Turkish Journal of Agriculture and Forestry

Volume 33 Number 4 Article 6

1-1-2009

Molecular characterization and genetic diversity analysis of citrus

cultivars by RAPD markers
M. N. R. BAIG

SAPNA GREWAL

SANTOSH DHILLON

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Recommended Citation
BAIG, M. N. R.; GREWAL, SAPNA; and DHILLON, SANTOSH (2009) "Molecular characterization and genetic
diversity analysis of citrus cultivars by RAPD markers," Turkish Journal of Agriculture and Forestry: Vol.
33: No. 4, Article 6. https://doi.org/10.3906/tar-0804-27
Available at: https://journals.tubitak.gov.tr/agriculture/vol33/iss4/6

This Article is brought to you for free and open access by TÜBİTAK Academic Journals. It has been accepted for
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 Turk J Agric For
33 (2009) 375-384
© TÜBİTAK
Research Article doi:10.3906/tar-0804-27

Molecular characterization and genetic diversity analysis of

citrus cultivars by RAPD markers

M.N.R. BAIG1, Sapna GREWAL2, Santosh DHILLON3,*

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University,

Wuhan, 430070, P. R. CHINA

2National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, 110012, New Delhi, INDIA

3
Department of Molecular Biology and Biotechnology, Department of Biochemistry
CCS Haryana Agricultural University, Hisar, Haryana 125004, INDIA

Received: 25.04.2008

Abstract: Random Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-
relationship among 18 citrus cultivars, including 13 species and 5 hybrids. Out of 40 decamer primers screened, 25 were
selected which produced 250 markers; of which 231 were polymorphic and some species or cultivar specific RAPD
markers. The Jaccard coefficient was used to calculate the genetic similarity. UPGMA was used to generate the
dendrogram which clearly separated Jatti-Khatti from all major clusters at a similarity coefficient of 0.61. The average
genetic similarity value observed across all the genotypes was 0.63, with the 2 sweet orange cultivars, Jaffa and Blood red,
showing maximum similarity (82%). The Jatti-Khatti and King Mandarin were found to be genetically most diverse. The
genetic variation between cultivars was quite high and revealed their different origins.

Key words: Citrus, RAPD, UPGMA, Dendrogram, Genetic diversity, PCR

Introduction Citrus is an economically important fruit crop,

Knowledge of genetic variation and genetic ranking almost as high as apples in world production
relationship among genotypes is an important and trade. The phylogeny and taxonomy of citrus fruit
consideration for classification, utilization of are complex, confusing and controversial due to the
germplasm resources and breeding. Without genetic heterogeneity of the genus, as well as its
determining the diversity reliably, it would not be polyembryonic nature and the long generation time
possible to identify molecular markers or qualitative needed to carry out selection and recombination
trait associations. Moreover the viability and purity of (Swingle, 1946; Nicolosi et al., 2000). Therefore,
rootstocks can be analyzed through the utilization of analysis of the genetic diversity of citrus fruit is
fingerprints based on molecular markers. This process crucial. To this end, DNA markers are being widely
can increase both quantity and quality of fruit used in studying polymorphism between species or in
production. populations. The application largely depends on the

* E-mail: santosh_dhillon@hau.ernet.in

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Molecular characterization and genetic diversity analysis of citrus cultivars by RAPD markers

type of markers employed, distribution of markers in has been attempted even with a large area under
the genome, type of loci they amplify, level of cultivation in India. In this study we used RAPD
polymorphism and reproducibility of products (Virk markers to characterize the citrus genotypes. The
et al., 2001; Fernandez et al., 2002). Techniques using objectives of the study were to achieve a better
Random Amplified Polymorphic DNA (RAPD) understanding of genetic variation and to investigate
markers are simple, fast, and sensitive. They require their inter-relationship.
no prior knowledge of the DNA sequence and can
amplify a large number of DNA fragments for
Materials and methods
reaction. The introduction of DNA markers based on
the polymerase chain reaction (PCR) technology has Plant material
led to the development of several novel genetic assays A total of 18 citrus genotypes (Table 1) used in this
that can be used for many purposes in plant genetic study were collected from the citrus orchard of
analysis such as cultivar identification and gene Chaudhary Charan Singh Haryana Agricultural
mapping. RAPD markers that result from the PCR University, Hisar, Haryana, India.
amplification of genomic DNA fragments using short
DNA isolation
oligonucleotide (usually 10-mers) of arbitrary
sequence as primers (William et al., 1990) provide a Total genomic DNA was isolated from fully
fast and easy approach for taxonomic classification expanded leaves using the CTAB
and cultivar-typing of fruit trees. This assay has the (hexadecyltrimethylammonium-bromide) method
advantage of being readily employed, requiring very (Murray and Thompson, 1980) with few
small amounts of genomic DNA, and eliminating the modifications. Briefly, 2 g of leaves were ground in
need for blotting and radio-active detection (Cipriani liquid nitrogen to a fine powder. The powder was
et al., 1996). For these reasons many fruit tree crops added to 6 mL of extraction buffer (100 mM Tris–HCl
have been successfully fingerprinted using RAPD pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2% (wv-1) CTAB,
-1
markers, e.g. grape (Huseyin and Sabitagaoglu, 2008), 2–mercaptoethanol 2% and 1% (wv ) PVP and
strawberry (Sugimoto et al., 2005), olive (Sanz-Cortes incubated at 65 °C for 30 min. The DNA was extracted
et al., 2001) and pineapple (Sripaoraya et al., 2001). with chloroform – octanol (24: 1). The DNA was
This technique, regardless of its sensitivity to reaction washed with 70% ethanol and dissolved in 100-400 μL
conditions, problems with repeatability, and of TE (10 mM Tris–HCl pH 8.0, 1 mM EDTA and 0.2
-1
mg mL RNase). The DNA concentration was
amplifying of non-homologous sequences, has been
determined spectrophotometrically at 260 nm. Stock
successfully used for the assessment of genetic
DNA samples were stored at –20 °C and diluted to
diversity in plants (Maria et al., 2008). Factors such as -1
20 ng uL when in use.
speed, efficiency and amenability to automation
make it one of the most suitable methods for PCR procedure
germplasm management with respect to estimating The RAPD primers were purchased from Operon
diversity, monitoring genetic erosion and removing Technologies Alameda, CA, USA. A total of 40
duplicates from germplasm collections (Khadari et al., decamer oligonucleotides of arbitrary sequence were
2003). tested for PCR amplification. The basic protocol
In citrus, RAPD markers have been used for reported by William et al. (1990) for PCR was
genetic diversity analysis (Abkenar and Ishhiki, 2003; performed in a total volume of 12.5 μL, containing 25
Mariniello et al., 2004; Campos et al., 2005; Novelli et ng of template DNA, 0.4 μM of single primer, 0.6 U
al., 2006; Shaaban et al., 2006; Shahsavar et al., 2007; Taq DNA polymerase (Bangalore Genei, India), 0.20
Hvarleva et al., 2008), chimeras (Sugawara et al., 2002) μM of each dNTP, 1.5 mM MgCl2, 10 mM Tris–HCl,
and phylogenetic analysis (Nicolosi et al., 2000). DNA and 50 mM KCl.
fingerprinting using PCR-based markers is very DNA amplification was carried out in a PTC-
important for breeding and taxonomy of citrus. No 10096V Thermocycler (MJ Research, Inc, USA) and
DNA-based markers approach to the study of citrus the thermal cycler conditions for PCR reactions were

376
 M.N.R. BAIG, S. GREWAL, S. DHILLON

Table 1. List of plant material used in this experiment.

S. No. Common Name Scientific Name (Swingle system)

Mandarins
1 Ponkan mandarin Citrus reticulata blanco
2 Shekwasha mandarin Citrus depressa hay.
3 King mandarin Citrus reticulata blanco
4 Satsuma mandarin Citrus unshui Marc
5 Pectinifera Citrus pectinifera cv Pectinifera
6 Cleopatra Citrus reshni cv Cleopatra
7 Kinnow hybrid (interspecific hybrids) Citrus nobilis ×
Citrus deliciousa
Sweet oranges
8 Jaffa Citrus sinensis cv jaffa
9 Blood Red Citrus sinensis cv Blood Red

Limes and Lemons

10 Rangpur lime Citrus limonia Osbeck
11 Bhadri lemon Citrus limon cv Bhadri lemon
12 Indian lime Citrus aurantifolia
13 Jatti Khatti Citrus jambhiri Lush cv Jatti Khatti
14 Jallundhari Citrus jambhiri Lush cv Jallundhari

Citranges and Hybrid

15 Troyer Citrus sinensis × P. trifoliata
16 Carrizo Citrus sinensis × P. trifoliate
17 C-639 Citrus sinensis × P. trifoliata
18 Swingle citrumello Citrus paradisi × P. trifoliata

an initial denaturation cycle of 1 min and 30 s at 94 °C (Rohlf, 1993). The dendrogram was constructed by
was followed by 45 cycles comprising 1 min at 94 °C, using a distance matrix using the unweighed pair-
1 min at 36 °C and 2 min at 72 °C. An additional cycle group method with arithmetic average (UPGMA)
of 7 min at 72 °C was used for final extension. sub-program of NTSYS-PC.
Amplification products were separated by
electrophoresis (8.3 V cm-1) in 1.5% agarose gels and
stained in ethidium bromide. A photographic record Results
was taken under UV illumination. Genetic polymorphism among citrus cultivars
Data analysis After screening 40 primers, 25 primers produced
Only clear and repeatable amplification products polymorphic and repeatable products. The banding
were scored as 1 for present bands and 0 for absent profile and polymorphism generated using one of the
ones. The specific bands useful for identifying species primers OPH-17 (Figure 1a) are shown. PCR
and cultivars were named with a primer number amplification of the DNA isolated from 18 selected
followed by the approximate size of the amplified citrus cultivars yielded a total of 250 amplified
fragment in base pairs. Polymorphism was calculated products, of which 231 were polymorphic and 19 were
based on the presence or absence of bands. The 0 or 1 monomorphic (Table 2). The total number of
data matrix was created and used to calculate the amplified DNA bands varied between 5 (primer OPA-
genetic distance and similarity using ‘Simqual’, a sub- 07) and 14 (primer OPA-17 and OPG-09) with the
program of the NTSYS-PC program (numerical average of 10 ± 0.51 bands per primer. The maximum
taxonomy and multivariate analysis system program) number of polymorphic bands (13) was obtained with

377
Molecular characterization and genetic diversity analysis of citrus cultivars by RAPD markers

primer OPG-09 and the minimum number (4) was

obtained with primer OPA-07. The polymorphism
percentage ranged from 72.7% (primer OPB-05) to as
high as 100% for 11 primers (OPQ-01, OPQ-07, OPA-
04, OPG-12, OPG-06, OPG-20, OPH-15, OPG-08,
OPA-17, OPA-16 and OPG-04). Average
a
polymorphism across all the 18 citrus cultivars was
found to be 92.4%. A significant co-relation (0.945, P
< 0.01) was observed between the total number of
bands and the number of polymorphic bands
amplified by the 25 primer pairs. Overall size of the
PCR amplified product bands ranged from 280 bp to
2800 bp. Polymorphism analysis was done for 7
mandarin cultivars and RAPD amplification within
these genotypes resulted in the total of 208 amplified
products of which 67 were monomorphic and 141
were polymorphic. Primer OPG–12 generated the
maximum number (10) of polymorphic alleles while
b
primer OPA–02 produced the minimum number (1)
of polymorphic alleles among these mandarin
genotypes. On average, 5.5 polymorphic bands were
produced per primer. The highest level of
polymorphism (100%) was detected by primers OPG-
12 and OPG-08, whereas primer OPA-02 detected the
lowest level of polymorphism (33.3%) among
mandarins.
A total of 112 amplified products were obtained
with DNA amplified from Jaffa and Blood Red. Out
c of these, 71 were monomorphic and only 41 were
polymorphic. Primer OPG-04 generated the
maximum number of polymorphic bands and showed
no polymorphism between these 2 genotypes. Two
primers OPG-01 and OPO-17 failed to amplify the
DNA. Polymorphism analysis was done for 5
genotypes belonging to the lime and lemon group
(Rangpur lime, Bhadri lemon, Indian lime, Jatti Khatti
and Jallundhari), resulting in a total of 190 amplified
Figure 1. a- Agarose gel showing RAPD amplification profile by products, of which 52 were monomorphic and 138
OPH-17 primer. M, 500 bp standard marker; 1) Ponkan
were polymorphic. Primer OPG-09 generated the
mandarin, 2) Satsuma mandarin, 3) King mandarin 4)
Shekwasha mandarin, 5) Pectinifera, 6) Cleopatra, 7) maximum number of polymorphic alleles (9) and
Kinnow, 8) Jallundhari, 9) Jatti khatti, 10) Indian lime, primer OPA-09 and OPQ-01 produced the minimum
11) Bhadri lemon 12) Rangpur lime, 13) Blood red, 14) number of polymorphic alleles (2) among lemon
Jaffa, 15) Troyer, 16) C-639, 17) Carrizo 18) Citrumello genotypes. The highest level of polymorphism was
b- Unique band for C-639 Citrange given by OPA-17 detected with primer OPG-12 (100%) whereas primer
(Genotypes are in the same order as Figure 1a) OPA-09 detected the least polymorphism (25%).
c- Unique band for Satsuma mandarin given by OPA- A total of 189 amplified products were obtained
07 (Genotypes are in the same order as Figure 1a)
with DNA amplified from 4 citrange genotypes

378
 M.N.R. BAIG, S. GREWAL, S. DHILLON

Table 2. DNA bands amplified and polymorphism generated in 18 citrus genotypes using 25 RAPD
markers.

S No Primer Code Total Bands Polymorphic Bands % of Polymorphism

1 OPA-02 7 6 85.70
2 OPG-05 7 6 85.70
3 OPQ-01 7 7 100.00
4 OPG-09 14 13 92.80
5 OPH-17 10 8 80.00
6 OPI-01 13 11 84.60
7 OPG-02 10 9 90.00
8 OPO-12 9 8 88.80
9 OPQ-07 11 11 100.00
10 OPA-04 10 10 100.00
11 OPG-12 10 10 100.00
12 OPO-06 9 9 100.00
13 OPA-07 5 4 80.00
14 OPG-20 10 10 100.00
15 OPB-05 11 8 72.70
16 OPB-14 11 10 90.00
17 OPH-15 8 8 100.00
18 OPG-08 7 7 100.00
19 OPO-17 9 9 100.00
20 OPA-09 13 12 92.30
21 OPH-20 13 12 92.30
22 OPA-16 7 7 100.00
23 OPG-04 14 14 100.00
24 OPA-17 14 12 85.70
25 OPB-18 11 10 90.90

Total 250 231

Mean 10 ± 0.51 9.24 ± 0.48 92.46 ± 1.61

(Troyer, Citrumello, C-639 and Carrizo) and Swingle were absent in sweet oranges. Primer OPQ-01 and
Citrumello. Out of these, 136 were polymorphic and OPB-05 amplified 1 allele unique to mandarins.
53 were monomorphic. Primer OPH-20 generated the Primer OPA-09 generated 1 specific allele for sweet
maximum number of polymorphic bands (11) oranges which was absent in mandarins. These
whereas each of the primers OPA-02, OPQ-01, OPG- primers thus were able to distinguish between
09, OPG-02, OPG-12, OPO-06, OPA-07, OPG-20, mandarin and sweet orange genotypes. Each of the
OPH-15, OPG-08, OPH-20 and OPA-16 generated primers OPB-05, OPB-18 and OPB-14 generated 1
only one monomorphic band. Primer OPQ-07 allele unique to mandarins, which was absent in lime
generated 100% polymorphism. and lemon genotypes. Primer OPA-17 generated a
Primers capable of differentiating genotypes specific allele which was present in lime and lemon
and absent in mandarin. Thus these 4 primers were
After analyzing all the data, 5 primers OPA-09, able to differentiate between citrus types. Primer OPI-
OPQ-01, OPG-09, OPB-05 and OPG-08 were found 01 resulted in the amplification of 1 allele for citranges
useful in differentiating between mandarin and sweet which was absent in mandarins. Two primers, OPA-
oranges. It was observed that 2 primers, OPA-09 and 09 and OPO-06, gave 1 allele each for mandarin
OPG-08 gave 2 unique alleles for mandarins which whereas primer OPG-05 amplified 2 alleles, one

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Molecular characterization and genetic diversity analysis of citrus cultivars by RAPD markers

unique to citranges and other to mandarins. Thus Unique bands in 18 citrus genotypes
these primers were found to differentiate between Some primers gave unique bands in a specific
mandarins and citranges. Five primers were found citrus genotype as shown in (Table 3). These primers
to be useful for distinguishing sweet oranges from produced a specific DNA band which distinguished
lemons. Each of the primers OPB-14, OPI-01, OPG- one genotype from the rest. Primer OPA-17 generated
12 and OPQ-07 generated 1 specific allele which was a unique allele for C-639 (Figure 1b). Primers OPG-
present in sweet oranges and absent in lemons. Each 12, OPA-07 and OPQ-01 generated a unique band for
of the primers OPQ-07 and OPO-06 gave 1 specific the Satsuma mandarin (Figure 1c). Primer OPG-20
allele which was absent in sweet oranges and present generated a unique allele for Troyer, whereas primer
in lemons. Therefore, these primers were able to OPG-09 gave it for kinnow. Primer OPO-17 and
distinguish between sweet oranges and lemons. Four OPB-14 generated respectively 2 and 3 specific bands
primers OPB-14, OPB-05, OPA-17 and OPA-04 for Jatti Khatti.
were found to distinguish between citranges and
sweet oranges. Three alleles, each generated by Genetic relationship between citrus genotypes
primers OPB-14, OPA-17 and OPA-04 were present Data of RAPD markers scanned from the 18
in sweet orange and absent in any genotypes genotypes with 25 reproducible primers was used to
belonging to citranges, while primer OPB-05 generate similarity coefficients. A maximum
amplified one allele present in citranges only. Primer similarity value of 0.82 was observed between 2
OPG-20 generated a unique allele for Troyer; Primer cultivars of sweet orange Jaffa and Blood Red, whereas
OPA-17 generated a unique allele for C-639, Jatti Khatti and King Mandarin were found to be
whereas primer OPG-09 generated it for kinnow. genetically most diverse (0.44). Average similarity
Primers OPO-17 and OPB-14 gave 2 and 3 specific value observed across all genotypes was 0.63. The 2
bands respectively for Jatti Khatti which were absent lemon genotypes followed by the Jatti Khatti were
in citranges, while primer OPB-05 gave 1 allele diverse having similarity value of 0.50. Troyer, a
specific for citranges which was absent in sweet Citrange showed its similarity with Cleopatra (0.72)
oranges. After analyzing the data generated by 25 a genotype of mandarin group. Rangpur lime showed
primers, it was concluded that 7 primers closeness with the sweet orange having similarity
differentiated citranges from lemon. Primer OPO- value of 0.74.
17 generated 3 specific alleles which were present in The cluster tree analysis (Figure 2) showed that
citranges and absent in lemon. Each of the primers the citrus genotypes were broadly divided into 3 main
OPH-20, OPG-04, OPA-09 and OPI-01 generated 1 groups representing mandarins, sweet oranges and
specific allele for citranges, which was absent in lemon. However, Jatti Khatti was found to be quite
lemon. Primer OPB-14 generated 2 alleles which divergent and did not fall in any of the major clusters.
were presents in lemon but absent in citranges as It separated from the above groups at a similarity
well as Swingle citrumello. coefficient of 0.61; 3 groups were further divided into

Table 3. List of RAPD primers producing unique alleles for specific citrus genotype.

S No Primers Number of alleles Genotypes

1 OPG-12 1 Satsuma mandarin

2 OPA-07 1 Satsuma mandarin
3 OPQ-01 1 Satsuma mandarin
4 OPG-20 1 Troyer
5 OPG-09 1 Kinnow
6 OPG-02 1 Box Orange
7 OPA-17 1 C-639

380
 M.N.R. BAIG, S. GREWAL, S. DHILLON

Citrumello

Ponkan-mandarin

Carrizo

Shekwasha

C-639

King

Satsuma

Jaffa

Blood-red

R.lime

Jallundhari

Bhandri-lemon

Indian-lime

Kinnow

Pectinifera

Cleopetra

Troyer

Jatti-khatti

0.61 0.66 0.72 0.77 0.82

Coefficient

Figure 2. Dendrogram illustrating genetic relationships among 18 citrus genotypes,

generated by UPGMA cluster tree analysis.

various sub-groups according to similarity between maximum similarity (0.82), Rangpur lime merged
them. Three main groups branched at similarity value with these at a similarity coefficient of 0.75.
of 0.65 into 2 major clusters. First major cluster Jallundhari entered the above sub-group at similarity
consisted of 2 sub-clusters, in which a high genetic value of 0.71.The second major cluster consisted of 2
similarity was observed between Citrumello and sub-groups. Two genotypes namely, Bhadri lemon
Ponkan mandarin (78%). Carrizo merged into this and Indian lime which were close to each other and
sub-group at different similarity coefficient value of showed similarity coefficient of 0.76 represented one
0.73. Shekwasha mandarin and C-639 had similarity subgroup the other subgroup included Kinnow and
coefficient of 0.78 which merged with the above Pectinifera, 2 genotypes of mandarin which showed
cluster at similarity value of 0.72. Two mandarin similarity coefficient of 0.73 and merged with Bhadri
genotypes Ponkan mandarin and Satsuma mandarin lemon sub-group at similarity value of 0.71. Cleopatra
showed 77% closeness and merged with the above and Troyer showed similarity coefficient of 0.72 and
major sub-group at similarity coefficient of 0.71. Jaffa merged with the above main sub-cluster at similarity
and Blood Red, 2 sweet orange cultivars showed coefficient of 0.67.

381
Molecular characterization and genetic diversity analysis of citrus cultivars by RAPD markers

Discussion al. (2000) also found few unique fragments for C.

Experiments with Citrus cultivars have indica (7) and C. succosa (1) of mandarin group.
demonstrated the potential of RAPD markers as a Twenty-five primers produced 219 alleles among
rapid, reproducible and useful method for 5 genotypes of lime and lemon group, of which 138
distinguishing among different cultivars and were polymorphic. Between 4 and 10 clear and
clustering genotypes in the Citrus species. A total of repeatable bands were obtained for each of these
250 amplified products were obtained using 25 RAPD primers, which is similar to the results (1 to 9 alleles)
primers which is quite consistent with that 212 obtained by Abkenar and Isshiki (2003) in 31 acid
RAPDs generated by using 23 primers reported by citrus species and cultivars. A search for unique bands
Coletta Filho et al. (1998) among 25 mandarin was made for all the species tested and it was found
accessions. Average polymorphism across all 18 citrus that primers OPO-17 and OPB-14 gave 2 and 3
genotypes was 86.35%. The high level of
unique bands respectively for Jatti-Khatti genotype.
polymorphism obtained in this study indicates greater
Nicolosi et al. (2000) also reported 2 unique bands in
genetic diversity between the genotypes selected
C. limonia and 1 unique band in C. aurantium in a
which includes genotypes that belong to 4 major
categories- Mandarins, Sweet oranges, Limes and study on phylogeny and genetic origin of Citrus.
lemons and Citranges including 1 hybrid. A Likewise, Abkenar and Isshiki (2003) also reported
significant correlation (0.945, P < 0.01) was obtained some unique RAPD fragments among acid citrus
between the total number of bands and number of species and cultivars, primers A12, A18, A20, B08,
polymorphic bands amplified by 25 RAPD primers. B11, E01 gave 1 unique band for ‘Myrtle-leaf orange,
The RAPD amplification within 7 genotypes of primer A12 gave 1 unique band for ‘China daidai’.
mandarins resulted in 208 amplified products. Across all 18 genotypes, Jaffa and blood Red, 2
Machado et al. (1996) obtained 111 amplification cultivars of sweet oranges were found to be most
products using 21 random primers among 39 closely related (0.82) whereas King mandarin and
Mediterranean genotypes. Out of 208, 131 were Jatti-Khatti showed the similarity coefficient value
polymorphic and 77 were monomorphic with (0.44), indicating their unrelatedness. Fang et al.
polymorphic percentage 66.65%.
(1998) reported similar results while working on 41
The number of DNA fragments amplified ranged samples of 33 cultivars, belonging to 3 groups i.e.
from 5 to 14 depending upon the primer and the Valencia, blood and navel, based on fruit traits. All of
DNA sample, which is quite consistent with 3-15 these cultivars had almost the same DNA fingerprints
amplified fragments, reported by Nicolosi et al. and isozymes and RFLP profiles as well. This further
(2000) among 36 accessions belonging to Pummelo, supports the view that a majority of sweet orange
Citron and mandarin genotypes. High similarity cultivars divided from a single ancestor by mutation.
coefficient value of 0.82 was found between two
cultivars of sweet oranges, similar to the result The citrus fingerprint data generated has revealed
obtained by previous studies (0.82-0.88) done by many genotype specific primers (Table 3) which have
Gulsen and Roose (2001), among 95 citrus potential to be used further in cultivar identification
accessions and for ISSR, SSR and isozymes analysis. and classification. These results can be further used to
Nine of 25 primers used were not able to manipulate genetic determinants of horticulturally
differentiate and gave monomorphic bands. The important traits and to characterize the basis of
high similarity (0.82) between the 2 cultivars of productivity of citrus. Though RAPD markers proved
sweet oranges is probably due to their common to be useful for germplasm characterization and
origin by mutation (Elisiario et al., 1999). diversity analysis in citrus in the present study, use of
The polymorphic RAPD markers present only in 1 other molecular marker techniques such as AFLP,
genotype were considered as unique allele for that ISSR and SSRs should be considered for finer
genotype. Primer OPG-12, OPA-07, OPQ-01 gave one molecular analysis of genotypes and to solve
unique allele each for Satsuma mandarin. Nicolosi et discrepancies left unresolved by RAPDs.

382
 M.N.R. BAIG, S. GREWAL, S. DHILLON

In this study we confirmed that RAPD markers, as accessions is required, the use of other molecular
a fast and simple technique, can detect enough marker techniques such as Amplification fragment
polymorphism to differentiate between citrus length polymorphisms (AFLPs) (Campos et al., 2005)
cultivars and to understand their inter-relationships, or inter-simple sequence repeat markers (ISSRs)
However, if a finer molecular analysis of Jatti Khatti (Shahsavar et al., 2007) should be considered.

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