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3秒自动关闭窗口Services on DemandArticleIndicatorsCited by SciELO Related linksSimilars in
SciELO ShareOn-line version ISSN Rev. Ciênc. Agron. vol.43 no.2 Fortaleza Apr./June 2012 http://dx.doi.org/10.-00014
FITOTECNIA
Molecular characterisation and similarity relationships among
iranian basil (Ocimum basilicum L.) accessions using inter simple
sequence repeat markers
Caracteriza&&o molecular de acessos de Ocimum
basilicum L. por meio de marcadores ISSR
Mohammad AghaeiI; Reza DarvishzadehII,; Abbas HassaniIII
IDepartment of Horticulture, Urmia University,
Urmia, Iran,
IIInstitute of Biotechnology,
Urmia University, Urmia, Iran,
IIIDepartment of Horticulture, Urmia University, Urmia, Iran,
The study of genetic relationships is
a prerequisite for plant breeding activities as well as for conservation of
genetic resources. In the present study, genetic diversity among 50 Iranian
basil (Ocimum basilicum L.) accessions was determined using inter simple
sequence repeat (ISSR) markers. Thirty-eight alleles were generated at 12 ISSR
loci. The number of alleles per locus ranged from 1 to 5 with an average of
3.17. The maximum number of alleles was observed at the A7, 818, 825 and 849
loci, and their size ranged from 300 to 2500 bp. A similarity matrix based on
Jaccard's coefficient for all 50 basil accessions gave values from 1.00-0.60.
The maximum similarity (1.00) was observed between the "Urmia" and "Shahr-e-Rey
II" accessions as well as between the "Urmia" and "Qazvin II" accessions. The
lowest similarity (0.60) was observed between the "Tuyserkan I" and "Gom II"
accessions. The unweighted pair- group method using arithmetique average UPGMA
clustering algorithm classified the studied accessions into three distinct
groups. All of the basil accessions, with the exception of "Babol III", "Ahvaz
II", "Yazd II" and "Ardebil I", were placed in groups I and II. Leaf colour was
a specific characteristic that influenced the clustering of Iranian basil
accessions. Because of this relationship, the results of the principal
coordinate analysis (PCoA) approximately corresponded to those obtained through
cluster analysis. Our results revealed that the geographical distribution of
genotypes could not be used as a basis for crossing parents to obtain high
heterosis, and therefore, it must be carried out by genetic studies.
Key words:
Basil. Cluster analysis. Molecular markers. Plant genetic diversity.
estudo das rela&&es gen&ticas & um pr&-requisito para atividades em reprodu&&o
de plantas assim como para conserva&&o de recursos gen&ticos. Neste trabalho a
diversidade gen&tica entre 50 acessos de Manejeric&o
Iraniano (Ocimum basilicum L.) foram determinadas usando marcadores de
Seq&&ncia Simples Repetida Interna (ISSR). Trinta e
oito alelos foram identificados utilizando-se 12 locos ISSR. O n&mero de alelos
por l&cus variou de 1 a 5 com uma m&dia de 3,17. O m&ximo n&mero de alelos foi
observado em A7; 818; 825 e 849 locos, e seus tamanhos variaram de 300 a 2500
bp. Uma matriz de similaridade baseada no coeficiente de Jaccard para todas as
50 acessos obteve valores de 1,00-0,60. A m&xima similaridade (1.00) foi
observada nos acessos "Urmia" e "Shahr-e-Rey II" assim
como entre os acessos "Urmia" e "Qazvin II". A menor
similaridade (0,60) foi observada entre os acessos
"Tuyserkan I" e "Gom II". O algoritmo de an&lise de agrupamentos foi o m&todo
da dist&ncia m&dia usando a m&dia aritm&tica (UPGMA) que classificou os acessos estudadas em tr&s distintos grupos. Todos os acessos do manjeric&o com exce&&o de "Babol III", "Ahvaz II", "Yazd II" e
"Ardebil I", foram localizadas nos grupos I e II. A cor da folha foi uma
caracter&stica espec&fica que influenciou o agrupamento dos acessos do Manjeric&o Iraniano. Devido a esta rela&&o, os resultados da
an&lise de coordenada principal (PCoA) aproximadamente corresponde & aquela
obtida pela an&lise de agrupamentos. Os resultados revelam que a distribui&&o
geogr&fica dos gen&tipos n&o pode ser usada como base para parentais cruzados
com alta heterosis e, portanto devem ser conduzidos estudos gen&ticos para tais
Palavras-chave: Manjeric&o. An&lise de agrupamentos. Marcadores moleculares.
Diversidade gen&tica de plantas.
Introduction
Ocimum basilicum L., known as common basil, is an annual aromatic
herb belonging to the Lamiaceae family. It is a dicotyledonous and diploid
plant with 2n = 48 chromosomes (PRAKASH, 1990). Among Ocimum spp.,
common basil is the most economic species to grow, and it is cultivated all
around the world (MAROTTI et al., 1996). Dried leaves of basil are used
to flavour many products in the food industry. Its essential oil is used in the
perfume and cologne, cosmetics, health, pharmaceutical and food industries
(HARISARANRAJ et al., 2008).
Based on Khosla's (1995) reports, the centre of basil
diversity is in the tropical and subtropical regions of Africa, Asia and South
America. Different chemotypes (plants of the same species that are chemically
different but otherwise indistinguishable) and morphotypes have been reported
in this species (MAROTTI et
al., 1996; PRAKASH, 1990).
To be most efficiently
managed and effectively utilised, germplasm collections must be well
characterised. In most cases, the identification of cultivars, lines and
hybrids is based on morphological traits. However, the number of these they are unstable and they do not always enable one to distinguish
between closely related accessions or cultivars (KONAREV et al., 2000). Cultivar identification based on phenotypic traits is often
influenced by the environment, making classification difficult.
On the other hand, basil taxonomy is complex because of
the occurrence of inter-/intra-specific hybridisation and morphological
similitude (DE MASI et al.,
2006). Sometimes, taxonomists defined a number of designations as synonymous
and confused different varieties as homonyms.
The emergence of
PCR-based molecular markers, such as randomly amplified polymorphic DNA
(RAPDs), microsatellite or simple sequence repeats (SSRs) and amplified
fragment length polymorphisms (AFLPs) has created the opportunity for
fine-scale genetic characterisation of germplasm collections (POWELL et al., 1996; SEMAGN et al.,
2006). The major limitations of these methods are the low reproducibility of
RAPD, the high cost of AFLP and the need to know the flanking sequences to
develop species-specific primers for SSR polymorphism.
The use of inter
simple sequence repeat (ISSR-PCR) markers is a molecular technique that
overcomes most of these limitations (PRADEEP REDDY et al.,
2002). It is rapidly being adopted by the research community in various fields
of plant improvement (GODWIN et al., 1997; RATNAPARKHE et al., 1998). This
technique is useful in the areas of genetic diversity, phylogenetic studies,
gene tagging, genome mapping and evolutionary biology and in a wide range of
crop species. ISSR techniques are nearly identical to RAPD techniques except
that ISSR primer sequences are designed from microsatellite regions, and the
annealing temperatures used are higher than those used for RAPD markers. Based on the published, unpublished and
in-progress studies that have been conducted using ISSR markers, it is clear
that ISSR markers have great potential for studies of natural populations
reviewed in (WOLFE et
al., 1998). ISSRs differentiate
mostly as dominant markers following simple Mendelian inheritance (PRADEEP
REDDY et al., 2002; WANG et al.,
1998). These markers, due to their reproducibility, are becoming more popular
and easier to use than other markers such as RAPD and AFLP (CHENNAOUI-KOURDA et al., 2007).
Previous researchers utilised RAPD-PCR for the identification
and characterisation of basil cultivars (DE MASI et al., 2006; HARISARANRAJ et al., 2008; IBTISAM, 2008;
VIEIRA et al.,
2003). Harisaranraj et al. (2008) investigated the relationship between seven species of basil using 15
RAPD primers. They found close relation between O. basilicum and O.
tenuiflorum.
The study of genetic relationships is a prerequisite for
plant breeding activities as well as for conservation of genetic resources. In
the present study, we report the genetic diversity among Iranian native basil
accessions originating from different regions based on ISSR markers.
Materials and methods
material and DNA extraction
Fifty basil accessions collected from different geographical regions of
Iran ( and ) were investigated. Basil accessions were grown in
controlled conditions, and single-leaf samples from 2-week-old seedlings
corresponding to 15 plants of each accession were taken for bulk DNA (DE MASI
et al., 2006), frozen in liquid nitrogen and then maintained at -80 &C. Samples
were ground to powder in the presence of liquid nitrogen. Genomic DNA was
extracted using a CTAB-based method (DE MASI et al.,
2006). DNA concentration was measured
at 260 nm in a spectrophotometer, and the quality of DNA was checked by running
of 1 µl of genomic DNA on an 0.8% agarose gel prepared in 0.5X TBE buffer (45
mM Tris base, 45 mM boric acid, 1 mM EDTA pH 8.0).
Polymerase chain reaction and data analysis
Polymerase chain reaction (PCR) was performed in a 25-&l volumes containing 12.5 &l of PCR master mix [200 mM Tris–HCl pH 8.55, 160 mM
(NH4)2SO4 0.1% (v/v), 3.0
mM MgCl2, 0.4 mM of
dNTPs, 1.0 U of Taq DNA
polymerase](CinnaGen Inc., Tehran, Iran), 2 &M ISSR primer (CinnaGen Inc., Tehran, Iran), 50 ng of
genomic DNA and ddH2O. DNA amplifictions were performed using a GeneAmp PCR System 9700
Thermocycler
(Perkin Elmer, Applied Biosystems, USA) programmed for a preliminary
step of 3 min at 95 &C, followed by 35 cycles of 93 &C for 30 s, 35-58 &C
(depending on primer sequence) for 45 s and 72 &C for 45 s. A final extension
step of 10 min at 72 &C was performed. The reaction products were then mixed
with an equal volume of formamide dye [98% (v/v) formamide, 10 mM EDTA, 0.05%
bromophenol blue and 0.05% xylene cyanol], resolved in a 1.5% (w/v) agarose gel
in 0.5X TBE, visualised with ethidium bromide (1.0 &gml-1) and photographed under UV light.
The PCR amplification products were scored
for the presence (1) or absence (0) of each band marker
across all 50 accessions, and the data were used to
construct a binary data matrix. Different methods
were used for constructing similarity matrices and
dendrograms. The efficiency of clustering algorithms
and their goodness of fit were determined based on the
cophenetic correlation coefficient. In addition to this
cluster analysis, a principal coordinate analysis (PCoA)
was used to confirm the results of the cluster analysis.
PCoA is a low-dimensional graphical plot that is used
to depict the relationships among the genotypes studied.
Data analyses were performed using NTSYS-pc version
2.11 software (ROHLF, 1998).
Results and discussion
Thirty-eight alleles were generated at 12 ISSR loci.
The number of alleles per locus ranged from one to five,
with an average of 3.17 (). The maximum number
of alleles was observed at the 'A7', '818', '825' and '849'
loci (), and their size ranged from 300 to 2500 bp.
Different methods were used for constructing
similarity matrices and dendrograms (). The
cophenetic correlation, a measure of the correlation
between the similarity represented on the dendrograms
and the actual degree of similarity, was calculated
for each dendrogram (). Among the different
methods, the highest value (r = 0.63; P ?? 0.05) was
observed for UPGMA based on Jaccard's coefficient
(). Therefore, the dendrogram constructed based on this method was used for depicting the
genetic diversity of accessions (). A similarity
matrix based on Jaccard's coefficient for all 50 basil
accessions gave values from 1.00-0.60. The average
pairwise genetic similarity was 0.73. The maximum similarity (1.00) was observed between the "Urmia"
and "Shahr-e-Rey II" accessions as well as between
the "Urmia" and "Qazvin II" accessions. The lowest
similarity (0.60) was observed between the "Tuyserkan
I" and "Gom II" accessions.
The UPGMA clustering algorithm classified
the studied accessions into three clusters (). The
first, second and third groups comprised 46%, 46%
and 8% of accessions, respectively (). All of the
basil accessions, with exception of "Babol III", "Ahvaz
II", "Yazd II" and "Ardebil I," were placed in group I
or II. In group I, the highest similarity value (1.00) was
observed between the "Urmia" and "Shahr-e-Rey II"
accessions as well as between "Urmia" and "Qazvin
II" accessions. Group I was further divided into two
subgroups. In group II, the highest similarity value
(0.96) was found between the "Babol II" and "Ahvaz
I" accessions. Group II was further divided into three
subgroups. In group III, the highest similarity value
(0.82) was found between the "Babol III" and "Ahvaz
II" accessions. Compared to group II and III, the pairwise similarity in group I was higher. In other
words, the accessions in group I were clustered together
at a higher similarity value.
Generally, accessions with purple leaves were
located in the first group (). Interestingly, the
accessions with green leaves located in this group
were not completely green-leaved but also included
accessions that have green leaves with purple veins
(). Accessions with green leaves were located
in the second group (). Our results showed that
accessions with purple and green leaves from the same
location were classified into two separate groups. For
instance, we studied two accessions from "Shahr-erey",
one with purple leaves and another with green
leaves. The purple-leaved accession was located in
group I, and the green-leaved accession was classified into group II. This was also seen for accessions from
"Kordestan", "Kerman", "Kermanshah", "Birjand",
"Dezful" and "Isfahan". De Masi et al. (2006) studied
the genetic similarity among 12 basil genotypes with
RAPD markers and identified two main groups, the
first of which consisted of purple-leaf cultivars. Purpleleaf
genotypes are used in food industries to aromatise
The clustering of a large number of genotypes in
two groups (I and II) revealed low genetic variability
that may be due to (a) the autogamous propagation
of cultivated basil, which reduces genetic variability
and (b) the biodiversity reduction in cultivars used in
agriculture, as growers use fewer genotypes for seed
production (DARRAH, 1980). In accordance with the
above, in northwest Iran (Azerbaijan and Maragheh),
the majority of cultivated genotypes have purple
leaves, whereas in the centre (Tehran and its suburbs),
green-leaved basil known as "Tehrani" basil is grown.
It was obvious that the genetic relationships
among the studied accessions did not have a strong
tendency to associate with their geographic origins.
Murthy and Arunachalam (1966) showed that genetic
drift and selection in different environments can cause
greater diversity among genotypes than geographic
distance. Therefore, selection of parental material in
breeding programmes simply based on geographic
diversity may not be rewarding. One possible reason for
the genetic similarity among germplasm from different
regions is that the materials might have originally been
introduced from the same region. Crossing between
clusters with maximum inter-cluster distance may
be result in high heterosis. It is well documented
that crosses between unrelated, and consequently
genetically distant parents, show greater hybrid vigour
than crosses between closely related genotypes (REIF et al., 2007; SOLOMON et al., 2007).
Principal coordinate analysis (PCoA) showed that
the first three PCs explained 38.14% of the cumulative
variation. These three PCs were then used to design
a principal coordinate plot to identify the diversity
pattern of studied genotypes (). The principal
coordinate plot showed the close genetic relationship
among the "Tabriz"(1), "Shahr-e-Rey II"(4), "Kerman
II"(14) and "Shiraz III"(40) accessions and among the
"Kordestan III"(9), "Kermanshah II"(16) and "Dezful
II"(20) accessions, which had also been observed
in cluster analysis (). Our results showed that
the PCoA data approximately corresponded to those
obtained through cluster analysis. This is in agreement
with other findings (DARVISHZADEH et al., 2010;
KUMAR et al., 2009; SORKHE et al., 2007).
Conclusions
1. In the present study, we characterised and identified
the genetic relationships of 50 Iranian basil
accessions using ISSR markers for the first time.
We found relatively acceptable genetic diversity
within available basil accessions. The studied
accessions were classified into three distinct groups.
Some accessions share specific characteristics that
2. Assessment of the genetic variability within plant
species is a prerequisite for plant breeding programs
and has an important role in the conservation of
plant genetic resources. It is particularly useful in
the characterisation of individual accessions and
cultivars, in detecting duplications of genetic material
in germplasm collections, and as a general guide in
the choice of parents for hybrid breeding programs.
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Recebido para publica&&o em 06/02/2011; aprovado
em 10/10/2011
Autor para correspond&ncia
Part of the Masters Thesis of the first author
presented at the Graduate Program in Medicinal Plant Production from the Urmia
University, Urmia, Iran