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Jessop and Its Relationship to Drimia Nagarjunae Using Phenotypic Traits and Molecular Markers

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Jessop and Its Relationship to Drimia Nagarjunae Using Phenotypic Traits and Molecular Markers Indian Journal of Experimental Biology Vol. 53, June 2015, pp. 412-416 Diversity of Drimia indica (Roxb.) Jessop and its relationship to Drimia nagarjunae using phenotypic traits and molecular markers Narendranath Alluri1, Shivakameshwari MN2, Manohar SH1 & Mala Majumdar1* 1Department of Biotechnology, Center for Post Graduate Studies, Jain University, Bangalore-560 011, India 2Department of Botany, Bangalore University, Bangalore-560 056, India Received 14 November 2014; revised 08 December 2014 In the present study, we explored the diversity of different accessions of Drimia indica and its relation to D. nagarjunae using phenotypic traits and molecular markers. Twenty populations of D. indica, from different parts of India, were compared with D. nagarjunae, an endangered medicinal plant collected from Andhra Pradesh, India. Two species showed appreciable phenotypic diversity in number of leaves, leaf indices, bulb circumference, bulb length and length of roots. The principal component analysis (PCA) performed on above 5 quantitative characters to determine relationship among populations, has distinguished D. nagarjunae from D. indica phenotypically. Genetic diversity was analysed using RAPD and ISSR primers which produced reproducible bands in 8 RAPD and 3 ISSR primers. A total of 89 amplicons were observed, of which 69 (77.53 %) were polymorphic. Cluster diagram and phylogenetic linkage showed that D. nagarjunae formed a separate cluster, showing no similarity with any of the populations of D. indica. The molecular marker data correlated with PCA of phenotypic traits. Current investigations have demonstrated that the statistical approach for phenotypic characters and molecular markers analysis can be applied to study diversity in Drimia species. Keywords: Indian squill, Population relationship, Species diversity, Squill, White squill The genus Drimia (Liliaceae), which includes about 100 (2n = 24, 32, 34, 36, 38, 46), whereas D. nagarjunae is species, is distributed widely around equatorial region diploid (2n = 20)3,9. Desai et al.10 also demonstrated the including India1. The genus is commonly known as polyploid nature of five Drimia species. Such species Squills and is used as cardiotonic2. With regard to delimitation of the genus Drimia indicates the need for taxonomical position of Drimia species in India, analysis of its genetic diversity using molecular markers. Hemadri and Swahari3 accounts for 9 in the sub- However, most of the studies in Drimia are limited to continent, whereas Deb and Dasgupta4 recognized only the phenotypic diversity10. Here, we investigated the 5 species. All Urginea species in India are included diversity of D. indica and its relation to D. nagarjunae under the genus Drimia5,6. using phenotypic and molecular markers. Drimia indica (Roxb.) Jessop serves as antiulcerous, antinematodal, antitumorous, anthelmintic, antiarthritic Materials and Methods as well as an antidote to scorpion sting6. It is known to Collection of Plant materials—About 20 accessions possess phenolic compounds such as coumarins, of D. indica were collected from different locations of flavonoids, salicylic acid, tannins and alkaloids, India, and D. nagarjunae from Andhra Pradesh, India steroids and glycosides7. D. indica exhibits variable (Table 1). The collected plants were grown in uniform morphological diversities and these variations have led field conditions in the Botanical garden, Department of to identifying new members of this species6. Botany, Bangalore University, India. Various An endangered species, Drimia nagarjunae Hemadri phenotypic traits viz., number of leaves, leaf length, et Swahari, was wrongly identified as D. indica leaf width, bulb circumference, bulb length and length initially3,8. D. nagarjunae has large bulb and broad of root of the accessions were measured quantitatively leaves whereas D. indica bear small bulbs and narrow during their vegetative phase in the month of leaves3. Also, D. indica is polyploidy in nature September, 2013. DNA isolation and PCR amplification—DNA was extracted from fresh or frozen bulbs, following the *Correspondence: 11 Phone: 080 43226500; Fax: 080 43226507 CTAB method with minor modifications . The DNA E-mail: [email protected] quality as well as quantity was checked by UV-VIS ALLURI et al.: ANALYSIS OF DRIMIA INDICA DIVERSITY AND ITS RELATIONSHIP TO DRIMIA NAGARJUNAE 413 Spectrophotometer. After quantification, the DNA presence and ‘0’ for absence in DNA samples was diluted with TE buffer to a working concentration amplified to create binary data matrix. of 25 ng mL-1 for PCR analysis. Statistical analysis—All the original data were RAPD and ISSR amplification was performed standardized to eliminate the difference in the according to the standard protocols with some variance of each character and PCA was performed modifications12-14. Initially, 40 RAPD and 20 ISSR using CAMO unscrambler software 10. Eigen values primers were screened; the primers (8 RAPD and 3 and contribution percentage of each PC axis were ISSR) that gave best results were considered for calculated using the correlation matrix among thirty further analysis. About 25 µl of reaction volume characters for 21 accessions15. The binary data was contained 2.5 µl assay buffer (100 mM Tris-HCl, pH analyzed using UPGMA module of Statistica to 9.0, 500 mM KCl, 15 mM MgCl2 and 0.1% gelatin), obtain a dendrogram and the similarity matrix. 2.5 µl of 200 mM of each dNTPs (dATP, dTTP, dCTP and dGTP), 2.5 µl of 15 ng primer, 0.4 µl of 0.5 Results unit of Taq DNA polymerase and 1 µl 25 ng of DNA Phenotypic diversity analysis—Apparent phenotypic template. The amplification reaction was carried out variability was observed in the number of leaves, leaf using a thermal cycler. The samples were subjected to length, leaf width, bulb length, bulb circumference and initial denaturation for 4 min at 94 ºC, followed by 40 root length among D. indica accessions and between cycles of 1 min at 94 ºC, 1 min at 35 ºC for RAPD D. indica and D. nagarjunae also (Table 1). and 53-56.5 ºC for ISSR, and extension for 1 min at D. nagarjunae possessed broader, more number of 72 ºC with final extension 5 min at 72 ºC. After leaves and large scaly bulbs whereas D. indica has completion of the PCR, the amplicons were separated narrow, less number of leaves and small bulbs by 1.2% agarose gel electrophoresis and visualized indicating clear distinction between the two species. under UV illuminator. The bands were scores ‘1’ for The scattering of 21 Drimia collection and component Table 1—List of plant species (S1-S20= Drimia indica; S21=Drimia nagarjunae) collection region, voucher information and morphological features Accession Voucher Number Leaf Indeces Bulb Bulb Length Region no. information of leaves (cm) (L x B) circumsphere (cm) length (cm) of root (cm) S1 Andhra Pradesh S&M 850 8 14×1 21.5 5.8 7.2 S2 Andhra Pradesh S&M 836 7 20×1 12.8 3.8 8.6 S3 Kerala S&M 818 5 29×1 15 4.5 9.4 S4 Tamilnadu S&M 844 9 23.6×1 15 5.5 12.1 S5 Tamilnadu S&M 822 7 23×2.4 10.1 6 4.6 S6 Tamilnadu S&M 819 9 9.1×1.2 7.4 5.1 5.7 S7 Jammu and Kashmir S&M 852 12 26×1 2.4 6.2 8.1 S8 Karnataka S&M 839 6 32×1.3 20.5 7.1 9.9 S9 Karnataka S&M 825 5 13×1.9 11.1 4.5 6.5 S10 Karnataka S&M 851 15 19×1.5 13.5 5.3 8 S11 Karnataka S&M 803 8 26×1 5.9 3.4 5.5 S12 Karnataka S&M 832 5 9×1.5 8.1 5.9 16.5 S13 Karnataka S&M 834 6 18×1.8 6.2 3.1 5.1 S14 Karnataka S&M 835 7 30×2.5 7.5 4 2.6 S15 Karnataka S&M 849 8 22.3×1 16.2 4.5 8.8 S16 Karnataka S&M 814 5 35×0.9 8.3 5.1 9.1 S17 Karnataka S&M 807 6 46×1.3 12.3 4.1 15.5 S18 Karnataka S&M 815 4 13×1 10.3 5.5 9.7 S19 Kerala S&M 842 8 19×2 8.4 3.1 1.8 S20 Karnataka S&M 809 7 14×2 11.5 3.6 10.1 S21 Andhra Pradesh SMPU/NADRI 14 47.3×4.9 45.5 9.9 20.5 /BNG/2013-14/83 414 INDIAN J EXP BIOL, JUNE 2015 scores of the first and the second components were ranged from 250 to 1,500 bp. Fig. 2 represents the depicted (Fig. 1). According to Table 2, 54%, 15%, banding profiles of RAPD OPE-2 primer. 11%, 9%, 5% and 4% are the percentage of variations explained by the first six components. Characters with higher coefficients on the PC axes should be considered more important and PC 1 had the highest coefficient (Eigen value) for all the traits15,16. D. nagarjunae which had a robust growth was segregated from the main cluster of D. indica (Fig. 1). The major cluster was that of D. indica which was scattered, as they showed large diversity among the collection. Genetic diversity analysis—Initially, 40 RAPD and 20 ISSR primers were screened and among them 11 primers were short listed (8 RAPD and 3 ISSR) as they gave reproducible and scorable bands (Table 3, Fig. 2). A total of 89 bands were formed, among them 69 (77.53%) were polymorphic. The average number of Fig. 1—Cluster diagram constructed on the basis the first two bands was 8.09 and average number of polymorphic principal component axes, which contain 70% of the total bands was 6.27. The size of amplified fragments variation Table 2—Proportions of variations by principal component analysis (PCA) and associated with first six PC axes of characters associated with 21 populations PCA-1 PCA-2 PCA-3 PCA-4 PCA-5 PCA-6 Explained proportion of variation (%) 54.54 15.50 11.67 9.65 4.83 3.81 Cumulative proportion of variation (%) 54.54 70.04 81.71 91.36 96.19 100.00 Eigen vector L 0.548 0.710 -0.038 -0.422 -0.126 0.019 LL 0.625 -0.311 0.649 -0.288 0.088 -0.032 LW 0.731 0.285 0.257 0.520 -0.152 -0.157 BD 0.897 -0.043 -0.075 0.167 0.074 0.394 BL 0.848 -0.006 -0.336 -0.022 0.355 -0.205 RL 0.725 -0.495 -0.304 -0.139 -0.334 -0.075 [L= Number of leaves; LL= Leaf length; LW= Leaf width; BD= Bulb diameter; BL= Bulb length; RL= Root length] Table 3—RAPD and ISSR primers used to detect polymorphism for analysis genetic diversity Scored Polymorphic Polymorphism rate Primers Sequence bands bands (%) OPD 1 5'-ACCGCGAAGG -3' 9 8 88.89 OPD 2 5'-GGACCCAACC-3' 8 6 75.00 OPD 3 5'-GTCGCCGTCA-3' 8 6 75.00 OPD 19 5'-CTGGGGACTT-3' 10 6 60.00 OPD 20 5'-ACCCGGTCAC-3' 9 7 77.78 OPE 2 5'-GGTGCGGGAA-3' 7 6 85.71 OPE 14 5'-TGCGGCTGAG-3' 7 5 71.43 OPE 16 5'-TGCGGCTGAG-3' 6 6 100.00 UBC 853 5'-TCTCTCTCTCTCTCTCAGT-3' 8 6 75.00 UBC 861 5'-ACCACCACCACCACCACC-3' 9 8 88.89 UBC 862 5'-AGCAGCAGCAGCAGCAGC-3' 8 5 62.50 Total no.
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