1. 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.
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2. 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/ company
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. Mruthyunjaya -2 Sasya Seeds
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6. Arka Megali 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(Male) 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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3. 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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