 1α+10θ+1γ+6δ |
| 4803 | 16 | 3.97±0.04 | 26.08 | 11α+5θ | |
| 4805 | 4 | 0.80±0.02 | 5.62 | 4θ+12δ | |
| 4806 | 4 | 2.77±0.04 | 14.87 | 1θ+3γ+14δ | |
| 4807 | 7 | 7.21±0.05 | 31.57 | 4γ+3θ+9δ | |
| C. incana | 4790 | 5 | 2.73±0.04 | 14.23 | 2θ+1α+2γ+12δ |
| 4801 | 9 | 3.57±0.06 | 17.07 | 3θ+4α+2γ+7δ | |
| 4804 | 3 | 0.65±0.03 | 2.88 | 2θ+1ρ+14δ | |
| 4809 | 3 | 0.60±0.05 | 2.53 | 2θ+1 Ω+15δ | |
| C. juncea | Local | 16 | 3.22±0.04 | 16.06 | 14α+2θ |
α=Band in centromeric region, γ=Band in whole chromosome, θ=Band in terminal region, ρ=Band in interstitial region, Ω=Band in whole short arm, δ=No band
In the case of C. pallida, heteromorphicity was found in chromosome pairs IV, V, and IX of Acc. No. 4250 (Fig. 21). One member of pairs IV and V, showed centromeric and terminal bands, respectively while no band was observed in their homolog suggesting probable deletion of the banded region from the respective chromosomes. In pair IX, one chromosome fluoresced entirely while its homolog member had a terminal CMA band on the short arm. This feature of heteromorphicity was also found in the same pair of the Acc. No. 4806 (Figs. 21, 24). In the Acc. No. 4803, heteromorphicity was observed in pair VIII. Here, a terminal band was found in a member of that pair, whereas the other member had an interstitial band. These banding features indicated a probable inversion either from terminal to interstitial or interstitial to the terminal region (Fig. 22). Heteromorphicity was observed in pairs V and VIII in Acc. No. 4805 (Fig. 23). One member of these pairs had a terminal band while their homolog member did not show band. A similar kind of heteromorphicity was observed in pair III of Acc. No. 4807 (Fig. 25). The reason for this heteromorphicity maybe the deletion of the banded region from the respective chromosomes.
In the case of C. incana, heteromorphicity was found in all germplasms after CMA-staining. In Acc. No. 4790 and 4804, heteromorphicity was observed in pair I. One member of pair I in Acc. No. 4790 had a centromeric band while no band was observed in its homolog (Fig. 26). Similarly, an interstitial band was observed in one member of pair I in Acc. No. 4804 without any band in its homolog (Fig. 28). The above feature suggested the probable deletion of the banded region from the respective chromosomes. In the case of the Acc. No. 4801 and 4809, heteromorphicity was found in chromosome pairs VI and VIII, respectively (Figs. 27, 29). In these pairs, one member had a terminal band while no band was present in the other member. The reason for this heteromorphicity maybe the deletion of the banded region from the respective chromosomes.
After DAPI-banding 10 germplasms of Crotalaria generated a total of 16 terminal bands which suggested that the terminal portions of the respective chromosomes were AT-rich. In addition, eight chromosomes were entirely fluoresced with DAPI where AT-rich sequences were distributed along the chromosomes. The probable reason for entire fluorescence was i) either these chromosomes were completely AT-rich by nature or ii) due to successive duplication of AT-rich repeats (Hiron et al. 2006, Mahbub et al. 2007, Sultana and Alam 2016).
The germplasm used in this study have a distinct DAPI-banding pattern. The number of DAPI-bands varied from two (Acc. No. 4809 of C. incana) to nine (Acc. No. 4804 of C. incana) among ten germplasms of Crotalaria (Figs. 11–20, 31–40, Table 3). The total length of DAPI band region ranges from 0.58±0.05 µm (Acc. No. 4809 of C. incana) to 5.35±0.04 µm (Acc. No. 4804 of C. incana). The percentage of AT-rich regions was lowest (2.45%) in the Acc. No. 4809 of C. incana and highest (24.49%) in the Acc. No. 4804 of C. incana (Table 3). No DAPI-band was found in two germplasms of C. pallida (Acc. No. 4805 and 4807) and C. juncea (Table 3).
| Species | Acc. No. | No. of DAPI-bands | The total length of DAPI-banded region (x̅±SD µm) | % of DAPI-banded region | DAPI banded karyotype formulae |
|---|---|---|---|---|---|
| C. pallida | 4250 | 5 | 4.20±0.10 | 18.44 | 1θ+3γ+1β+13δ |
| 4803 | 3 | 1.35±0.13 | 8.50 | 2φ+1ϕ+13δ | |
| 4805 | — | — | — | 16δ | |
| 4806 | 6 | 1.69±0.03 | 9.0 | 5θ+1γ+12δ | |
| 4807 | — | — | — | 16δ | |
| C. incana | 4790 | 6 | 2.47±0.03 | 12.87 | 5θ+1γ+11δ |
| 4801 | 4 | 2.49±0.03 | 11.73 | 3θ+1 Ω+12δ | |
| 4804 | 9 | 5.35±0.04 | 24.49 | 4α+3γ+2λ+8δ | |
| 4809 | 2 | 0.58±0.05 | 2.45 | 2θ+16δ | |
| C. juncea | Local | — | — | — | 16δ |
α=Band in centromeric region, γ=Band in whole chromosome, θ=Band in terminal region, λ=Band in both terminal and centromeric region, ϕ=band on whole chromosome without centromere, β=Band in whole long arm, φ=Band in half of long arm, Ω=Band in whole short arm, δ=No band
In the case of C. pallida, after DAPI-staining, heteromorphicity was found in chromosome pairs IV and VII of Acc. No. 4250 (Fig. 31). In pair IV, one chromosome fluoresced entirely while its homolog member had DAPI-bands along with a long arm. In this case, AT-rich sequences of one member may be tandemly duplicated along the length of the respective chromosomes. A terminal band was found in a member of pair VII while the other member had no band. The reason for this heteromorphicity maybe the deletion of the banded region from the respective chromosomes. In Acc. No. 4803, heteromorphicity was observed in pair V (Fig. 32). In this pair, one chromosome fluoresced entirely except centromere while its homolog member had no DAPI-band. In this case, AT-rich sequences of one member may be tandemly duplicated along the length except the centromere of the respective chromosomes. Heteromorphicity was observed in pair IX in Acc. No. 4806 (Fig. 34). In this pair, one chromosome fluoresced entirely while its homolog member had a terminal DAPI-positive band on its short arm. In this case, AT-rich sequences of one member may be tandemly duplicated along the length of the respective chromosomes.
In the case of C. incana, heteromorphicity was found in three germplasm after DAPI-staining. In Acc. No. 4790, heteromorphicity was observed in pair VII (Fig. 36). One chromosome fluoresced entirely while its homolog member had no DAPI-band in this pair. This type of heteromorphicity was also found in pair IV of Acc. No. 4804 (Fig. 38). In this case, AT-rich sequences of one member may be tandemly duplicated along the length of the respective chromosomes. On the other hand, in pair VI of Acc. No. 4790, a chromosome had a terminal band and no band was observed in its homolog suggesting probable deletion of the banded region from the respective chromosome (Fig. 36). A similar kind of heteromorphicity was found in pair IV of Acc. No. 4801 (Fig. 37). Besides this, heteromorphicity was also found in pair VIII of Acc. No. 4801. A chromosome of this pair had the band on the whole short arm while no band was observed in its homolog suggesting probable deletion of the banded region from the respective chromosome.
In Acc. No. 4803 of C. pallida, a member of pair V fluoresced entirely with DAPI except centromere (Figs. 12, 32, arrow). In Acc. No. 4804 of C. incana, both members of chromosome pair I had DAPI-bands at the centromere and the terminal region (Figs. 18 and 38, arrow). These chromosomes are unique since absent in the rest of germplasms and thus could be used as a marker for these germplasms.
In Acc. No. 4790 and 4804 of C. incana, 2n=17 chromosomes were observed after CMA and DAPI-stainings. The same chromosome number was reported previously by Warasy et al. 2021 after orcein staining. In addition, about 70% of pods of this two germplasm were seedless. The above features indicated the probable aneuploid nature of germplasm. This aneuploidy might be trisomy with basic chromosome number 8 (2n=16+1=17) or monosomy with basic chromosome number 9 (2n=18−1=17).
Diversification and reshuffling of CMA- and DAPI-positive banded regions were observed in these ten germplasms of Crotalaria studied. The number, location, and distributions of GC- and AT-rich repeats were specific for each germplasm. Therefore, each germplasm has its characteristic CMA- and DAPI-banding pattern. This information would be helpful for a future breeding program.
This research is dedicated by the authors to Late Professor Dr. Sheikh Shamimul Alam for his eminent contribution to the Cytogenetics Laboratory, University of Dhaka, Bangladesh. This research was partly supported by a grant from the Prime Minister’s Research and Higher Education support fund.
