Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 81-88, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-580 Citation: T.B. Jha, P.S. Saha, S. Jha (2020) A comparative karyo-morpho- metric analysis of Indian landraces of Sesamum indicum using EMA-giemsa and fluorochrome banding. Caryologia 73(2): 81-88. doi: 10.13128/caryolo- gia-580 Received: August 1, 2019 Accepted: March 12, 2020 Published: July 31, 2020 Copyright: © 2020 T.B. Baran Jha, P.S. Saha, S. Jha. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum using EMA-giemsa and fluorochrome banding Timir Baran Jha1,*, Partha Sarathi Saha2, Sumita Jha2 1 Department of Botany, Maulana Azad College, Rafi Ahmed Kidwi Road, Kolkata- 700113, West Bengal, India 2 CAS, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kol- kata-700019, West Bengal, India *Corresponding author. E-mail: tbjha2000@yahoo.co.in Abstract. Sesamum indicum commonly known as ‘Sesame’, ‘Til’ or ‘Gingli’ is an age- old high valued oil crop. With distinct seed and floral diversity and no detailed chro- mosomal analysis is available on Indian landraces of S. indicum (2n= 26). The present study demonstrates standardization of enzymatic maceration and air drying (EMA) method of chromosome preparation and comparative karyometric analysis in four Indian landraces of S indicum. All the landraces were characterized by very small chro- mosomes, length ranging from 1.24 ± 0.02 to 2.87 ± 0.09 µm. The EMA- Giemsa based karyotype analysis revealed nine pairs of chromosomes with nearly median primary constriction, three pairs were submedian and a single satellite pair in each of the stud- ied landrace. The CMA staining of Sesamum chromosomes revealed the presence of distinct CMA positive (CMA+ve) signals in all the studied landraces. The Black seeded til (BT) and White seeded til (WT) were characterized by six chromosomes with dis- tal CMA+ve signal on short arm, while the Dark brown seeded til (DBT) showed ten chromosomes with distal CMA+ve signal on short arm. The Light brown seeded til (LBT) was characterized by eight chromosomes with distal CMA+ve signal on short arm. The results obtained from the scatter plot of A1 versus A2 and PCA analysis pro- vide a strong relationship with that of the fluorochrome banding analysis. The present research offers an explicit karyo-morphometric characterization of four Indian lan- draces of S. indicum for the first time. Keywords: fluorochrome banding, karyotype, sesame, Sesamum indicum, small chro- mosomes, til. INTRODUCTION Sesamum indicum L. commonly known as ‘Sesame’, ‘Til’ or ‘Gingli’ is an age-old high valued oil crop. As per the Index Kewensis the genus belongs to the family Pedaliaceae and comprises 36 species. However, S. indicum is the only cultivated species of this genus (Nayar and Mehra 1970). Sesame seeds are also known as the ‘Queen of the oil seeds’ and the first oil known to be 82 Timir Baran Jha, Partha Sarathi Saha, Sumita Jha consumed by human (Bedigian and Harlan 1986). Ben- eficial effects exhibited by sesame as antioxidant, antimi- crobial, anti-inflammatory, antidiabetic, anticancer on human health has recently renewed the interest in this crop (Amoo et al. 2017, Zhang et al. 2013). The species was domesticated in India long back (Bedigian 2003; 2010) and now ranked first in production and export of this crop (IOPEPC Kharif 2017). Cultivated S. indicum has highly variable genotypes and distinct differences have been noted in floral and seed colour morphology within the cultivated landraces (Raghavan et al. 2010). Chromosome analysis has played an important role in genetics and plant breeding for conservation of genetic diversity and improvement of crops. It is felt that chro- mosome analysis still provides foundational pieces of genomic information (Soltis 2014) and considered “the quickest, cheapest, and easiest way to get any substan- tial information about the genome of a species which is not possible by any other methods” (Guerra 2008). Chromosome analysis in this cultivated species (2n=26) was reported long back by Morinaga et al. (1929), Raghavan and Krishnamurthy (1947) and Kobayashi (1949). Raghavan and Krishnamurthy (1947) reported that all 13 small chromosome pairs have terminal con- strictions, while Mukherjee (1959) reported presence of five types of somatic chromosomes. However, Kobayashi (1949; 1991) in his analyses noted five pairs of median and eight pairs of sub median chromosomes. Zhang et al. (2013) reported three pairs median, eight pairs sub median and two pairs sub-terminal chromosomes in S. indicum cv. Yuzhi 11. It appears from the earlier reports that karyometric analysis of Indian sesame deserves priority as detailed chromosomal analysis is not avail- able on S. indicum along with their important landrac- es. Thus, the present communication for the first time details the standardization of enzymatic maceration and air drying (EMA) method of chromosome preparation and comparative karyometric analysis using non-fluo- rescent Giemsa and fluorescent DAPI and CMA stains in four distinct Indian landraces of S indicum. MATERIALS AND METHODS Plant materials The present study included four Indian landraces of S. indicum namely Black seeded till (BT), Dark brown seeded till (DBT), Light brown seeded till (LBT) and White seeded till (WT). Among these four landraces, seeds of BT, WT and LBT were collected from differ- ent parts of West Bengal and DBT was collected from Mangalore, Karnataka. All the collected seeds were ger- minated, grown in earthen pots and maintained under natural environment. Voucher specimens were prepared for all the collected samples. Somatic chromosome preparation and karyo-morphometric analysis Nearly 20- 25 seeds from each landrace were imbibed in water for overnight and germinated in dark on moist filter papers to harvest their root tips. A minimum of ten healthy root tips of each sample were pre-treated sepa- rately in saturated solution of p-dichlorobenzene (PDB) at 14- 16ºC for 4- 5 hrs, fixed overnight in glacial acetic acid: methanol (1:3) and finally stored therein at - 20 °C. Enzy- matic maceration and air-drying (EMA) method was car- ried out following our earlier published protocol (Jha and Yamamoto 2012; Jha et al. 2015; Jha and Saha 2017) with required modifications of enzyme digestion time (55 min- 90 min). Completely air-dried slides were stained with 2% Giemsa solution (Merck; Germany) in 1/15th phosphate buffer solution (pH 6.8) for 10-30 min at room tempera- ture. After 4- 5 times washing with ddH2O, the slides were air dried, mounted with xylene and observed (a minimum of 20 well scattered metaphase plates for each landrace). They were examined and photographed under Carl Zeiss, Axio. Lab. A1 microscope fitted with CCD Camera using Axiovision L. E4 software. For karyo-morphometric analysis, different karyo- logical parameters viz. length of long arm (l) and short arm (s), absolute chromosome length (CL), relative chromosome length (RL) and total diploid chromatin length (TCL) were used. Five somatic metaphase plates were used for karyometric analysis as well as to pre- pare ideogram. The centromeric index (CI) was used to classify the chromosomes according to Levan et al. (1964) [metacentric (m) (1.00–1.70), submetacentric (Sm) (1.70–3.00), subtelocentric (St) (3.00–7.00) and telocen- tric (t) (7.00–α)]. The karyotype asymmetry was esti- mated using intra-chromosomal asymmetry index (A1) and inter-chromosomal asymmetry index (A2) (Zarco 1986), asymmetric karyotypes percent (AsK%), asymme- try Index (AI) (Paszko 2006), total form percent (TF%), coefficient of variation of chromosome length (CVCL), coefficient of variation of centromeric index (CVCI), coef- ficient of variation of arm ratio (CVr) and categories of Stebbins (1971). Fluorochrome staining with DAPI and CMA Giemsa stained slides of each landrace were de- stained with 70% methanol for 40 min and air dried. 83A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum DAPI and CMA staining was carried out separately fol- lowing the protocol of Kondo and Hizume (1982) with required modifications. For DAPI staining, slides were kept for 30 min in Mcllvaine buffer and then stained with 0.1µg ml-1 solution of DAPI for 10- 30 min, mount- ed in non-fluorescent glycerol and observed under Carl Zeiss Axio Lab A1 fluorescent microscope using carl zeiss DAPI filter cassette. For CMA staining, the same slides were de-stained air-dried and then kept in Mcll- vaine buffer for 30 min followed by McIlvaine buffer with 5mM MgCl2 for 10 mins. Slides were stained with 0.1mg ml-1 solution CMA for 30- 60 mins followed and rinsed with Mcllvaine buffer containing 5mM MgCl2. Finally, slides were mounted with non-fluorescent glyc- erol and kept for maturation at 40C for 72 hrs. CMA stained chromosomes were observed under the above- mentioned fluorescent microscope fitted with Carl Zeiss FITC filter cassette and signals were analyzed using soft- ware Prog Res 2.3.3. Statistical analysis Descriptive statistics including mean values were analyzed for all measured parameters and variability in the data was expressed as the mean ± standard devia- tion (S.D.). One-way analysis of variance (ANOVA) was performed to detect significant differences (p ≤ 0.05) in the mean (Rohlf 1998). Duncan’s multiple range test (DMRT) was used for post hoc analyses using SPSS v 16.0 statistical package. To study the karyotypic relation- ships among the collected landraces of S. indicum, scat- ter diagram of A1 versus A2 was drawn following the descriptions of Paszko (2006). In order to further clarify the chromosomal relationship between each of the stud- ied landrace, principal components analysis (PCA) was conducted according to McVean (2009). In this study, nine karyological variables (A1, A2, TF%, AsK%, CVCL, CVCI, CVr, AI and TCL) were used to plot the principal components using the InfoStat version 2013d (free ver- sion). RESULTS In the present study, four landraces of S. indicum differing in seed coat colour viz., Black seeded til (BT), Dark brown seeded til (DBT), Light brown seeded til (LBT) and White seeded til (WT) were used for karyo- type analysis (Fig. 1). Nearly 99% seeds of each sesame landrace germinated within 3- 6 days after imbibition. Distinct diversity in the floral morphology pertaining to four different landraces of Sesamum was noted. The length of the corolla was 15- 20 mm with characteris- tic pigmentations on the lower lip. The flowers in black seeded til (BT) showed intense purple pigmentation in lower lip of corolla while in other landraces (DBT, LBT and WT) the intensity of the pigmentation ranged from pale lavender/ purple to light pink to white respectively (Fig. 1). Figure 1. Flower and seed morphology of four Indian landraces of S. indicum. a & e) Black seeded til; b & f ) Dark Brown seeded til; c & g) Light Brown seeded til; d & h) White seeded til. Dotted arrows indicate pigmentation patterns in lower lip of the corolla. 84 Timir Baran Jha, Partha Sarathi Saha, Sumita Jha Karyo-morphometric analysis Standardization of enzymatic maceration of root tip cells at 37 °C is the most crucial step to obtain well scattered metaphase chromosomes. In the present study, enzymatic maceration of root tips of all the collected landraces was performed for 55- 90 min and finally the time was optimized to 85- 90 min to obtain cytoplasm free well scattered chromosomes. The giemsa stain- ing was done for 20 min. For each landrace, at least 20 countable metaphase plates were studied to determine diploid chromosome number. Somatic chromosome number of 2n= 26 was observed in all the studied landraces of S. indicum (Fig. 2; Table 1). All the landraces were characterized by small sized chromosomes ranging from 1.24 ± 0.02 to 2.87 ± 0.09 µm (Fig. 2; Table 1). A significant variation in the total chromatin length was observed among the stud- ied accessions. Black seeded til (BT) was characterized by highest total chromatin length (52.75 ± 0.24 µm), while the lowest (44.85 ± 0.35 µm) being found in Dark brown seeded til (DBT). The detailed karyotype analysis revealed nine pairs of chromosomes with nearly median primary constriction, three pairs with sub median pri- mary constrictions and a single satellite pair in each of the studied landraces (Fig. 2). The ordering of satellite (sat) bearing pair was found to be constant (5th pair) in all the landraces having identical haploid karyotype for- mula: 3Sm + 9m + 1Sm.Sat (Fig. 2; Table 1). In the present study, several karyo-morphometric variations were also noted among the studied landraces. Low values of intra-chromosomal asymmetry index (A1) and inter-chromosomal asymmetry index (A2) were observed in all the studied landraces (Table 2). Asym- metric Index (AI), the product of coefficient of variation in chromosome length (CVCL) and coefficient of vari- ation in centromeric index (CVCI) was found to be low (ranging from 1.464 to 1.964) in all studied accessions of Sesame (Table 2). Whereas, Ask% and TF% showed moderate values for all the four Sesamum landraces (Table 2). Analysis of the karyotype asymmetric indi- ces also revealed that all studied landraces except Black seeded til (BT) belong to the group 2A of Stebbins classi- fication while Black seeded til (BT) belongs to group 2B (Table 2). Fluorochrome banding analysis In the present study, fluorochrome staining of Sesa- mum somatic chromosomes using DAPI and CMA was standardized for the first time. Chromosomes were stained properly with DAPI when incubated for 30 min, while for CMA, staining time was optimized at 60 min. The CMA staining of Sesamum chromosomes revealed the presence of distinct CMA positive (CMA+ve) signals/ zones in all the studied landraces However, the number of chromosomes showing CMA+ve signals varied among them (Table 3). Based on the CMA signalling patterns, chromosomes were grouped into two basic types: type A [chromosomes with no CMA+ve signals] and type B [chromosomes (including one pair of sat-bearing chro- mosomes) with distal CMA+ve signal on short arm]. Both the type A and B chromosomes were present in all the four landraces of Sesamum while, the number of each type was found to be landrace specific (Table 3). The Black seeded til (BT) and White seeded til (WT) were characterized by six chromosomes with distal CMA+ve signal on short arm (Fig. 3b and 3k), while the Dark brown seeded til (DBT) showed ten chromosomes with distal CMA+ve signal on short arm (Fig. 3e). The Light brown seeded til (LBT) was characterized by eight chro- mosomes with distal CMA+ve signal on short arm (Fig. 3h). However, we could not detect DAPI +ve/-ve signals on chromosomes of any of the landraces studied (Table 3). Figure 2. Panel A: EMA based giemsa stained mitotic metaphase plates of four Indian landraces of S. indicum showing 2n= 26 chro- mosomes. a) Black seeded til; b) Dark Brown seeded til; c) Light Brown seeded til; d) White seeded til. Arrows indicate secondary constricted chromosomes. Bar= 5 µm. Panel B: Comparative ideo- grams of the studied four landraces. Also showing positive CMA fluorescent band on respective chromosomes, Bar= 1 µm. 85A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum CMA+ signals in four studied landraces are incorporated in the Idiogram (Fig. 2) Scatter plot and principal component (PCA) analyses The scatter diagram of A1 versus A2 revealed that Dark brown seeded til (DBT) and Light brown seeded til (LBT) were placed close to each other, thereby forming a cluster, while the Black seeded til (BT) and White seed- ed til (WT) were positioned away from the cluster (Fig. 4). In the present study, PCA was further conducted to clarify the karyotypic relationship between the landrac- es using different karyo-morphometric parameters. In this eigenvector-based multivariate analysis, the compo- nent 1 (PC1) was found to be 59.6% of the total varia- tion whereas component 2 (PC2) was 33.4% (Fig. 5). The obtained cophenetic correlation was 0.998, indicating a good fit between the eigenvalues and eigenvectors dis- tance matrix. The PCA plot (Fig. 5) displayed the close positioning of Dark brown seeded til (DBT) with Light brown seeded til (LBT), which was similar to that of the scatter plot (Fig. 4). On the other hand, the Black seeded Table 1. Chromosome morphometric analysis of four Indian landraces of S. indicum*. S. indicum landraces Zygotic chromosome number (2n) Length of longest chromosome (μm) Length of shortest chromosome (μm) Total chromatin length (μm) (Mean ± S.D.) Ordering no. of SAT bearing pair Karyotype formulae (n)Absolute (Mean ± S.D.) Relative (Mean ± S.D.) Absolute (Mean ± S.D.) Relative (Mean ± S.D.) Black seeded Til 26 2.87 ± 0.09b 5.44 ± 0.14b 1.36 ± 0.02 c 2.58 ± 0.05a 52.75 ± 0.24c 5th 3Sm+9m+ 1Sm.Sat Dark Brown seeded Til 26 2.20 ± 0.12a 4.90 ± 0.24a,b 1.24 ± 0.02a 2.77 ± 0.06b 44.85 ± 0.35a 5th 3Sm+9m+ 1Sm.Sat Light Brown seeded Til 26 2.15 ± 0.14a 4.76 ± 0.28a 1.28 ± 0.02a 2.84 ± 0.07b 45.20 ± 0.42a 5th 3Sm+9m+ 1Sm.Sat White seeded Til 26 2.62 ± 0.07b 5.42± 0.06b 1.33± 0.03b 2.76 ± 0.03b 48.29 ± 0.74b 5th 3Sm+9m+ 1Sm.Sat *Values followed by same letter are not significantly different according to Duncan’s multiple range tests test (P=0.05). Table 2. Comparative karyometric analysis of four Indian landraces of S. indicum*. S. indium landraces A1 A2 TF% AsK% CVCL CVCI CVr AI Stebbin’s group Black seeded Til 0.603 0.004 37.061 62.293 22.496 8.732 15.639 1.964 2B Dark Brown seeded Til 0.619 0.007 37.747 61.360 19.056 10.218 17.877 1.947 2A Light Brown seeded Til 0.628 0.009 38.228 60.886 17.369 8.433 14.562 1.464 2A White seeded Til 0.636 0.015 38.163 61.007 21.370 7.523 12.311 1.607 2A *Values followed by same letter are not significantly different according to Duncan’s multiple range tests test (P=0.05). A1: Intra-chromo- somal asymmetry index; A2: Inter-chromosomal asymmetry index; TF%: Total form percent; AsK%: Asymmetric karyotype percent; CVCL: Coefficient of variation of chromosome length; CVCI: Coefficient of variation of centromeric index; CVr: Coefficient of variation of arm ratio; AI: Asymmetry index. Table 3. Fluorescent banding patterns in four Indian landraces of S. indicum. S. indicum landraces Maximum no. of chromosomes with CMA+ve bands Position of CMA+ve bands in chromosome CMA karyotypes (n) Maximum no. of chromosomes with DAPI+ve/ve- bands Black seeded Til 6 Distal part of short arm 20A+6B Nil Dark Brown seeded Til 10 Distal part of short arm 18A+8B Nil Light Brown seeded Til 8 Distal part of short arm 18A+8B Nil White seeded Til 6 Distal part of short arm 20A+6B Nil 86 Timir Baran Jha, Partha Sarathi Saha, Sumita Jha til (BT) and White seeded til (WT) were located at a sig- nificant distance from each other (Fig. 5). DISCUSSION The present study demonstrates a comprehensive karyo-morphometric analysis of four Indian landraces of S. indicum based on giemsa, CMA and DAPI band- ing analysis. Due to its characteristic life forms and immense nutritive value, S. indicum has attracted the attention of researchers and breeders to plan a successful conservation strategies and improvements in breeding programs. However, only a few reports are available on the cytogenetics of Indian varieties of Sesamum till date may be owing to very small size of the chromosomes (< 3 µm) and technical limitations (Raghavan and Krishna- murthy 1947; Mukherjee 1959). In the present study, we have adopted the EMA based chromosome analysis to obtain cytoplasm free well scattered metaphase chro- mosomes of the species. The combination of enzymatic maceration and air dying methods is a very useful tech- nique to analyze chromosome morphology, constrictions and types of chromosomes in detail (Fukui 1996). This method was instrumental in obtaining uniformly spread chromosomes against a cytoplasm free background in several crop species with small and medium sized chro- mosomes (Kurata and Omura 1978; Moscone 1996; Yamamoto 2007; Jha 2014; Jha and Halder 2016; Jha et al. 2017; Jha and Saha 2017; Ghosh et al. 2018). The results obtained from the present analysis offers several insights into the karyological characterization of different S. indicum landraces viz. Black seeded til (BT), Dark brown seeded til (DBT), Light brown seed- ed til (LBT) and White seeded til (WT). The diploid chromosome number (2n= 26) in all studied landraces Figure 5. Principal component analysis (PCA) plot showing group- ing of four Indian landraces of S. indicum based on nine karyo- morphometric variables. Figure 3. Somatic metaphase chromosomes (2n= 26) of four Indi- an landraces of S. indicum stained with Giemsa, CMA followed by DAPI. a- c) Black seeded til; d- f ) Dark Brown seeded til; g- i) Light Brown seeded til; j- l) White seeded til. Bar= 5 µm. Arrows indicate the chromosomes showing CMA+ve signals/ zones when stained with CMA fluorochrome. Figure 4. Scatter diagram of intra-chromosomal asymmetry index (A1) versus inter-chromosomal asymmetry index (A2) of four Indi- an landraces of S. indicum. 87A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum of S. indicum is in agreement with the earlier reports (Morinaga et al. 1929; Kobayashi 1949; Raghavan and Krishnamurthy 1947). Raghavan and Krishnamur- thy (1947) identified 13 pairs of somatic chromosomes with terminal constrictions in this species, while Kob- ayashi (1991) classified five pairs of median and eight pairs of submedian chromosomes including one pair (10th pair) of sat-bearing chromosomes in S. indicum. In the present study, a significant variation in chromo- some size (ranging from 1.24 μm- 2.87 μm) has been scored among the Sesamum landraces. Mukherjee (1959) reported two pairs of chromosomes having second- ary constrictions in S. indicum, while the present EMA based analysis clearly revealed the presence of nine pairs of chromosomes with nearly median primary constric- tion, three pairs were submedian and a single pair (5th pair) of sat-bearing chromosomes in all the studied lan- draces and which can be considered as the modal karyo- type for Indian Sesame. In addition to the EMA based giemsa staining, the fluorochrome banding patterns are documented here for the first time in four studied Indian landraces of Sesam- um. The application of nucleotide specific fluorochromes i.e. GC-specific CMA, AT-specific DAPI in chromosome analysis has been reported to be very expedient in prop- er karyological characterization of many plant species (Schweizer 1976; Moscone et al. 1996). In the present study, CMA banding analysis provides a comprehensive cytogenetic characterization of four Indian landraces of S. indicum. Based on both karyomorphology and CMA signalling patterns, distinct homologies could be estab- lished between the studied landraces. Presently, we could not locate DAPI+ve bands in any of the studied samples. However, the differences in distribution of CMA+ve sig- nals/ zones clearly delimit each of the studied landraces of Sesamum. In the present study, all the collected landraces of S. indicum exhibited symmetrical karyotypes based on categories of Stebbins (1971). However, the analyses of scatter diagram of A1 versus A2 and PCA plot unam- biguously delimit each of the studied landrace (Fig. 4 and 5). PCA is a true eigenvector-based multivariate analysis, which can be used to project samples onto a series of orthogonal axes and to statistically clarify the genetic relationship among the studied samples (McVean 2009). The results obtained from scatter plot of A1 vs A2 and PCA analysis provide a strong relationship with that of the fluorochrome (CMA) banding analysis. The Dark brown seeded til (DBT) and Light brown seeded til (LBT) exhibited maximum CMA+ve signals/ zones and appeared close to each other, while both the Black seeded til (BT) and White seeded til (WT) characterized by minimum CMA+ve signals (i.e. six chromosomes with distal CMA+ve signal on short arm) positioned distantly in the scatter diagram of A1 versus A2 and PCA plot. As a whole, the present study involving EMA based giemsa staining techniques demonstrates an explicit kar- yo-morphometric characterization of four Indian lan- draces of S. indicum for the first time. Distinct landrace- specific variation in the distribution of CMA+ve signals/ zones in somatic chromosomes was also established in the species. The grouping of the studied landraces was also corroborated by the analysis of scatter diagram of A1 versus A2 and PCA plot which revealed a strong rela- tionship with that of the fluorochrome banding analysis. 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