Inter-simple sequence repeat (ISSR) analysis was performed in seven accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the applicability of this analysis for assessing the intraspecific variation. The value of similarity indices ranged from 0.375 to 0.125. The similarity result indicates the presence of high level of genetic diversity among the accessions of Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear grouping among the populations. The primers showed abilities in detecting genetic diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus, ISSR-PCR technology can be used to study genetic variation and genetic relationships in the genus Solanum xanthocarpum Schard. and Wendl.
Article Citation:
Ajoy Kumar Das, Sailendra Prasad Borah.
Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker.
Journal of Research in Plant Sciences (2012) 1(2): 146-152.
Full Text:
http://plantsciences.co.in/documents/PS0035.pdf
Título original
Intraspecific Variation in Solanum Xanthocarpum Schard. and Wendl. revealed by ISSR marker
Inter-simple sequence repeat (ISSR) analysis was performed in seven accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the applicability of this analysis for assessing the intraspecific variation. The value of similarity indices ranged from 0.375 to 0.125. The similarity result indicates the presence of high level of genetic diversity among the accessions of Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear grouping among the populations. The primers showed abilities in detecting genetic diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus, ISSR-PCR technology can be used to study genetic variation and genetic relationships in the genus Solanum xanthocarpum Schard. and Wendl.
Article Citation:
Ajoy Kumar Das, Sailendra Prasad Borah.
Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker.
Journal of Research in Plant Sciences (2012) 1(2): 146-152.
Full Text:
http://plantsciences.co.in/documents/PS0035.pdf
Inter-simple sequence repeat (ISSR) analysis was performed in seven accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the applicability of this analysis for assessing the intraspecific variation. The value of similarity indices ranged from 0.375 to 0.125. The similarity result indicates the presence of high level of genetic diversity among the accessions of Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear grouping among the populations. The primers showed abilities in detecting genetic diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus, ISSR-PCR technology can be used to study genetic variation and genetic relationships in the genus Solanum xanthocarpum Schard. and Wendl.
Article Citation:
Ajoy Kumar Das, Sailendra Prasad Borah.
Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker.
Journal of Research in Plant Sciences (2012) 1(2): 146-152.
Full Text:
http://plantsciences.co.in/documents/PS0035.pdf
Inter-simple sequence repeat (ISSR) analysis was performed in seven accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the applicability of this analysis for assessing the intraspecific variation. The value of similarity indices ranged from 0.375 to 0.125. The similarity result indicates the presence of high level of genetic diversity among the accessions of Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear grouping among the populations. The primers showed abilities in detecting genetic diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus, ISSR-PCR technology can be used to study genetic variation and genetic relationships in the genus Solanum xanthocarpum Schard. and Wendl. 146-152 | JRPS | 2012 | Vol 1 | No 2
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www.plantsciences.info Journal of Research in Plant Sciences An International Scientific Research Journal Authors: Ajoy Kumar Das 1 , Sailendra Prasad Borah 2 .
Institution: 1. Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India, 781 039.
2. Department of Botany, Gauhati University, Guwahati, Assam, India, 781 014.
Corresponding author: Ajoy Kumar Das.
Email: ajoykdas@iitg.ernet.in
Phone No: +91 99579 96983.
Web Address: http://www.plantsciences.info documents/PS0035.pdf.
Dates: Received: 15 Aug 2012 Accepted: 15 Sep 2012 Published: 13 Dec 2012 Article Citation: Ajoy Kumar Das, Sailendra Prasad Borah. Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker. Journal of Research in Plant Sciences (2012) 1(2): 146-152 Original Research Journal of Research in Plant Sciences J o u r n a l
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An International Scientific Research Journal INTRODUCTION The genus Solanum xanthocarpum Schard. and Wendl. is an important medicinal perennial herb belonging to the family Solanaceae. This prickly herb is a good source of important alkaloids such as Solasodine, solasonine, solamargine and diosgenin. Solasodine extracted from this plant has antispermatogenic activity. Solamargine found in this herb has anticancer properties. Diosgenin extracted from Solanum xanthocarpum Schard. and Wendl. is found to be effective in suppressing FAS expression in HER 2 over expressing breast cancer cells (Singh and Singh, 2010). Glucoalkaloid solanocarpine, sterol carpesterol, solanocarpidine and traces of isochlorogenic, neochronogenic, chronogenic and caffeic acids are also found in the fruits of this plant. In India, this plant is used for curing various ailments since ancient times. Powder made from this herb is very useful in asthma, breathlessness and allergies of respiratory tracts. It is also used as carminative, digestive, alternate and astringent. As the herb is a stimulant to the heart and is a blood purifier, it is extremely beneficial in the treatment of cardiac diseases associated with edema. The fruit is useful as an aphrodisiac in males and the seeds, in women for irregular menstruation and dysmenorrheal. It also promotes conception in females. The roots of this plant are largely used in catarrhal and febrile affection, cough, chest pain, flatulence, sore throat, toothache and constipation and in bronchial diseases. Leaves of this plant are also used in rheumatic pain. Solanum xanthocarpum Schard. and Wendl. is non toxic and safe for human use and is regarded as a valuable plant in both ayurvedic and modern drug development areas for its versatile medicinal uses. The taxonomic description of the genus Solanum, the largest and hyper diverse genus among the members of the family Solanaceae accumulated so far is mainly base on morphometrical characters and has left many issues unresolved. The nomenclature of S. xanthocarpum had remained controversial in the past. Phenotypically this species is highly polymorphic. Morphological character such as presence of hairs on the stem and petiole, point of origin of spines, arrangement of spines, length of filaments, flower colour etc. of this plant shows polymorphism. Even, many taxonomists confused this species with other Solanum species. The status of this critical taxa can be justified by using molecular tools. Again, the existence of genetic variability among the species is very important for any plant improvement program. The analysis of genotypes derived from different geographical areas is important to study genetic diversity. Though morphological characters are used for distinguishing the plants but it is often restricted as Das and Borah, 2012 147 Journal of Research in Plant Sciences (2012) 1(2): 146-152 Table 1 Plant materials used in the study Name of the species Acc. No. Locality Geographical location(Altitude/latitude) Solanum xanthocarpum Schard. And Wendl. 1 = Sx01 Jalukbari, Assam 268'38.8914''N and 9039'41.3809''E 2 = Sx02 Goalpara, Assam 2610'0''N and 9037'0''E 3 = Sx03 Golaghat, Assam 2630'36''N and 9385'0''E 4 = Sx04 Rani, Assam 263'43.84872''N and 9136'3.90852''E 5 = Sx05 Khetri, Assam 2610'00''N and 9204'00''E 6 = Sx06 Shillong, Meghalaya 2534'0''N and 9153'0''E 7 = Sx07 Karbi Anglong, Assam 2533' N and 9210' E Figure 1 Ethidium bromide stained agarose gel showing genomic DNA of seven accessions of Solanum xanthocarpum Schard. and Wendl. M = Marker. theses character may not be evident at all stages of development. Now days, variety of genetic markers are used to assess the genetic variation and identification of species. Molecular tools provide valuable data in exploiting the genetic variability that exist among different genotypes through their ability to detect variation at DNA level. The use of molecular markers has been developed as powerful tools, for diversity analysis in establishing relationship between cultivars, by many workers all over the world (Awasthi et al., 2004, Rangan et al., 2008). Among other molecular markers, random amplified polymorphic DNA (RAPD) markers and inter - simple sequence repeats (ISSR) markers are widely used to exploit the variation existed among the plants species. But though, RAPD is technically simple and less expensive, ISSR is highly reproducible, more reliable and very useful for population genetics, variety identification, gene tagging, germplasm evaluation and phylogenetic studies, conservation genetics. Due to these advantages, it might provide valuable data in exploring Das and Borah, 2012 Journal of Research in Plant Sciences (2012) 1(2): 146-152 148 Table 2 Characters of Primers used in the study Marker Primer code Sequence (5- 3) Annealing Temperature(C) ISSR UBC 812 GAGAGAGAGAGAGAGAA 50.9 UBC 814 CACACACACACACACACAA 55.0 UBC 840 GAGAGAGAGAGAGAGAAT 58.0 UBC 848 CACACACACACACACAAG 50.9 Figure 2 Polymorphism on the band profiles of genotypes of Solanum xanthocarpum Schard. and Wendl. revealed by the PCR Amplification generated by UBC 812 (A), UBC 814(B), UBC 840 (C) and UBC 848 (D) resolved on 2.0% gel. M = Marker.
the genetic variation and distinction among the genotypes of S. xanthocarpum. In this study, four ISSR molecular markers were used in the evaluation of the genetic diversity of seven accessions of S. xanthocarpum Schard. and Wendl. of Assam and the efficiency of ISSR markers in the discrimination of accessions was evaluated.
MATERIALS AND METHODS Plant material Seven accessions of Solanum xanthocarpum Schard. and Wendl. were collected from the different parts of Assam. Information about the collected plants is included in Table-1. Fresh, healthy green leaves of these samples were used for DNA isolation and molecular fingerprinting analysis. DNA isolation Genomic DNA was extracted from 0.5 g of youn g fr esh l ea ves usi n g CTAB (Hexadecyltrimethylammoniumbromide) method (Lassner et al.,1989). The purity of genomic DNA was evaluated by measuring absorbance (A260 nm/A280 nm ratio) with a Double Beam UV spectrophotometer. The Purity and integrity of the DNA isolated were determined by agarose gel (0.8%) electrophoresis stained with ethidium bromide and using ladder DNA cleaved as a size standard. Primer used in PCRs For ISSRs, ten primers obtained from UBC primer set 100/9 (University of British Columbia) were initially screened for their repeatable amplification with seven accessions of Solanum xanthocarpum Schard. and Wendl. After screening, four primers were selected for further analysis based on their ability to detect distinct, clearly resolved polymorphic amplified products. To ensure reproducibility, the primers generating weak products were discarded. Characters of the selected primers are given in Table-2. ISSR amplification ISSR amplification reactions were carried out in 20 l volume containing 50 ng template DNA, 0.5 U Taq DNA polymerase, 10 mM dNTP, 10 M primer in 1 reaction buffer that contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , and 0.01% gelatin. Amplification was performed PCR amplification was carried out in a Mini Thermal Cycler (Applied Biosystems 9700). Amplification conditions were one cycle at 94C for 5 min., and 36 cycles each with 94C Das and Borah, 2012 149 Journal of Research in Plant Sciences (2012) 1(2): 146-152 Table 3 Degree of polymorphism, percentage of polymorphism (Pol %), polymorphic information content (PIC) and marker index (MI) for ISSR primers in the seven genotypes of Solanum xanthocarpum Schard. and Wendl. Marker Primer code Total No. of bands Total No. of polymorphic bands POL% PIC MI(Pol% x PIC) ISSR UBC 812 9 9 100 0.42 42.0 UBC 814 10 10 100 0.42 42.0 UBC 840 12 11 91.6 0.49 43.9 UBC 848 8 8 100 0.48 49.0 Sx01 Sx02 Sx03 Sx04 Sx05 Sx06 Sx07 Sx01 1.000 Sx02 0.375 1.000 Sx03 0.333 0.227 1.000 Sx04 0.360 0.370 0.308 1.000 Sx05 0.227 0.200 0.125 0.233 1.000 Sx06 0.227 0.304 0.286 0.276 0.364 1.000 Sx07 0.316 0.333 0.250 0.296 0.273 0.333 1.000 Table 4 Similarity matrices of seven genotypes of Solanum xanthocarpum based on ISSR profiling. for 30 sec., 42C for 30 sec., and 72C for 1 min. The amplified products were loaded on 2% agarose gel and separated in 0.5 TBE buffer at 75 V and documented using a gel documentation and image analysis system (Make: Gel Doc. 2000, Bio Red). Data scoring and analysis Duplicate samples from each individual were tested and, only well resolved and reproducible bands amplified in both cases, were considered for the scoring and data analysis. The numbers of polymorphic and monomorphic amplification products were determined for each primer for seven accessions of Solanum xanthocarpum Schard. and Wendl. Scoring was done as 1 for the presence and 0 for the absence across the genotypes. The similarity matrix was computed to estimate all pair wise differences in similarity matrices of the amplification product, using sequential, hierarchical clustering option of the SPSS version 11.0 software package (Information Technology Science Centre, Lingnan University, 2002). Level of similarity among species was established as percentage of polymorphic bands and a matrix of genetic similarity was compiled using the Dices coefficient (Dice, 1945). The program also generated a dendrogram which grouped the species on the basis of Nei Genetic distance (Nei, 1978) using unweighted pair group method with arithmetic average (UPGMA) cluster analysis (Sneath and Sokal, 1973).
RESULTS Genomic DNA isolation The genomic DNA of nine genotypes of Solanum xanthocarpum Schard. and Wendl. was extracted. The genomic DNA extracted were pure and intact bands of each sample was obtained when run in 0.8% agarose gel (Figure 1). ISSR amplification The total number of DNA bands amplified with the genotypes as well as the number of polymorphic bands along with percentage of polymorphic bands (POL%), polymorphic information content (PIC) and marker Index (MI) is presented in Table 3. A total of 39 mappable ISSR markers were generated by four primers. Out of these, 38 were polymorphic (97.43%).The number of amplification products obtained per primer is in the range of 8 to 12, with the primer UBC 848 producing the minimum number (8) and UBC 840 producing the maximum number (12) of bands. Except primer UBC 840 all the primers showed 100% POL percentage. The PIC value ranged from 0.42 to 0.49. Marker index (MI) value was found to be ranging from 42.0 to 49.0. Primer UBC 848 showed maximum value of MI i.e. 49.0 (Table 3). The amplification products obtained by all the primer used in the study are exhibited in Figure 2, which exemplified the typical ISSR banding patterns observed. Gene diversity analysis with ISSR marker The ISSR data revealed that the genetic similarity indices ranged from 0.125 to 0.375 (Table No. 4). The minimum similarity (0.125) was found between Solanum xanthocarpum (Sx03) collected from Golaghat and Sx05 collected from Khetri. Maximum level of similarity indices (0.375) was found between Sx01 and Sx02 collected from Jalukbari and Goalpara respectively (Table 4). The genetic similarity value ranged from 0.125 to 0.375 suggesting a wide genetic base within the genotypes used in the present investigation. Dendrogram based on the similarity Das and Borah, 2012 Journal of Research in Plant Sciences (2012) 1(2): 146-152 150 Figure 3 UPGMA dendrogram based on ISSR data for the studied population matrices of ISSR-PCR banding patterns clearly distinguished the samples of Solanum xanthocarpum Schard. and Wendl. collected from different parts of Assam into two clusters (Figure 3). Cluster-I has four accessions comprising Sx01, Sx02, Sx04, and Sx03 collected from Jalukbari, Goalpara, Rani and Golaghat. While three accessions (Sx05, Sx06 and Sx07) that were collected from Khetri, Shillong and Karbi Anglong formed cluster-II. A close association was observed between Sx02 and Sx04 and in between Sx05 and Sx06.
DISCUSSION Assessment of genetic variability within a germplasm is of interest, for practical applications such as, for conservation of genetic resources management and for breeding purposes, to predict the ability to combine or to rapidly verify the breeding material. Again, identification of the species is of fundamental importance in diversity studies in a variety of ways. For evaluation of species diversity, it is essential that individuals can be classified accurately. The identification of taxonomic units, whose genetic constitution is distinct from their more abundant relatives, is important in the development of appropriate conservation strategies. Most of the important members of Solanum show high polymorphism in morphological character possessing taxonomic confusion. ISSR molecular markers have been used successfully in germplasm bank characterization (Sudupak, 2004, Carvalho et al., 2005, Essadki et al., 2006 and Terzopoulos and Bebeli, 2008) especially in the assessment of the differences among species or varieties belonging to the same genus. The present study revealed the genetic diversity within a collection of Solanum xanthocarpum Schard. and Wendl. germ-plasm representing different geographical regions of Assam using ISSR marker. The ISSR data generated in these population insights into the existing diversity of the species. In this study, ISSR markers revealed moderate levels of polymorphism with an average of nine polymorphic bands per primer. Similarity indices ranged from 0.125 to 0.375, also emphasizing the effectiveness of ISSR markers. Cluster analysis based dendrogram prediction using ISSR markers clearly distinguished the experimental species. This indicates that the markers have high discrimination ability of the groups of Solanum xanthocarpum species. Evaluation of diversity would also have immense significance for in situ conservation of this medicinally important species especially for further genetic improvement programmes. Moreover, the results suggest that the application of molecular fingerprinting enables a rapid and sensitive method in detecting genetic variations among the different populations of S.m xanthocarpum. Our results confirm that DNA analysis by ISSR is an efficient method for the exploration of genetic diversity in S. xanthocarpum populations. To our knowledge, this is the first report by ISSR markers to assess the great variability that exist among germplasm of members of Solanum xanthocarpum from Assam.
CONCLUSION Genetic diversity that refers to the total number of genetic characteristics in the genetic makeup of a species is the most fundamental component of biological diversity. Its analysis is a key element for any kind of genetic improvement and conservation programme. Again, maintaining diversity gives the population a buffer against change, providing the flexibility to adapt. Moreover, the results suggest that DNA analysis by ISSR marker can be considered as a valuable method for discrimination of S. xanthocarpum. And this study might help in formulating new drugs in the field of pharmacology. To our knowledge, this is the first report to assess the variability among germplasm of Solanum xanthocarpum by using ISSR markers from Assam.
Das and Borah, 2012 151 Journal of Research in Plant Sciences (2012) 1(2): 146-152 REFERENCES Awasthi AK, Nagaraja GM, Naik GV, Kanginakudru S, Thangavelu K and Nagaraju J. 2004. Genetic diversity and relationships in mulberry (genus Morus) as revealed by RAPD and ISSR marker assays. BMC Genet., 5: 1-7.
Carvalho A, Matos M, Lima-Brito J, Guedes-Pinto H and Benito C. 2005. DNA fingerprint of F1 interspecific hybrids from the Triticeae tribe using ISSRs. Euphytica., 143:93-99.
Dice LR. 1945. Measures of the amount of ecologic association between species. Ecol., 26: 297-302.
Essadki M, Ouazzani N, Lumaret R and Moumni M. 2006. ISSR variation in olive-tree cultivars from Morocco and other western countries of the Mediterranean Basin. Genet Res Crop Evol., 53:475-482.
Lassner MW, Peterson P and Yoder JI. 1989. Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol Biol Rep., 7:116-128.
Nei M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics., 89:583-590.
Rangan L, Das A, Kesari V, Agarwal S and Sarma GC. 2008. Use of DNA barcodes to identify Curcuma species of Northeast India. International conference of emerging technologies and applications in engineering, technology and sciences, Rajkot, 2116-2120.
Singh OM and Singh TP. 2010. Phytochemistry of Solanum xanthocarpum : an amazing traditional healer. JSIR., 69:732-740.
Sneath PHA and Sokal RR. 1973. Numerical Taxonomy. W.H. Freeman and Company, San Francisco. 230-234. Sudupak MA. 2004. Inter and intra-species Inter Simple Sequence Repeat (ISSR) variations in the genus Cicer. Euphytica., 135:229-238.
Terzopoulos PJ and Bebeli PJ. 2008. DNA and morphological diversity of selected Greek tomato (Solanum lycopersicum L.) landraces. Sci Hortic., 116(4):354-361.
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