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Genetic integrity of somaclonal variants in tea (Camellia sinensis (L.) O Kuntze) as revealed by inter simple sequence repeats
Journal of Biotechnology 123 (2006) 149–154
Genetic integrity of somaclonal variants in tea (Camellia sinensis (L.) O Kuntze) as revealed by inter simple sequence repeats Jibu Thomas ∗ , Deepu Vijayan, Sarvottam D. Joshi, S. Joseph Lopez, R. Raj Kumar Plant Physiology and Biotechnology, UPASI Tea Research Institute, Nirar Dam BPO, Valparai, Coimbatore District, Tamil Nadu 642 127, India Received 8 May 2005; received in revised form 18 October 2005; accepted 9 November 2005
Abstract Adoption of inter simple sequence repeats (ISSR) technique to analyze the genetic variability of somatic embryo derived tea plants was evaluated. Morphological characterisation of the field grown plants revealed no identical character aligning with the parent, UPASI-10. Out of 40 primers, 15 exhibited concurrent polymorphism were selected for the study. Genetic variability of somaclones derived from single line cotyledonary culture ranged from 33.0 to 55.0%. A unique fragment of 1.2 Kb was visible in majority of the accessions whereas the fragments below the length of 0.6 Kb were noticed only in 50% of the variants. Out of 120 interactions attempted using Pearson’s coefficient correlation, only 9.2% of somaclones exhibited significant similarity at genetic level. Dendrogram constructed based on simple matching coefficient revealed a distance of 2.257–3.317 between the final clusters. This strengthens the existence of wide genetic variation among the somaclones. © 2005 Elsevier B.V. All rights reserved. Keywords: Camellia sinensis; Genetic integrity; ISSR; Somaclonal variants
1. Introduction South Indian tea (Camellia spp.) germplasm is known for its inherent genetic variations which could be attributed due to their free natural hybridization within and between the species (Saravanan et al., 2005). ∗ Corresponding author. Tel.: +91 4253 235301; fax: +91 4253 235302. E-mail address: [email protected] (J. Thomas).
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.11.005
Certain trait specific elite plants are propagated vegetatively and commercially exploited as “Clones” which in turn narrow down the vast germplasm resources. In order to conserve the trait specific germplasm and to widen the genetic reservoir, in recent years, somatic hybridization has been attempted in tea besides the conventional breeding programme (Palni et al., 1991). Somaclonal variation occurs in plants regenerated from somatic tissues and has been observed for morphological, physiological, biochemical and genetic
150
J. Thomas et al. / Journal of Biotechnology 123 (2006) 149–154
traits. Although these prototypes are not commercially exploited, they provide a reservoir of important genes which deserve introgression on genetic relationship within the variants and among the common cultivars. Somatic embryogenesis is comparatively feasible and easy to access economically in comparison to gene transfer techniques. Diversity studies on somaclonal variants have been carried out using physiological/biochemical tools that too, with a small population of plants which exhibited morphological variations (Raj Kumar et al., 2001). Moreover, conventional method of selection is a laborious and time consuming process which seeks an alternative avenue to identify the elite prototype within a stipulated time frame. In the biotechnological era, to analyze the characteristic features of available tea germplasm, molecular markers such as AFLP, RAPD and ISSR were employed which in turn offer the possibility of observing genetic diversity (Devarumath et al., 2002; Balasaravanan et al., 2003). When compared to other crop plants, genetic diversity studies in South Indian tea germplasm using DNA based marker system are limited. Since, the somaclones exhibited very low level of morphological variations, ISSR technique can be employed with an advantage to analyze the genetic diversity than other molecular marker tools where the differentiation may be difficult to discern (Rostiana et al., 1999). ISSR consists of tandemly arranged repeats of several nucleotides that are distributed through the whole genome and are flanked by highly conserved sequences (Chambers and MacAvoy, 2000). Some types of microsatellites seem to be specific or much more present for a certain group of plants (Morgante and Vogel, 1994). Advantages of this marker system are that it is technically simpler than many other molecular marker systems for investigation of genetic instabilities and also provides reproducible results that generate abundant polymorphism even at the early stages of in vitro culture (Tsumura et al., 1996). They also seem to be suitable for phylogenetic studies, the evaluation of genetic diversity and cultivar identification (Jain et al., 1999; Raina et al., 2001). Even though, Jha et al. (1992) attempted chromosomal study of somatic embryo derived tea plants, no marker studies on the genetic integrity of somatic embryo derived plants of Camellia has been reported.
In this context, the present investigation was aimed to determine the genetic variability existed among the somaclones using ISSR technique, which was developed and popularized by Zietkiewicz et al. (1994) and Wolfe et al. (1998).
2. Materials and methods 2.1. Plant material In vitro studies with particular reference to induction of somatic embryoids from cotyledonary tissues of a “China” hybrid clone UPASI-10, germination and successive field transfer were carried out between 1997 and 2001 at United Planters Association of Southern India (UPASI) Tea Research Institute located in the Anamallais, Tamil Nadu, India (Raj Kumar, 2001). All cultivation practices were carried out uniformly according to standard recommendations of UPASI (Hudson et al., 2002). 2.2. Morphological characterization Leaf characteristics such as colour, texture, venation, serration, leaf tip and angle were studied in accordance with National Bureau of Plant Genetic Resources (NBPGR), New Delhi to compare the morphological similarity of variants. 2.3. Molecular characterization 2.3.1. DNA extraction Leaf samples were collected from all the 15 field grown somaclonal variants after a period of 3 years from planting. Genomic DNA was extracted with cetryl trimethyl ammonium bromide (CTAB) using the modified protocol of Kobayashi et al. (1998). Obtained DNA pellet was washed three times with 70% ethanol, vacuum dried and dissolved in 100 l of TE buffer (10 mM Tris–HCl and 1mM EDTA (pH 8.0)). After treating with 5 l of RNaseA (10 mg/ml), the quantity and quality of the DNA was checked with spectrophotometer (Genesys 10UV, USA) and agarose gel (0.8%) electrophoresis. Absorbance ratio between 260 and 280 nm was computed and the quality of the genomic DNA was ◦ confirmed. Final sample was stored at 4 C for downstream applications.
J. Thomas et al. / Journal of Biotechnology 123 (2006) 149–154
2.3.2. ISSR analysis ISSR primers (Sigma Genosys, USA) were synthesized based on di- and tri-nucleotide repeats (GA, GT, CT and GTC) as a core sequence with a Tm value range of 40.0–55.0. Screening was carried out with 40 primers. Out of which 15 primers, which gave clear banding pattern were used for confirmatory studies. Choice of nucleotides was based on the probability of its relative abundance in tea genome (Mondal, 2002). The reaction mixture included 0.1 mM of each dNTP, 150 nM primer, PCR master mix (MBI Fermentas, USA) supplied with enzyme and 50 ng DNA. Reaction mixture without DNA served as negative control. Amplification was carried out in a programmable peltier thermocycler PTC 200 (MJ Research, USA). Amplification protocol includes initial denaturation for 4 min at 94 ◦ C followed by 42 successive cycles of 40 s denaturation at 94 ◦ C, annealing for 2 min at respective Tm values of the selected primers and 1.45 min elongation at 72 ◦ C. Final elongation was performed for 8 min at 72 ◦ C. Amplicons were checked by separating on 1.4% agarose gel electrophoresis for 3 h at a constant temperature of 75 V with 1× TAE (TRIS base 4.84 g, glacial acetic acid 1.14 and 2.0 ml EDTA (0.5 M, pH 8.0) in 1000 ml of distilled water) running buffer. Finally the gel was stained with ethidium bromide (0.5 g/ml), visualized under ultraviolet rays and documented. The analysis was performed for all the samples at least three times with each selected primers. 2.3.3. Data analysis Scanned gel image was analyzed using special software Total Lab, version 3.1 (Amersham Bioscience, USA) for fragment length calibration. Amplified fragments were scored manually for the band presence (1) and absence (0). To characterize the ability of each primer to detect the polymorphic loci, the ratio of the number of polymorphic bands to the total number of bands revealed per primer reaction was computed to detect the percentage of polymorphism. From the binary data, the similarity coefficient values between the variants were derived based on the probability that amplified fragment from one variant will also be present in another with the Nei’s estimate (Nei and Li, 1979). Similarity based relationship between the variants were presented in the form of the dendrogram, developed following unweighted pair group method
151
with arithmetic mean algorithm (UPGMA) using SAHN cluster analysis of NTSYS-pc version 2.0 (Rohlf, 1992). Principal component analysis (PCO) was performed using statistical package for social sciences (SPSS) version 7.5 for Windows. First two components of PCO were used to plot the graph to determine the relationship among the variants.
3. Results and discussion Except the variants SE5, SE13 and SE14, all others possessed pale to yellowish green leaves while a very few variants were eliptic in their leaf shape and with smooth venation resembling the source plant, UPASI10 (Table 1). Except SE13, all other variants had acute leaf tip and majority of them had horizontal leaf posture. Besides SE5, leaves of all other variants were closely/double serrated. Majority of the variants had medium size leaves followed by large size and only one variant (SE7) had small leaves. In all the cases, texture of the mature leaves was found to be brittle. Although variants were derived from “China” hybrid, UPASI-10, a very few variants possessed unique “Chinery” character (elliptic, dark green, smooth, erect to semierect, small/medium size leaves) while others exhibited “Assam” characters (broad/ovate, pale to yellowish green, rugose, horizontal, medium to large leaves). However, no variant showed identical morphological characters aligning with the parent. Since the variants possessed both “Assam” and “China” characteristics, these could not be categorized into their respective groups. In the present study, among the primers tested, 15 primers which resulted in polymorphic amplifications with scorable PCR products (Table 2) were selected. Size of the polymorphic amplified fragments ranged from 450 to 1200 bp (Fig. 1). Results indicated that the ISSR has high resolution in the genotype identification, confirming the findings of Yang et al. (1996) and Esselman et al. (1999). Even though the morphological variations of the somaclones were negligible, the banding pattern of these primers on an average showed about 50% polymorphism (Table 2). Specificity and reproducibility of ISSR amplifications as reported by Bornet and Branchard (2001) was taken into account when designing the reactions. Amplified fragments of 1.2 and 0.65 kb were present in almost all accessions
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Table 1 Morphological variations in leaf characteristics of the somaclonal variants derived from UPASI-10 clone Variant number
SE1 SE2 SE3 SE4 SE5 SE6 SE7 SE8 SE9 SE10 SE11 SE12 SE13 SE14 SE15 UP-10 a
Leaf characteristics Colour
Shape
Venation
Tip
Posture
Serration
Size (cm2 )a
Yellowish Yellowish Yellowish Yellowish Dark Pale Yellowish Pale Pale Pale Pale Yellowish Dark Dark Pale Dark
Ovate Ovate Ovate Ovate Eliptic Eliptic Ovate Ovate Ovate Ovate Ovate Eliptic Ovate Eliptic Ovate Eliptic
Rugose Rugose Rugose Rugose Smooth Smooth Rugose Rugose Rugose Rugose Rugose Rugose Rugose Smooth Rugose Smooth
Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Blunt Acute Acute Blunt
Horizontal Horizontal Horizontal Horizontal Semierect Semierect Horizontal Horizontal Horizontal Horizontal Horizontal Semierect Semierect Erect Semierect Erect
Close Close Close Close Distant Close Close Close Close Close Close Close Close Close Close Distant
35–50 35–50 35–50 35–50 >50 35–50 <25 35–50 35–50 35–50 >50 35–50 >50 >50 >50 >50
<25 (small); 35–50 (medium); >50 (large).
attempted whereas uniqueness of 0.5 kb was restricted to the variant, SE7. Pearson’s co-relation coefficient matrix revealed that SE1 had a significant similarity with SE2, SE6 and SE9 at 1.0% probability while it had significant relation with SE4 and SE14 at 5.0% level. SE15 had distinct similarities with SE2, SE8, SE11 and SE12 at 5.0% level. Similar trend was observed between SE9 and SE13 while SE8 and SE10 exhibited very high Table 2 Details of the ISSR primers used and polymorphism exhibited by analysis Code
Sequence
Polymorphism (%)
SGIP1 SGIP2 SGIP3 SGIP4 SGIP6 SGIP7 SGIP8 SGIP10 SGIP14 SGIP15 SGIP16 SGIP17 SGIP18 SGIP20 SGIP23
GAGAGAGAGAGAGAGAT GAGAGAGAGAGAGAGYG GAGAGAGAGAGAGAGARGY GAGAGAGAGAGAGAGARGY GAGAGAGAGAGAGAGAC ACACACACACACACACYT ACACACACACACACACYG GTGTGTGTGTGTGTGTYR CACACACACACACAAC CACACACACACACAGT CACACACACACACAAG CACACACACACACAGG GAGAGAGAGAGAGG GAGAGAGAGAGACC GAGGAGGAGGC
78.0 68.0 62.0 60.5 65.0 50.0 50.0 45.0 33.0 30.5 38.0 40.0 65.0 60.5 62.0
where R = A + G and Y = C + T.
coefficient value (1.00) which is significant at 1.0% probability. Out of 120 interactions attempted, only 9.2% revealed significant similarity at molecular level. This strengthens the existence of wide genetic variation among the somaclonal plants. Dendrogram was constructed based on simple matching coefficients, taking into account the presence or absence of the bands and ignoring their intensities. Individual similarity within the clusters was analyzed by hierarchical cluster and the principal component analysis segregated the 15 accessions into three distinct groups. Distance between final cluster centeroids varied from 2.257 to 3.317. Dendrogram provides an idea about the genetic relationship between the individuals and relation among the groups (Fig. 2).
Fig. 1. Electrophoretic pattern of amplified DNA using primer SGIP3. Lane 1–200 bp ladder (MBI Fermentas, USA); Lane 2–14: Samples SE1 to SE15.
J. Thomas et al. / Journal of Biotechnology 123 (2006) 149–154
Fig. 2. Hierarchial cluster analysis of somaclonal variants.
Earlier, diversity among the germplasm resource has been demonstrated using morphological, physiological and biochemical characterization. In spite of asexual propagation, somatic embryogenesis is accompanied by a difference in morphologic characteristics allowing the selection for genetic improvement (Raj Kumar et al., 2001). Molecular markers allow the identification of variation at the genomic level (Bornet et al., 2003; Mondal and Chand, 2002) and permit detection of genetic mutation as in the case of tissue culture induced variations (Sanchez-Teyar et al., 2003). Development of molecular markers using AFLP in tea was first attempted by Rajsekaran (1997). Present study confirms that the ISSR analysis can be used effectively for the initial assessment of partitioning of genetic variation within plant species, where only a little genetic diversity information available. This corroborates with the findings of Devarumath et al. (2002). Results of present investigation confirmed the variability in genetic nature of the regenerants and also
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unique molecular markers in the somaclones. It is therefore envisaged that the genetic variation observed in the somaclonal variants are due to the chances of inadvertent manipulations that occur at the cellular and subcellular level and have been fixed in the genotypes contributing to the genetic uniqueness of the south Indian germplasm. Even though the true rate of somaclonal variation is difficult to ascertain due to the involvement of multiple genes, these can be used for plant improvement. Since somatic embryogenesis is a process controlled by multi-factors, the subtle interactions among the conditions to be satisfied for optimum results right from somatic embryo production up to germination, which differs from, clone to clone and different explants needs attention. Limited samples utilized in the study revealed the presence of genetic reservoir to explore the possibility to identify superior genotypes with correlation to the environment. Under evaluation, prototype which exhibits desired agronomic characters can be integrated in future breeding programme for conservation and commercial exploitation. Identification of unique regions can serve as a source of ISSR markers. They are suitable for the evaluation of genetic integrity and cultivar identification. Unique fragments thus obtained may be transformed into sequence characterized amplified region (SCAR) for marker aided selection.
Acknowledgements Authors are thankful to Dr. N. Muraleedharan, Director, UPASI Tea Research Foundation for the support extended during the course of study. The financial grant received from Department of Biotechnology (DBT), Government of India is gratefully acknowledged.
References Balasaravanan, T., Pius, P.K., Raj Kumar, R., Muraleedharan, N., Shasany, A.K., 2003. Genetic diversity among south Indian tea germplasm (Camellia sinensis C. assamica and C. assamica spp. lasiocalyx) using AFLP. Plant Sci. 165, 365–372. Bornet, B., Branchard, M., 2001. Non anchored inter simple sequence repeat (ISSR) markers: reproducible and specific tools for genome fingerprinting. Plant Mol. Biol. Rep. 19, 209– 215.
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Bornet, B., Balatti, P.A., Branchard, M., 2003. Inter simple sequence repeat (ISSR) markers as a tool for the assessment of both genetic diversity and gene pool origin in common bean (Phaseolus vulgaris L.). Euphytica 132, 297–301. Chambers, G.K., MacAvoy, E.S., 2000. Microsatellites: consensus and controversy. Comp. Biochem. Physiol. 126, 455– 476. Devarumath, R.M., Nandy, S., Rani, V., Marimuthu, S., Muraleedharan, N., Raina, S.N., 2002. RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type). Plant Cell Rep. 21, 166–173. Esselman, E.J., Jianqiang, L., Crawford, D.J., Windus, J.L., Wolfe, A.D., 1999. Clonal diversity in the rare Calamagrostis porteri ssp. inseperata (Poaceae): comparative results for allozymes and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. Mol. Ecol. 8, 443– 451. Hudson, J.B., Durairaj, J., Muraleedharan, N., 2002. Guidelines on Tea Culture in South India. Allied Publishers Private Limited, Chennai, India. Jain, A., Apparanda, C., Bhalla, P.L., 1999. Evaluation of genetic diversity and genome fingerprinting of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 714–719. Jha, T.B., Jha, S., Sen, S.K., 1992. Somatic embryogenesis from immature cotyledon of an elite Darjeeling tea clone. Plant Sci. 84, 209–213. Kobayashi, N., Horikoshi, H., Katsuyama, H., Handa, T., Takayanagi, K., 1998. A simple and efficient DNA extraction method for plants, especially woody plants. Plant Tissue Cult. Biotech. 4, 76–80. Mondal, T.K., 2002. Assessment of genetic diversity of tea (Camellia sinensis (L.) O Kuntze) by inter-simple sequence repeat polymerase chain reaction. Euphytica 128, 307–315. Mondal, T.K., Chand, P.K., 2002. Detection of genetic instability among the miocropropagated tea (Camellia sinensis) Plants. In vitro Cell Dev. Biol. 37, 1–5. Morgante, M., Vogel, J., 1994. Compound microsatellite primers for the detection of genetic polymorphism. U.S. Patent Application 08/326456. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 74, 5267–5273. Palni, L.M.S., Sood, A., Chand, G., Sharma, M., Rao, D.V., Jain, N.K., 1991. Tissue culture studies in tea (Camellia sinensis (L). O. Kuntze). In: Proceedings of the International Symposium on Tea Science, Shizuoka, Japan.
Raina, S.N., Rani, V., Kojima, T., Ogihara, Y., Singh, K.P., Devarumath, R.M., 2001. RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species. Genome 44, 763–772. Raj Kumar, R., 2001. Development of a protocol for efficient somaclonal plants using drought tolerant UPASI clones as source of explants. Final Technical Report. National Tea Research Foundation, Kolkata, India. Raj Kumar, R., Balasaravanan, T., Jayakumar, D., Vinod Haridas, Marimuthu, S., 2001. Physiological and biochemical features of field grown somaclonal variants. Bull. UPASI Tea Res. Found. 54, 73–81. Rajsekaran, P., 1997. Development of molecular markers using AFLP in tea. In: Varghese, J.P (Ed.), Molecular Approaches to Crop Improvement. Proceedings of the National Seminar on Molecular Approaches to Crop Improvement. Kottayam, Kerala, India, December 29–31. Rohlf, F.J., 1992. NTSYS-PC Numerical Taxonomy and Multivariate Analysis System, Version 2.0. State University of New York, Stony Brook, NY. Rostiana, O., Niwa, M., Marubashi, W., 1999. Efficiency of intersimple sequence repeat PCR for detecting somaclonal variation among leaf-cultureregenerated plants of horseradish. Breed. Sci. 49, 245–250. Sanchez-Teyar, L.F., Quiroz-Figueroa, F., Loyola-Vargas, V., Infante, D., 2003. Culture induced variation in plants of Coffea arabica cv. Caturra rojo, regenerated by direct and indirect somatic embryogenesis. Mol. Biotechnol. 23, 107–115. Saravanan, M., Maria John, K.M., Raj Kumar, R., Pius, P.K., Sasikumar, R., 2005. Genetic diversity of UPASI tea clones (Camellia sinensis (L.) O Kuntze) on the basis of total catechins and their fractions. Phytochemistry 66 (5), 561–565. Tsumura, Y., Ohba, K., Strauss, S.H., 1996. Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudosuga menziesii) and sugi (Cryptomeria japonica). Theor Appl. Genet. 92, 40–45. Wolfe, A.D., Xiang, Q.-Y., Kephart, S.R., 1998. Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands. Mol. Ecol. 7, 1107–1125. Yang, W., de Oliveira, A.C., Godwin, I., Scheritz, K., Bennetzen, J.L., 1996. Comparison of DNA marker technologies in characterizing plant genome diversity: variability in Chinese sorghums. Crop Sci. 36, 1669–1676. Zietkiewicz, E., Rafalski, A., Labuda, D., 1994. Genome fingerprinting by simple sequence repeat (SSR)—anchored polymerase chain reaction amplification. Genomics 20, 176–183.
Genetic integrity of somaclonal variants in tea (Camellia sinensis (L.) O Kuntze) as revealed by inter simple sequence repeats Jibu Thomas ∗ , Deepu Vijayan, Sarvottam D. Joshi, S. Joseph Lopez, R. Raj Kumar Plant Physiology and Biotechnology, UPASI Tea Research Institute, Nirar Dam BPO, Valparai, Coimbatore District, Tamil Nadu 642 127, India Received 8 May 2005; received in revised form 18 October 2005; accepted 9 November 2005
Abstract Adoption of inter simple sequence repeats (ISSR) technique to analyze the genetic variability of somatic embryo derived tea plants was evaluated. Morphological characterisation of the field grown plants revealed no identical character aligning with the parent, UPASI-10. Out of 40 primers, 15 exhibited concurrent polymorphism were selected for the study. Genetic variability of somaclones derived from single line cotyledonary culture ranged from 33.0 to 55.0%. A unique fragment of 1.2 Kb was visible in majority of the accessions whereas the fragments below the length of 0.6 Kb were noticed only in 50% of the variants. Out of 120 interactions attempted using Pearson’s coefficient correlation, only 9.2% of somaclones exhibited significant similarity at genetic level. Dendrogram constructed based on simple matching coefficient revealed a distance of 2.257–3.317 between the final clusters. This strengthens the existence of wide genetic variation among the somaclones. © 2005 Elsevier B.V. All rights reserved. Keywords: Camellia sinensis; Genetic integrity; ISSR; Somaclonal variants
1. Introduction South Indian tea (Camellia spp.) germplasm is known for its inherent genetic variations which could be attributed due to their free natural hybridization within and between the species (Saravanan et al., 2005). ∗ Corresponding author. Tel.: +91 4253 235301; fax: +91 4253 235302. E-mail address: [email protected] (J. Thomas).
0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.11.005
Certain trait specific elite plants are propagated vegetatively and commercially exploited as “Clones” which in turn narrow down the vast germplasm resources. In order to conserve the trait specific germplasm and to widen the genetic reservoir, in recent years, somatic hybridization has been attempted in tea besides the conventional breeding programme (Palni et al., 1991). Somaclonal variation occurs in plants regenerated from somatic tissues and has been observed for morphological, physiological, biochemical and genetic
150
J. Thomas et al. / Journal of Biotechnology 123 (2006) 149–154
traits. Although these prototypes are not commercially exploited, they provide a reservoir of important genes which deserve introgression on genetic relationship within the variants and among the common cultivars. Somatic embryogenesis is comparatively feasible and easy to access economically in comparison to gene transfer techniques. Diversity studies on somaclonal variants have been carried out using physiological/biochemical tools that too, with a small population of plants which exhibited morphological variations (Raj Kumar et al., 2001). Moreover, conventional method of selection is a laborious and time consuming process which seeks an alternative avenue to identify the elite prototype within a stipulated time frame. In the biotechnological era, to analyze the characteristic features of available tea germplasm, molecular markers such as AFLP, RAPD and ISSR were employed which in turn offer the possibility of observing genetic diversity (Devarumath et al., 2002; Balasaravanan et al., 2003). When compared to other crop plants, genetic diversity studies in South Indian tea germplasm using DNA based marker system are limited. Since, the somaclones exhibited very low level of morphological variations, ISSR technique can be employed with an advantage to analyze the genetic diversity than other molecular marker tools where the differentiation may be difficult to discern (Rostiana et al., 1999). ISSR consists of tandemly arranged repeats of several nucleotides that are distributed through the whole genome and are flanked by highly conserved sequences (Chambers and MacAvoy, 2000). Some types of microsatellites seem to be specific or much more present for a certain group of plants (Morgante and Vogel, 1994). Advantages of this marker system are that it is technically simpler than many other molecular marker systems for investigation of genetic instabilities and also provides reproducible results that generate abundant polymorphism even at the early stages of in vitro culture (Tsumura et al., 1996). They also seem to be suitable for phylogenetic studies, the evaluation of genetic diversity and cultivar identification (Jain et al., 1999; Raina et al., 2001). Even though, Jha et al. (1992) attempted chromosomal study of somatic embryo derived tea plants, no marker studies on the genetic integrity of somatic embryo derived plants of Camellia has been reported.
In this context, the present investigation was aimed to determine the genetic variability existed among the somaclones using ISSR technique, which was developed and popularized by Zietkiewicz et al. (1994) and Wolfe et al. (1998).
2. Materials and methods 2.1. Plant material In vitro studies with particular reference to induction of somatic embryoids from cotyledonary tissues of a “China” hybrid clone UPASI-10, germination and successive field transfer were carried out between 1997 and 2001 at United Planters Association of Southern India (UPASI) Tea Research Institute located in the Anamallais, Tamil Nadu, India (Raj Kumar, 2001). All cultivation practices were carried out uniformly according to standard recommendations of UPASI (Hudson et al., 2002). 2.2. Morphological characterization Leaf characteristics such as colour, texture, venation, serration, leaf tip and angle were studied in accordance with National Bureau of Plant Genetic Resources (NBPGR), New Delhi to compare the morphological similarity of variants. 2.3. Molecular characterization 2.3.1. DNA extraction Leaf samples were collected from all the 15 field grown somaclonal variants after a period of 3 years from planting. Genomic DNA was extracted with cetryl trimethyl ammonium bromide (CTAB) using the modified protocol of Kobayashi et al. (1998). Obtained DNA pellet was washed three times with 70% ethanol, vacuum dried and dissolved in 100 l of TE buffer (10 mM Tris–HCl and 1mM EDTA (pH 8.0)). After treating with 5 l of RNaseA (10 mg/ml), the quantity and quality of the DNA was checked with spectrophotometer (Genesys 10UV, USA) and agarose gel (0.8%) electrophoresis. Absorbance ratio between 260 and 280 nm was computed and the quality of the genomic DNA was ◦ confirmed. Final sample was stored at 4 C for downstream applications.
J. Thomas et al. / Journal of Biotechnology 123 (2006) 149–154
2.3.2. ISSR analysis ISSR primers (Sigma Genosys, USA) were synthesized based on di- and tri-nucleotide repeats (GA, GT, CT and GTC) as a core sequence with a Tm value range of 40.0–55.0. Screening was carried out with 40 primers. Out of which 15 primers, which gave clear banding pattern were used for confirmatory studies. Choice of nucleotides was based on the probability of its relative abundance in tea genome (Mondal, 2002). The reaction mixture included 0.1 mM of each dNTP, 150 nM primer, PCR master mix (MBI Fermentas, USA) supplied with enzyme and 50 ng DNA. Reaction mixture without DNA served as negative control. Amplification was carried out in a programmable peltier thermocycler PTC 200 (MJ Research, USA). Amplification protocol includes initial denaturation for 4 min at 94 ◦ C followed by 42 successive cycles of 40 s denaturation at 94 ◦ C, annealing for 2 min at respective Tm values of the selected primers and 1.45 min elongation at 72 ◦ C. Final elongation was performed for 8 min at 72 ◦ C. Amplicons were checked by separating on 1.4% agarose gel electrophoresis for 3 h at a constant temperature of 75 V with 1× TAE (TRIS base 4.84 g, glacial acetic acid 1.14 and 2.0 ml EDTA (0.5 M, pH 8.0) in 1000 ml of distilled water) running buffer. Finally the gel was stained with ethidium bromide (0.5 g/ml), visualized under ultraviolet rays and documented. The analysis was performed for all the samples at least three times with each selected primers. 2.3.3. Data analysis Scanned gel image was analyzed using special software Total Lab, version 3.1 (Amersham Bioscience, USA) for fragment length calibration. Amplified fragments were scored manually for the band presence (1) and absence (0). To characterize the ability of each primer to detect the polymorphic loci, the ratio of the number of polymorphic bands to the total number of bands revealed per primer reaction was computed to detect the percentage of polymorphism. From the binary data, the similarity coefficient values between the variants were derived based on the probability that amplified fragment from one variant will also be present in another with the Nei’s estimate (Nei and Li, 1979). Similarity based relationship between the variants were presented in the form of the dendrogram, developed following unweighted pair group method
151
with arithmetic mean algorithm (UPGMA) using SAHN cluster analysis of NTSYS-pc version 2.0 (Rohlf, 1992). Principal component analysis (PCO) was performed using statistical package for social sciences (SPSS) version 7.5 for Windows. First two components of PCO were used to plot the graph to determine the relationship among the variants.
3. Results and discussion Except the variants SE5, SE13 and SE14, all others possessed pale to yellowish green leaves while a very few variants were eliptic in their leaf shape and with smooth venation resembling the source plant, UPASI10 (Table 1). Except SE13, all other variants had acute leaf tip and majority of them had horizontal leaf posture. Besides SE5, leaves of all other variants were closely/double serrated. Majority of the variants had medium size leaves followed by large size and only one variant (SE7) had small leaves. In all the cases, texture of the mature leaves was found to be brittle. Although variants were derived from “China” hybrid, UPASI-10, a very few variants possessed unique “Chinery” character (elliptic, dark green, smooth, erect to semierect, small/medium size leaves) while others exhibited “Assam” characters (broad/ovate, pale to yellowish green, rugose, horizontal, medium to large leaves). However, no variant showed identical morphological characters aligning with the parent. Since the variants possessed both “Assam” and “China” characteristics, these could not be categorized into their respective groups. In the present study, among the primers tested, 15 primers which resulted in polymorphic amplifications with scorable PCR products (Table 2) were selected. Size of the polymorphic amplified fragments ranged from 450 to 1200 bp (Fig. 1). Results indicated that the ISSR has high resolution in the genotype identification, confirming the findings of Yang et al. (1996) and Esselman et al. (1999). Even though the morphological variations of the somaclones were negligible, the banding pattern of these primers on an average showed about 50% polymorphism (Table 2). Specificity and reproducibility of ISSR amplifications as reported by Bornet and Branchard (2001) was taken into account when designing the reactions. Amplified fragments of 1.2 and 0.65 kb were present in almost all accessions
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Table 1 Morphological variations in leaf characteristics of the somaclonal variants derived from UPASI-10 clone Variant number
SE1 SE2 SE3 SE4 SE5 SE6 SE7 SE8 SE9 SE10 SE11 SE12 SE13 SE14 SE15 UP-10 a
Leaf characteristics Colour
Shape
Venation
Tip
Posture
Serration
Size (cm2 )a
Yellowish Yellowish Yellowish Yellowish Dark Pale Yellowish Pale Pale Pale Pale Yellowish Dark Dark Pale Dark
Ovate Ovate Ovate Ovate Eliptic Eliptic Ovate Ovate Ovate Ovate Ovate Eliptic Ovate Eliptic Ovate Eliptic
Rugose Rugose Rugose Rugose Smooth Smooth Rugose Rugose Rugose Rugose Rugose Rugose Rugose Smooth Rugose Smooth
Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Acute Blunt Acute Acute Blunt
Horizontal Horizontal Horizontal Horizontal Semierect Semierect Horizontal Horizontal Horizontal Horizontal Horizontal Semierect Semierect Erect Semierect Erect
Close Close Close Close Distant Close Close Close Close Close Close Close Close Close Close Distant
35–50 35–50 35–50 35–50 >50 35–50 <25 35–50 35–50 35–50 >50 35–50 >50 >50 >50 >50
<25 (small); 35–50 (medium); >50 (large).
attempted whereas uniqueness of 0.5 kb was restricted to the variant, SE7. Pearson’s co-relation coefficient matrix revealed that SE1 had a significant similarity with SE2, SE6 and SE9 at 1.0% probability while it had significant relation with SE4 and SE14 at 5.0% level. SE15 had distinct similarities with SE2, SE8, SE11 and SE12 at 5.0% level. Similar trend was observed between SE9 and SE13 while SE8 and SE10 exhibited very high Table 2 Details of the ISSR primers used and polymorphism exhibited by analysis Code
Sequence
Polymorphism (%)
SGIP1 SGIP2 SGIP3 SGIP4 SGIP6 SGIP7 SGIP8 SGIP10 SGIP14 SGIP15 SGIP16 SGIP17 SGIP18 SGIP20 SGIP23
GAGAGAGAGAGAGAGAT GAGAGAGAGAGAGAGYG GAGAGAGAGAGAGAGARGY GAGAGAGAGAGAGAGARGY GAGAGAGAGAGAGAGAC ACACACACACACACACYT ACACACACACACACACYG GTGTGTGTGTGTGTGTYR CACACACACACACAAC CACACACACACACAGT CACACACACACACAAG CACACACACACACAGG GAGAGAGAGAGAGG GAGAGAGAGAGACC GAGGAGGAGGC
78.0 68.0 62.0 60.5 65.0 50.0 50.0 45.0 33.0 30.5 38.0 40.0 65.0 60.5 62.0
where R = A + G and Y = C + T.
coefficient value (1.00) which is significant at 1.0% probability. Out of 120 interactions attempted, only 9.2% revealed significant similarity at molecular level. This strengthens the existence of wide genetic variation among the somaclonal plants. Dendrogram was constructed based on simple matching coefficients, taking into account the presence or absence of the bands and ignoring their intensities. Individual similarity within the clusters was analyzed by hierarchical cluster and the principal component analysis segregated the 15 accessions into three distinct groups. Distance between final cluster centeroids varied from 2.257 to 3.317. Dendrogram provides an idea about the genetic relationship between the individuals and relation among the groups (Fig. 2).
Fig. 1. Electrophoretic pattern of amplified DNA using primer SGIP3. Lane 1–200 bp ladder (MBI Fermentas, USA); Lane 2–14: Samples SE1 to SE15.
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Fig. 2. Hierarchial cluster analysis of somaclonal variants.
Earlier, diversity among the germplasm resource has been demonstrated using morphological, physiological and biochemical characterization. In spite of asexual propagation, somatic embryogenesis is accompanied by a difference in morphologic characteristics allowing the selection for genetic improvement (Raj Kumar et al., 2001). Molecular markers allow the identification of variation at the genomic level (Bornet et al., 2003; Mondal and Chand, 2002) and permit detection of genetic mutation as in the case of tissue culture induced variations (Sanchez-Teyar et al., 2003). Development of molecular markers using AFLP in tea was first attempted by Rajsekaran (1997). Present study confirms that the ISSR analysis can be used effectively for the initial assessment of partitioning of genetic variation within plant species, where only a little genetic diversity information available. This corroborates with the findings of Devarumath et al. (2002). Results of present investigation confirmed the variability in genetic nature of the regenerants and also
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unique molecular markers in the somaclones. It is therefore envisaged that the genetic variation observed in the somaclonal variants are due to the chances of inadvertent manipulations that occur at the cellular and subcellular level and have been fixed in the genotypes contributing to the genetic uniqueness of the south Indian germplasm. Even though the true rate of somaclonal variation is difficult to ascertain due to the involvement of multiple genes, these can be used for plant improvement. Since somatic embryogenesis is a process controlled by multi-factors, the subtle interactions among the conditions to be satisfied for optimum results right from somatic embryo production up to germination, which differs from, clone to clone and different explants needs attention. Limited samples utilized in the study revealed the presence of genetic reservoir to explore the possibility to identify superior genotypes with correlation to the environment. Under evaluation, prototype which exhibits desired agronomic characters can be integrated in future breeding programme for conservation and commercial exploitation. Identification of unique regions can serve as a source of ISSR markers. They are suitable for the evaluation of genetic integrity and cultivar identification. Unique fragments thus obtained may be transformed into sequence characterized amplified region (SCAR) for marker aided selection.
Acknowledgements Authors are thankful to Dr. N. Muraleedharan, Director, UPASI Tea Research Foundation for the support extended during the course of study. The financial grant received from Department of Biotechnology (DBT), Government of India is gratefully acknowledged.
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