Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 65-77, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-919 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy (2021) Anal- ysis of CMA-DAPI bands and prepara- tion of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris. Cary- ologia 74(2): 65-77. doi: 10.36253/caryo- logia-919 Received: April 24, 2020 Accepted: May 19, 2021 Published: October 08, 2021 Copyright: © 2021 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy. This is an open access, peer-reviewed article published 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. ORCID TBJ: 0000-0003-0900-8167 BKB: 0000-0001-6029-1098 Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Timir Baran Jha1,*, Biplab Kumar Bhowmick2, Partha Roy1 1 Department of Botany, Maulana Azad College, Kolkata -700013, West Bengal, India 2 Department of Botany, Scottish Church College, 1&3, Urquhart Square, Kolkata- 700006, West Bengal, India *Corresponding Author. E-mail: tbjha2000@yahoo.co.in Abstract. India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of pheno- typically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochrome dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cul- tivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes cap- tured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culi- naris Medik. cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes. Keywords: lentils, CMA-DAPI, chromosomal bands, fluorescent karyotype, hetero- chromatin. INTRODUCTION Lentil is one of the richest protein containing domesticated ancient crop with only one globally cultivated species Lens culinaris Medik. India is the second highest producer and biggest consumer of Lentils. The genus belongs to the largest subfamily (Papilionoideae) of Fabaceae (Azani et al. 2017), along with many economically important genera producing pulses and beans. Being the single cultivated species, large number of cultivars is in cultivation in our country. The characterization of Indian germplasm is 66 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy needed to sustain conservation and programmable uti- lization of resources. Chromosomal characterization is a cost effective method to provide foundational infor- mation on the genome and genetic conservation for any future breeding program of particular crop plants. Cytogenetic studies of Indian Lentils through conven- tional method failed to provide uniformity on chromo- some morphometric parameters (Bhattacharjee 1953; Sharma and Mukhopadhyay 1963; Sinha and Acharia 1972; Naithani and Sarbhoy 1973; Lavania and Lavania 1983; Nandanwar and Narkhede 1991). On the other hand, we have published detailed karyotype analysis of more than thirty L. culinaris cultivars obtained from the Indian Institute of Pulses (Jha et al. 2015, 2017; Jha and Halder 2016) through EMA based Giemsa staining method. Our results were found to have near similarities with the results obtained by Ladizinsky (1979). However, Lens chromosomes (2n=14) are nearly similar in mor- phology. Considering the status of research, we ques- tion i) is there any karyotype variability across cultivars beyond chromosome number, morphology and ploidy? ii) is it possible to find visible chromosomal landmarks in accordance with the germplasm diversity? and iii) whether we can step forward towards molecular kay- otype database for Indian Lentils. As EMA based chro- mosome analysis (Fukui 1996) is the basis of molecular cytogenetics, we decided to carry forward our work with two contrasting fluorescent stains DAPI and CMA on the same cultivars. Having affinity towards specific base pairs of DNA, these fluorescent dyes reliably identify heterochromatin rich sectors on chromosomes, differen- tiate morphologically alike chromosomes and improve karyotype characterization (Schweizer 1976; Guerra et al. 2000; Yamamoto 2012; Weiss‐Schneeweiss and Sch- neeweiss 2013). So, our objective is to address chromo- somal behavior after application of base specific fluoro- chromes and compile cultivar specific fluorescent band- ing profiles. The present paper considers a fluorescent karyotype dataset of 30 Indian L. culinaris cultivars for the first time, as an important kit for Lentil breeders and genome researchers. MATERIALS AND METHODS Chromosome preparation and fluorochrome staining The fluorescent karyotype analysis was carried out on 30 cultivars of Lens culinaris presented in Table 1. Except for two (Barasat, Micro type and Barasat, Macro type, Table 1), all the cultivars of Lentil were obtained from the Indian Institute of Pulse Research (IIPR), Kanpur. Germination of seeds and chromosome pro- cessing through enzymatic maceration and air drying (EMA) was carried out as per our earlier protocol (Jha and Yamamoto 2012; Jha et al. 2015, 2017, 2020). For fluorescent staining with DAPI and CMA, we followed our protocol (Jha 2019) with required modifications. For DAPI staining, slides were kept for 30 min in McIl- vaine buffer, stained with 0.1µg ml-1 solution of DAPI for 10 min, counterstained with 0.25mg/ml of Actino- mycin D (AMD) for 15min and then mounted in non- fluorescent glycerol and observed under Carl Zeiss Axio Lab A1 fluorescence microscope using Carl Zeiss DAPI filter cassette. Chromosome images were captured with CCD camera attached with microscope. The slides were destained and air-dried. The same slides were placed in McIlvaine buffer for 30 min followed by incubation in McIlvaine buffer with 5mM MgCl2 for 10 mins and then stained with 0.1mg ml-1 CMA solution for 45-50 mins. The slides were again washed in McIlvaine buffer with 5mM MgCl2 and finally mounted with non-fluorescent glycerol and kept for maturation at 40C for 48-72 hrs. CMA stained slides were observed under the above- mentioned f luorescence microscope fitted with Carl Zeiss FITC filter cassette, images captured with attached CCD camera and signals were analyzed using the soft- ware Prog Res 2.3.3. Statistical analysis of karyotype relations Karyotype relations among the cultivars was evalu- ated with the help of cluster analysis for data matrix normalization by unweighted pair group method with arithmetic averages (UPGMA) based on Euclidean dis- tance using Info Stat 2017d (free version). Here, only the fluorochrome banding pattern of the cultivars viz. types and numbers of CMA and DAPI bands were utilized to draw the phenogram. RESULTS Fluorochrome banding pattern in cultivars of L. culinaris Medik. Somatic chromosome analysis of the 30 Lentil culti- vars based on fluorescence banding patterns has provid- ed an interesting catalogue of chromosome diversity. The chromosomes took up DAPI stain within 10 minutes of incubation while the incubation time for CMA staining was about 45-50mins. The same CMA and DAPI stain- ing protocol was followed for all the 30 cultivars of L. culinaris. Interestingly, we have obtained different types of DAPI and CMA banding patterns within the studied 67Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Figure 1. Somatic metaphase chromosomes of Lens culinaris cultivars stained with CMA: (a) DPL-15, (b) DPL-62, (c) IPL-81, (d) IPL-406, (e) IPL-316, (f ) JL-1, (g) HUL-57, (h) KLS-210, (i) EC-70394, (j) EC-70403, (k) EC-70404, (l) EC-78452, (m) EC-78455, (n) EC-78461, (o) EC-78475, (p) EC-78498, (q) EC-78542-A, (r) EC–223188, (s) EC – 255491, (t) EC–267526, (u) EC-267569-A, (v) EC–267590, (w) EC–267877, (x) Barasat Micro type. White arrows indicate CMA+ bands and arrowheads indicate CMA0 bands. Bars 5µm. 68 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy cultivars. At least 10 plates stained with DAPI and CMA for each cultivar was considered for analysis of band- ing types. Secondary constriction marked the nucleolar organizing region (NOR) of most of the cultivars, show- ing CMA+ bands with different intensities while some NORs remained neutral and termed CMA0 as per Bar- ros e Silva and Guerra (2010). DAPI staining in most of the cultivars resulted a clear gap (DAPI-) corresponding to CMA+ band. However, few exceptional cultivars yield- ed DAPI+ band in the NOR regions. Based on the CMA and DAPI fluorescent banding, we have categorized fol- lowing types of somatic chromosomes. The chromo- somes with CMA+/DAPI- band in the nucleolar region is termed type ‘A’. Type ‘B’ has CMA+/DAPI- nucleolar constriction followed by a DAPI+/CMA0 band below cen- tromere. The ‘C’ type nucleolar chromosome has a dis- tinct CMA+/ DAPI+ secondary constriction. The fourth type ‘D’ has neutral CMA band in secondary constric- tion. Chromosomes with centromeric CMA+/DAPI0 bands are termed type ‘E’ while those with centromeric DAPI+/CMA0 bands are termed type ‘F’. Type ‘G’ chro- mosome contains intercalary DAPI+/CMA0 band. The chromosomes having no detectable bands were termed as type ‘H’. Distribution of different types of fluoro- chrome bands among the cultivars is summarized in Table 1. A detailed analysis of the fluorochrome stained metaphase plates (Figures 1-3) was carried out to formu- late the diagrammatic fluorescent karyotypes of the 30 cultivars under study (Figures 4 and 5). CMA-DAPI banding patterns have revealed that in majority of L. culinaris cultivars (Table 1), the marker secondary constrictions with CMA+ signals are present in the 4th pair of chromosomes. However, the same in some cultivars are present in the 3rd and exceptionally in the 5th and 2nd pairs, as in two cultivars (EC-70394, EC-78542-A). The most abundant CMA+ satellites (type A chromosomes) are found among 50% of the presently studied cultivars. In addition to CMA+ satellites, exist- ence of type B chromosomes is found in 8 different cul- tivars (HUL-57, EC-70403, EC-78542-A, EC-267526, EC – 267877, Barasat Micro type, PL -1406, EC -78410, Table 1) and type D chromosomes in 5 different cultivars (DPL15, JL-1, EC-78452, EC – 70306, EC – 78473, Table 1). Of special mention, are the two cultivars (EC-70404, EC- 267569-A, Table 1) with CMA+/DAPI+ satellite (type C chromosome). Three cultivars (IPL -316, EC-70394, EC – 267877) had centromeric CMA+ bands (type E) (Table 1). One of them (IPL -316) shows centromeric CMA+ bands (type E) in every chromosome except the nucleolar pair (Table 1). On the other hand, EC -78410 shows intense centromeric DAPI+ bands (type F) in all non-nucleolar chromosome pairs (Table 1). Centromeric DAPI+ bands are consistently found in the 2nd or the 3rd pair of chro- mosomes in 5 cultivars (HUL-57, EC-70403, EC-267526, Barasat, Macro type, PL -1406, Table 1). Intercalary DAPI+ band (type G) is seen only in IPL-406 (Table 1). Comparative statistical assessment of fluorochrome banding pattern Statistical evaluation of karyotype relations among the 30 Lentil cultivars was carried out using Euclidean distance matrix on the basis of CMA and DAPI bands. The UPGMA phenogram presented relative karyotype affinities and distances with a cophenetic correlation of 0.986 as a good fit between the cophenetic value matrix and the average Euclidean distance matrix (Figure 6). There are three separate groups in the UPGMA phe- nogram of which Group I consisted of cultivars that do not have close affinity with each other (Figure 6). Within this group, EC -78410 and IPL -316 have fluo- rescent banding pattern that are in contrast to each other. Also, existence of intercalary DAPI+ band makes IPL-406 distinct, placed at the extreme end of the phe- nogram. The next noticeable cultivars are EC-70404 and EC-267569-A with CMA+/DAPI+ secondary constriction (Table 1) (Figure 6). The Group II is large, composed of three subgroups mainly differentiated by nucleolar banding pattern in their marker chromosomes. The first subgroup comprised of 5 cultivars with neutral CMA- DAPI bands in their satellites (type D) (Table 1, Figure 6). The second subgroup is largest, comprising of 13 cultivars with CMA+/DAPI- satellite (type A). Here, two cultivars (EC-70394 and Barasat, Macro type) show little distance from rest of the cultivars, because of different types of centromeric bands (Table 1, Figure 6). The third subgroup comprises of 7 cultivars with ‘B’ type nucleo- lar chromosomes. This subgroup shows heterogeneity because of variations in centromeric bands (Table 1, Fig- ure 6). DISCUSSION Cytogenetics of L.culinaris is traditionally acknowl- edged for species delimitations, crossing behavior, con- servation and utilization of plant genetic resources (Ladizinsky 1979; Tadmor et al. 1987; Ladizinsky et al. 1990; Ladizinsky 1999; Mishra et al. 2007). With the present approach, we have entered the modern karyotyp- ing system to study chromosomal specialization in Indi- an Lentils. The diversity of fluorescent karyotypes can be indisputably attributed to the differences in underlying chromosomal heterochromatin of the samples since i) 69Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Figure 2. Somatic metaphase chromosomes of Lens culinaris cultivars stained with DAPI: (a) DPL-15, (b) DPL-62, (c) IPL-81, (d) IPL-406, (e) IPL-316, (f ) JL-1, (g) HUL-57, (h) KLS-210, (i) EC-70394, (j) EC-70403, (k) EC-70404, (l) EC-78452, (m) EC-78455, (n) EC-78461, (o) EC-78475, (p) EC-78498, (q) EC-78542-A, (r) EC–223188, (s) EC – 255491, (t) EC–267526, (u) EC-267569-A, (v) EC–267590, (w) EC–267877, (x) Barasat Micro type. White arrows indicate DAPI+ bands and arrowheads indicate DAPI- and DAPI0 bands. Bars 5µm. 70 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy Figure 3. Somatic metaphase chromosomes of Lens culinaris cultivars. CMA stained plates: (a) Barasat Macro type, (b) PL-1406, (c) EC-70306, (d) EC -78410, (e) EC-78451-A, (f ) EC-78473. DAPI stained plates: (g) Barasat Macro, (h) PL-1406, (i) EC-70306, (j) EC -78410, (k) EC-78451-A, (l) EC-78473. White arrows indicate CMA+ or DAPI+ bands and arrowheads indicate CMA0 or DAPI0 and DAPI- bands. Bars 5µm. 71Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Figure 4. Fluorescent ideograms of Lens culinaris cultivars based on CMA/DAPI banding pattern: (a) DPL-15, (b) DPL-62, (c) IPL-81, (d) IPL-406, (e) IPL-316, (f ) JL-1, (g) HUL-57, (h) KLS-210, (i) EC-70394, (j) EC-70403, (k) EC-70404, (l) EC-78452, (m) EC-78455, (n) EC-78461, (o) EC-78475, (p) EC-78498, (q) EC-78542-A, (r) EC–223188, (s) EC – 255491, (t) EC–267526, (u) EC-267569-A, (v) EC–267590, (w) EC–267877, (x) Barasat Micro type. CMA+, DAPI+, CMA+/DAPI+ and CMA0 bands are highlighted with green, blue, red and grey colors on the chromosomes, respectively and the types are indicated above the chromosome diagrams. Bars 5µm 72 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy we have applied the same fluorochrome staining proto- col for every cultivar, ii) the method is repeated a num- ber of times before ascertaining banding pattern in a cultivar and iii) at least 5 best metaphase plates of each cultivar with scorable signals were considered for estab- lishing the fluorescent karyotype. Considering the nature of nucleolar chromosomes, molecular banding technique has shed light on chro- mosomal landmarks and possible differences in NORs that were previously found to be similar in Lens (Mehra et al. 1986; Jha et al. 2015, 2017; Jha and Halder 2016). The marker nucleolar chromosomes (4th, along with the 3rd, 2nd and 5th in few cases) have been confirmed with characteristic CMA-DAPI signals, corroborating to our previous report (Jha et al. 2017). The CMA+ signals are generally accepted as the GC heterochromatic elements of the NORs in plant groups (Guerra et al. 2000; Barros e Silva and Guerra 2010; Yamamoto 2012; Olanj et al. 2015) and so in Papilionoids such as Vicia (Fuchs et al. 1998), Cicer (Galasso et al. 1996) and Crotalaria (Mon- din and Aguiar-Perecin 2011). Previously, 18S-5.8S-25S rDNA probes had been localised in a single pair of L. culinaris, near the centromere (Balyan et al. 2002), cor- roborating to the observation of CMA+ signals in our present study. However, we found that the intensity of the nucleolar CMA signals (type A) varies in certain cultivars, suggesting differences in NORs that influence affinity towards the stain. Intraspecific rDNA variation has been thoroughly worked out in Phaseolus (Moscone et al. 1999; Pedrosa-Harand et al. 2006) and Vigna (Bor- toleti et al. 2012; She et al. 2015, 2020) of Papilionoide- ae. A number of factors such as transposition, unequal crossing over, inversion or locus duplication, had been suggested to drive NOR variation in plant groups, including Papilionoideae (Moscone et al. 1999; Chung et al. 2008; Raskina et al. 2008). We consider similar possi- bilities in the Indian Lentils, subject to future confirma- tion by AgNOR staining or rDNA FISH. The ty pe D chromosomes have satellites that respond indifferently to the CMA stain. The CMA0 sat- ellites indicate GC neutral nature of heterochromatin (Barros e Silva and Guerra 2010). The type D satellites are in sharp contrast to type A bands, marking culti- var distinction. The other unusual type was the CMA+/ DAPI+ satellites (type C). Previously, the CMA+/DAPI+ satellites were suggested to be a ‘less common’ or ‘rare’ type of heterochromatin (Barros e Silva and Guerra 2010), breaking the generality of GC rich composition of plant NORs (Schweizer 1976; Guerra et al. 2000). We document the occurrence of CMA+/DAPI+ satellites for the first time in Lens of Papilionoideae. Co-localized CMA+/DAPI+ satellites are so far reported in Allium nigrum (Maragheh et al. 2019) and Cestrum (Fernandes et al. 2009). It is difficult to ascertain the heterochro- matin composition of this type. There is a possibility of having AT and GC rich segments to be placed so close that the different chromatin bands cannot be distin- guished in condensed mitotic chromosomes (Maragheh et al. 2019). However, nucleolar heterochromatin compo- sition of Indian Lens culinaris displays considerable vari- ation, perhaps due to enormous cultivation practice and artificial hybridization, which is a yet unaddressed field of study. Cultivar specific differences were also accentu- ated by the non-nucleolar DAPI+ and CMA+ bands. The type E centromeric CMA bands are unique type Figure 5. Fluorescent ideograms of Lens culinaris cultivars based on CMA/DAPI banding pattern: (a) Barasat Macro type, (b) PL-1406, (c) EC-70306, (d) EC -78410, (e) EC-78451-A, (f ) EC-78473. CMA+, DAPI+, CMA+/DAPI+ and CMA0 bands are highlighted with green, blue, red and grey colors on the chromosomes, respectively and the types are indicated above the chromosome diagrams. Bars 5µm. 73Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris of heterochromatin rarely reported in plants. However, non-nucleolar GC-rich heterochromatin was previously characterized in centromeric as well as pericentromeric regions of Papilionoid species belonging to Dioclea (Sou- za and Benko-Iseppon 2004), Psophocarpus (Chaowen et al. 2004), Crotalaria (Mondin and Aguiar-Perecin 2011), Vigna (Bortoleti et al. 2012; She et al. 2015, 2020), Phaseolus (Bonifácio et al. 2012), Lablab (She and Jiang 2015), and Canavalia (She et al. 2017). She et al. (2020) suggested the centromeric or pericentromeric GC- het- erochromatin to be a relic of genomic evolution in the subfamily Papilionoideae. Other even rare heterochro- matin blocks were the centromeric (type F), pericen- tromeric (type B) and intercalary (type G) DAPI bands, constituting landmarks to differentiate karyotypes of certain Lentil cultivars. Terminal or intercalary DAPI+ bands were documented in few plants (Vanzela and Guerra 2000; Divashuk et al. 2014), including few species of Cucurbitaceae (Bhowmick and Jha 2015, 2019). Ter- minal DAPI bands are found in Crotalaria (Mondin and Aguiar-Perecin 2011) of Papilionoideae. Centromeric DAPI bands are yet rare to encounter. However, in case of L. culinaris and related species, AT heterochromatic regions were mapped by repetitive sequence probe FISH (Galasso et al. 2001; Galasso 2003). Also in Papilionoide- ae, AT rich heterochromatin at centromere and pericen- tromeric regions are reported in Vigna (Bortoleti et al. 2012; She et al. 2020), Lablab (She and Jiang 2015) and Figure 6. UPGMA dendrogram derived from average Euclidean distance based on fluorochrome banding pattern of 30 Indian Lentil culti- vars, cultivar names in the left of their serial numbers. 74 Timir Baran Jha, Biplab Kumar Bhowmick, Partha Roy Ta bl e 1. A na ly si s of C M A a nd D A PI fl uo re sc en t b an ds in th ir ty I nd ia n cu lti va rs o f L en s cu lin ar is ( 2n = 1 4) . Sl . N o. C ul tiv ar s O rd er o f nu cl eo la r pa ir C M A b an ds D A PI b an ds To ta l n o. of b an ds / 2n Fl uo re sc en t K ar yo ty pe fo rm ul a (2 n) Fi gu re no . N o. C hr om os om e pa ir /s Ty pe */ in te ns ity Fi gu re n o. N o. C hr om os om e pa ir /s Ty pe */ in te ns ity Fi gu re n o. 1 D PL 15 4t h 2 4t h D / ne ut ra l 1a 0 - - 2a 2 2D +1 2H 4a 2 D PL -6 2 4t h 2 4t h A /lo w 1b 0 - - 2b 2 2A +1 2H 4b 3 IP L -8 1 4t h 2 4t h A /lo w 1c 0 - - 2c 2 2A +1 2H 4c 4 IP L- 40 6 3r d 2 3r d A /h ig h 1d 2 6t h G /h ig h 2d 4 2A +2 G +1 0H 4d 5 IP L -3 16 4t h 2 12 4t h 1s t -3 rd , 5 th -7 th A /h ig h E/ hi gh 1e 0 - - 2e 14 2A +1 2E 4e 6 JL -1 4t h 2 4t h D / ne ut ra l 1f 0 - - 2f 2 2D +1 2H 4f 7 H U L- 57 3r d 2 3r d B / lo w 1g 2 2 3r d 2n d B /lo w F/ hi gh 2g 4 2B +2 F+ 10 H 4g 8 K LS -2 10 4t h 2 4t h A /h ig h 1h 0 - - 2h 2 2A +1 2H 4h 9 EC -7 03 94 5t h 2 2 5t h 4t h A /h ig h E/ hi gh 1i 0 - - 2i 4 2A +2 E+ 10 H 4i 10 EC -7 04 03 4t h 2 4t h B /h ig h 1j 2 2 4t h 2n d B /h ig h F/ hi gh 2j 4 2B +2 F+ 10 H 4j 11 EC -7 04 04 4t h 2 4t h C /h ig h 1k 2 4t h C /lo w 2k 2 2C +1 2H 4k 12 EC -7 84 52 4t h 2 4t h D / ne ut ra l 1l 0 - - 2l 2 2D +1 2H 4l 13 EC -7 84 55 4t h 2 4t h A /h ig h 1m 0 - - 2m 2 2A +1 2H 4m 14 EC - 78 46 1 4t h 2 4t h A /h ig h 1n 0 - - 2n 2 2A +1 2H 4n 15 EC -7 84 75 3r d 2 3r d A /lo w 1o 0 - - 2o 2 2A +1 2H 4o 16 EC – 78 49 8 4t h 2 4t h A /h ig h 1p 0 - - 2p 2 2A +1 2H 4p 17 EC -7 85 42 -A 2n d 2 2n d B /h ig h 1q 2 2n d B /lo w 2q 2 2B +1 2H 4q 18 EC -2 23 18 8 4t h 2 4t h A /h ig h 1r 0 - - 2r 2 2A +1 2H 4r 19 EC -2 55 49 1 4t h 2 4t h A /h ig h 1s 0 - - 2s 2 2A +1 2H 4s 20 EC -2 67 52 6 4t h 2 4t h B /h ig h 1t 2 4 4t h 1s t , 3 rd B /lo w F/ lo w 2t 6 2B +4 F+ 8H 4t 21 EC -2 67 56 9- A 3r d 2 3r d C /lo w 1u 2 3r d C /lo w 2u 2 2C +1 2H 4u 22 EC -2 67 59 0 4t h 2 4t h A /h ig h 1v 0 - - 2v 2 2A +1 2H 4v 23 EC - 2 67 87 7 4t h 2 2 4t h 5t h B /h ig h E/ lo w 1w 2 4t h B /h ig h 2w 4 2B +2 E+ 10 H 4w 24 B ar as at , M ic ro ty pe 3r d 2 3r d B /h ig h 1x 2 3r d B /lo w 2x 2 2B +1 2H 4x 25 B ar as at , M ac ro ty pe 4t h 2 4t h A /h ig h 3a 2 3r d F/ hi gh 3g 4 2A +2 F+ 10 H 5a 26 PL - 14 06 4t h 2 4t h B /h ig h 3b 2 2 4t h 2n d B /lo w F/ lo w 3h 4 2B +2 F+ 10 H 5b 27 EC - 7 03 06 4t h 2 4t h D / ne ut ra l 3c 0 - - 3i 2 2D +1 2H 5c 28 EC - 78 41 0 4t h 2 4t h B /h ig h 3d 2 12 4t h 1s t - 3r d , 5t h - 7t h B /h ig h F/ hi gh 3j 14 2B +1 2F 5d 29 EC – 7 84 51 -A 4t h 2 4t h A /h ig h 3e 0 - - 3k 2 2A +1 2H 5e 30 EC - 7 84 73 4t h 2 4t h D / ne ut ra l 3f 0 - - 3l 2 2D +1 2H 5f *T yp es o f n uc le ol ar b an ds - A : C M A + / D A PI - sa te lli te , B : C M A + / D A PI - s at el lit e an d D A PI + / C M A 0 ba nd in lo ng a rm , C : C M A + / D A PI + sa te lli te , D : C M A 0 sa te lli te ; c en tr om er ic b an ds - E: C M A + / D A PI 0 , F: D A PI + / C M A 0 ; in te rc al ar y ba nd G : D A PI + / C M A 0 ; H : n o ba nd s. 75Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Arachis (Silvestri et al. 2020). Nonetheless, occurrence of centromeric CMA+ or DAPI+ bands along with nucleolar CMA+/DAPI+ or CMA0 bands certainly advocate atypi- cal heterochromatin composition in Lens. The non-uni- form composition and rearrangements of heterochro- matin had been observed repeatedly in Papilionoideae species (Moscone et al. 1999; Souza and Benko-Iseppon 2004; Pedrosa-Harand et al. 2006; Mondin and Aguiar- Perecin 2011; She et al. 2020), which becomes apparent in our study once again. In view of the diversity in fluorochrome banding pattern, we attempted to resolve karyotype relation- ships by the UPGMA method. Identification of distinct subgroups has opened further scopes to complement marker assisted analysis of genetic diversity across var- ied range of Indian cultivars with valuable agronomic traits. Application of fluorochrome banding method has therefore helped to i) break the perception of an over- all similar karyotype of cultivated Lentils as observed in Giemsa plates (Jha et al. 2015, 2017; Jha and Halder 2016) ii) serve as the chromosomal blueprint for culti- var discrimination, ii) statistically represent the status of chromosomal relationships, iii) highlight the uniqueness of certain Indian cultivars by means of unconventional banding pattern, and v) construct a fluorescent karyo- type dataset of Indian Lentil cultivars. CONCLUSION Being a crop ‘as old as agriculture’ (Sandhu and Sin- gh 2007), an exclusive chromosomal database of Lentils is essential to complement genomic research databases like Legume Information System (Dash et al. 2016) and KnowPulse (Sanderson et al. 2019). As an extension of our study involving Lentil cytogenetics, we have delved into the first molecular karyotypes of the country’s native cultivars. Notably, the cultivars are hosted by world’s second largest ex situ Lentil germplasm stock i.e. IIPR of NBPGR, the first being ICARDA (Muehlbauer and McPhee 2005; Coyne and McGee 2013). In future, molecular cytogenetic study of wild Lens species of India can be expected to strengthen the base of chromosom- al evolution in Papilionoideae. In face of stern climatic changes that affect future cultivation, the Indian culti- vars with interesting karyotype features and relation- ships can be fluently tested for performance and pro- ductivity. Thus, our findings complement traditional or marker assisted breeding and would undoubtedly bridge up the lacuna for a systematic chromosomal database of Indian Lentils. ACKNOWLEDGEMENTS TBJ acknowledges Dr. S. Dutta, Principal, Maulana Azad College, Kolkata for the facilities provided and Indian Institute of Pulses Research, Kanpur for provid- ing certified Lentil cultivars. The grant was provided by the UGC, Govt. of India. 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