International Journal of Biodiversity and Conservation Vol. 3(3), pp. 92-100, March 2011 Available online http://www.academicjournals.org/ijbc ISSN 2141-243X ©2011 Academic Journals Full Length Research Paper Generic relationship among Cassia L., Senna Mill. and Chamaecrista Moench using RAPD markers Vivek Tripathi1* and Sandhya Goswami2 1Delhi Technological University,Shahbad, Daulatpur Bawana Road,Delhi-110042, India. 2Betel Vine Biotechnology Laboratory,National Botanical Research Institute,Lucknow-226 001, India. Accepted 8 February, 2011 Generic relationships were examined among twenty-four species belonging to genus Cassia L., Senna Mill. and Chamaecrista Moench using RAPD marker. Total 80 primers were initially screened, 514 amplification products obtained with 38 informative primers, of which 514 were polymorphic. A vary high degree of polymorphism (100%) was observed among them. UPGMA cluster analysis of genetic similarity indices grouped all the species into three major clusters. Cluster I included four species of Cassia L., Cluster II included eighteen species of Senna Mill. and Cluster III included two species of C. Moench. Highest similarity (0.9%) was observed between Cassia fistula L. and Cassia fistula with nodded filaments and least (0.001%) between Cassia fistula L. and Senna splendida. The Polymorphic information contents (PIC) of the twenty-four species with RAPD marker varied from 0.08 to 0.49 with an average of 0.005. The result confirms the statement of Irwin and Barneby, they divided the genus Cassia L. into three subgenera; Cassia L., Senna Mill. and C. Moench on the basis of morphological characters. The results obtained from the present study support the previous taxonomic classification of the genus Cassia L. and showed large diversity among the species of three newly created genera. Our results suggested that RAPD marker is a sensitive, precise and efficient tool for genomic analysis of Cassia L. that may be useful in future studies by assigning new unclassified germplasm to specific taxonomic groups and reclassify previously classified species and genera. Key words: Cassia, Senna, Chamaecrista, genetic relationship, RAPD. INTRODUCTION Cassia L. is the largest genus in the subfamily and raised the subgenera Senna Mill. and C. Moench to Caesalpiniodeae of the Caesalpinioceae. It contains genetic level. about 600 flowering species which are distributed in most The plants of Cassia L. are used as fodder, purgatives, continents (Singh, 2001). Cassia L. species show large timber and medicine (Tomlinson, 1981; Tiwari, 1983). diversity related to habit, ranging from delicate, annual The taxonomy and nomenclature of Cassia L. species herbs to tall trees. Bentham (1871) divided the genus are quite complex and intriguing. They are not easily Cassia into three genera and nine sections. Britton and differentiated from closely related species due to the Rose (1930) splited the genus Cassia into twenty-eight large variation in similarities range causing mis- genera. Recently Irwin and Barneby (1982) splited the identification and misinterpretation of the components. genus Cassia L. into three subgenera; Cassia L., Senna The cultivars identification and assessing of diversity Mill. and Chamaecrista Moench. Irwin and Baneby (1981 using phenotypic markers have several limitations to 1882) realizing diversity and complexity and proposed especially in perennial crops (reference). In plants as an improved classification proposing new delimitation general, due to the overlapping of morphological based on persistent suit of characters. They have raised characters, a great amount of confusion persists for the the genus Cassia L. to the levels of subtribe (Cassiinae) selection of ideal plant. Generally morphological characters are influenced by environmental changes, and the changes are not constant in the species to species and differ from place to place. *Corresponding author. E-mail: [email protected]. Tel: Structural changes in DNA, that is, translocation, +91 11-27871018. Fax: +91 1127871023. deletion, inversion mutation have been able to Tripathi and Goswami 93 Table 1. Collected Cassia, Senna and Chamaecrista species, accessions no. and their location. S/N Accessions no. Accessions code. Accessions name Location 1 200382 Cf1 C. fistula Utterkashi (Uttranchal) 2 200390 Cf2 C. fistula (white) NBRI, Lucknow India 3 200435 Cj C. javanica NBRI, Lucknow India 4 200436 Cr C. roxburghii BSI Jodhpur (Rajasthan) India 5 200437 St S. tora Kanpur (U.P) India 6 200438 So S. obtusifolia Unnao (U.P) India 7 200439 Su S. uniflora Karnataka university Dharwad India 8 200440 Sb S. biflora Lucknow (U.P) India 9 200441 Ss S. sophera Karnataka university Dharwad India 10 200442 Sp S. purpurea Metha (U.P) India 11 200443 Ss1 S. spetabilis Karnataka collage Dharwad India 12 200444 Se S. excelsa NBRI, Lucknow India 13 200415 Ss2 S. sulfurea Tehari garhwal (Uttranchal) India 14 200432 Ss3 S. surattensis Aligarh (U.P) India 15 200447 Ss4 S. splendida Madgavo Karnataka India 16 200448 So1 S. occidantalis Bhopal (M.P) India 17 200449 Sa S. auriculata BSI Jodhpur (Rajasthan) India 18 200450 Ss5 S. siamea Faizabad (U.P) India 19 200451 Si S. italica BSI Jodhpur (Rajasthan) India 20 200452 Sa1 S. alexandrina CIMAP Lucknow 21 200453 Sh S. hirsuta Karnataka collage Dharwad India 22 200454 Sa2 S. alata Lucknow (U.P) 23 200455 Chm C. mimosoides K.U Dharwad India 24 200456 Cha C. absus K.U Dharwad India change the genomic constitution of species. In decades, Chamaecrista were collected from different ecological locations of these changes in the genome of species, separate the Uttrakhand, Uttar Pradesh, Madhya Pradesh, Karnataka, Rajasthan particular species from their ancestors. To assess these and Himachal Pradesh, India (Table 1). Out of which some species are cultivated due to the medicinal and ornamental properties and changes, parameters based on morphological characters some are wild. are not sufficient. Now it is possible to single out differences on the basis of molecular markers, which is authentic and less affected by environmental factors. Preparation of genomic DNA Hence characterization of species at the genetic level Total genomic DNA from the leaves of Cassia, Senna and supplemented efficient conservation, maintenance and Chamaecrista species was extracted using the protocol proposed utilization of the existing genetic diversity. by Doyle and Doyle (1987) with slight modification. 1 g of leaves In the present study, RAPD markers were used to around to powder with liquid nitrogen in a morter and pestle, then assess genetic relationship among the species of Cassia, transferred to a 30 ml centrifuge tube containing 12 ml of CTAB Senna and Chamaecrista by using a single arbitrary buffer (2% CTAB, 100 mM Tris-Cl, 20 mM EDTA and 1.4 M NaCl) primer (10-mer) and amplifying DNA by polymerase chain and 120 μl β-mercaptoethanol mixed thoroughly and incubated in water bath at 68°C for 3 h. The tube was cool at room temperature, reaction (PCR) as the resulting DNA marker can be mix with equal volume of chloroform and tube was gently inverted easily separated on an agarose gel by electrophoresis repeatedly. The tube was centrifuged at 10,000 rpm for 10 min. The (Williams et al., 1990). The advantage of RAPD is its supernatant was transferred to a corex tube and 0.7% volume of 2- simplicity, rapidity and requirement for only a small propanol was added. Tube was put at -20°C for 2 h. The tube was quantity of DNA, and the ability to generate numerous centrifuged at 12000 rpm 10 min at 4°C to collect precipitated DNA. polymorphism (Stebbins, 1957; Cheng et al., 1997; The pellet was resuspended with 700 μl TE (10 mM, Tris-Cl pH-8.0, 1 mM EDTA) and incubated with 2 μl (10 mM DNase free RNase A) Khanuja et al., 1998). for 40 min at 37°C. The RNase A and the remaining protein were extracted with equal volume of phenol: chloroform 1:1 and centrifuged at 12000 rpm for 15 min at room temperature. The MATERIALS AND METHODS supernatant was transferred to a new tube and the DNA was precipitated by the addition of 0.7 volume of 2-propanol. Plant materials Precipitated DNA was collected by centrifugation at 12000 rpm for 15 min at 4°C washed with 70% ethanol twice and dried before Twenty-four species belongs to three genera; Cassia, Senna and redissolving in 100 μl of TE. DNA yield were calculated by 94 Int. J. Biodvers. Conserv. fluorometer (DyNa Quant 200) (Amersham pharmacia Biotech, Figure 1. A total 514 bands were scored from PCR USA). amplification of genomic DNA with all the species. The RAPD markers showed 100% polymorphism (Table 2). RAPD reactions Homology of the RAPD bands with the same molecular weight was confirmed using EcoRI and Hind III digested Sixty-eight decamer Oligonucleotide primers (Operon, USA) were marker. Average number of 14 bands was obtained per screened by polymerase chain reaction (PCR). PCR reactions were primer and amplification product ranged in size from 100 performed by using a 20 μl mixture, conataing 50 ng template bp to 4.2 kb. Maximum numbers of 21 amplification (genomic DNA), dNTPs (0.2 mM each), 0.5 mM primer, 1.5 U Taq products were obtained with primer OPC-19 followed by DNA polymerase, 25 mM MgCl2 and remain Mili Q water. The DNA thermocycler (Applied Biosystem, 9700) was programmed as 20 products with primer OPAP-1 and OPAP-20. Minimum follow: Incubation at 94°C for 2 min, 44 cycles at 94°C for 1 min. numbers of RAPD products were generated with primers 36°C for 1.30 min and 72°C for 1.30 min followed by OPAP-1, OPAP-17 and OPAP-16. The polymorphic electrophoresis in 1.4 % (w/v) agarose gel with 0.5 × TBE stained information contents (PIC) ranged from 0.08 to 0.49 with with diluted ethidium bromide (10 mg/ml) and photographed in gel an average of 0.005.