2019 Volume 84 Issue 2 Pages 147-151
Comparative chromosome analysis by conventional staining with orcein and differential staining with CMA and DAPI was carried out in three Amaranthus species viz. A. viridis, A. tricolor, and A. lividus. Although, the three species were found to possess 2n=34 chromosomes, differed in respect of total length of chromosome complement. Each Amaranthus species showed a characteristic fluorescent banding pattern based on distribution and amount of CMA- and DAPI-bands. In the three species, out of 22 CMA-banded and 27 DAPI-banded chromosomes, seven and eight were entirely fluoresced with CMA and DAPI, respectively. The compiled data gathered from comparative chromosome analysis on orcein, CMA-and DAPI-banding will be useful for cytogenetical characterization of Amaranthus species.
Amaranthus L. belonging to Amaranthaceae is consisting of more than 60 species (Akin-Idowu et al. 2016). These are annual herbs and commonly known as Amaranths (Ahmed et al. 2008, Akin-Idowu et al. 2016). It is one of the old cultivated crops originated in the American continent (Das 2012). It is also an excellent versatile crop which can grow over a wide range of agro-climatic zones showing resistance to various environmental stresses and thereby may readily adapt to new environmental conditions. Thus, the species of this genus are distributed worldwide with an interesting diversity of landraces (Das 2012, Akin-Idowu et al. 2016).
In Bangladesh, nine species of Amaranthus have been reported viz. A. blitum L., A. caudatus L., A. dubius Mart., A. graecizans L., A. lividus L., A. spinosus L., A. tenuifolius Willd., A. tricolor L. and A. viridis L. (Ahmed et al. 2008). Among these species A. viridis, A. tricolor, and A. lividus are most widely distributed in Bangladesh with promising economic, nutritional and medicinal values and are known as Notay Shak, Lal Shak and Data Shak, respectively (Ahmed et al. 2008). On the other hand, though Amaranth is a self-pollinated crop, wide variation in genotypes exists due to frequent interspecific hybridization (Ray and Roy 2009, Akin-Idowu et al. 2016). It also exhibits tremendous diversity related to their wide adaptability to different eco-geographic distributions (Lee et al. 2008). Genotype identification in Amaranth had been a long term challenge due to the close relationship that exists within the genus (Wax 1995, Akin-Idowu et al. 2016).
Public and private sectors, Bangladesh Agricultural Research Institute (BARI) and BRAC Seed and Agro Enterprise have been trying to develop improved cultivars of A. tricolor and A. lividus in Bangladesh. They have been trying to develop improved cultivars by classical breeding and selection. In this circumstance, BARI has been able to release several cultivars of A. tricolor and A. lividus. These cultivars are characterized solely on the basis of their morphological features. However, this kind of identification is not reliable and sometimes creates confusions since phenotypic plasticity may change the morphology in different environments (Zaman and Alam 2009). Therefore, genomic characterization of Amaranthus spp. is essential to initiate any breeding programme (Paroda and Arora 1991).
For authentic identification, reliable and stable parameters are required. Chromosome analysis could be regarded as an excellent source of information for evolutionary studies within different species (Dash et al. 2017, Sultana and Alam 2007). According to previous chromosome records, the chromosome number of A. viridis, A. tricolor, and A. lividus is 2n=34 (Kihara et al. 1931, Krishnaswamy and Raman 1949, Grant 1959b, Pal 1964, Sharma and Banik 1965, Tandon and Tawakley 1970, Behera and Patnaik 1974, Bir and Sidhu 1980). Most of the previous works were confined to the chromosome count only. However, chromosome counting is not enough to provide detail genomic information for authentic characterization. Fluorescent banding is an admirable tool for chromosome study. It provides information regarding the distribution of adenine-thymine (AT)- and guanine-cytosine (GC)- rich segments or bands in the genome (Schweizer 1976, Kondo and Hizume 1982, Alam and Kondo 1995, Sultana and Alam 2016). Staining with DNA-base specific binding fluorochromes such as CMA to GC-rich sequence and DAPI to AT-rich sequence is a simple and reproducible method for chromosome study (Schweizer 1976, Kondo and Hizume 1982, Alam and Kondo 1995, Sultana and Alam 2016). The fluorescent banding is quite satisfactory for detail and critical chromosome analysis such as identification of individual chromosome, on the basis of the determination of number and location of AT- and GC-rich segments or bands in chromosomes.
In this study, chromosome analysis by a combination of conventional and fluorescent stainings was performed for authentic cytogenetical characterization of A. viridis, A. tricolor and A. lividus for the first time in Bangladesh.
Twenty plants of A. viridis were collected from Botanical garden, Department of Botany, University of Dhaka. Seeds of A. tricolor and A. lividus were collected from Bangladesh Agriculture Development Corporation (BADC), Dhaka and BRAC Seed and Agro Enterprise, respectively. Then, seeds were germinated and maintained in the Botanical garden, Department of Botany, University of Dhaka, Bangladesh.
Cytogenetical studyHealthy root tips (RTs) were collected and pretreated with 2 mM 8-hydroxyquinoline for 1 h at 20–25°C followed by 15 min fixation in 45% acetic acid at 4°C. The pretreated RTs were hydrolyzed for 45 s at 65°C in a mixture of 1 M HCl and 45% acetic acid (2 : 1). One drop of 1% acetic orcein was added to the materials and then the slides are kept in an acetic acid chamber for overnight.
For CMA- and DAPI-bandings, a method of Alam and Kondo (1995) was used with slight modification. After hydrolysis, the dissected RTs were squashed in 45% acetic acid. The cover glasses were removed by a dry ice method and allowed to air-dry for at least 24 h before staining. The air-dried slides were incubated in McIlvaine buffer (pH 7.0) for 30 min followed by 0.1 mg mL−1 distamycin A treatment for 10 min. The slides were rinsed mildly in the buffer supplemented with 5 mM MgSO4 for 15 min. One drop of 0.1 mg mL−1 CMA was added to the materials for 4 h in a humid chamber and then rinsed with the buffer with MgSO4 for 10 min. Slides were mounted in 50% glycerol and kept at 4°C overnight before observation. These were observed under a fluorescent microscope (Eclipse 50i, Nikon) with a blue–violet filter cassette. In DAPI-staining, the air-dried slides were incubated in the buffer for 30 min and treated 0.25 mg mL−1 actinomycin D for 10 min in a humid chamber. The slides were immersed in 0.01 mg mL−1 DAPI solution for 4 h and then rinsed with the buffer for 10 min. Finally, the slides were mounted with 50% glycerol. These were observed under the fluorescent microscope with a UV filter cassette.
At least 50 mitotic metaphase cells from 20 different individuals of three Amaranthus species were observed for each staining. A. viridis, A. tricolor, and A. lividus possessed 2n=34 chromosomes with basic chromosome number, x=17 (Figs. 1–3, Table 1). The present chromosome numbers in three species confirmed the previous reports (Kihara et al. 1931, Krishnaswamy and Raman 1949, Grant 1959b, Pal 1964, Sharma and Banik 1965, Tandon and Tawakley 1970, Behera and Patnaik 1974, Bir and Sidhu 1980).
| Species | 2n | Range of chromosome length (µm) | Total length of chromosome complement (µm) | No. of CMA-bands | Length percentage of CMA-bands | No. of DAPI-bands | Length percentage of DAPI-bands | No. of satellites |
|---|---|---|---|---|---|---|---|---|
| A. viridis | 34 | 1.59–2.69 | 69.85±2.30 | 6 | 10.26±1.66 | 11 | 20.54±0.76 | — |
| A. tricolor | 34 | 1.47–2.83 | 74.68±3.19 | 7 | 11.03±1.26 | 6 | 9.43±1.84 | 2 in CMA |
| A. lividus | 34 | 1.52–3.47 | 84.57±3.80 | 9 | 16.70±0.98 | 10 | 14.95±0.92 | 2 in CMA and 8 in DAPI |
In Amaranthus, four chromosome numbers were recorded for species groups such as 2n=28 in A. tenuifolius (Pal et al. 2000, Prajitha and Thoppil 2018), 2n=32 in 17 species (Murray 1940a, b, Heiser and Whitaker 1948, Mulligan 1957, Grant 1958, 1959a, b, 1967, Pal 1964, Subramanyam and Kamble 1966), 2n=34 in 15 species (Kihara et al. 1931, Krishnaswamy and Raman 1949, Grant 1958, 1959a, b, 1967, Mulligan 1961, Murray 1940a, b, Pal 1964, Pal and Khoshoo 1965, Sharma and Banik 1965, Tandon and Tawakley 1970, Behera and Patnaik 1974, Bir and Sidhu 1980) and 2n=38 in A. emarginatus (Mitra 1964). Therefore, according to chromosome number records, the Amaranthus is characterized by multiple basic chromosome numbers (x=14, 16, 17, 19).
Comparative chromosome analysisAlthough the three Amaranthus species shared 2n=34 chromosomes, differed in respect of total chromosome length (Figs. 1–3, Table 1). The total length of diploid chromosome complement of A. viridis, A. tricolor, and A. lividus was 69.85±2.30 µm, 74.68±3.19 µm and 84.57±3.80 µm, respectively (Table 1). In this regard, A. lividus showed a somewhat larger chromosome complement than the other two species (Table 1). The range of chromosome length was an insignificant i.e., difference between the shortest and the longest chromosomes were about 1.00 µm in A. viridis and A. tricolor (Table 1). These features indicated that these two species showed homogeneity in chromosome size. In contrast, the longest chromosome was more than double (3.47±1.52 µm) from the shortest one (1.52±1.04 µm) in A. lividus (Table 1). As a result, less homogeneity in chromosome size was observed in A. lividus. The centromeric position of most of the chromosomes in three species was not clearly visible. Therefore, it was quite difficult to prepare centromeric formulae for each species. No satellite or secondary constriction was clearly detectable in these three species after orcein-staining (Figs. 1–3).
Fluorescent banding with CMA and DAPIA total of six CMA-positive banded chromosomes were observed in A. viridis (Fig. 4, Table 1). Two chromosomes were entirely fluoresced with CMA whereas four chromosomes showed CMA-fluoresced region at the middle portion (Fig. 4, Table 1). The total length of the CMA-banded region was 7.16±0.64 µm which covered about 10.26±1.66% of total chromosome length (Table 1). After DAPI-staining, A. viridis was found to possess 11 DAPI-banded chromosomes (Fig. 7, Table 1). In this species, AT-rich sequences were occupied about a half portion of a chromosome. Four chromosomes showed terminal DAPI-bands. Three chromosomes were entirely fluoresced with DAPI. Three chromosomes were found to possess AT-rich bands at the middle portion (Fig. 7). The total length of DAPI-banded region was 14.35±1.21 µm which occupied about 20.54±0.76% of the total chromosome length (Table 1). No satellite was observed after CMA- and DAPI-banding (Figs. 4, 7).
Reversible banding pattern (Schweizer 1976) was observed in A. tricolor (Figs. 5, 8). The number of CMA-bands was 7 (Fig. 5, Table 1). Terminal CMA-bands were present in four chromosomes (Fig. 5). In contrast, CMA-bands were distributed throughout the entire length of two chromosomes. One chromosome showed CMA-band at the middle portion of the chromosome. The total length of the CMA banded region was 8.24±0.99 µm which occupied about 11.03±1.26% of the total chromosome length (Table 1). A total of six DAPI-banded chromosomes were observed in A. tricolor (Fig. 8, Table 1). After DAPI-staining, three entirely fluoresced chromosomes were observed. About half portions of a chromosome were brightly fluoresced with DAPI. However, in the above mentioned DAPI-banded regions, no band was observed with CMA-banding (Figs. 5, 8). Two chromosomes showed DAPI-band at the middle portion of chromosomes (Fig. 8). However, the middle portion of one of these chromosomes was fluoresced with both CMA and DAPI which indicated the possible co-localization of GC- and AT-rich regions in chromosomes (Figs. 5, 8, asterisk). Presence of both GC- and AT-rich portions in the same locus of chromosomes was reported earlier in Allium species (Mahbub et al. 2014). The total length of DAPI-banded region was 7.04±0.65 µm which covered about 9.43±1.84% of total chromosome length (Table 1). Two satellites were observed after CMA-banding whereas no satellite was found after DAPI-banding in this species (Figs. 5, 8, arrow).
After CMA-staining, A. lividus was found to possess nine CMA-banded chromosomes (Fig. 6, Table 1). Approximately one-third portions of two chromosomes were fluoresced with CMA. Terminal CMA-bands were present in four chromosomes (Fig. 6). Three chromosomes were entirely fluoresced with CMA (Fig. 6). The total length of CMA-banded region was 14.12±1.28 µm which occupied about 16.70±0.98% of the total chromosome length (Table 1). After DAPI-staining, A. lividus was found to possess 10 DAPI-banded chromosomes (Fig. 9, Table 1). Two entirely fluoresced and one almost fully fluoresced DAPI-banded chromosomes were observed. Two chromosomes showed DAPI-bands approximately at the middle portion of the chromosome. Terminal DAPI-bands were observed in five chromosomes of which two were larger. The total length of the DAPI banded region was 12.64±0.84 µm which occupied about 14.95±0.92% of the total chromatin length (Table 1). In A. lividus, two and eight satellites were found after CMA- and DAPI-banding, respectively (Figs. 6, 9, arrow).
Out of 22 CMA-banded chromosomes, seven were entirely fluoresced with CMA in three species of Amaranthus (Figs. 4–6). In these entirely fluoresced chromosomes, GC-rich sequences were not confined rather distributed along the chromosomes. The possible reason for these entirely fluoresced chromosomes was rich in GC-rich sequences. Similarly, out of 27 DAPI-banded chromosomes, eight were entirely fluoresced with DAPI in these species (Figs. 7–9). The possible reason for these entirely fluoresced chromosomes was rich in AT-rich sequences. These whole banded chromosomes could be used as marker chromosomes and thus useful for chromosome analysis among different Amaranthus species.
In spite of similar chromosome number, diversification and reshuffling of CMA- and DAPI-positive banded regions were observed in three Amaranthus species. The fluorescent banding pattern indicated chromosomal diversity among the three Amaranthus species based on distribution and percentage of GC- and AT-rich sequences. The compiled data gathered from comparative chromosome analysis after orcein, CMA- and DAPI-banding will be useful for cytogenetical characterization of Amaranthus species.
This research was partly supported by a grant from the Ministry of Science and Technology, People’s Republic of Bangladesh.
