Assessing the robustness of IRAP and RAPD marker systems to
study intra-group diversity among Cavendish (AAA) clones of
By M. S. SARASWATHI*, S. UMA, K. PRASANYA SELVAM, S. RAMARAJ, P. DURAI and
M. M. MUSTAFFA
National Research Centre for Banana (ICAR),Thogamalai Road,Thayanur (Post),
Tiruchirapalli – 620 102,Tamil Nadu, India
(Accepted 24 August 2010)
Bananas belonging to the Cavendish sub-group (AAA) are cultivated commercially for their high yield, short
cropping cycle,and high economic returns.The field performance of exotic Cavendish cultivars such as ‘Grand Naine’,
‘Williams’,and ‘Dwarf Cavendish’ is superior due to their sturdy stature,high yield potential,and better bunch quality.
However, morphological similarities among the dwarf, medium, tall, and giant types of Cavendish clones make
identification difficult under field conditions. Multiplication by tissue culture further aggravates this problem by
inducing somaclonal variants. In the present study, ten IRAP (Inter-Retrotransposon Amplified Polymorphism)
primer pairs and 30 decamer RAPD (Randomly Amplified Polymorphic DNA) primers were tested to examine intra-
group diversity in Cavendish bananas. The average level of polymorphism exhibited by RAPD markers was 67.8%,
and by IRAP markers the level was 81.3%,indicating that there was substantial variation at the DNA level among the
19 accessions tested.All dwarf Cavendish-type cultivars such as ‘Williams’,‘Dwarf Cavendish’,‘Singapuri’,‘Jahaji’,and
‘Manjahaji’ clustered together using IRAP markers. Similarly, all medium-tall types of Cavendish such as ‘Harichal’,
‘Robusta’,‘Shrimanti’, and ‘Pedda Pacha’, and giant Cavendish types such as ‘Gandevi Selection’,‘Grand Naine’, and
‘Madhukar’ grouped separately. These results suggest that IRAP markers are more robust than RAPD markers for
studying intra-group diversity in the Cavendish sub-group. Furthermore, the use of appropriate primer combinations
could enable the development of DNA fingerprints for genetic fidelity testing within Cavendish clones.
worldwide on an area of 2.5 million ha, producing 123.33
million metric tonnes of fruit (FAOSTAT, 2009). They
form the basis for the livelihoods and nutritional security
of millions of people worldwide. Extensive diversity
exists in the Musa genus, with several groups and sub-
groups (Uma et al., 2005). Clones of the Cavendish sub-
group such as ‘Grand Naine’,‘Williams’,‘Poyo’,‘Novaria’,
and ‘Valery’ dominate the global banana export industry,
depending on geographical location.The Cavendish sub-
group has two major morphotypes, namely normal and
giant types, and it is difficult to discriminate between
them, except for minor phenotypic variations. All
Cavendish clones have a common genetic make-up and
have arisen from one another through point mutations.
Although this mutation-induced variability has been
exploited effectively in Cavendish clones, under some
stress conditions they tend to revert back to their parental
type, as reported by Agrawal et al. (2009). This poses a
major problem when Cavendish clones are multiplied
through tissue culture, leading to unstable off-types, or
somaclonal variations under field conditions. This is
mainly due to the insertion or deletion of transposable
elements at novel sites in the genome (Courtial et al.,
2001). Therefore, morphotaxonomic characterisation,
ananas and plantains are crops of international
significance grown in approx. 150 countries
complimented by molecular characterisation, is likely to
facilitate the identification of authentic clones within a
sub-group. In this context, two DNA marker systems,
Randomly Amplified Polymorphic DNA (RAPD) and
(IRAP), both of which are dominant, simple, and cost-
effective in nature, were compared to assess their
robustness for studying intra-group variability within
Cavendish clones. This work would enable the
identification of a robust marker system for future use in
genetic fidelity testing of tissue-cultured plants.
The RAPD technique uses short primers of arbitrary
nucleotide sequence to amplify genomic DNA
fragments. Polymorphism results from changes in the
primer binding sites on genomic DNA sequences. In
IRAP, polymorphism is due to the insertion of
retroelements into the genome,which can be detected by
PCR using primers facing outwards from their distal
sequences. Because of the high copy number of the
conserved retro-element sequences, and their presence
within distances that can be amplified by PCR, a single
PCR amplification can generate 5 – 20 fragments
between different elements from a particular genomic
DNA in Musa spp. or clones.
The assessment of genetic diversity and phylogenetic
relationships is a basic requisite for genetic improvement
through conventional breeding and can be best achieved
through the use of molecular markers. RAPD markers
*Author for correspondence.
Journal of Horticultural Science & Biotechnology (2011) 86 (1) 7–12
Intra-group diversity among Cavendish banana clones
are an easy, economical, and rapid technique widely
applied for varietal identification and classification in
banana (Howell et al., 1994). RAPDs also permit
discrimination between plants of different genomic
composition (Pillay et al., 2000). Furthermore, RAPDs
facilitate the early detection and elimination of
somaclonal variants (Damasco et al., 1996; Maria and
Garcia, 2000). The major disadvantage of the RAPD
technique is its low reproducibility.
Insertional polymorphisms due to retrotransposons can
be detected by a variety of PCR-based techniques
(Kalendar et al., 1999). Teo et al. (2005) exploited the
repetitive and dispersed nature of many Long Terminal
Repeat (LTR)-retrotransposon families in order to
characterise genome compositions and to classify cultivars
in the genus Musa. Nair et al. (2005) used IRAP markers
for the genomic classification of banana cultivars from
southern India. IRAP has also been used by Muhammad
and Othman (2005) to characterise somaclonal variants of
the banana cultivar ‘Rasthali’ (AAB).
In this paper, we report on the application of 30
RAPD and ten IRAP primers to discriminate between
19 accessions (clones) of Cavendish-group bananas and
to examine intra-group diversity.
MATERIALS AND METHODS
The Musa accessions tested included 19 Cavendish
clones which were morphologically similar, except for a
few traits such as plant height, pseudostem colour, fruit
apex, and appearance of the rachis.The accessions were
collected from varied agro-ecological regions of the
Indian sub-continent and formed a subset of a larger
collection maintained at the Musa field genebank at the
National Research Centre for Banana (NRCB), Trichy,
Young “cigar” leaves collected from trees in the field
genebank of NRCB were used for the isolation of high
molecular weight, genomic DNA using the cetyl
trimethyl ammonium bromide (CTAB) method (Gawel
and Jarret, 1991) with minor modifications. Fresh leaf
samples (approx. 2 g each) were frozen in liquid
nitrogen, ground to a fine powder, and mixed with 10 ml
of pre-heated (65ºC) CTAB extraction buffer [1.5%
(w/v) CTAB in 20 mM EDTA, 1.4 M NaCl, 10 mM Tris-
HCl (pH 8.0), and 1 – 2% (v/v) ?-mercaptoethanol].The
mixture was then shaken gently for 30 min at 65ºC. An
equal volume (10 ml) of 24:1 (v/v) chloroform:isoamyl
alcohol was added and gently mixed for 15 min, then the
sample was centrifuged at 10,000 ? g for 20 min at 27ºC.
The aqueous phase was transferred to a clean 50 ml
polypropylene tube and an equal volume (10 ml) of ice-
cold 100% (v/v) iso-propanol was added and mixed
gently to precipitate the DNA.The DNA precipitate was
recovered with 70% (v/v) ethanol,then dried,and finally
dissolved in nuclease-free water and stored at –20ºC.
After treatment with RNase A (Sigma-Aldrich,St.Louis,
MO, USA) and purification, the concentration of DNA
was determined using a UV/VIS spectrophotometer
(Lambda 25; Perkin-Elmer, Beaconsfield, UK). The
integrity and concentration of the DNA was checked by
0.8% (w/v) agarose gel electrophoresis. Only DNA of
high quality was used for PCR amplification.
RAPD and IRAP marker amplification
The purified DNA samples were diluted to 10 ng ml–1
for PCR amplification. The protocol of Williams et al.
(1990) was followed for RAPD analysis. Thirty random
primers (10-mers) belonging to the OPA, OPB, OPC,
OPD,OPE,and OPV series were obtained from Operon
Technologies Inc.(Alameda,CA,USA) and were used in
Insertional polymorphisms caused by retrotransposon
elements were studied in DNA from the same set of
Musa accessions using ten published IRAP primer pairs.
IRAP analysis was carried out using LTR primers
derived from the barley (Hordeum vulgare) genome (n =
7 chromosomes; Kalendar et al., 1999; 2000; Boyko et al.,
2002). Two additional IRAP primers (reverse TY1and
reverse TY2) were designed facing outward from the
highly-conserved reverse transcriptase priming sites of
the banana TY1-Copia-like retrotransposons (Teo et al.,
2001). The protocol of Nair et al. (2005) was used for
IRAP analysis, except for minor modifications in the
amounts of enzyme (2.0 vs. 1.0 Units) and primers (5.0
vs. 10.0 pmol) used.
PCR was carried in 25 µl RAPD or 20 µl IRAP
reactions containing 50 ng genomic DNA, 1X PCR
buffer (Genei,Bangalore,India),200 µmol of each of the
Details of the Cavendish banana (Musa sp.) accessions used in this study
Name of accession (AAA)
‘NRCB - 0498’
‘NRCB - 1621’
NRCB Acc. No.
Site of collection
M. S. SARASWATHI, S. UMA, K. PRASANYA SELVAM, S. RAMARAJ, P. DURAI and M. M. MUSTAFFA
four dNTPs, 1.5 Units Taq DNA polymerase (Genei),
and 0.2 µmol of each random (RAPD) primer or 5 pmol
of each of the IRAP primers pairs. Reproducibility was
determined by running different PCR reactions
containing the same DNA samples and primers.
All PCR amplifications were performed using a
Master Cycler ESP gradient S (Eppendorf, Hamburg,
Germany). The programme for RAPD analyses was an
initial denaturation at 94ºC for 5 min, followed by 40
cycles of denaturation at 94ºC for 60 s, annealing at 37ºC
for 1 min, followed by extension at 72ºC for 1 min,
terminated by a final extension at 72ºC for 15 min,
followed by incubation at 4ºC.
For IRAP analysis, the PCR programme consisted of
an initial denaturation at 94ºC for 5 min, followed by 30
cycles of denaturation at 94ºC for 30 s, annealing at a
specific annealing temperature (which varied with the
primer combination) for 1 min, followed by extension at
72ºC for 1 min, terminated by a final extension 72ºC for
15 min, followed by incubation at 4ºC.
Gel electrophoresis and photography
The amplified RAPD and IRAP products were
fractionated by gel electrophoresis in 1.5% (w/v) agarose
gels in 1X TAE buffer (40 mM Tris-acetate, 1.0 mM
EDTA, pH 8.0) and the gels were stained with 0.5 mg
ml–1ethidium bromide. The banding patterns were
documented using an Alpha Innotech Image Analyser
(Alpha Innotech Corp., Santa Clara, CA, USA). The
molecular weights of the amplified fragments were
calculated by comparing the molecular weights of DNA
markers (500 bp DNA ladder) using Alpha Imager
Version 4.0 software. The percentage of polymorphism
was calculated using the following formula:
No. of Polymorphic bands
No. of Amplified bands
Polymorphism (%) = ———————————— ? –––
The PCR amplified products from individual samples
were scored as being either present (1) or absent (0). A
Genetic Similarity (GS) matrix was then used in a
hierarchical cluster analysis using the unweighted pair
group method with arithmetic averages (UPGMA) and
sequential agglomerative hierarchical and nested
(SAHN) clustering methods (NTSYS statistical package;
Rohlf, 1998) to produce a dendrogram.
RESULTS AND DISCUSSION
The average level of polymorphism exhibited by
RAPD markers was 67.8%, and that by IRAP markers
was 81.3%, indicating that there was substantial
variation among the 19 Musa accessions tested at the
DNA level. The Clusters discussed in the present study
were derived by dissecting the dendrogram at a
similarity coefficient of 80%.
Of the 30 RAPD primers tested, 29 produced
reproducible PCR bands (amplicons) and were
considered for further genetic analysis. A total of 281
PCR fragments were detected, with an average of 9.37
bands per primer. Among the amplified fragments,
67.8% were polymorphic and the rest were
monomorphic (Table II), indicating the existence of
considerable genetic variation among the Cavendish
clones.The maximum level of polymorphism (100%) was
observed using the RAPD primers OPB-11, OPC-09,
OPD-10,and OPE-20;while a low level of polymorphism
(< 50%) was observed with the RAPD primers OPA-18,
OPC-05, OPC-20, OPD-18, OPD-20, and OPE-18.
Primer OPB-20 failed to produce any PCR fragments.
This failure was not due to failure of the PCR reaction as
this was repeated three times.Although PCR amplicons
of the same size co-migrate electrophoretically,they may
not represent the same region of genomic DNA, which
introduces uncertainties. The limitations of the RAPD
method can be overcome by carefully adjusting the
reaction conditions (Colosi and Schaal, 1997).
Some RAPD primers, such as OPE-17 and OPV-06,
produced unique bands specific for the accessions,
NRCB-1621 and 2390-2 (Figure 1). Bands of 871 bp and
494 bp produced by OPE-17 and OPV-06, respectively,
were specific for NRCB-1621. Similarly, the RAPD
primers OPE-17 and OPV-06 produced bands of 1,029
bp and 1,234 bp, respectively, which were specific for
2390-2. The amplification of polymorphic RAPD
markers could be attributed to deletions or insertions at
the random priming sites, as reported by Damasco et al.
(1996). Sequencing of these unique bands might enable
the establishment of unique fingerprints for the various
commercial Cavendish clones.
All ten pairs of IRAP primers amplified bands that
were clearly scorable. All the primer pairs were
consistent and informative, and hence were considered
Polymorphisms exhibited by 29 RAPD primers across 19 Cavendish
banana (Musa sp.) clones
Code No.RAPD primerbands
24 OPV-01 16
Intra-group diversity among Cavendish banana clones
for further analysis.A total of 122 IRAP-PCR fragments
were scored, with an average of 12.2 bands per primer.
The numbers of bands generated by IRAP is normally
equivalent to the number of retroelements that are
situated close enough to one another to allow
amplification. On average, 81.3% of the amplified IRAP
fragments were polymorphic, and the rest were
monomorphic (Table III). The maximum polymorphism
(100%) was exhibited by the primer pair LTR 6149 and
Nikita, followed by 5’ LTR2 and Sukkula (90.9%), 3’
LTR and REV TY2(86.7%), and 3’ LTR and REV TY1
(84.6%). The minimum level of polymorphism (71.4%)
was exhibited by Nikita and LTR 6150, followed by
3’LTR and LTR 6150 (71.5%).Possible reasons for these
variations could be the appearance of new alleles of
increased or decreased size, or insertions or deletions in
the repeat units.This is the first report of IRAP markers
being used to investigate genetic relatedness in
Cavendish group (AAA) Musa spp.. The average
polymorphism (81.3%) observed by IRAP was approx.
15% higher than that seen using RAPD markers,
indicating the improved potential of IRAP to distinguish
between Cavendish clones.
RAPD cluster analysis
Cluster analysis resulted in a dendrogram with two
major Clusters, with all clones, except 2390-2, grouped in
one Cluster (Figure 2).
Cluster I: This included five sub-Clusters, coded from Ia
to Ie.All the accessions grouped together,with only 20%
Sub-Cluster Ia: Nine accessions including ‘Jahaji’,
‘Manjahaji’, ‘Singapuri’, and ‘Harichal’ grouped with >
94% similarity. ‘Borjahaji’ exhibited 9% dissimilarity
with the other accessions which could be attributed to its
barren male axis, while the other members possessed
persistent male flowers and bracts. Surprisingly,
‘Singapuri’, the most dwarf type among the Cavendish
clones, was grouped with these accessions. This result
requires confirmation.‘Williams’, an exotic introduction,
grouped with the indigenous ‘Shrimanthi’ with > 98%
similarity and both shared 95% similarity with
‘Peddapacha Arati’. ‘Grand Naine’ also grouped with
these accessions,with 91% similarity. The 9%
dissimilarity exhibited by ‘Grand Naine’ might be due to
its high yielding nature.
Sub-Cluster Ib: Although ‘Dwarf Cavendish’, ‘Robusta’,
and ‘Gandevi’ belong to different Cavendish sub-groups,
respectively, they only exhibited 6% dissimilarity which
might have grouped them together. ‘Lacatan’ and
GCTCV-215 clustered together in Ib, with 8% and 10%
dissimilarity from the above members.This might be due
to their exotic origin.
and giant Cavendish,
Sub-Cluster Ic: This was a uni-member sub-Cluster
containing only GCTCV 119.
Sub-Cluster Id:NRCB-1621 and ‘Madhukar’ were the two
members of this sub-Cluster,sharing ≤84% similarity.This
unusual grouping of members from two different
Cavendish sub-groups merits further investigation.
Sub-Cluster Ie: This was a uni-member sub-Cluster with
only NRCB-0498.NRCB-0498 is a giant type Cavendish,
like ‘Madhukar’, ‘Gandevi’, and ‘Borjahaji’; however it
Examples of DNA polymorphisms revealed by RAPD primer OPE17 (Panel A) or IRAP primers 3’LTR and Nikita (Panel B) using genomic DNA
from 19 accessions of Cavendish clones of banana (Musa sp.). Lanes M, DNA size markers. Lanes 1–19, DNA from the 19 clones coded as shown
in Table I.
Polymorphisms exhibited by ten IRAP primer pairs across 19 Cavendish
banana (Musa sp.) clones
No. IRAP primer pairbands
1 Sukkula and LTR 6149
2 Sukkula and LTR 6150
3 Sukkula and 5’LTR2
4 LTR 6149 and Nikita
5 LTR 6149 and 3’LTR
6Nikita and LTR 6150
73’LTR and Nikita
8 3’LTR and LTR 6150
93’LTR and REV TY1
103’LTR and REV TY2
M. S. SARASWATHI, S. UMA, K. PRASANYA SELVAM, S. RAMARAJ, P. DURAI and M. M. MUSTAFFA
clustered independently and far from the other giant
Cavendish types.This result should be clarified.
Cluster-II: This was a uni-member Cluster containing
only 2390-2. This might be attributed to the orange-
coloured pulp which is unique among Cavendish clones.
IRAP cluster analysis
The dendrogram derived through cluster analysis
showed two major Clusters with all dwarf, medium, and
tall types in one Cluster,and the giant Cavendish types in
another Cluster (Figure 2).
The similarity coefficients ranged from 80 – 98% when
the Cavendish clones were characterised using RAPD
and IRAP markers, indicating that genetic diversity
within the Cavendish clones was minimal.IRAP markers
grouped the tissue-culture variants of ‘Giant Cavendish’
(GCTCV 119 and GCTCV 215) in the same Cluster with
95% similarity, while RAPD markers placed them apart
in different Clusters. This contradicts earlier reports by
Damasco et al. (1996) who reported RAPDs to be
efficient for detecting variants from their parental clones
in the Cavendish group, and Muhammad and Othman
(2005) who reported RAPDs to be more polymorphic
than IRAP markers in detecting somaclones of
All dwarf Cavendish types such as ‘Williams’, ‘Dwarf
clustered together using IRAP markers. Similarly, the
medium-tall types of Cavendish (‘Harichal’, ‘Robusta’,
‘Shrimanti’, and ‘Pedda Pacha’) and the giant Cavendish
types (‘Gandevi Selection’, ‘Grand Naine’,
‘Madhukar’) were grouped separately. ‘Singapuri’ and
‘Dwarf Cavendish’ are morphologically similar
considering their dwarf stature and IRAP markers
grouped them in the same sub-Cluster.However,RAPD
polymorphism was observed in morphologically similar
accessions and they were placed in different sub-Clusters
(Table IV). This could be attributed to the insertion or
deletion of nucleotides at the PCR priming sites, which
need not necessarily control a specific trait such as
Our results suggest that IRAP is a more robust
marker system than RAPD markers for studying intra-
group diversity among Cavendish clones. Clustering by
IRAP was most similar to the morphotaxonomic
characterisation. This clustering pattern also had
correlations with the geographical origin of the
accessions. Most of the indigenous accessions were
classified as dwarf types and grouped together, except
for ‘Williams’ and ‘Dwarf Cavendish’, both of which
were exotic introductions. However, because of their
‘Jahaji’, and ‘Manjahaji’
‘NRCB - 1621’
‘NRCB - 0498’
‘NRCB - 1621’
‘NRCB - 0498’
Dendograms showing the level of genetic diversity and/or relatedness among 19 Cavendish banana clones (Musa sp.) using RAPD (Panel A) or IRAP
(Panel B) markers. The lower scales in both Panels indicate similarity coefficients ranging from 60 – 100%. The Clusters in each Panel were
derived by dissecting the dendograms at a similarity coefficient of 80%.
Composition of Clusters and sub-Clusters generated by RAPD and IRAP markers
Names of the Musa accessions
Borjahaji, Jahaji, Manjahaji, Singapuri, Harichal, Peddapacha Arati,Williams, Shrimanti, Grand Naine
Robusta, Dwarf Cavendish, Gandevi Collection, Lacatan, GCTCV-215
NRCB - 1621, Madhukar
NRCB - 0498
Jahaji, Manjahaji, Singapuri,Williams, Dwarf Cavendish
Harichal, Robusta, Shrimanti, Pedda Pacha Arati
NRCB - 1621, 2390-2
Lacatan, GCTCV-215, GCTCV-119
Borjahaji, Madhukar, Grand Naine, Gandevi Collection, NRCB - 0498
IRAP IIa (Dwarf)
Giant IRAP II
0.60 0.700.800.90 1.00
0.60 0.70 0.80 0.901.00
Intra-group diversity among Cavendish banana clones Download full-text
stature, they grouped with the other dwarf types in sub-
Cluster Ia.‘Harichal’,‘Robusta’,‘Shrimanti’,and ‘Pedda
Pacha Arati’ are indigenous, medium-statured, and
originated from Central India, and grouped together in
sub-Cluster Ib.Those accessions exhibiting a tall stature
formed two Clusters (Ic and Id) with respect to their
geographical origin. Most of the indigenous accessions
such as ‘Borjahaji’, ‘Madhukar’, and ‘Gandevi’
belonging to the giant-type, grouped together, except
for ‘Grand Naine’ and NRCB-0498, both of which were
exotic introductions. Due to their stature, they grouped
with the other giant types in IRAP Cluster II.
Use of appropriate IRAP primer combinations such
as Sukkula and 5’LTR2,LTR 6149 and Nikita,3’LTR and
REV TY1, or 3’LTR and REV TY2 might therefore
enable the development of diagnostic fingerprints for
use in genetic fidelity testing among Cavendish clones.
The authors acknowledge the infrastructure facilities
provided by The Director, NRCB,Trichy, India.
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