Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 11-22, 2019 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-754 Citation: A. Naik, A.K. Patel, S.K. Mishra, A. Nag, J. Panigrahi (2019) Characterization of intraspecific hybrid in Clitoria ternatea (L.) using morpho- physiological, cytogenetic, metabolic and molecular markers. Caryologia 72(3): 11-22. doi: 10.13128/caryolo- gia-754 Published: December 13, 2019 Copyright: © 2019 A. Naik, A.K. Patel, S.K. Mishra, A. Nag, J. Panigrahi. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, 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. Characterization of intraspecific hybrid in Clitoria ternatea (L.) using morpho- physiological, cytogenetic, metabolic and molecular markers Aparupa Naik1, Amiya K. Patel1, Sujit K. Mishra1, Atul Nag2, Jogeswar Panigrahi1,2,* 1 Plant Biotechnology Laboratory, Department of Biotechnology and Bionformatics, Sam- balpur University, Jyoti Vihar-768019, Sambalpur, Odisha, India 2 Department of Biotechnology, Central University of Rajasthan, NH-8, Bandarsindri, Kishangarh, Rajasthan *Corresponding author: drjpanigrahi@gmail.com Abstract. Clitoria ternatea (L.) is a medicinal plant possessed with bioactive molecules such as taraxerol, delphinidin, kaempoferol and quercetin etc. For the development genotype with higher content of these bioactive molecules, marker-assisted breeding is one of the best strategies and it initiates with the development of F1 hybrids. Thus, an intraspecific F1 hybrid was raised involving two contrasting genotypes of C. terna- tea acc. CtB3-SL1 (Blue flowered) and C. ternatea acc. CtW2-BL1 (white flowered). The hybridity of the F1 plant was confirmed by assessing the phenotypic traits, such as col- our of the petal, pod shape and seed coat colour, 100 seed weight, and the content of taraxerol and delphinidin. The pollen mother cells in the F1 hybrid showed eight biva- lents with preponderance of ring bivalents and 8I:8I segregation at metaphase-I and Anaphase-I, respectively. SDS-PAGE seed albumin and globulin detected three pollen parent-specific polypeptides (Mw 31.62, 22.38 and 18.81KDa), and were inherited to F1 hybrids, which evidenced the hybridity of putative F1 plants. Further, DNA mark- er analysis also showed the inheritance of 11 RAPD, six SCoT and one ISSR markers to putative F1 plant, which affirmed the hybrid nature of the F1 plant. This study also evidenced that combined use of morphophysiological, cytogenetic, protein and DNA marker analyses could be effective for precise characterization of intra-specific hybrids in C. ternatea. These F1 hybrid and its derived future progenies could also be used for mapping of QTLs or genes contributing higher accumulation of taraxerol and delphini- din in different plant parts. Keywords.Hybridity, Meiosis, Seed protein profile, DNA marker, Taraxerol, Delphini- din. INTRODUCTION Clitoria ternatea (L.) belongs to the family-Leguminosae (Fabaceae) with somatic chromosome count 2n=2x=16 and was distributed worldwide 12 Aparupa Naik et al. (Joson and Ramirez 1991; NPGS2008). The C. ternatea is also known as Shankhpushpi, Butterfly pea, Aparajita, Gokarni, Girikarnika (Bishoyi et al. 2014). The plant is elongated, slender, climbing herbaceous vine with five leaflets, white to purple flowers, and has deep roots. But- terfly pea is predominantly a self-pollinated species, but sometimes considered as often cross-pollinated due to the appearance of segregating genotypes in some popu- lations (Cook et al. 2005). This plant has been considered as important medicinal plant, and the phyto-chemical studies explored several bioactive metabolites such as alkaloids, triterpenoids, flavonoids, glycosides, antho- cyanins and lactones etc. in this species (Mukherjee et al. 2008; Sethiya et al. 2009). Thus the plant parts based extracts have been prepared and used as a memory enhancer, nootropic, anti-stress, anxiolytic, anticonvul- sant, tranquilizing and sedative agent (Parrotta 2001; Prajapati et al. 2003; Khare 2004; Kapoor 2005; Mar- gret et al. 2015). Among the bioactive metabolites, con- tent of kaempferol, delphinidin and taraxerol having anti-cancer and anti-tumour properties have also been documented in this species (Braig et al. 2005; Chen and Kong 2005; Niering et al. 2005; Singletary et al. 2007; Swain et al. 2012). The white-flowered genotypes of C. ternatea were found to be medicinally rich in taraxerol and kaempferol, whereas the blue flowered genotypes were affluent in delphinidin, quercetin and isoquerce- tin etc. For the development of improved genotype with higher content of these bioactive metabolites marker assisted breeding (MAB) is one of the finest strategy, where F1 hybrid serves as the starting material for all future breeding efforts for the genetic improvement. Thus intra-specific F1 hybrid involving blue f lowered and white flowered genotype can be used as the start- ing material for widening genetic base of this medici- nal species in term of bioactive metabolites composition and production of advanced breeding lines containing desired metabolites of pharmaceutical importance. How- ever, interaction between both the genome in either of the parental cytoplasmic background often led to genetic variation due to genetic recombination, differential gene action, penetrance and expressivity. F1 hybrid being the starting material for all breeding efforts, its precise identification at early stage is mandatory. Thus morpho- logical, cytological, biochemical and molecular markers have been effectively used to ascertain hybridity of F1 plants in many species. But the reproducibility of mor- phological, cytological and biochemical markers in con- sonance to environmental variation and developmen- tal regulations limits their applicability. Therefore seed protein and DNA sequence based marker analysis could also be utilized to screen and identify F1 hybrids at an early stage because of their stability, uniformity, reliabil- ity and reproducibility across the environment and are also free from penetrance and expressivity. The seed protein markers have been effectively employed for cultivar characterization, genetic diversity assessment, and verification of hybridity in many species (Mohanty et al. 2001; Panigrahi et al. 2007; Jisha et al. 2011; Mishra et al. 2012). Similarly DNA markers, such RAPD, ISSR and SSR have been used for characterization of hybrids in different species (Lima-Brito et al. 2006; Muthusamy et al. 2008; Goldmann et al. 2008; Hemala- tha et al. 2010; Bianco et al. 2011; Mishra et al. 2012; Mishra et al. 2017). In case of C. ternatea, few of these DNA markers have only been used for genetic diversity studies (Chandra 2011; Swati et al. 2011; Ganie et al. 2012; Ali et al. 2013: Bishoyi et al. 2014). However, no report has been made so far on the development of intra-specific hybrid in C. ternatea and its characterization. In the present study, an intra-specific F1 hybrid of C. ternatea, involving blue flowered [C. ternatea acc. CtB3- SL1] and white flowered [C. ternatea acc. CtW2-BL1] genotype, was raised and its hybridity was affirmed by simultaneous use of morpho-physiological and cytoge- netic analyses, estimation of bioactive metabolite, profil- ing of seed protein and DNA based markers. MATERIALS AND METHODS Plant materials Genotypes of C. ternatea (including of five white genotypes, six blue genotypes, and four bipetaloid blue genotypes) were collected from Sambalpur and Bargarh districts of Odisha and maintained at the experimental garden, School of Life Sciences, Sambalpur University, Odisha, India (Table 1). Among these genotypes, two genotypes of C. ternatea acc. CtB3-SL1 and C. ternatea acc. CtW2-BL1 were identified on the basis of their bio- active metabolites (Delphinidin and Taraxerol) content, and used as seed parent and pollen parent, respectively for the development of F1 hybrid. Morpho-physiological traits characterization Different morpho-physiological traits like colour of standard petal, flower length, flower breadth, floral bud size, anther size, style length, stigma length, seed coat colour and 100 seeds weight were studied for three F1 plants along with their parents. The morpho-physiolog- ical traits unique to pollen parent were used as visual DUS marker for the characterization of F1 hybrid. 13Characterization of intraspecific hybrid in Clitoria ternatea Cyto-genetic characterization To study the chromosome homology, mitotic and meiotic analysis of F1hybrids and their parents were car- ried out following Behera et al. (2010). Well-developed roots (1-3 cm) obtained from the seedlings at 8.00-8.30 a.m. and incubated with pre-chilled p-dichlorobenzene (PDB) solution for two hours at 20ºC, followed by fixa- tion in 1:3 aceto-alcohol and kept overnight at room temperature. Subsequently, the root tips were transferred to 70% ethanol and stored at 40C. Hydrolysis of the root tips was carried out in preheated 1N HCl at 600C for 10 min followed by staining with the help of 1.5% aceto- orcein for one hour and squashed with 45% propionic acid. For meiotic analysis, the flower buds of appropriate size were fixed in 1:3 aceto-alcohol and kept overnight at 25 ± 2oC and then it was transferred to 70% ethanol and stored at 4oC. The anthers of suitable size were squashed in a drop of 1.5% acetocarmine, and the meiotic behav- iour of chromosomes at diakinesis, metaphase-I and anaphase-I were observed. Suitable stages of mitosis and meiosis were observed under a compound micro- scope (Unilab, India) and were documented using Nikon Coolpix-4500 camera. Bioactive metabolites characterization Estimation of taraxerol in root tissues: Roots of C. ternatea genotypes and their F1 hybrid were collected after 30 days of initiation of flowering, air dried under shade and ground to f ine powder. Powder of each sample (appx. 20gm) was subjected to extraction with 70% alcohol for 5h at 60ºC, filtered (using Whatmann No.1 filter paper) and dried under vacuum in a rotary evaporator (RV-10, IKA, Germany). The hydroalco- holic extract (appx. 8.4 g) was suspended in water and sequentially extracted using hexane, chloroform, ethyl acetate and n-butanol as described by Kumar et al. (2008). The hexane and chloroform fractions were sub- jected to chromatography using Chlorofom: methanol (1:1, v/v) as eluent and the eluted fractions were further chromtographed using hexane: ethyl acetate (80:20, v/v) as eluent and yielded the delphinidin as described ear- lier (Kumar et al. 2008). This compound was dissolved in ethanol (1mg.ml-1) and 10 µl aliquot of each sample was used for HPTLC assay along with standard tarax- erol solution (10-100µg.ml-1) as described by Kumar et al. (2008). Thin layer chromatography was carried out using aluminum backed HPTLC plates (100cm2; 0.2 mm thickness) of silica gel 60 F254 (Merck, Germany) in a HPTLC system (CAMAG, Switzerland) consisting of Linomat-IV sampler, twin plate development cham- ber and CAMAG TLC scanner 3 with WINCATS soft- ware. The derivatized plates were scanned under vis- ible light and the content of taraxerol was estimated densitometrically by measuring absorption at 420 nm by TLC scanner-3 integrated with WINCATS v 1.4.2 software (slit dimension- 6 mm x 0.45 mm; scanning speed- 20 mm.s-1). Estimation of delphinidin in f lowers: The f low- ers were collected, air dried and extracts of petals were prepared following Fukui et al. (2003). The petal Table 1. Fourteen accessions of C. ternatea with their flower colour, petal configuration and the geographical coordinates of collection sites located in Odisha, India. Sl. no. Accession (acc._) Collection Site Latitude Longitude Mean Sea Level(m) Flower Colour & Petal structure 1 CtW1-BG1 Bargarh 21⁰22’50”N 83⁰44’48”E 186 White unipetaloid 2 CtW2-BL1 Sriram vihar (Burla) 21⁰28’46”N 83⁰53’5”E 172 White unipetaloid 3 CtW3-BL2 Burla Town 21⁰28’46”N 83⁰53’5”E 172 White unipetaloid 4 CtW4-BGK1 Kandahata (Bargarh) 21⁰15’57”N 83⁰39’54”E 186 White unipetaloid 5 CtW5-BG2 Bargarh 21⁰22’50”N 21⁰22’50”E 186 White unipetaloid 6 CtW6-BG3 Bargarh 21⁰22’50”N 21⁰22’50”E 186 White unipetaloid 7 CtB1-KL1 kuchinda 21⁰37’34”N 83⁰19’0”E 254 Blue unipetaloid 8 CtB2-BGK2 Kandahata (Bargarh) 21⁰15’57”N 83⁰39’48”E 186 Blue unipetaloid 9 CtB3-SL1 Sambalpur 21⁰46’81”N 83⁰97’54”E 151 Blue unipetaloid 10 CtB4-PL1 Padampur (Bargarh) 21⁰0’0”N 83⁰3’46”E 205 Blue unipetaloid 11 CtB5-PL2 Padampur (Bargarh) 21⁰0’0”N 83⁰3’46”E 205 Blue unipetaloid 12 CtBB1-PL3 Padampur (Bargarh) 21⁰0’0”N 83⁰3’46”E 205 Blue bi-petaloid 13 CtBB-PL4 Padampur (Bargarh) 21⁰0’0”N 83⁰3’46”E 205 Blue bi-petaloid 14 CtBB-PL5 Padampur (Bargarh) 21⁰0’0”N 83⁰3’46”E 205 Blue bi-petaloid 14 Aparupa Naik et al. extracts were dissolved in 0.2 ml of 6 N HCl and kept at 100 ºC for 20 min. The hydrolyzed anthocyanidins were extracted with 0.2 ml of 1-pentanol. HPLC was performed using an ODS-A312 column as described by Katsumoto et al. (2007) using acetic acid : metha- nol : water (15 : 20 : 65) as solvent with flow rate 1ml per min, and the delphinidin content was estimated by measuring absorbance from 400-600 nm on photo- diode array detector (SPD-M10A; Shimadzu Co., Ltd). Under these HPLC conditions, the λmax of delphinidin and retention time were 540 nm and 4 min, respectively which were validated with those of delphinidin chloride (Sigma-Aldrich). Proteomic characterization using seed protein profiling The albumin and globulin fraction of the seeds were extracted and denatured as described by Panigrahi et al. (2007). Protein samples (appx. 25.0 μg) were separated under discontinuous sodium dodecyl sulphate poly- acrylamide gel electrophoresis (SDS-PAGE; Laemmli et al. 1970) using 10 % resolving gel (0.375 M Tris–HCl, pH 8.8) and 4% stacking gel (0.125 M Tris–HCl, pH 6.8). Tris-glycine (0.1% SDS, 25 mM Tris-glycine, pH 8.3) was used as running buffer, and electrophoresis was carried out at 1.5 mA per well constant current until tracking dye reaches the separating gel, and then cur- rent supply was increased to 2 mA per well till track- ing dye reach bottom of the gel. The molecular weight marker-PMWM (Genei Pvt. Ltd.) was used as a stand- ard, and the size of polypeptides was estimated by standard curve method. Genomic characterization using DNA markers The genomic DNA from F1 hybrid and its par- ents were isolated using the modified CTAB method (Sivaramakrishnan et al. 1997) and purified (Mishra et al. 2012). DNA was dissolved in 2.0 ml TE (Tris- EDTA) buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and stored at -20°C. Purity and concentration of the DNA sample were measured using a UV-Vis spectro- photometer (UV 1601, Shimadzu, Kyoto, Japan) by taking TE buffer as the blank. The quantification of DNA was validated by analyzing the purified DNA on 0.8 % (w/v) agarose gel by taking diluted, uncut phage lambda DNA as standard. The DNA samples were equilibrated to 10 ngμl−1 in TE buffer. For RAPD, SCoT and ISSR marker analysis, the PCR amplifica- tion of 25ng of genomic DNA was carried out using 30 random decamer oligonucleotide primers (OPA-01-20 and OPB-01-10; Operon Technologies, Alameda, CA, USA), 36 SCoT primers (SCoT -1 to SCoT -36; Collard and Mackill 2009) and seven ISSR primers from the set 100/9 (UBC-861, UBC-865, UBC-868, UBC-873, UBC-872, UBC-808, UBC-807; University of British Columbia, Vancouver, Canada), respectively. The PCR amplification reaction (25μl) contained 25 ng template DNA, 2.5μl of 10X assay buffer [100 mM Tris-Cl, pH 8.3; 0.5 M KCl; 0.1 % (w/v) gelatin], 1.5 mM MgCl2, 200 μM of each dNTP, 0.25μM primer, 1.0 units Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Banga- lore, India). For R APD, ISSR and SCoT analysis, the amplification were carried out using a thermal cycler (GENEAMP-9700; Applied Biosystems, Foster City, USA), and conditions are as below: Amplification Conditions RAPD ISSR SCoT Initial Denaturation 94 °C for 5 min PCR cycles 45 cycles 40 cycles 35 cycles Cyclic Denaturation 94 °C for 60 s 94 °C for 30 s 94 °C for 30 s Annealing of primers 37 °C for 60 s 40-60 °C for 60 s 50 °C for 60 s Elongation 72 °C for 2 min 72 °C for 2 min 72 °C for 2 min Final Elongation 72 °C for 5 min 72 °C for 5 min 72 °C for 5 min Storage of sample 4 °C for ∞ min The amplified products were mixed with gel load- ing buffer [20 % (w/v) sucrose; 0.1 M EDTA, 1.0 % (w/v) SDS; 0.25 % (w/v) bromo-phenol blue; 0.25 % (w/ v) xylene cyanol] and separated in 1.4 % (w/v) agarose gel containing 0.5μgml−1 ethidium bromide in TAE buffer (40 mM Tris acetate, pH 8.0; 2 mM EDTA) at 50V con- stantly. The separated DNA fragments were documented using gel documentation system (Gel Doc XR system, Biorad, USA), size of amplified fragments was estimated using TL-120 software (Non-linear Dynamics, Total Lab Ltd., Newcastle Upon Tyne, UK) and 250 bp step-up lad- der (Bangalore Genei Pvt. Ltd.) as standard. RESULTS In this study, the putative intra-specific F1 hybrids were raised by convetional hybridization and were char- acterized by using morpho-physiological and cytogenetic analysis, estimation of bioactive metabolites, seed pro- tein (albumin and globulin) profiling and DNA marker analysis. 15Characterization of intraspecific hybrid in Clitoria ternatea Morpho-physiological and metabolite characterization of the intraspecific F1 hybrid Morpho-physiological traits including flower colour, pod beak, and seed coat colour distinguish the blue flow- ered parental genotype C. ternatea (acc. CtB3-SL1) from white flowered one (C. ternatea acc. CtW2-BL1). The size of flower and 100 seed weight were also distinguishes both the parents. In many such morpho-physiological traits, the F1 hybrid was intermediate between the parents with predominance of the characters of C. ternatea, acc. CtW2- BL1, such as seed coat colour, petal colour being the pollen parent, and their appearance in the intermediate form was also very vivid for the identification of the F1 hybrid (Table 2; Fig. 1a, b). In most of the quantitative traits, such as leaf size, flower size, and 100 seed weight, the F1 plant resided well around the mid-parental value (Table 2). The raised F1 hybrids were assessed along with their parents for two important bioactive metabolites (Tarax- erol and Delphinidin). Taraxerol was obtained mainly from root tissues whereas delphinidin was obtained from the petals of the f lowers. The F1 hybrid con- tains 0.856±0.031 mg.g-1 taraxerol in its root tissue and 0.372±0.019 mg.g-1 delphinidin in its flowers. On com- parison with its parents taraxerol content in root tissue of F1 hybrid was almost at par with the donor parent (C. ternatea acc. CtW2-BL1) whereas delphinidin content was intermediate between both the parents (Table 2; Fig. 1c). Cyto-genetical characterization of the intraspecific F1 hybrid Appropriate stages like metaphase, anaphase, diaki- nesis, metaphase-I, anaphase-I were observed in the Table 2. Morpho-physiological and metabolite characterization of intra-specific F1 hybrid of C. ternatea. Morphological traits C. ternatea acc. CtW2-BL1 C. ternatea acc. CtB3-SL1 F1 hybrid Shape of the leaflets Lanceolate Lanceolate Ovate-lanceolate Base of the leaflets Cuneate Oblique Oblique Flower length (cm) 5.23±0.15 5.03±0.15 5.08±0.1 Flower breadth (cm) 3.2±0.1 3.04±0.06 3.07±0.06 Average days to flowering 56 days 64 days 64 days Colour of the Petal White Blue Intermediate Nature of the ovary & style Pubescent Glabrous Glabrescent Seed colour Black Brown Blakish brown Number of seeds per pod 4-5 5-6 4-5 100 Seed weight (g) 5.82 ± 0.19 5.16 ± 0.2 5.26 ± 0.18 Taraxerol Content (mg.g-1) 0.821 ± 0.026 0.377 ± 0.014 0.856 ± 0.031 Delphinidin Content (mg.g-1) 0.104 ± 0.02 0.514 ± 0.019 0.372 ± 0.019 Fig. 1. Characterization of intra-specific F1 hybrid along with its parents (C. ternatea acc. CtB3-SL1 and C. ternatea acc. CtW2-BL1). Floral attributes including petal colour (a), Mature seeds showing colour of the seed coat and aril (b); Graphical representation of bio- active metabolites (taraxerol and delphinidin) content. 16 Aparupa Naik et al. PMCs of both the parents and F1 hybrid. Chromosome analysis of both the parents revealed 16 chromosomes at metaphase in each of them, and the separation at ana- phase occurs in a normal fashion. The mitotic metaphase of F1 hybrid showed 16 distinct chromosomes similar to its parents (Fig. 2a). As expected, the PMCs of the F1 hybrid showed formation of eight bivalents (II) at diaki- nesis and metaphase-I (Fig. 2b, c) and 8II: 8II separa- tion at anaphase-I (Fig. 2d). The pollen fertility in the F1 hybrid was almost 86% and was equivalent to its parents. Proteomic and genomic characterization of the intraspecific F1 hybrid SDS-PAGE of seed albumins of two parental geno- types, including C. ternatea acc. CtB3-SL1 and acc. CtW2-BL1, and their F1 hybrids led to the detection of 33 polypeptide bands with molecular weight 12.59 to 84.14 KDa. Out of which 30 polypeptides were monomorphic, and rest three were varied for their expression (Table 3; Fig. 3). The putative F1 hybrid possessed with three poly- morphic polypeptides (Mw 31.62, 22.38 and 18.81 KDa) specific to pollen parent C. ternatea acc. CtW2-BL1 along with the monomorphic polypeptides (Table 3; Fig. 3). Since C. ternatea acc. CtW2-BL1 was used as pollen par- ent, the appearance of these unique albumin polypep- tides in the F1 hybrid can potentially be used as markers for identification of hybrids involving at least C. ternatea acc. CtW2-BL1 as pollen parent. In the present study, all three kinds of DNA mark- ers showed polymorphism at par. Thirty RAPD prim- ers have amplified 127 RAPD fragments ranging from 130 to 2389 bp, and among them, 22 amplified frag- ments (17.32%) showed parental polymorphism (Table 4). Contrasting to this amplification with nine ISSR and 36 SCoT primers generated 39 and 224 fragments rang- ing from 437 to 3154 bp and 116 to 3916 bp, respec- tively (Table 4, 5). Both ISSR and SCoT analysis showed lower polymorphism (2.56% and 5.80%) in comparison to RAPD markers. Among the parental polymorphic markers 22 RAPD, one ISSR and 13 SCoT markers were inherited to the putative F1 hybrid, and among them 11 RAPD and six SCoT markers, unique to pollen par- ent (CtW2-BL1), were very vivid in its appearance for the identification of F1 hybrid (Fig. 4). The total number of fragments amplified, percentage of polymorphism, inheritance of polymorphic fragments to the F1 hybrid were shown in Table 4 and 5. Fig. 2. Cytogenetic characterization of F1 hybrid showing 2n=2x=16 chromosome configuration at mitotic metaphase (a), association of eight bivalents at diakinesis (b) and metaphase-I (c), and segrega- tion of chromosome at anaphase-I (d). Fig. 3. Inheritance of seed albumin markers to intra-specific F1 hybrid (C. ternatea acc. CtB3-SL1 X C. ternatea acc. CtW2-BL1) intra-specific F1 hybrid; Lane ‘M’ represents Mol. Weight Marker (PMW-M, GENEI, India), and arrow indicates on right hand side indicate the polypeptides inherited to the F1 hybrid (MW in kDa). 17Characterization of intraspecific hybrid in Clitoria ternatea DISCUSSION The F1 hybrids have been considered as start- ing material for all breeding endeavours including the development of mapping population, mapping, and tag- ging of traits, marker-aided selection and generation of advanced breeding lines before the release of cultivars. Thus, identification and characterization of F1 hybrids at an early stage during hybridization programme is quite essential (Lima-Brito et al. 2006; Mishra et al. 2012). In this study, the putative intra-specific F1 hybrid was char- acterized by using morpho-physiological and cytogenetic analyses, estimation of bioactive metabolites (taraxerol and delphinidin), seed protein (albumin and globulin) profiling, and DNA marker analysis. The F1 hybrid was intermediate between the parents (C. ternatea acc. CtB3-SL1 and acc. CtW2-BL1),in term of morpho-physiological traits distinguish the parents, such as flower colour, pod beak and seed coat color, size of flower and 100 seed weight. Although no pervi- ous report is available in C. ternatea, in several species the character(s) distinguish the parents were appeared in its intermediate form in F1 hybrid (Mishra et al. 2012; Mishra et al. 2017) and in some cases the pollen parent specific traits were also predominated as observed in the present study. Either the appearance of pollen par- ent specific traits in the F1 or appearance of traits in intermediate form could be used vividly for the identi- fication of hybridity. The consistency of metabolites in the F1 hybrid of any medicinal plant in consonance to their parents is very vital in the perspective of their use as source material either to harvest therapeutics com- pounds or to generate breeding lines. C. ternatea plant parts are source of many important bioactive metabo- lites, and also have wide range of biological and pharma- cological activities (Mukherjee et al. 2008; Sethiya et al. 2009). In view of this, the raised F1 plants were assessed along with their parents for two important bioactive metabolites, Taraxerol and Delphinidin, mostly used for the treatment of various kind cancer and tumours (Braig et al. 2005; Chen and Kong 2005; Niering et al. 2005; Singletary et al. 2007; Swain et al. 2012). Taraxerol was obtained mainly from root tissues whereas delphi- nidin was obtained from the petals of the flowers. The F1 hybrid contains 0.856±0.031 mg/g Taraxerol in its root tissue and 0.372±0.019 mg/g delphinidin in its flow- ers. On comparison with its parents taraxerol content in root tissue of F1 hybrid was almost at par with the pollen parent (0.821±0.026) whereas delphinidin content was intermediate between both the parents (C. ternatea acc. Fig. 4. Inheritance of pollen parent specific fragments (→) to C. ternatea acc. CtB3-SL1 X C. ternatea acc. CtW2-BL1 intra-specific F1 hybrid, generated by RAPD primers (a), ISSR primers (b) and SCoT primers (c). (M: 250 bp step-up ladder, B: C. ternatea acc. CtW2-BL1, F: F1 hybrid and S: C. ternatea acc. CtB3-SL1). Table 3. Details of seed albumin and seed globulin markers inherited to the intraspecific F1 hybrid. Marker Total no of bands Range of Molecular Weight (KDa) No. of polymorphic polypeptides No. of polymorphic bands inherited to F1 from Parents specific bands in F1 from (kDa) CtW2-BL1 CtB3-SL1 CtW2-BL1 CtB3-SL1 Seed albumin 20 14.13-66.83 03 (15.0%) 03 -- 31.6, 22.4 & 18.8 -- Seed globulin 13 12.59-84.14 -- -- -- -- -- Total 33 13.34-112.2 03 (9.09%) 03 -- 31.6, 22.4 & 18.8 18 Aparupa Naik et al. Table 4. Details of RAPD and ISSR markers used for characterization of intraspecific F1 hybrid showing the inheritance of parent specific markers to the intraspecific F1 hybrid. Primer Sequence (5 ’ → 3’) No. of fragments amplified Range of amplified fragments (bp) Polymorphic bands Percentage of polymor-phism (%) No. of Non- parental fragments* in F1 Parent specific polymorphic bands (bp) in F1 from CtW2-BL1 CtB3-SL1 RAPD Marker OPA-01 CAGGCCCTTC 7 176-1500 --- -- OPA11500 --- --- OPA-02 TGCCGAGCTG 3 432-1233 OPA-021233 33.33 --- --- OPA-021233 OPA-03 AGTCAGCCAC 4 273-968 --- 0 --- --- --- OPA-04 AATCGGGCTG 9 170-1500 OPA-04170 OPA-04314 22.22 --- OPA-04170 OPA-04314 OPA-05 AGGGGTCTTG 6 506-1233 OPA-05 506 16.66 --- --- OPA-05 506 OPA-06 GGTCCCTGAC 8 276-2045 OPA-061516 OPA-06612 25 --- --- OPA-061516 OPA-06612 OPA-07 GAAACGGGTG 5 530-1937 --- --- --- --- --- OPA-08 GTGACGTAGG 4 130-1019 OPA-07895 25 --- OPA-07895 --- OPA-09 GGGTAACGCC 5 461-1401 --- --- --- --- --- OPA-10 GTGATCGCAG 2 277-911 --- --- --- --- --- OPA-11 CAATCGCCGT 6 139-2389 OPA-111079 16.66 --- OPA-111079 --- OPA-12 TCGGCGATAG 5 330-2333 OPA-122333 20 --- OPA-122333 --- OPA-13 CAGCACCCAC 3 758-1144 --- --- --- --- --- OPA-14 TCTGTGCTGG 2 599-717 --- --- --- --- --- OPA-15 TTCCGAACCC 9 284-2250 OPA-152250 OPA-15622 22.22 --- OPA-152250 OPA-15622 --- OPA-16 AGCCAGCGAA 9 299-2187 OPA-162167 OPA-16622 22.22 --- OPA-162167 OPA-16622 OPA-17 GACCGCTTGT 1 437 --- 0 --- --- --- OPA-18 AGGTGACCGT 2 569-974 OPA-18974 50 --- OPA-18974 --- OPA-19 CAAACGTCGG 1 1193 OPA-191193 100 --- --- OPA-191193 OPA-20 GTTGCGATCC 1 1000 --- 0 --- --- --- OPB-01 GTTTCGCTCC 3 777-1417 OPB-011417 OPB-01976 66.66 --- OPB-011417 OPB-01976 --- OPB-02 TGATCCCTGG 2 759-898 --- --- --- --- --- OPB-03 CATCCCCCTG 2 637-1689 --- --- --- --- --- OPB-04 GGACTGGAGT 2 942-2187 --- --- --- --- --- OPB-05 TGCGCCCTTC 6 539-1653 --- --- OPB-05539 --- --- OPB-06 TGCTCTGCCC 6 750-1575 --- 0 -- --- --- OPB-07 GGTGACACGG 2 741-1377 OPB-07741 50 --- OPB-07741 --- OPB-08 GTCCACACGG 8 520-1520 OPB-081386 OPB-081000 OPB-08870 OPB-08520 50 OPB-091530 OPB-091164 --- OPB-081386 OPB-081000 OPB-08870 OPB-08520 OPB-09 TGGGGGACTC 0 --- --- 0 --- --- --- OPB-10 CTGCTGGGAC 4 956-2156 --- 0 --- --- --- Total 127 130-2389 22 17.32 4 11 11 ISSR Marker UBC-861 (ACC)6 4 --- --- --- --- --- --- UBC-865 (CCG)6 6 445-1000 --- --- --- --- --- UBC-868 (GAA)6 10 539-1889 UBC-8681486 10.0 --- --- UBC-8681486 UBC-873 (GACA)4 6 505-1541 --- --- --- --- --- UBC-872 (GATA)4 2 1250-3038 --- --- --- --- --- UBC-808 (AG)8C 12 429-2036 --- -- UBC8081058 UBC808981 --- --- UBC-807 (AG)8T 3 592-924 --- --- --- --- --- 39 437-3154 2 2.56 2 0 1 19Characterization of intraspecific hybrid in Clitoria ternatea CtB3-SL1: 0.104±0.02; acc. CtW2-BL1: 0.514±0.019). This variation might be attributed to the genetic recombina- tion favouring conglomeration of suitable alleles, expres- sion of genes producing key enzymes of metabolic path- ways and growth environment which probably neces- sitated the production and accumulation of more tarax- erol as reported for different bioactive metabolites in several medicinal species (Amoo and Van Staden 2013). Mitotic analysis of F1 hybrid revealed its chromo- some count as 2n=16 similar to its parents. Sixteen dis- Table. 5. Details of SCoT markers used for characterization of intraspecific F1 hybrid showing the inheritance of parent specific markers to the intraspecific F1 hybrid. Primer Sequence (5 ’ → 3’) No. of fragments amplified Range of amplified fragments (bp) Polymorphic bands Percentage of polymor- phism (%) No. of Non- parental fragments* in F1 Parent specific polymorphic bands (bp) in F1 from CtW2-BL1 CtB3-SL1 SCoT -01 CAACAATGGCTACCACCA 5 389-1077 --- --- --- --- --- SCoT -02 CAACAATGGCTACCACCC 7 293-1218 --- --- --- --- --- SCoT-03 CAACAATGGCTACCACCG 12 341-1593 --- --- SCoT-03341 --- --- SCoT -04 CAACAATGGCTACCACCT 4 684-2437 --- --- --- --- --- SCoT -05 CAACAATGGCTACCACGA 3 500-1250 --- --- --- --- --- SCoT -06 CAACAATGGCTACCACGC 8 151-1706 --- --- --- --- --- SCoT -07 CAACAATGGCTACCACGG 6 967-2096 --- --- SCoT-07539 --- --- SCoT-09 CAACAATGGCTACCAGCA 5 394-2260 --- --- --- --- --- SCoT-10 CAACAATGGCTACCAGCC 5 1666-3916 --- --- --- --- --- SCoT-11 AAGCAATGGCTACCACCA 4 509-1255 --- --- --- --- --- SCoT-12 ACGACATGGCGACCAACG 3 366-676 --- --- --- --- --- SCoT-13 ACGACATGGCGACCATCG 2 310-607 --- --- --- --- --- SCoT-14 ACGACATGGCGACCACGC 6 313-1351 --- --- --- --- --- SCoT-15 ACGACATGGCGACCGCGA 2 257-358 --- --- --- --- --- SCoT-16 ACCATGGCTACCACCGAC 5 550-1658 --- --- --- --- --- SCoT-17 ACCATGGCTACCACCGAG 3 590-1546 --- --- --- --- --- SCoT-18 ACCATGGCTACCACCGCC 8 316-1805 --- --- --- --- --- SCoT-19 ACCATGGCTACCACCGGC 14 341-2231 SCoT-19583 7.14 SCoT-19548 SCoT-19480 --- 583 SCoT-20 ACCATGGCTACCACCGCG 5 500-2210 --- --- --- --- --- SCoT-21 ACGACATGGCGACCCACA 5 231-1023 --- --- --- --- --- SCoT-22 AACCATGGCTACCACCAC 7 269-1132 --- --- --- --- --- SCoT-23 CACCATGGCTACCACCAG 4 275-977 --- --- --- --- --- SCoT-24 CACCATGGCTACCACCAT 6 480-1669 SCoT-24562 16.66 --- --- SCoT-24562 SCoT-25 ACCATGGCTACCACCGGG 8 369-2654 SCoT-251308 12.5 --- SCoT-251308 --- SCoT-26 ACCATGGCTACCACCGTC 7 294-963 SCoT-26566 14.28 --- --- SCoT-26566 SCoT-27 ACCATGGCTACCACCGTG 8 527-2386 SCoT-27647 12.5 --- SCoT-27647 --- SCoT-28 CCATGGCTACCACCGCCA 10 474-1552 SCoT-281431 SCoT-28981 20.0 --- --- SCoT-281431 SCoT-28981 SCoT-29 CCATGGCTACCACCGGCC 14 335-2523 --- 0 --- --- --- SCoT-30 CCATGGCTACCACCGGCG 8 390-2954 --- 12.5 SCoT-30438 --- --- SCoT-31 CCATGGCTACCACCGCCT 11 326-2477 SCoT-31774 9.09 SCoT-31326 SCoT-31774 --- SCoT-32 CCATGGCTACCACCGCAG 9 116-2000 SCoT-321030 SCoT-32830 22.22 --- SCoT-321030 SCoT-32830 --- SCoT-33 CCATGGCTACCACCGCAG 5 210-1000 --- 0 --- --- --- SCoT-34 ACCATGGCTACCACCGCA 4 339-1176 --- 0 --- --- --- SCoT-35 GCAACAATGGCTACCACC 6 210-2555 SCoT-35 1430 SCoT-35210 33.33 --- SCoT-351430 SCoT-35210 --- SCoT-36 GCAACAATGGCTACCACC 5 449-899 SCoT-36899 20.0 --- --- SCoT-36899 Total 224 13 5.80 6 7 6 20 Aparupa Naik et al. tinguished chromosomes were also observed in the mitotic metaphase and they were separated in normal fashion during anaphase. Meiotic analysis revealed for- mation of eight bivalents at diakinesis and metaphase-I and 8II: 8II separation at anaphase-I, which might be due to homology between the parents. As result the pol- len fertility of F1 hybrid is almost equivalent to its par- ents. These cytological observations along with morpho- physiological traits could be helpful for the characteriza- tion of the F1 hybrids of C. ternatea as reported in many species. The homologous multigene families control the expression seed protein profile across the species, thus the seed protein marker exhibits monogenic segrega- tion where the presence of polypeptide being com- pletely dominant over absence, and in some cases, co- dominance for molecular weight variants also noticed (Osborn 1988). Mutations or deletions of structural genes coding for these polypeptides or their regulatory loci might lead to lack of expression of the concerned polypeptides (Panigrahi et al. 2007). This kind of vari- ations in seed protein marker profiling led the use this as as reliable markers for verification of hybridity of inter-varietal crosses (Bennet et al. 1991), and inter-spe- cific (Panigrahi et al. 2001, Jisha et al. 2011, Mishra et al. 2012). In the present study SDS-PAGE of seed albumins revealed inheritance of three polymorphic polypeptides (Mw 31.62, 22.38 and 18.81 KDa) specific to pollen par- ent C. ternatea acc. CtW2-BL1 in the F1 hybrid. Since C. ternatea acc. CtW2-BL1 was used as pollen parent, the appearance of these unique albumin polypeptides in the F1 hybrid can potentially be used as markers for iden- tification of hybrids involving at least C. ternatea acc. CtW2-BL1 as pollen parent as reported in Cajanus cajan (Panigrahi et al. 2007) RAPD, SCoT and ISSR marker analysis relies on dif- ferential enzymatic amplification of targeted DNA frag- ments on the basis of primer annealing sequence of the genome. RAPD is being random in nature, this kind of DNA markers were ubiquitously distributed through- out the genome, and capable of detecting a high level of polymorphism. Whereas ISSR is simple sequence repeat specific and SCoT is the specific to the conserved sequence around the initiating codon of the gene. These markers have also been successfully utilized in several crop species for diverse breeding efforts including iden- tification and characterization of the hybrids. In the present study, RAPD, ISSR and SCoT markers showed 17.32, 2.56 and 5.80% polymorphism among the par- ents. Both ISSR and SCoT analysis showed lower poly- morphism (2.56% and 5.80%) in comparison to RAPD markers in the present study. There are some contradic- tory reports on detection of polymorphism by RAPD and ISSR markers, ISSR markers showed more polymor- phism than RAPD markers (Godwin et al. 1997; Lima- Brito et al. 2006; Nagaoka et al. 1997; Zietkiewicz et al. 1994) and vice versa (Muthusamy et al. 2008). This con- tradiction might be due to the use of different decamer oligonucleotides or SSR motifs as primers, and varied primer-annealing site in the genomes. Again, ISSR and SCoT polymorphism depends on the frequency of SSR motifs and conserved sequence around the initiating codon, respectively (Depeiges et al.1995; Collard and Mackill 2009) which vary within a species or even vari- eties targeted. Identification of inter and intra-specific hybrids has been carried out in several species using either RAPD or ISSR markers individually, or in combi- nation (Goldmann et al. 2008; Jisha et al.2011; Bianco et al. 2011, Mishra et al. 2012). In this study 22 RAPD, one ISSR and 13 SCoT markers were found to be inherited to the putative F1 hybrid, and among them 11 RAPD and six SCoT markers, unique to pollen parent (C. ternatea, acc. CtW2-BL1), were very vivid in its appearance for the identification of F1 hybrid. In the present study, several non-parental fragments have also been amplified in the F1 hybrid, and this might be due to either DNA recom- bination followed by minor genomic reorganization dur- ing the hybridization (Huchett et al. 1995), or loss of priming sites due to chromosomal crossing over during meiosis (Smith et al.1996). As the objectives is to identify the hybrids and to confirm the hybrid nature of putative seedlings at the juvenile stage, screening of the putative F1 hybrids using pollen parent-specific RAPD, ISSR and SCoT markers contribute economic significance to this medicinal plant. The findings from the present study, it has been assorted that use of seed protein profiling and DNA marker analysis complements the characterization of intra-specific F1 hybrid along with morpho-physiolog- ical traits and cytogenetic analyses more precisely. Fur- ther, these inherited seed albumin and DNA markers could also be used for further studies in gene mapping, marker-assisted breeding involving intra-specific hybrid- ization in C. ternatea aiming at enhanced metabolite content of therapeutic importance. ACKNOWLEDGEMENTS The authors are highly thankful to DST, Govt. of Odisha for providing Biju Patnaik Research Fellowship to the author (AN) for pursuing the doctoral degree, and acknowledge the laboratory facility provided by the Vice Chancellor, Sambalpur University. 21Characterization of intraspecific hybrid in Clitoria ternatea FUNDING Research work funded by Department of Science and Technology, Govt. of Odisha and UGC, Govt. of India. REFERENCES Ali Z, Ganie S, Narulaa A, Sharma M, Srivastava P. 2013. Intra-specific genetic diversity and chemical profiling of different accessions of Clitoria ternatea L. Indus- trial Crops Prod. 43: 768-773 Amoo SO, Van Staden J. 2013. 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