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

© 2010, INSInet Publication

Varietal Characterization of Tomato Cultivars Based on RAPD Markers

Vishwanath, K., Ananthararayanan, T.V., Pallavi, H.M., Ramegowda,1 2 1 1

Rajendra Prasad, S., Prasanna, K.P.R. Shailaja Hittalamani1 1 3

Department of seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore1

Division of plant genetic Recourses, Indian Institute of Horticultural Research, Hesaraghatta,2

Bangalore

Department of genetics and plant breeding, University of Agricultural Sciences, GKVK, Bangalore, 3

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

INTRODUCTION

The ability to distinguish and clearly identification

of varieties are fundamental for the operational aspects

in the seed trade. With 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 . With more and more players[1]

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

recognition of new laboratory tests (Electrophoresis,

RFLP, RAPD, AFLP, SSR, STMS, 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) . Although tomato being widely[3]

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.

MATERIALS AND METHODS

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 Meng et. al. (1998). Seeds

were kept for germination by following top of the

paper (TP) method in petri plates . 0.2 g of one week[5 ]

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

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 Technogies Inc.) wereT M

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

2KCl, 1.9 mM MgCl , 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 94 C (1 min), 35 C (1 min), 72 C (2 min.),0 0 0

then followed by final-extension at 72 C for 7 min.0

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.

Table 1: Cultivars of tomato used for varietal characterization

Sl No. Cultivar Developed institute /company Sl No. Cultivar Developed institute/ com pany

1. Arka Alok IIHR 13. Nandi UASB

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2. Arka Vikas IIHR 14. Sankranthi UASB

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3. Arka Ahuti IIHR 15. Vybhav UASB

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4. Arka Ashish IIHR 16. NS - 2535 Namdhari Seeds

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5. Arka Abha IIHR 17. M ruthyunjaya -2 Sasya Seeds

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6. Arka M egali IIHR 18. US -618 U.S. Agriseeds

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7. Arka Saurab IIHR 19. J.K. Desi J.K. Agrigenetics

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8. Arka Shresta IIHR 20. J.K. Asha J.K. Agrigenetics

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9. Arka Abijeet IIHR 21. Ronco Bejo Seeds

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10. Pusa Ruby IARI 22. A -32/63(Female) Indosem Seeds

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11. Pusa Early Dwarf IARI 23. 128/M 131(M ale) Indosem Seeds

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12. PKM -1 TNAU 24. M -03/868* (F1) Indosem Seeds

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.

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. While, 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

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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 primers and their level of polymorphism in different tomato cultivars

Sl. No. Primer Total No. of bands Total No. of polymorphic bands Per cent polym orphism

1 OPB-01 9 8 88.88

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2 OPB-10 9 9 100.0

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3 OPD-11 7 7 100.0

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4 OPD-12 4 3 75.00

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5 OPD-18 8 8 100.0

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6 OPD-19 13 13 100.0

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7 OPD-20 4 3 75.00

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8 OPC-02 11 10 90.90

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9 OPC-08 14 14 100.0

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10 OPC-18 7 6 85.71

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11 OPC-19 14 13 92.85

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Pooled 100 94 1008.34

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mean 9.09 8.54 89.39

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