DOI: 10.35407/bag.2020.31.02.01

ARTÍCULOS ORIGINALES

First karyotype report on Colocasia oresbia: a comparative cytogenetic study between two varieties

Primer reporte del cariotipo de Colocasia oresbia: un estudio citogenético comparativo entre dos variedades

 

Begum Kazi N.¹
Dash Chandan K.²
Sultana Syeda S.² *

 

¹ Department of Botany, Jagannath University, Dhaka-1100, Bangladesh.
² Cytogenetics Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

 

* Corresponding author: Syeda Sharmeen Sultana syedasharmeensultana@du.ac.bd
ORCID 0000-0002-6196-0844

 

ABSTRACT
Karyotypes of two Colocasia oresbia botanical varieties from Bangladesh were analyzed and compared with orcein, chromomycin A3 (CMA) and 4´-6 diamidino-2-phenylindole (DAPI). Both varieties had 2n=2x=26 chromosomes (karyotypic formula: 20m+6sm) and a pair of satellites each. Total chromosome length was 144.18 ± 2.45 μm in C. oresbia var. oresbia and 133.02 ± 2.75 μm in C. oresbia var. stolonifera. The karyotype of Colocasia oresbia var. oresbia is 2A whereas that of C. oresbia var. stolonifera is 1A. Six CMA and four DAPI bands were observed in C. oresbia var. oresbia and eight CMA and six DAPI bands in C. oresbia var. stolonifera. However, in these two morphologically distinct C. oresbia varieties of two different ecological zones, the same somatic chromosome number, diversification in various karyotypic parameters and CMA/DAPI-banding patterns were observed. In addition to taxonomic characters, the studied karyotype features will contribute to the characterization of these two C. oresbia varieties and to establish a base for future research.
Key words: Chromosome banding; CMA; DAPI; Karyotype

 

RESUMEN
Se analizaron y compararon los cariotipos de dos variedades botánicas de Colocasia oresbia de Bangladesh con orceína, chromomicina A3 (CMA) y 4-6 diamidino-2-phenilindol (DAPI). Ambas variedades presentaron 2n=2x=26 cromosomas (fórmula cariotípica: 20m+6sm) y un par de satélites cada una. La longitud total de cromosomas fue 144,18 ± 2,45 μm en C. oresbia var. oresbia y 133.02 ± 2.75 μm en C. oresbia var. stolonifera. El cariotipo de Colocasia oresbia var. oresbia es 2ª, y 1ª el de C. oresbia var. stolonifera. Se observaron seis bandas CMA y cuatro DAPI en C. oresbia var. oresbia y ocho bandas CMA y seis DAPI en C. oresbia var. stolonifera. Sin embargo, en estas dos variedades morfológicamente distintivas de C. oresbia de dos zonas ecológicas diferentes se observó el mismo número cromosómico somático, diversificación en varios parámetros cariotípicos y en patrones de bandeo CMA/DAPI. En adición a los caracteres taxonómicos, las características de los cariotipos estudiados contribuirán a la caracterización de estas dos variedades de C. oresbia y a establecer una base para futuras investigaciones.
Palabras clave: Bandeo cromosómico; CMA; DAPI; Cariotipo

 

Received: 03/15/2020
Revised version received: 06/06/2020
Accepted: 06/18/2020

 

 

INTRODUCTION

 

 

The genus Colocasia Schott belonging to the Araceae family, comprises about 20 species over the world (Li and Boyce, 2010). A total of nine of these species has been reported for Bangladesh so far, such as C. affinis Schott, C. esculenta (L.) Schott, C. fallax Schott, C. gigantea (Blume) Hook. f., C. heterochroma H. Li et Z.X. & Wei, C. lihengiae C.L. Long et K.M. Liu, C. mannii Hook. f., C. oresbia A. Hay and C. virosa Kunth. (Ara and Hassan, 2019). This genus is popular because it is edible and has medicinal, ornamental and cultural importance. Ara and Hassan (2019) reported and differentiated two varieties of C. oresbia from Bangladesh viz. C. oresbia A. Hay var. stolonifera H. Ara & M.A. Hassan, var. nov. and C. oresbia A. Hay var. oresbia based on several prominent morphological features. In fact, most species of this genus are morphologically distinct although the morphological features of a few of them are very confusing. In those cases, karyo-morphological information can open a new direction for evaluating the relationship among them. The nature and degree of karyotype differences obtained from conventional and fluorescent banding techniques could be useful to discuss plant phylogeny. In addition, the cytogenetical information will be useful for development of successful breeding programs in this crop. So far C. oresbia is unexplored cytogenetically. Therefore, in the present study, a combination of morphological and cytogenetical analyses with orcein, chromomycin A3 (CMA) and 4´-6 diamidino-2-phenylindole (DAPI) were carried out for the first time to present karyotype data from two varieties of C. oresbia viz. Colocasia oresbia var. stolonifera and Colocasia oresbia var. oresbia to determine chromosomal relationships among them.

 

 

MATERIALS AND METHODS

 

 

Two varieties of Colocasia oresbia viz. C. oresbia A. Hay var. stolonifera H. Ara & M.A. Hassan, var. nov. and C. oresbia A. Hay var. oresbia were studied. Colocasia oresbia var. oresbia was collected from Chittagong, Cox’s Bazar, Khagrachari, Moulvibazar, Rangamati, Kaptai, Rajbari area, Shubalong and Dhaka (flat regions) of Bangladesh whereas C. oresbia var. stolonifera was found and collected only from Rangamati district (hilly regions), Bangladesh. For cytogenetic investigation, healthy roots of ten individuals of each variety were collected and pretreated with 2 mM 8-hydroxyquinoline for 3 h at room temperature followed by 15 min fixation in 45 % acetic acid at 4 °C, then hydrolyzed in a mixture of 1 N HCl and 45% acetic acid (2:1 v/v) at 60 °C for 3 min. The root tips were stained and squashed in 1% aceto-orcein. For CMA- and DAPI-banding, Alam and Kondo’s (1995) method was used with slight modifications. Slides were observed under a Nikon (Eclipse 50i) fluorescent microscope with a blue violet (BV) filter cassette for CMA and an ultraviolet (UV) one for DAPI-banding. CMA binds with GC (Guanine-Cytosine)-rich repetitive sequences of the genome expressing yellow fluorescence, and DAPI binds to AT (Adenine-Thymine)-rich repeats giving a characteristic blue color (Schweizer, 1976). For every staining, at least 50 cells were observed in each variety. The idiograms were made on the basis of chromosome size in decreasing order. Levan et al. (1964) was followed for determining centromeric type of chromosomes. Karyotype asymmetry index (AI) was also calculated to determine the degree of karyotype heterogeneity (Paszko, 2006).

 

 

RESULTS AND DISCUSSION

 

 

Morphological investigation

 

 

The two studied varieties of Colocasia oresbia show some prominent morphological dissimilarities. Colocasia oresbia var. stolonifera has stolons, which are absent in C. oresbia var. oresbia. They also show differences in inflorescence formation: the inflorescence of C. oresbia var. stolonifera is normally formed in group of up to 3 but in C. oresbia var. oresbia inflorescence occurred in group of up to 8 (never less than 4).

 

 

Somatic chromosome number and karyotype analysis

 

 

This present study provides detailed chromosomal information of C. oresbia for the first time. The two varieties are found to possess 2n=26 chromosomes (Figure 1A, B; Table 1).

 

Figure 1. Metaphase chromosomes and idiograms of two Colocasia oresbia varieties. A. Orceinstained mitotic metaphase of C. oresbia var. oresbia, B. Orcein-stained mitotic metaphase of C. oresbia var. stolonifera, C. CMA-stained mitotic metaphase of C. oresbia var. oresbia, D. CMAstained mitotic metaphase of C. oresbia var. stolonifera, E. DAPI-stained mitotic metaphase of C. oresbia var. oresbia, F. DAPI-stained mitotic metaphase of C. oresbia var. stolonifera, G. Idiogram of C. oresbia var. oresbia, H. Idiogram of C. oresbia var. stolonifera. Arrows indicate satellites. Bars=10 μm.

 

Table 1. Comparative cytogenetical analysis of two Colocasia oresbia varieties. 2n=Somatic chromosome number; KF=Karyotypic formula; TCL=Total chromosome length; RCL=Range of chromosomal length; RL=Relative length of chromosome; ACL=Average chromosome length; AI=Asymmetry index of karyotype.

 

Somatic chromosome numbers 2n=28 and 42 have been reported for most of the studied species of this genus. Besides, some infrequent records such as 2n=26 in C. gigantea and C. esculenta, 2n=38 in C. antiquorum, and 2n=56 (tetraploid) in C. esculenta have also been reported (Wang et al., 2017). Previous literature has stated the basic chromosome number of Colocasia is x=14 since most of the species belonging to this genus have 2n=28 chromosomes (Yang et al., 2003). Other researchers have suggested that chromosomal variation regarding ploidy levels and aneuploidy occurred frequently in this genus (Fedorov, 1974; Kumar and Subramanian, 1979; Cao and Long, 2004; Huang et al., 2012). Moreover, the presence of euploid and aneuploid cytotypes in different species represents inconstancy in the basic chromosome number. The reported basic chromosome numbers are x=13, 14, 19, present in 2x, 3x, and 4x cytotypes (Wang et al., 2017). Previous studies concerning genus Colocasia showed that x=14 should be considered as ancestral basic chromosome number (Yang et al., 2003; Wang et al., 2017). In two varieties of C. oresbia of the present study, the basic chromosome number is x=13. Other previously reported basic chromosome numbers of x=13 and x=19 indicate that these two basic numbers probably originated from x=14 by secondary modifications (Leong-Škorničková et al., 2007). Both varieties of C. oresbia display relatively homogeneous karyotype arrangement with metacentric and submetacentric chromosomes with a KF of 20m+6sm, and have one pair of satellites in chromosome pair III (Figure 1G, H). However, these two varieties show differences in other karyotype parameters. Colocasia oresbia var. oresbia and C. oresbia var. stolonifera has TCL of 144.18±2.45 μm and 133.02±2.75 μm, respectively (Table 1).

The ACL is lower in C. oresbia var. stolonifera (5.12 μm) than C. oresbia var. oresbia (5.55 μm). The RCL is 4.23-7.02 μm in C. oresbia var. oresbia and 4.05-6.75 μm in C. oresbia var. stolonifera. The RL is 2.93-4.87% in C. oresbia var. oresbia whereas 3.04-5.07% in C. oresbia var. stolonifera. When evolutionary positions are taken into consideration in relation to the karyotypic nature, symmetric karyotypes are usually regarded as primitive and asymmetrical as advanced, since karyotype asymmetry can be considered to be the dynamic force behind speciation (Stebbins, 1971). Furthermore, a higher AI value represents more asymmetric karyotypes (Paszko, 2006). The studied asymmetry index of karyotype reveals that the karyotype of C. oresbia var. oresbia is more asymmetric than the karyotype of C. oresbia var. stolonifera. Thus, C. oresbia var. oresbia is more advanced from an evolutionary point of view. Chromosome number and size along with karyotypic features are subjected to evolutionary change (Lavia et al., 2009). Chromosome evolution can take place either by increasing or decreasing chromosomal length (Brandham and Doherty, 1998; Martel et al., 2004). In this case, the total length of the chromosome complements increase in the course of evolution, since both varieties have similar 2n numbers and karyotype formula. Colocasia oresbia var. oresbia and C. oresbia var. stolonifera have 2A and 1A karyotypes, respectively, which also correlate with the asymmetric index (Table 1).

 

 

Fluorescent banding

 

 

Each variety exhibited distinct CMA-banding pattern (Figure 1C, H; Table 1). Six and eight CMA-bands were found in C. oresbia var. oresbia and C. oresbia var. stolonifera, respectively, with 5.31% GC-rich repeats in C. oresbia var. oresbia and 6.79% in C. oresbia var. stolonifera. Six chromosomes (pairs VII, X and XII) of C. oresbia var. oresbia and four chromosomes (pairs II and VI) of C. oresbia var. stolonifera exhibited terminal CMA-bands. In addition, two chromosomes (pair I) of C. oresbia var. stolonifera had a peculiar CMA-banding pattern. In this variety, two chromosomes possess a pair of interstitial bands that may be used as chromosome markers. Four and six DAPI-bands were observed in C. oresbia var. oresbia and C. oresbia var. stolonifera, respectively. The DAPI-banded regions are 3.12% and 5.41% of the total chromosome complements in C. oresbia var. oresbia and C. oresbia var. stolonifera, respectively. Four terminal DAPI-bands (pairs VI and XI) in C. oresbia var. oresbia and two terminal DAPI-bands (pair VIII) in C. oresbia var. stolonifera were found. In addition, two centromeric (pair IX) and two intercalary DAPI-bands (pair VII) were also observed in C. oresbia var. stolonifera (Figure 1G, H). The mentioned findings suggest that each variety has a characteristic CMA and DAPI banded pattern with different number, location, total banded regions and percentage of GC- and AT-rich segments. Most of the bands are present at the terminal regions of the short arms of the respective chromosomes (Figure 1C, H). The presence of terminal bands indicated the tendency of accumulating GC- and AT-rich repetitive sequences at the chromosomal ends. Even though both varieties have the same chromosome number, diversification in karyotypic features and reshuffling of GC- and AT-rich banded regions were observed. The variation in karyotype indices and fluorescence banding patterns may be the result of inversions, deletions or unequal translocations, among other chromosomal aberrations. The diversity in karyotypes of these two varieties may have arisen due to the exposure to different environmental conditions. This research is the first cytogenetical report for C. oresbia. The findings of the present study would be useful for future breeding programs and a contribution to the systematics of the species.

 

ACKNOWLEDGEMENT

 

The authors are grateful to Dr. Hosene Ara, Ex- Director (In-charge), Bangladesh National Herbarium, Chiriakhana Road, Mirpur-1, Dhaka-1216, for providing the research materials and necessary information about Colocasia oresbia.

 

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