Molecular Biodiversity of Selected Mango Cultivars Based on DNA Sequences of Internal Transcribed Spacer Region
Shahkila Mohd. Arif
Azman Abd. Samad
The mango (Mangifer indica L.) is an important species
of the family Anacardiaceae and is one of the most important crops cultivated
commercially in many parts of the world. Hence, a better understanding of the
phylogeny in this species is crucial as it is the basis knowledge of improving
its genetic resources which is beneficial for breeding programs. Phylogenetic
relationships among 13 mango cultivars from Indonesia, Malaysia and Taiwan were
carried out by comparing DNA sequence data sets derived from the Internal Transcribed
Spacer (ITS) region pf nuclear ribosomal DNA (nrDNA). Analysis using parsimony
method showed that the cultivars were classified into three major groups. The
first group composed almost Malaysian cultivars although with low bootstrap
value, the second group consisted of mainly Taiwan cultivars and the last group
included mostly Indonesia one. The results indicated that some cultivars have
a close relationships with each other even it is originated from different countries.
With regards to the relationship among these cultivars, this gives better insight
for generating new cultivar.
Received: March 10, 2013;
Accepted: March 16, 2013;
Published: April 16, 2013
Mango (Mangifera indica L.) is one of the most prominent members of
the family Anacardiaceae that is grown commercially in many parts of the world,
particularly tropical and subtropical countries (Rajwana
et al., 2008, 2011). This is due to several
characteristics possess by this climacteric fruit such as stupendous quality
(attractive appearance, great taste and adorable flavour) and excellent nutritional
composition (diverse amount of fibre, minerals, vitamins and various antioxidant
Its genotypes are classified into two categories, namely monoembryonic which
is mostly from subtropical regions (Indian types) and polyembryonic which is
from tropical regions (Southeast Asian types) (Luo et
al., 2011). Over 4000 years ago, mango is originated from India and
Burma and has been spread to Eastern Asia, Eastern Africa and Malaysia (Luo
et al., 2011). Malaysia is among countries that cultivates mango
for commercial production in Southeast Asia. There are several cultivars of
mango grown in Malaysia, including Chokanan (MA 224), Harumanis (MA 128), Maha
65 (MA 165) and Nam Doc Mai (223).
In terms of production, it is ranked as the fifth major fruit grown throughout
the world after apples, bananas, grapes and oranges and the second most vital
tropical fruit (Bally et al., 2009). Generally,
there are numbers of mango cultivars that arise from naturally occurred open-pollinated
seedlings (Iyer and Degani, 1997). As for commercially
grown cultivars, seedling selections with respect to characteristics like colour,
flavour, size and taste has been done and subsequently, these cultivars are
propagated and cultivated in a huge area (Ravishankar et
According to Ravishankar et al. (2004), information
with regards to genetic diversity is one of the most crucial parts in designing
breeding programmes in order to maintain mango production. Furthermore, another
factor that contributes to the effective production of hybrids is the well-defined
phylogenetic relationship between the parents (Nishiyama
et al., 2006). However, the phylogenetic relationship among numbers
of mango cultivars is poorly studied and remains unclear as there are numbers
of mango cultivars and sometimes different or synonym names are used referring
to a single cultivar. Additionally, the morphological based approach has led
to confusion on cultivars identification. Thus, creating a conflict in classification
of mango cultivars.
Rapid advances of molecular techniques such as Polymerase Chain Reaction (PCR)
bring an impact for the use of DNA sequences in molecular phylogenetic studies
(Topik et al., 2005). DNA sequencing-based method
is very reliable due to the reproducibility of the results and the scope of
application. Duneman (1994) has mentioned that DNA-based
markers are very advantageous in characterizing and studying genetics similarities
among cultivars, land races and varieties.
Now-a-days, various DNA markers namely Restriction Fragment Length Polymorphism
(RFLP) (Ravishankar et al., 2004), Random Amplified
Polymorphic DNA (RAPD) (Karihaloo et al., 2003;
Ravishankar et al., 2004), Amplified Fragment
Length Polymorphism (AFLP) (Yamanaka et al., 2006)
and Simple Sequence Repeats (SSRs) (Viruel et al.,
2005; Schnell et al., 2006) have been utilized
to determine taxonomic identity (Schnell et al.,
2006), estimate genetic diversity (Viruel et al.,
2005) and infer evolutionary histories of mango (Yamanaka
et al., 2006).
In this study, phylogenetic relationship of selected mango cultivars was inferred
by using DNA sequences of Internal Transcribed Spacer (ITS) region of nuclear
ribosomal DNA (nrDNA), a widely used DNA marker in order to resolve phylogenetic
relationship at various taxa (Karehed et al., 2008).
This DNA marker is very useful due to several features such as high copy number,
small in size, possess highly conserved flanks, rapid concerted evolution and
universality of primers (Baldwin et al., 1995).
MATERIALS AND METHODS
In this study, a total of five mango cultivars namely Chokanan (MA 224), Harumanis
(MA 128), Maha 65 (MA 165), Mangga Epal (MA 194) and Nam Doc Mai (MA 223) were
chosen. The sampling of young and fresh mango leaves were carried out in Institute
Penyelidikan dan Kemajuan Pertanian Malaysia (MARDI), Serdang, Selangor with
the help of Mr. Ahmad Najib, research officer at MARDI. The collected samples
were labeled and kept in a proper storage bag and stored in -20°C freezer
to maintain its freshness. Furthermore, the DNA sequences of ITS region of mango
cultivars from Indonesia and Taiwan were retrieved from Topik (Unpublished data).
The genomic DNA was extracted from young, flushing mango leaves by using Qiagen
DNeasy Plant Mini Kit with a slight modification. The ITS region was amplified
by using Polymerase Chain Reaction (PCR) with a set of primers namely AB101
as the forward primer and AB102 as the reverse primer (Table 1).
About 20 μL of amplicon for each sample was inserted to 1.5 mL Eppendorf
tube and sealed using parafilm. The amplicons were sent for sequencing at First
BASE Laboratories, Seri Kembangan, Selangor.
The DNA sequences obtained were combined with the DNA sequence of ITS region
for Indonesian cultivars, Taiwanese cultivars and the outgroup, Mangifera
oblongifolia (Topik, Unpublished data) and saved in FASTA format. After
that, multiple sequence alignment was performed using ClustalX and the sequences
were adjusted manually. Subsequently, phylogeny reconstruction analysis based
on the maximum parsimony method was performed to the aligned DNA sequences using
PAUP version 5.10 (MEGA 5.1 Beta 3). The bootstrap test with 1000 replicates
was conducted to assess the degree of support for each branch with the consensus
tree option of retaining groups with frequency >50%.
RESULTS AND DISCUSSION
In phylogenetic study, the construction of phylogenetic tree is very important,
as it function as a tool that helps in inferring and elucidating evolutionary
relationships among organism. As for this study, relationships among selected
mango cultivars were analyzed based on the DNA sequences obtained.
The phylogenetic analysis revealed that the 13 cultivars were classified into
three major groups (Fig. 1). The first group consisted of
Harumanis (MA 128), Chokanan (MA 224), Mangga Epal (MA 194) and Nam Doc Mai
(MA 223) with 54% BS value. The second group included Irwin, Liar, Haden
and Saigon with moderate BS (72%). Remain cultivars are grouped in group 3.
As shown in the consensus tree (Fig. 1), it is suggested
that Mangga Epal (MA 194) is a sister cultivar to Nam Doc Mai (MA 223). A similar
pattern of relationship among cultivars such as Harumanis (MA 128) and Chokanan
(MA 224), both Mangga Epal (MA 194) and Nam Doc Mai (MA 223) are supported by
fruit morphology and characteristics such as tender in texture, yellowish orange
colour of flesh, aromatic and sweet in flavour. However, there are few differences
between these cultivars such as the shape of the fruit, Mangga Epal (MA 194)
is round in shape whereas Nam Doc Mai (MA 223) has oblong shape, the peel of
Mangga Epal (MA 194) is yellowish green and sometimes a bit red while Nam Doc
Mai (MA 223) has yellowish peel colour (Rajwana et al.,
Group 2 is predominant by Taiwanese cultivars. Member of this group shares
reddish peel colour.
||Strict consensus tree from the parsimony analysis of ITS region
(2 most parsimonious tree, Tree length: 471 steps, CI: 0.883, RI: 0.556).
The value in the branch represents the bootstrap support (BS)
Saigon has yellowish red peel colour, Haden and Irwin has bright yellow with
crimson or red blush peel with numerous large yellowish and whitish glands (dots)
and Liar has reddish peel colour. In addition, Saigon, Haden and Liar
shared similar flesh texture, which is moderate. On contrary, Irwin possesses
soft and tender flesh texture (Rajwana et al., 2008;
Bally et al., 2009).
Group 3 comprises of mostly Indonesian cultivars, with one Malaysian cultivar
(Maha 65). They are related since they share similarities in the fruit morphology
and characteristics such as oval to oblong in shape and sweet in flavour (Rajwana
et al., 2008; Bally et al., 2009).
From the phylogenetic tree constructed, the evolutionary pattern of the cultivars
can be determined. The tree suggested that both group 2 dan 3 has evolved earlier
as compared to the group 1 that consisted of cultivars from Malaysia only based
on the length of the nodes that branch out from the outgroup. This finding was
in line with the previous study that most of Malaysian cultivars were originated
from India, Indonesia and Thailand (Yamanaka et al.,
2006). However, Maha 65 (MA 165) is Malaysian local cultivar which is derived
from Kelantan and evolved earlier than Delima, Arumanis 143, Gedong Gincu of
Indonesia. This is supported by Bally et al. (2009)
which indicated that some of the Mangifera species are originated from
Generally, some cultivars are formed by hybridization between two cultivars
in order to improve its genetic resources. For instance, hybridization of Arumanis
143 with Delima has increased the attractiveness and quality of Arumanis 143
(Bally et al., 2009). It is suggested that hybridization
is possible among different cultivars from the same group rather than between
two different groups. This is because hybridization is the result of combination
of two or more attributes from different cultivars. Therefore, the cultivars
in the same group share similar genetic pattern and tends to possess the same
evolutionary pathway (Baldwin et al., 1995).
There are several recommendations that can be applied for the betterment of
this study in the future. Firstly, it is recommended to employ molecular marker
which derived from different genome of the plant such as rbcL and matK
that is derived from chloroplast (cpDNA) and protein-coding genes of mitochondria
(mtDNA) in order to determine the most appropriate genetic marker to reconstruct
phylogenetic relationship among mango cultivars. Moreover, it is suggested to
increase number of mango cultivars samples which enables confirmation of good
tree topology and increase the robustness of the tree, thus the relationship
among cultivars is predicted accurately.
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