Karyotype Variation in Eight Cultivars of Indian Dessert Banana (Musa acuminata L.) of Section Eumusa From Odisha, India

Banana (Musa spp.) cultivars especially dessert banana are important cash crop with high market demand all over the world as an integral part of the diet. The need for assessment of cytogenetic characters in Musa cultivars is inevitable as out of thousands of cultivars, cytogenetic characterization of most of them remains unresolved due to difficulties like small chromosome size, diversity in ploidy levels and high cultivar diversity which behave differently to standardized cytogenetic protocols. In this report, somatic chromosome number, detailed karyotype analysis including total chromosome length, volume, form percentage, Interphase Nuclear Volume (INV) were accessed on eight dessert type of Musa accessions from different places of Odisha. All the cultivars studied were found triploid (2n = 33) with a basic chromosome number of x=11. The karyotype formulae were assigned to each cultivar by grouping the chromosome according to their shared characteristics. The total chromosome length ranged from 54.95 μm in cv. Robusta to 81.5 μm in cv. Kathia with symmetric karyotype in all the studied cultivar. Karyotype formula revealed structural alteration of chromosome with Total Form percentage (TF%) variation from 35.65% in cv. Amritapani to 41.68% in cv. Patakpura that confirms more number of nearly median constricted chromosome as compared to sub-median chromosome. The total chromosome volume recorded from 10.78 μm3 in cv. Robusta to 15.99 μm3 in cv. Khatia and the INV varied from 1336.44 μm3 in cv. Dwarf Cavendish to 2048.37 μm3 in cv. Patakpura. The recorded structural variation might be due to differential genome specific condensation of chromosome. Chromosome length and volume found statistically significant among the cultivars.


INTRODUCTION
Banana (Musa spp.) belongs to family Musaceae is an important monocot plant used as staple food and cash crop for millions of people that provide nutrition and minerals with high calorific value. Cultivated banana are distinguished into dessert or simply called banana and cooking banana or plantain. Banana is cultivated primarily for its highly nutritious fruit beside it has good fiber content obtained from its pseudostem, leaves are used as disposable leaf plates and inflorescence are used for food with high potassium (50.08 mg g -1 ), calcium (3.78 mg 1 -1 ) and phosphorus (3.66 mg g -1 ) content in dry weight basis (Fingolo et al. 2012).
There are over a thousand domesticated Musa cultivars with a very high genetic diversity (Stover and Simmonds 1987;Perrier et al. 1990). However, due to difficulty of genetic makeup, and sterility of the crop, the development of new varieties through hybridization, mutation or transformation was not very successful in Musa till date (Heslop-Harrisons and Swarzacher 2007). The ploidy level determination of different varieties of Musa is economically important as well as preliminary requisite to facilitate breeding programme from existing genetic diversity of the country for future quantitative and qualitative morphological trait targeted breeding programme. Inter and intra specific hybridization of two wild diploid (2n = 2x = 22) Musa species, Musa acuminata (AA) containing 'A' genome and Musa balbisiana (BB) having 'B' genome gave rise to most of the natural banana cultivars with different genomic and ploidy levels i.e. AA, AAA, AAB, ABB, AAAB, AABB and ABBB. The cultivated banana are mostly triploid (2n = 3x = 33) with a limited varieties/species with diploid or tetraploid constituents. Various cultivars of banana have been originated from independent sources in the wild, so the hybridization events and mutations giving rise to seedless and parthenocarpic characters have occurred many hundreds of times (Simmonds and Shepherd 1955;Heslop-Harrisons and Swarzacher 2007). Where fertile plants occur together, hybridization continues to produce new diversity (Pollefeys et al. 2019) and parental combinations, hence, structural analysis of chromosome is important. Simmonds (1962) considered five plant characteristics that lead to farmers for picking plant vigour, yield, seedlessness, hardiness and fruit quality, the first four of which are related to polyploidy (triploidy). Karyotype analysis provides valuable information related to the mechanisms of genome evolution. Several types of banana out of thousands of cultivars are adapted to the agro-climatic condition of Odisha. Traditionally the economically important cultivars grown in the state are Silk (Patkapura), Poovan (Champa), Cavendish group. Recently, there has been a trend towards the cultivation of Amritpani due to high productivity and consumer acceptability (Maharana et al. 2017). Some of the earlier reports confirmed chromosome number with karyotypes, still data are scanty for different cultivars of banana (Cheesman and Larter 1935;Das and Das 1997). In this study, a detailed karyotype analysis and chromosome number determination has been carried out for further structural analysis of chromosome which is the prerequisite for localization of specific marker gene of interest on to the chromosome through Fluorescence in situ Hybridization (FISH) for genome analysis in eight triploid cultivars of dessert banana cultivated in different parts of Odisha.

MATERIALS AND METHODS
Eight cultivars of M. acuminata namely cv. Amritapani, cv. Champa, cv. Chini Champa, cv. Dwarf Cavendish, cv. Grand Naine, cv. Kathia, cv. Patkapura, cv. Robusta were collected from different parts of Odisha and maintained in green house of Department of Botany, Utkal University, Bhubaneswar (Table 1). Actively growing root tips were pre-treated in half saturated Para dichlorobenzene (pDB) and aesculin mixture (1:1) for 3½ h at 18 o C in refrigerator and then fixed in 1:3 acetic acid : ethanol overnight at room temperature. Fixed roots were treated in 45% glacial acetic acid for 15 min. Chromosome staining of fixed roots were done with 2% aceto-orcein preceded by cold hydrolysis with 5N HCl at 4 o C for 5 min. Chromosome squash preparation were made using 45% glacial acetic acid. Squashed slides were observed under Olympus BX-53 microscope and number of chromosomes were calculated. Digital microphotographs were taken in Micro Publisher 5.0 RTV camera observed under Olympus BX-53 microscope for detail analysis of chromosomes and karyotype.
Total chromosome length was estimated by adding the length of all chromosomes in the karyotype and total chromosome volume by applying formula πr 2 h, where 'r' is the radius and 'h' is the length of the chromosome respectively. Analysis of the chromosome type was conducted according to the classification system of Levan et al. (1964), and that of the karyotype in accordance with the classification standard of Stebbins (1971) modified by Das and Mallick (1993). Form percentage (F %) of individual chromosome was calculated.
Interphase Nuclear Volume (INV) was calculated following the formula of sphere i.e. 4/3πr 3 , where r is the radius of interphase nucleus. Results were analysed from 5-6 well spread metaphasic plates each obtained from the eight Musa cultivars. In order to ascertain the significant differences of different genomic parameters among eight cultivars of banana, if any, the one-way ANOVA test (Sokal and Rohlf 1973) was carried out with Tukey's Honest Significant Difference (HSD) test among the cultivars (Tukey 1949). Correlation co-efficient 'r' of different chromosomal parameters were made following 't' test to compare the significant cytological variation, if any, among the studied cultivated desert banana cultivars.

RESULTS
Chromosome numbers of all the eight cultivars found to be 2n = 3x = 33. The chromosome size varied from small to large. All the somatic chromosomes are classified as Type A with comparatively large chromosomes having nearly median (NM) primary and secondary constrictions. Type B with medium to large sized chromosomes having nearly sub-median (NSM) primary constriction and nearly sub terminal (NST) secondary constriction. Type C with medium size chromosome having nearly median primary constriction (NM) and Type D with small to medium size chromosomes having nearly sub-median (NSM) primary constriction ( Fig. 1). Although all the cultivars showed 2n = 33 chromosomes, the number variation of different Types of chromosomes in the karyotype formulae were found among the genotypes showing definite differences in their chromosome structure (Figs. 2, 3, Tables 2, Supplementary Table 1).
The total chromosome length ranged from 55.68 µm in cv. Chini Champa to 81.50 µm in cv. Kathia. Predominance of nearly median chromosomes is a characteristic   of the eight studied cultivars in which the TF% varied from 35.65% in cv. Amritapani to 41.68% in cv. Patakpura. The total chromosome volume was found lowest in cv. Robusta (10.78 µm 3 ) and highest in cv. Kathia (15.99 µm 3 ). The interphase nuclear volume ranged from 1336.44 µm 3 in cv. Dwarf Cavendish to 2048.37 µm 3 in cv. Patakpura. Presence of secondary constricted chromosomes varied among cultivars from 12 in cv. Amritapani to 3 in cv. Grand Naine and Robusta. Karyotype of all the cultivars showed Type A secondary constricted chromosome except cv. Patakpura while B Type secondary constricted chromosomes were found in cv. Amritapani and cv. Patakpura. Other related cytological parameters against each cultivars have been given in Table 2. Statistical analysis showed significant differences among the cultivars of banana (Table 3). The chromosome length and volume found significantly correlated with a coefficient value of r = 0.99. However, chromosome length and volume have no such significant correlation with nuclear volume which were -0.287 and -0.288 respectively. Tukey's Honest Significant Difference (HSD) test confirmed that significant differences of total chromosome length and INV were recorded among the studied varieties (data not shown). The chromosome volume varied significantly among the varieties without having any significant variations between cv. Chini Champa and cv. Champa, cv. Dwarf Cavendish and cv. Grand Naine, cv. Champa, cv.

DISCUSSION
Cultivated bananas are scientifically interesting, as there is no genetic exchange during reproduction and selection is mostly depends on random mutations (Oladosu et al. 2016). Knowledge of chromosomal characters of the edible cultivars is valuable in order to know banana genetics in details. Edible bananas have 2n = 2x 22, 33 or 44 chromosomes for diploid, triploid and tetraploid cultivars respectively (Stover and Simmonds 1987). These cultivars have a wide range of genome permuta-  Musa cultivars were studied and no numerical changes in the somatic chromosomes was observed in the genome that reconfirmed x = 11 ( Table 2). Majority of the chromosomes in each karyotype were found to be in the group of the medium-sized chromosome with median primary constriction. All the 4 Types of chromosomes were present in cv. Amritapani whereas rest of cultivars has only 3 Types of chromosomes. Type C and D were common in all the cultivars with different doses whereas Type B was present in cv. Patakpura only and rest cultivars had Type A chromosomes. The dose of nearly median constricted chromosomes were found more in all the cultivars except cv. Dwarf Cavendish and cv. Grand Naine that showed 12 Type D chromosomes in the karyotype. Numbers of secondary constricted chromosomes found variable among the cultivars. The total chromosome length varied from 54.95 µm in cv. Robusta to 81.50 µm in cv. Kathia and TF% varied from 35.65% in cv. Amritapani to 41.68% in cv. Patakpura among the studied cultivars. Chromosome volume also found significantly different among the cultivars ranged from 10.78 µm 3 in cv. Robusta to 15.99 µm 3 in cv. Kathia that might be due to genome specific differential condensation of the heterochromatin and euchromatic region of the chromosomes during metaphase. Thus, variety specific chromosome condensation and volume variation might be an indication of genome size variation which need further experimentation.
Differences in chromosome length or chromosome volume may be due to differential condensation and spiralization of the chromosome arms. In addition, the species-specific compaction of DNA threads along with nucleosomes with altered non-histone proteins (Das and Mallick 1989). The alteration in the TF% might be due to chromosomal alteration due to break and reunion of the chromosome arms in early stages of evolution in the genome rather than the methodological defect of chromosome squash preparation. Furthermore, translocation mediated structural alteration played a crucial role in chromosome evolution (Lysak et al. 2006;Luiz et al. 2009) besides heteromorphicity in centromeriac position among the chromosomes of Allium localizing GC-and AT-rich repeats by CMA-and DAPI-banding patterns (Mahbub et al. 2014). The dissymmetrical coefficient of the karyotype through FISH in Hibiscus mutabilis f. mutabilis, L. confirms relatively advanced type over plants with symmetrical chromosomes of the primitive type with respect to evolution (Li et al. 2015). Duplication of chromosomes or translocation between the chromosomes with or without secondary constrictions at a very early stage of evolution might be the reason for the structural alteration of the chromosome morphology as well as the variation of secondary constricted chromosomes in the above cultivars (Das and Das 1994;Rai et al. 1997;Ghosh et al. 2013;Das et al. 2015Das et al. , 2020Dehery et al. 2020).
Cultivars with reported AAA genome like cv. Amritapani, cv. Dwarf Cavendish, cv. Grand Naine and cv. Robusta found to have Type A, C and D found common with 12 numbers of Type D chromosomes each of cv. Dwarf Cavendish and cv. Grand Naine and 3 Type A each of cv. Grand Naine and cv. Robusta showing interrelationships among them having close affin- Robusta genome might be more stable with less chances of chromosomal alteration due to break and reunion of the chromosomes in karyotypes during micro-evolution. But cv. Amritapani differs from others with the presence of more number of secondary constriction and the karyotype is comparatively more fragile and karyotype asymmetry analysis might through some light on karyotype evolution in banana . Cultivars recorded AAB genome types like cv. Champa, cv. Cheni Champa, cv. Patakpura and cv. Kathia with 3 types of chromosomes where Type C and D were common in all the 4 cultivars. In this genotypic group cv. Patakpura showed 6 Type of B chromosomes. In contrary, cv. Chini Champa and cv. Kathia showed each of 9 Type A chromosomes that with less number of Type D chromosomes in cv. Kathia than cv. Chini Champa. Evidently, all the members of AAB group might close genetic relationship and decrease of median constricted Type C chromosomes and increase of Type D chromosomes in cv. Champa and cv. Chini Champa clearly indicates their close genetic affinity in this genotypic group.
High TF% in all the cultivars except cv. Amritapani indicate the alteration of chromosome structure in the genome. These factors indicate greater genome stability conferring resistance to the cultivars against biotic or abiotic environmental stresses which is a characteristic feature of cultivars with B genome that need to confirm in future by fluorescent in situ hybridization (FISH) or genomic in situ hybridization (GISH) as shown in other cultivars of banana using BAC clones (D'Hont et al. 2000;Doležel et al. 2004;D'Hont 2005;Jeridi et al. 2011).
Chromosomes with median, nearly median, submedian or nearly sub-median position of centromere are prevalent in karyotypes reported in this work. Significant variations in the chromosome were not noted while analyzing the karyotypes of the eight cultivars studied as the eight triploid varieties known to have been derived from hybridization of the wild species have almost similar combinations of chromosomes with median and sub-median constrictions, with minute variations. Although a significant variation in genome length, volume and INV was recorded (Table 3). The small size of the chromosomes and the difficulty in obtaining a sufficient number of cells containing metaphase chromosomes makes it tedious rather difficult for the studies of the karyotype of bananas and plantain represented by many cultivars and subgroups in nature need to be analyzed with FISH applying genome specific probes of transposable element for evolution among the cultivars. A positive high correlation was noted between chromosome length and chromosome volume (r = 0.99) that might be due to genome specific genetic control of chromosome condensation and packaging of histone protein. Evolution of karyotype in species of identical chromosome number belongs to a distinct phylogenetic group is a long-standing issue that could be addressed by comparative chromosome painting to reconstruct karyotype evolution as evident in Crucifer species of Brassicaceae (Mandáková and Lysak 2008) and Orchidaceae (Medeiros-Neto et al. 2017).