CompCytogen 9(1): | 19-132 (2015) COMPARATIVE A veerrerewet open-access over doi: 10.3897/CompCytogen.v9i 1.9012 Kan Cyto genetics http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics Heterochromatin distribution and comparative karyo-morphological studies in Vigna umbellata Thunberg, 1969 and V. aconitifolia Jacquin, 1969 (Fabaceae) accessions Anju Shamurailatpam!', Latha Madhavan’, Shrirang Ramachandra Yadav’, Kangila Venkatraman Bhat*, Satyawada Rama Rao! | Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong-793022, India 2 National Bureau of Plant Genetic Resources, Regional Station, Vellanikkara, Thrissur-680654, India 3 Department of Botany, Shivaji University, Kolhapur-416004, India 4 National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa, New Delhi-110012, India Corresponding author: Satyawada Rama Rao (srrao22@yahoo.com) Academic editor: L. Peruzzi | Received 24 November 2014 | Accepted 21 January 2015 | Published 31 March 2015 http://zoobank.org/E63C4064-560F-41FB-941 C-B8220BA5C3CE Citation: Shamurailatpam A, Madhavan L, Yadav SR, Bhat KV, Rao SR (2015) Heterochromatin distribution and comparative karyo-morphological studies in Vigna umbellata Thunberg, 1969 and V. aconitifolia Jacquin, 1969 (Fabaceae) accessions. Comparative Cytogenetics 9(1): 119-132. doi: 10.3897/CompCytogen.v9i1.9012 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 V. aconitifolia Jacquin, 1969, a species of arid and semi-arid regions in desert plains of Western India. Karyo-morphological studies in both species reveal 27 = 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 chromo- somes, rather than numerical changes. Heterochromatin distribution as revealed by fluorochrome binding pattern using CMA, and DAPI, confirms the occurrence of relatively more GC content in V. aconitifolia as compared to V. umbellata. However, AT content was found to be comparatively higher in V. umbellata which perhaps play a role in species interrelationships. Keywords Asymmetry index, C-heterochromatin, Fabaceae, karyotype, NOR-chromosomes, Vigna Copyright Anju Shamurailatpam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 120 Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) Introduction The pantropical genus Vigna Savi, 1824 (Fabaceae) includes 104 described species (Lewis et al. 2005). Among its subgenera, only Ceratotropis Marechal, 1978 is known for its rich species diversity in Asia (Verdcourt 1970, Marechal et al. 1978, Tateishi 1996). Tomooka et al. (2002) recognized 21 species in the subgenus Ceratotropis, out of which six species are domesticated: azuki bean (V. angularis Willdenow, 1969), mung bean (V. radiata Linnaeus, 1954), black gram (V. mungo Linnaeus, 1956), rice bean (V. umbellata Thunberg, 1969), moth bean (V. aconitifolia Jacquin, 1969) and creole bean (V. reflexo-pilosa var. glabra Marechal, 1911). The genetic resources and diversity in cultivated and wild forms of subgenus Ceratotropis occurring in Indian subcontinent are extremely rich and interesting (Bisht et al. 2005). The domesticated V. aconitifolia is confined only to the tropical region of India, while V. umbellata is widely domesticated across the South-east Asia. The origin of V. umbellata is con- sidered to be Indo-China region and also to a certain extent from South-east Asia (Marechal et al. 1978, Baudoin and Marechal 1988). 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 and Rao 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 fluores- cence 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 Hoch- stetter, 1871 (Forni-Martins et al. 1994, Forni-Martins and Guerra 1999); Vicia faba Linnaeus, 1753 (Greilhuber 1975); Vigna ambacensis Welwitsch, 1978 (Gal- asso et al. 199Gb). A certain degree of chromosomal variation at inter-specific level of the genus Vigna has been documented using cytogenetic approaches by earlier workers (Rao and Chandel 1991, Shamurailatpam et al. 2012). Hence, it will be quite significant to see the extent of variation among the domesticated species of Vigna (Ceratotro- pis). 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. Analy- sis 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. Heterochromatin distribution and comparative karyo-morphological studies... 121 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 Agricul- tural 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 micro- photographs 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), subtelocen- tric (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 Eroglu (2013): Coefficient of Varia- tion of Chromosome Length (CV_,) and Mean Centromeric Asymmetry (M_.,). For heterochromatin characterization, root-tips were digested in 2% cellulase and 20% pectinase solution for 180 min at 37°C. Meristems were washed in dis- tilled water, squashed in a drop of 45% acetic acid, and frozen in liquid nitrogen. The slides were stained with DAPI (2 pg/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, (0.5 mg/ml, 1 h) and DAPI (2 pg/ml, 30 min), mounted in MclIlvaine’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 mi- croscope and photographs were carried out with different filter combinations using Leica CCD camera. Results The somatic chromosome number of all the accessions had consistently 27 = 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 122 Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) : > a 2s . a> oe . t wean ras ~ ae | ¥ + at ~ 7 ome < - a 1 b 7 z we ~ =~ * oe ree oe = Sze Sr ~~ a c ~* “Yo d ~ - ~ ~ . - z \ y=: = 4 * = en “tn 3 — we’ e wP ns f wa ¢ an ww & ~™ = =. - + —~: » ew z = = ° oe % mS gS « . * a 2 = ~~ h .~» as re = oF z. 7 CE DSS fs ‘> *. r pa a me ~ rc i * j - ieee Figure |. Mitotic complements of 10 accessions of V. umbellata. a-j: a BKSB 205 b TRB 160 ¢ RBS 35 d IC 551699 e BKSB 192 f RBS 53 g IC 55440 h IC 176563 i EC 97882 j BKSB 194. Bar = 5 um. Heterochromatin distribution and comparative karyo-morphological studies... 1px) — > - od 7 ee,” =« ™ -* * “a = ° x : = ‘ i 2 - “st > : - > kk os A> i ots o">,, wi coh pts ——$ —$____—. ta Figure |. Continued. k-t: k IC 36157 | VDV 6175 m IC 472147 n RM 040 o IC 39809 p IC 285159 q IC 36592 r IC 472173 s IC 39713 t IC 36562. Bar = 5 pm. Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) 124 Trl + AS Ce St ial ue Sz ZISIE Ol wyofinuow A | (OT T91 + AO CTT C1Z6E DI pyofinuow A | 61 18+ API CUP ELITL¥ OL «| Uyofiazuosy 7 | gi TZI + AOI 97 Z6S9€ Ol yyofinuow A | LT IZ + AOI Ce Ze 6SIS8TOI =| Byafzszuos A | OT T8I + AP Oe OOF Tall WG wEZC aia eee 6086€ OI wyofinuow A | CT (T+ I81 + AZ CF : 9071 |eGL | eve 0r0 WY yyofiuow A | FH IZI + AOL CTC © | sz TC LYITLV OL =| yofiszuosy 7 | €I (7+ T91+ AP GZ rad CLIO AGA | By oftgzu0sv A | ZI T91 + AO Ri LS19E OL yyofinUuow A | TT TOI + ATI Oe 761 ASNd mympaquin ‘A OI TrI + AS ey. 78826 OA MIDI Iquin ‘A 6 TrI+ AS 97 C9S9LI OI myvpaquin ‘A 8 T9T + A9 Ore OFFS Ol mvpaquin ‘A ys TOI + ATI Sz eo Sau myvpaquin ‘A 9 19 + AST 07 761 ASN mmyaquin ‘A C TOI + AZI OY 6691S DI myvpaquin ‘A y TOL + AZI 87 ce Saul myvyaquin ‘A ¢ TOI + AZI Cz 7 091 ALL mymyaquin ‘A Z OZ + AZ VT Ze coz dSNa mvpaquin ‘A I PpNUIIOF smOsouosyS 3S9}.0YSs 4 d f adAjoAre yy pur jsa3uo] Jo oney (O1eI $/'TJ) Yisu2] We suTOsoWOIY) Shige ae ae tas ws ‘PUSLA JO Xe) PoIpMis UT ONeI we pue Asojoydiowodrey *| BIqe_L Heterochromatin distribution and comparative karyo-morphological studies... 125 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. Submeta- centric 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 karyo- type 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. Heteromor- phic chromosomes were observed in some of the V. umbellata accessions: BKSB 205°C" pair, ios 2a) TRBY160-Candi2™ pair-Fis..2b), IG 55:1699° (1 *-pair, Fig. 2d), 1C55440 (1* pair, Fig. 2g) and IC 176563 (1" pair, Fig. 2h). In V. aconitifolia heteromorphic chromosomes were found in IC 36157 (5" pair, Fig. 2k), VDV 6175 (1* pair, Fig. 21), IC 472147 (3" pair, Fig. 2m), RM040 (3” pair, Fig. 2n), IC 36592 (1* pair, Fig. 2q) and IC 472173 (1* and 2™ pair, Fig. 2r) accessions. Nucleolar Organizing Regions (NORs), as a secondary constriction/ satellites, were observed in V. umbellata accessions RBS 35 (1* pair, Fig. 2c), IC 551699 (2™ pair, Fig. 2d), and EC 97882 (3 and 4" pair, Fig. 2i). Vigna umbel- lata was characterized by the presence of both metacentric and submetacentric chromosomes and two V. aconitifolia accessions (VDV 6175 and RM 040) were characterized by the presence of distinct subtelocentric chromosome, though their position differed in karyotype. The remaining accessions were devoid of any sub- telocentric chromosome. According to the scatter plot obtained by CV vs.uM_...BKSB 192 (V. umbel- lata) and EC 97882 (V. umbellata) showed the lowest (2.81) and highest (55.07) M., respectively (Fig. 4). Furthermore IC 285159 (V. aconitifolia) and RM 040 (V. aconiti- folia) showed lowest (10.8) and highest (22.88) M_., values. In V. umbellata TRB 160 and IC 551699 exhibited lowest (19.59) and highest (37.4) CV, values. Among V. aconitifolia accessions IC 36157 and IC 472173 had shown lowest (19.92) and highest (42.64) CV. values. A comparative account of heterochromatin distribution pattern within the chro- mosome complements in V. umbellata and V. aconitifolia has been summarized in Table 3 and the data have been illustrated in Fig. 3. The CMA,* and DAPI* bind- ing 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 126 Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) iE 1) sk 08 8 fe tk os tk om te BR XE BR ORR 32 BF AR RA Re gp ce b C2 33 ok ORS Gu 4a os gs Sa ne ®& « i +} 2 92 82 89 T2 Sa as ny ke S338 tf BE ue 32 Bt ts oe gs ef (8 at 98 af ae M8 os 8s ote ee oe [2 G8 Ra oun da ts st ue te ge tee if SA MM SK wx aR ok 83 Bs 2k as h ch Go Cf 8 83 as Gh Ae ue oe oa i Sl 32 58 af 8S 88 oe et ee tg ej te ee 0 fr | 2 2 es AB AD O03 AR 64 Ba th 68 se ee 88 1 6 88 8 o8 88 oe 8a 66 88 se 88 m 83 cS gh os su 88 Ee a8 Ge 35 of n ft §6 68 a8 Oe G0 @8 88 O88 se ot ° ap 68 @2 oe 88 08 @8 om 68 eos @~ p Al BS £2 Se 62 0f 88 4h 46 Be ae 8 Ch U2 Abra es R$ me 68 8h Bt og ot ee ea 8S pRB AD 9B 83 BR =£E EER ED On s Sa nt 82 66 68 88 S88 on 28 oe pe tt Figure 2. Photo-idiograms of a-j 10 accessions of V. umbellata a BKSB 205 b TRB 160 ¢ RBS 35 d IC 551699 e BKSB 192, f RBS 53 g IC 55440 h IC 176563 i EC 97882 j BKSB 194 k=t 10 acces- sions of V. aconitifolia k IC 36157 | VDV 6175 m IC 472147 n RM 040 o IC 39809 p IC 285159 q IC 36592 r IC 472173 s IC 39713 t IC 36562. Heteromorphic groups marked above the short arm and nucleolar groups are marked below the long arm. 127 Heterochromatin distribution and comparative karyo-morphological studies... C'S 89°€T (608 #) PE'EY (Lvl ¥) 779 Z9S9E OI 07 CO'6I bY CC (S8°9 ¥) LV'0F (871 ¥) LS CIL6€ Ol 61 rrr ph (Clow | orcyos [| Sy | re | w [| eittvor | rad OC cone | ose [| oe | om | « | coor | A 801 68°97 (ZL'S ¥) EN EH (STI ¥) 89°F 6S1S87 OI 91 TOI Z10€ (L1'9 ¥) VOTH (70°C F) 7L'9 6086€ OI CI STC COLE (S79 F) SL°8E (Z9'I ¥) 60°9 0v0 Wa I 1S°91 SET Gostioty | Oligo | CTT 5-6 SE |. L¥IEZV OL | EI CCST CTLT (0'L7)9L0F | STF) 89S | CC y-0l zz | ‘sWIOSOWOTYD Jo YISUdT UvIU ‘TD ‘yISus] sUIOSOWOIYD IsadUO] By) TT ‘YISUIT sUTOSOWTOIYD Isd1IOYS BY] DS ‘VUIZA JO EXE] PIIpNs oY) UT SoNsTIOIOEIeYO pue seNuTIOS adAMOAIeY *7 BIQUL 128 Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) Figure 3. Differentially stained mitotic chromosomes complements a=b V. umbellata c—d V. aconitifolia. Arrows indicate CMA* and DAPI sites. Scale bar = 5 pm in all the figures. Table 3. Distribution of CMA‘ and DAPI sites in the chromosomes of Vigna species. Mean+ SD of CMA* Mean+ SD of DAPI* Species sites in chromosomes | sites in chromosomes Range of CMA‘ | Range of DAPI* sites Terminal | sites Interstitial Interstitial | Terminal | Interstitial 2.1+0.8 18+06 |] 3.1+1.9 1.7+0.8 241.2 Def LOT | Act OS 2.9+1.3 Terminal V. umbellata | 1.7 + 0.8 V. aconitifolia 2.1 40.8 species. On the other hand, in V. aconitifolia the heterochromatin block comprised more of CMAt 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 num- ber of chromosomes showing CMA‘ sites ranged from 3-7, while those showing the DAPI’ sites ranged from 3-8. Heterochromatin distribution and comparative karyo-morphological studies... 129 40 ce 0 T — T —— 3 - 3 TT ~ I 0 10 20 30 40 50 60 Mea Figure 4. Scatter plot based on the karyotype parameters M_., (x axis) vs. CV., (y axis) a BKSB 192 b RBS 53 c RBS 35 d BKSB 194 e IC 55440 f IC 551699 g TRB 160 h IC 472173 i IC 285159 j IC 176563 k IC 36592 I BKSB 205 m IC 36562 n IC 36157 0 IC 39809 p IC 472147 q VDV 6175 r IC 39713 s RM 040 t EC 97882. Discussion The present data, combined with the chromosome counts available from the literature confirm the somatic chromosome number of 27 = 22 for both species, V. umbel- lata 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. umbel- lata had a higher degree of karyotype asymmetry as compared to V. aconitifolia, sug- gesting structural rearrangements in karyotypes. Hence, the observed karyotype varia- tion is likely to have originated by structural changes in chromosomes vs. duplication, deletions, interchanges and inversions (Stebbins 1971, Rao and Chandel 1991). Thus, structural alteration of the chromosomes involving centric fusion and centromere re- positioning might have influenced the speciation in genus Vigna. 130 Anju Shamurailatpam et al. / Comparative Cytogenetics 9(1): 119-132 (2015) 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 heterochro- matin 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 configura- tion, that might have been involved in diversification of this genus. Vigna umbel- lata, 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 differen- tiation of plant groups. Acknowledgements The present work is supported by a grant from the World Bank funded by Indian Council of Agriculture Research (ICAR) project received through National Agricul- ture Innovation Program (NAIP). We thank the Head, Department of Biotechnology and Bioinformatics, North Eastern Hill University (NEHU), Shillong for providing facilities. Sincere thanks are also due to members of the Plant Biotechnology Labora- tory for their help. The generous support for germplasm of V. umbellata extended by Dr. A. 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