Heterochromatin distribution and comparative karyo-morphological studies in Vigna umbellata Thunberg, 1969 and V. aconitifolia Jacquin, 1969 (Fabaceae) accessions

Abstract Chromosome studies along with heterochromatin distribution pattern analysis have been carried out in two domesticated species of Vigna Savi, 1824 which grow in contrasting geo-climatic conditions of India: Vigna umbellata Thunberg, 1969, a legume well acclimatized to subtropical hilly regions of North-east India and Vigna aconitifolia Jacquin, 1969, a species of arid and semi-arid regions in desert plains of Western India. Karyo-morphological studies in both species reveal 2n = 22 chromosomes without any evidence of numerical variation and the overall karyotype symmetry in chromosome morphology suggest that the diversification at intraspecific level in genus Vigna has occurred through structural alteration of chromosomes, rather than numerical changes. Heterochromatin distribution as revealed by fluorochrome binding pattern using CMA3 and DAPI, confirms the occurrence of relatively more GC content in Vigna aconitifolia as compared to Vigna umbellata. However, AT content was found to be comparatively higher in Vigna umbellata which perhaps play a role in species interrelationships.

The structure and morphology of the chromosomes are of vital importance when studying the origin, evolution and classification of taxa (Yang et al. 2005) as well as distance or relatedness among diverse genomes (Stace 2000, Kumar andRao 2002). Quite a few number of reports dealing with such studies are available for Vigna species (Rao and Chandel 1991, Rao and Raina 2004, Shamurailatpam et al. 2012.
Chromosome location and characterization of C-heterochromatin by fluorescence staining procedures which preferentially stain GC-rich DNA and DAPI, which localised AT-rich regions has been successfully applied in a large number of Fabaceae taxa including Cicer arietinum Linnaeus, 1753 (Galasso et al. 1996a); Phaseolus calcaratus Roxburgh, 1832 (Zheng et al. 1991); Sesbania tetraptera Hochstetter, 1871(Forni-Martins et al. 1994, Forni-Martins and Guerra 1999; Vicia faba Linnaeus, 1753 (Greilhuber 1975); Vigna ambacensis Welwitsch, 1978(Galasso et al. 1996b. A certain degree of chromosomal variation at inter-specific level of the genus Vigna has been documented using cytogenetic approaches by earlier workers Chandel 1991, Shamurailatpam et al. 2012). Hence, it will be quite significant to see the extent of variation among the domesticated species of Vigna (Ceratotropis). V. umbellata is a species domesticated extensively in the subtropical hilly and moist regions of North-east India. On the other hand, V. aconitifolia has been adapted to the arid and semi-arid region of tropical Western plain of India. Analysis of karyo-morphological details in V. umbellata and V. aconitifolia, adapted to extremely contrasting environmental conditions, may ultimately help us to define their chromosome variation. Meaningful propagation programs can be developed from such information.

Materials and methods
Karyo-morphological studies were undertaken in ten accessions each of V. umbellata and V. aconitifolia. The germplasm has been obtained from Indian Council of Agricultural Research (ICAR), Baranapi, Meghalaya and also from National Bureau of Plant Genetic Resources (NBPGR), New Delhi. Actively growing root tips of about 1-2 cm long were excised from germinating seeds on moist filter paper in Petri dishes at 25 ± 2 °C, pre-treated with 0.025% colchicine (Himedia) for 3 h at room temperature (20 ± 2 °C). The root tips after pre-treatment were fixed in freshly prepared ethanol-acetic acid (v/v, 3:1) and subsequently stored at 4 °C until required. For slide preparation, the root tips were washed twice in distilled water, hydrolyzed in 1N HCl at 60 °C for 8 min and stained in Feulgen stain (leuco-basic fuchsin) for 45 min. The stained root tips were thoroughly washed and subsequently squashed in 1% acetocarmine. The microphotographs of the metaphase plates were taken from both temporary and permanent preparations. At least 10-15 clear preparations of chromosome complements of each species were analyzed. Photo-idiograms were prepared from photomicrographs by cutting out individual chromosome and arranging them in descending order of their length and matching on the basis of morphology, the chromosomes were resolved into 11pairs. The standard method of chromosome classification given by Battaglia (1955) classification of metacentric / median (V), submetacentric/ submedian (L), subtelocentric (J) and telocentric (I) based on the arm ratio of 1:1, >1:1<1.3, >1:3<1:0 and 1:0 respectively was employed for comparison. The degree of asymmetry was estimated by means of the parameters proposed by Peruzzi and Eroğlu (2013): Coefficient of Variation of Chromosome Length (CV CL ) and Mean Centromeric Asymmetry (M CA ).
For heterochromatin characterization, root-tips were digested in 2% cellulase and 20% pectinase solution for 180 min at 37°C. Meristems were washed in distilled water, squashed in a drop of 45% acetic acid, and frozen in liquid nitrogen. The slides were stained with DAPI (2 µg/ml): glycerol (1:1, v/v) solution to allow selection of the best plates. Subsequently, they were destained in ethanol: glacial acetic acid (3:1, v/v) for 30 min and transferred to absolute ethanol for 1 h, both at room temperature. Slides were air-dried and aged for 3 days at room temperature. The slides were stained with CMA 3 (0.5 mg/ml, 1 h) and DAPI (2 µg/ml, 30 min), mounted in McIlvaine's buffer (pH 7.0): glycerol (1:1, v/v), and stored for 3 days (Schweizer and Ambros 1994). Slides were analyzed under Leica DM 4000 B microscope and photographs were carried out with different filter combinations using Leica CCD camera.

Results
The somatic chromosome number of all the accessions had consistently 2n = 2x = 22 (Fig. 1). The chromosome complements were resolved into 11 pairs which formed a graded series from longest to shortest within the idiograms. A noticeable difference in   length between the longest and the shortest chromosomes within the complement was recorded (Table 1). The longest chromosome of the haploid complement was almost 2.5 times longer than the shortest one in V. aconitifolia accessions, while it was 2 times longer than the shortest one in V. umbellata accessions. Further investigated accessions belonging to V. umbellata and V. aconitifolia had metacentric, submetacentric and subtelocentric chromosomes in their respective chromosome complements. Submetacentric chromosomes outnumbered the metacentric ones in V. aconitifolia accessions while metacentric chromosomes outnumbered the submetacentric chromosomes in the case of V. umbellata accessions.
Various accessions of these species have shown distinctive variation in the karyotype with respect to number of metacentric and submetacentric chromosomes (Fig.  2). Subtelocentric chromosomes were found in V. aconitifolia but not in V. umbellata accessions. Heteromorphic chromosome and nucleolar chromosomes are recorded in the accessions of both V. umbellata and V. aconitifolia.
Telocentric chromosomes were absent in both the taxa studied. Heteromorphic chromosomes were observed in some of the V. umbellata accessions: BKSB 205 (1 st pair, Fig. 2a According to the scatter plot obtained by CV CL vs. M CA , BKSB 192 (V. umbellata) and EC 97882 (V. umbellata) showed the lowest (2.81) and highest (55.07) M CA respectively (Fig. 4) A comparative account of heterochromatin distribution pattern within the chromosome complements in V. umbellata and V. aconitifolia has been summarized in Table 3 and the data have been illustrated in Fig. 3. The CMA 3 + and DAPI + binding sites were found either in terminal or in interstitial regions, in both the taxa studied. V. umbellata had more of DAPI + sites 3.1(± 1.9) in the interstitial region of the chromosomes and the terminal binding sites were 1.8(± 0.6). The number of chromosomes showing different CMA + and DAPI + sites also ranged from 2-7 in this    species. On the other hand, in V. aconitifolia the heterochromatin block comprised more of CMA + binding sites 2.9(± 1.3), which were found in the terminal region of the chromosomes while 2(± 1.2) binding sites were interstitial in position. The number of chromosomes showing CMA + sites ranged from 3-7, while those showing the DAPI + sites ranged from 3-8.

Discussion
The present data, combined with the chromosome counts available from the literature confirm the somatic chromosome number of 2n = 22 for both species, V. umbellata and V. aconitifolia. Such observation received support from reports of Singh and Roy (1970), Rao and Chandel (1991), Rao and Raina (2004), Shamurailatpam et al. (2012). The presence of subtelocentric chromosomes in V. aconitifolia accessions is in agreement with the earlier report of Sinha and Roy (1979). All the accessions of V. umbellata and V. aconitifolia have shown no deviation in somatic chromosome numbers and overall karyotype appearance. However, V. umbellata had a higher degree of karyotype asymmetry as compared to V. aconitifolia, suggesting structural rearrangements in karyotypes. Hence, the observed karyotype variation is likely to have originated by structural changes in chromosomes vs. duplication, deletions, interchanges and inversions (Stebbins 1971, Rao andChandel 1991). Thus, structural alteration of the chromosomes involving centric fusion and centromere repositioning might have influenced the speciation in genus Vigna.
Due to the very small size of chromosomes accompanied by technical difficulties, the nucleolus organisers among the chromosome complements could not be clearly resolved. Other cytogenetic techniques such as silver staining and fluorescence in situ hybridization (FISH) can be useful in detecting NOR-loci on chromosomes.
The DAPI + binding sites in chromosomes, which are indicative of AT-rich region, were recorded in the interstitial regions of chromosomes in V. umbellata. However CMA + sites, found mostly in V. aconitifolia chromosomes, suggest that the heterochromatin blocks were rich in GC base composition at terminal regions of chromosomes. The higher distribution of AT-and GC-repetitive sequence in heterochromatin blocks is probably reflecting the processes of divergent evolution of repetitive sequences, in heterochromatin regions of Vigna species (Shamurailatpam et al. 2014).
In the course of evolution, most of the heterochromatin regions tend to increase (Ikeda 1988), this phenomenon is also observed in Vigna (Shamurailatpam et al. 2015). Certain genera such as Vicia, Phaseolus, Sesbania, Cicer and Vigna (Greilhuber 1975, Zheng et al. 1991, Forni-Martins et al. 1994, Galasso et al. 1996a, b, Forni-Martins and Guerra 1999 showed a heterochromatin-rich chromosome configuration, that might have been involved in diversification of this genus. Vigna umbellata, which is domesticated extensively in the sub tropical hilly and moist regions of North-east India, had its heterochromatin blocks rich in AT content with fewer GC base pairs. On the contrary, more GC content in heterochromatin blocks was observed in V. aconitifolia, which is acclimatized to the arid and semi-arid region of tropical Western plains of India, helping the species to overcome adverse climatic conditions of Indian desert. Our observations in this regard constitute a first attempt to probe the role of heterochromatin distribution pattern, if any, in species differentiation of plant groups.