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Research Journal of Agriculture and Biological Sciences, 6(6): 713-715, 2010 © 2010, INSInet Publication

Varietal Characterization of Tomato Cultivars Based on RAPD Markers 1

Vishwanath, K., 2 Ananthararayanan, T.V., 1 Pallavi, H.M., 1 Ramegowda, 1 Rajendra Prasad, S., 1 Prasanna, K.P.R. 3 Shailaja Hittalamani


Department of seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore 2 Division of plant genetic Recourses, Indian Institute of Horticultural Research, Hesaraghatta, Bangalore 3 Department of genetics and plant breeding, University of Agricultural Sciences, GKVK, Bangalore, Abstract: Present study characterized 24 popular tomato cultivars using eleven decamer primers. Eleven primers produced 100 bands out of which 89.39 were polymorphic and each cultivar had one or more novel sequences which were not found in other cultivar. These distinguish banding patterns can be successfully used as genetic markers for identification of these cultivars by using specific primer. Primers OPC-02, OPC-19, OPD-19, OPD-18 and OPC-08 were found to be the most effective in generating unique bands. All these primers produced total of 13 unique bands in 10 cultivars. It was observed that combination of OPB-10 either with OPC-19 or OPB-08 is sufficient to identify all the 24 tomato cultivars in the course of Varietal characterization. Key words: Tomato, Varietal Characterization, RAPD recognition of new laboratory tests (Electrophoresis, RFLP, RAPD, AFLP, SSR, STM S, SNP etc.) for varietal identification is considered with the aim to face the problem of new biotechnological varieties. New standard and rules are expected by the different Intergovernmental organizations involved (EEC, UPOV, ISTA, AOSA, OECD)[3 ] . Although tomato being widely studied crop a systematic studies in varietal characterization and test for hybridity is lacking especially for newly developed promising varieties and hybrids. Thus characterization of varieties and hybrids which are of wider acceptance by farming community need to be studied in order to regulate their genetic purity during their multiplication and seed quality evaluation.

INTRODUCTION The ability to distinguish and clearly identification of varieties are fundamental for the operational aspects in the seed trade. W ith the introduction of Indian legislation on “Protection of plant varieties and Farmer’s Rights”, the new varieties developed in agricultural and horticultural crops should be distinct from other existing varieties. The assessment of varietal identity and genetic purity is also a critical component of seed production and certification procedures. The success of high yielding cultivars depends upon the availability of seed with assured genetic purity. Therefore, it is important to assess the purity of commercial cultivars before they reach the farmers’ fields particularly in case of hybrids to exploit full potential of heterosis[1 ] . W ith more and more players involved in seed trade, it becomes imperative for the seed companies to supply quality seeds to the farmers in order to stand in the business. The traditional way to assess the genetic purity of seed is grow out test (GOT), where the crop is grown and rigorous observations on morphological traits of crop at different phases of growth is done with aid of morphological descriptors available for that variety. Normally the hybrid is similar to one of the parents therefore; use of morphological characteristics for its identification is difficult. Present rules for varietal identification at domestic, EEC and international level are examined in the framework of varietal registration, seed certification and plant breeding rights. The official

M ATERIALS AND M ETHODS Genomic DNA Extraction: Twenty four cultivars which are under cultivation in India were collected from different government institutions and private seed companies (table 1). DNA extraction from seedling was based on procedures outlined by M eng et. al. (1998). Seeds were kept for germination by following top of the paper (TP) method in petri plates [5 ] . 0.2 g of one week old seedlings was collected from incubated petri plates, surface sterilized with alcohol and washed with sterile distilled water. 0.2 g of seedling was homogenized in liquid nitrogen with 3.5 ml CTAB buffer and 14 mL of b-Mercaptoethanol and incubated at 65<C for 15

Corresponding Author: Vishwanath, K., Department of seed Science and Technology. 713

Res. J. Agric. & Biol. Sci., 6(6): 713-715, 2010 KCl, 1.9 mM MgCl 2, 0.1 mg/ml BSA, 0.1mM dNTP, 0.2 µl primer, 0.02-0.04 units/µL Taq DNA Polymerase, 0.1-4.0 ng/µL genomic DNA. Thermal Cycler was used and programmed for 40 cycles of 940 C (1 min), 35 0 C (1 min), 72 0 C (2 min.), then followed by final-extension at 72 0 C for 7 min. PCR products were used for electrophoresis on 1.4 per cent agarose gels stained with ethidium bromide at 50 volts for first 30 minutes then at 100 volts for next 3.5 hrs. After the completion of electrophoresis, the DNA profile was documented using BIO-VIS gel documentation unit. The bands of DNA fragments were scored as present (1) or absent (0). The amplification profiles for all primers were compared with each other and unique bands and characteristic profiles were identified using 0, 1 matrices.

minutes. Then 400ìL 24:1 of chloroform:isoamyl alcohol mixture was added and blended thoroughly for 5 min. After centrifugation (5 min, 13 000 rpm), aqueous layer was pipetted into a new eppendorf tube and an approximately equal volume of cold ethanol was added. After storage at -20 EC for 30-60 min, precipitated DNA was centrifuged, vacuum dried and finally stored in TE buffer. PCR Amplification: A set of sixty decamer primers (kit B,C and D; Operron T M Technogies Inc.) were preminary screened with pooled DNA of all 24 cultivars. Eleven of these which produced clear banding pattern with good polymorphism were then utilized in screening of all the accessions. The reaction mixture for amplification consisted of 11 mM Tris-HCl, 50 mM

Table 1: Cultivars of tom ato used for varietal characterization Sl N o. Cultivar D eveloped institute /com pany Sl N o. Cultivar D eveloped institute/ com pany 1. Arka Alok IIH R 13. N andi U ASB --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2. Arka Vikas IIH R 14. Sankranthi U ASB --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3. Arka Ahuti IIH R 15. Vybhav U ASB --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4. Arka Ashish IIH R 16. N S - 2535 N am dhari Seeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5. Arka Abha IIH R 17. M ruthyunjaya -2 Sasya Seeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6. Arka M egali IIH R 18. U S -618 U .S. Agriseeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7. Arka Saurab IIH R 19. J.K. D esi J.K. Agrigenetics --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8. Arka Shresta IIH R 20. J.K. Asha J.K. Agrigenetics --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------9. Arka Abijeet IIH R 21. Ronco Bejo Seeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10. Pusa Ruby IA RI 22. A -32/63(Fem ale) Indosem Seeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------11. Pusa Early D warf IA RI 23. 128/M 131(M ale) Indosem Seeds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------12. PKM -1 TN AU 24. M -03/868* (F1) Indosem Seeds

The highest numbers of bands (14) were recorded with primer OPC-19 and OPD-08 while the least (04) was obtained with primer OPD-12 and OPD-20. The number of bands using the same primer is not always identical among the cultivars but a few primers shared the same behavior. Primer OPD-02 produced similar banding pattern among 19 cultivars. W hile, only two cultivars showed similar banding pattern among each other with OPB-10. However, pooled profiles of all 11 primers were different among each other for 24 cultivars. The number of total bands also varied between cultivars, where highest number was 52 in cultivar US-618 and the least was 31 bands in Mruthyunjaya-2. Reports have found that a change of one base pair in the target sequence of the genome might result in completely different RAPD profile [6 ]. Since each 10 bp oligonucleotide primer only covers a

RESULTS AND DISCUSSION Among primers used eleven primers produced distinguishable polymorphic bands in each of the DNA samples studied. Out of total 100 bands produced 89.39 were polymorphic bands in the data set were sufficient to discriminate among all 24 cultivars (table 2); every cultivar had one or more novel sequences which was not found in other cultivar. These bands can be successfully used as genetic markers for identification of these cultivars. Primers OPC-02, OPC-19, OPD-19, OPD-18 and OPC-08 were found to be the most effective in generating unique bands. All these primers produced total of 13 unique bands in 10 cultivars. These unique bands are more useful for cultivar identification to differentiate specific cultivar from others. 714

Res. J. Agric. & Biol. Sci., 6(6): 713-715, 2010 very limited part of the genome, important differences located on non-amplified region could be missed. In the event of similar profiles obtained from two different cultivars using a particular primer could lead to a false conclusion that the two cultivars are same. Thus it is important use a series of primers for any sample to be tested. The combination of 11 RAPD primers profiles helped for clear identification of all the 24 cultivars studied. However, OPC-19, OPC-08 and OPB-10 are more useful than other primers since they generated high number of RAPD markers with higher polymorphism and consequently more cultivars were

identified. The primer OPB-10 produced characteristic profiles for 22 cultivars, OPC-19 for 8 cultivars, OPC-08 for 16 cultivars, OPC-18 for 11 cultivars, OPD-18 for 17 cultivars while, primer OPB-01, OPC-02, OPD-11 and OPD-12, OPD-20 and OPD-19 produced characteristic profiles for 12, 13, 12 and 3, 7 and 18, respectively. Among the primers used in this study, OPB-10 produced distinct band for 22 cultivars followed by OPC-19 (18 cultivars) and OPC-08 (16 cultivars). Therefore, these three primers can be used to identify all the 24 tomato cultivars in varietal characterization.

Table 2: Selected prim ers and their level of polym orphism in different tom ato cultivars Sl. N o. Prim er Total N o. of bands Total N o. of polym orphic bands Per cent p olym orphism 1 O PB-01 9 8 88.88 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2 O PB-10 9 9 100.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3 O PD -11 7 7 100.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4 O PD -12 4 3 75.00 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5 O PD -18 8 8 100.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6 O PD -19 13 13 100.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7 O PD -20 4 3 75.00 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8 O PC-02 11 10 90.90 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------9 O PC-08 14 14 100.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10 O PC-18 7 6 85.71 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------11 O PC-19 14 13 92.85 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Pooled 100 94 1008.34 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------m ean 9.09 8.54 89.39




AGARW AL, R.L., 1980. Seed Technology. Oxford and IBH Publishing Co., New Delhi. COOKE, R.J., 1984. The use of SDS polyacrylamide gel electrophoresis in varietal identification. Biochemical tests for cultivar identification. Proceedings of an ISTA symposium held at the National Institute of Agricultural Botany, Cambridge, UK, on 12-15. pp: 107-108. ZAULI, G. AND P.G. BIANCHI, 1993. Varietal identification in the framework of seed rules. Rules in force and expected changes. Sementi Elette, 39(3-4):5-8.




M ENG, X.D., Y.Y. W EI, H. MA, ZHANG, AND J.R. LI, 1998. Identification of wax gourd and chich-qua cultivars using RAPD markers. Acta Agriculurae shanghai, 12: 45-49. ISTA., 1996. International Rules for Seed Testing.(Suplliment) Seed Science & Technology, 29: 1-135. W illiams, J.G., J.A. Rafalski and S.Y. Tingey, 1992. Genetic analysis using RAPD markers. Methods in Enzymology, pp: 66.

16 varietal characterization of tomato cultivars based on rapd markers