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Genetic Diversity Study in Annona squamosa by

Morphological, Biochemical and RAPD Markers

Article in Acta horticulturae · July 2009

DOI: 10.17660/ActaHortic.2009.839.84

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Genetic Diversity Study in Annona squamosa by Morphological,
Biochemical and RAPD Markers
S.G. Bharad1, P.L. Kulwal2 and S.A. Bagal1
1
Department of Horticulture, 2Biotechnology Center
2
Department of Agricultural Botany
Dr. Panjabrao Deshmukh Agricultural University
Akola-444 104
India

Keywords: Annona squamosa, diversity, RAPD, isozyme

Abstract
Morphologically different 11 genotypes of sugar apple (Annona squamosa L.)
were studied for diversity at molecular level using isozyme and RAPD markers. The
four random primers yielded a total of 26 scorable markers, of which 19 were
polymorphic. RAPD analysis revealed that three genotypes viz., AKCa 05, AKCa 07
and AKCa 10 were divergent and the rest of eight genotypes were almost similar to
each other and these 11 genotypes were grouped into three distinct groups.

INTRODUCTION
Deficit of water is the single most important factor which limits the crop yield. In
rainfed agriculture delay in onset of monsoon and long dry spells during the crop growth
are decisive in reducing the yields and quality of traditional annual crops. The recurrent
droughts and resultant crop failure are observed in several parts of India in recent times.
Despite of the good progress made in dryland agricultural research, it is a reminder of the
fact that a shift in land use strategies is essential. The shift from usual crop farming to
growing of fruit orchards in the drylands will have beneficial consequences both,
economically and environmentally.
In order to achieve this, it is necessary to opt for niche crops, which will have to
be selected on the basis of regional edapho-meteorological conditions, water sources,
socio-economic conditions and market availability. This will have to be followed by
selecting appropriate species which can thrive well under adverse climatic and soil
conditions based on comparative evaluation. While considering all these facts, the
annonaceous fruit seems to be very suitable for such areas and specially sugar apple
(Annonna squamosa L.) which is deciduous in nature and synchronizes the flowering and
fruiting during water available periods.
Annona is a multipurpose tree. The fruits are consumed widely and the tree is also
a source of medicinal and industrial products. The fruits contain vitamin C and minerals
such as calcium, phosphorous and potassium. Fruits are also an excellent source of energy
being high in carbohydrate. Annona trees can give an average fruit yield of 50-100 kg/tree
and the commercial life of a tree is about 15 years. They are generally small trees or
shrubs, which makes maintenance and fruit harvesting easy. The trees are easy to
cultivate, require comparatively little care and do not suffer from serious pests and
diseases (SCUC, 2006).
Naturally a large intra- and inter-specific variability exists in Annonaceae family.
Annona plantations owe their origin to vast populations of seedlings that have originated
in nature from scattering of seed. As a result of this, they exhibit great diversity in quality
and bearing tendency. This has offered ample scope for studying the genetic variation in
Annona (Venkataratham and Satyanarayanswamy, 1958). This variability comprises of
about 500 species (Popenoe, 1974). Amongst them sugar apple (Annona squamosa L.) is
most preferred for arid and semiarid fruit production in India. Within this species, various
cultivars are available but barring few, none of the available cultivars have got the status
of commercial cultivar due to seed propagation, which results in considerable variability
(Rathore and Sanyal, 2002). To expedite the crop improvement programme, it is

Proc. 1st IS on Biotechnol. of Fruit Species 615

Eds.: M.-V. Hanke et al.
Acta Hort. 839, ISHS 2009
necessary to trap the natural variability through surveys and the variability should be
conserved ex situ and in situ to utilize for further hybridization programmes. For primary
screening, the morphological characterization is effective and to trace true genetic
variation, molecular characterization is essential. Biochemical marker and DNA
fingerprinting techniques are quick and accurate which excludes the managemental and
environmental effects unless there are strong genetic mutations. Thus, truly divergent
genotypes could be further collected and utilized in crop improvement programmes.
Studies on the use of morphological and biochemical markers have been reported on
members of Annonaceae (Escribano et al., 2004; Perfectti and Pascual, 2004; Cautin and
Agusti, 2005) including those by Ronning et al. (1995), Rahman et al. (1997, 1998),
Kwapata (2007) and Narvaez-Trujilla (2007) wherein RAPD, SSR and AFLP markers
have been used to study the genetic diversity of Annona species. However, there are very
few reports of use of biochemical and molecular markers for the study of diversity in
sugar apple.
In the present investigation, which is a sort of pilot study, diversity among 11
sugar apple genotypes, which were found divergent on earlier morphological analysis,
were compared using peroxidase enzyme and random amplified polymorphic DNA
(RAPD) analysis.

MATERIAL AND METHODS

For collecting the elite sugar apple germplasm, the survey was undertaken in five
districts of the Vidarbha region of Maharashtra State (Fig. 1). In all, 21 genotypes were
collected from this region. The physico-chemical study of all these types was undertaken
and 10 diverse genotypes (named as AKCa 01 to AKCa 10) were selected and planted for
further evaluation at the main campus of Dr. Panjabrao Deshmukh Agricultural
University, Akola. In addition to this, one commercially grown cultivar ‘Balanagar’ was
also included in the present study, thus making a total of 11 genotypes.
Akola is situated in sub-tropical region between 22.42°N latitude and 77.02°E
longitude. The altitude of the place is 307.42 m from MSL. The climate of Akola is
semiarid and characterized by three distinct seasons i.e., hot and dry summer from March
to May, warm humid and rainy monsoon from June to October and mild cold winter from
November to February.

Field Observations
The data on growth observations viz. plant height, plant spread (North-South and
East-West), plant volume, stem diameter and branches per plant was recorded as per the
standard procedures. The variance and covariance matrix was computed and the D2 values
were obtained following Mahalanobis (1936). The grouping of the genotypes was done by
Tocher’s method as described by Rao (1952) and the clustering was done.

Biochemical Marker Study

Isozyme was analyzed for biochemical marker study. Peroxidase enzyme group
was analyzed by native polyacrylaminde gel electrophoresis (PAGE) as per Maniatis et al.
(1989) with little modifications. The gel image was captured using Gel Documentation
System (Bio-Rad, USA). The migration of the isozyme was expressed as the ratios of the
band from the top of the lane to the total length of the lane also called as relative front
(Rf). The experiment was repeated thrice to confirm the reproducibility of the results. The
cluster analysis was performed from similarity matrix using UPGMA analysis using Gel
Doc EQ 4.5.0 programme (Bio-Rad USA).

Molecular Marker Study

Eleven random primers (Table 1) from Operon Technologies, Alameda, USA
were used in this study.
1. Extraction of DNA. Fresh leaf samples from new flush were collected from the
selected genotypes and genomic DNA was extracted using the CTAB method suggested

616
by Sharma et al. (2002) with little modifications. The genomic DNA was quantified on
0.8% agarose gel for further RAPD analysis.
2. PCR Amplification. DNA amplification was carried out using 25 μl reaction volume
consisting of 10 mM DNTP mix, 25 mM MgCl2, 30 ng primer, 30 ng of DNA, 5 units/μl
Taq DNA polymerase, 10X PCR buffer and sterile water. Amplification reaction was
carried out following the profile: hot start of 94°C for 3 min. and 45 cycles consisting of
93°C for 1 min, 40°C for 1 min, 72°C for 2 min and final extension at 72°C for 10 min in
a GeneAmp PCR System 2700 of Applied Biosystems make. In case of RAPD analysis
also, the experiment was repeated thrice to confirm the reproducibility of the results.
3. Electrophoresis. PCR products were electrophoresed on 2% agarose gel prepared
using 1 x TBE buffer and stained by pre-added ethidium bromide (0.2%). The gel image
was visualized using Gel Documentation System of Bio-Rad, USA. The banding patterns
were recorded as 1 for presence and 0 for absence of a band. The cumulative data from all
the primers was subjected to NTSys PC analysis software and the Dendrogram was
constructed.

RESULTS AND DISCUSSION

Diversity Based on Morphological Data

Based on the morphological data as recorded on various parameters (data not
shown), the clustering was done and the 11 sugar apple genotypes were grouped in four
different clusters (Table 2; Fig. 2). This revealed that the 11 genotypes studied were
polymorphic. The variation observed amongst the genotypes in respect of biometric traits
in studies like the present one, were attributed most of the times to either genetic or the
environmental or managemental effects or integration of these factors. It should be noted
that, most of the fields were not uniform and there exits a within field variation in soil
characteristics, topography, drainage, microclimate, etc. which may cause the variation in
biometric behavior of the same genotypes (Kumar and Sailaja, 2006). However, all the
custard apple genotypes in the present study were planted on the same site with the
similar management practices and therefore it can be considered that the variation may be
due to genetic polymorphism only. These results advocate the use of molecular markers
for confirming the diverse genotypes.

Diversity Based on Isozyme Data

The peroxidase polymorphism revealed that 11 sugar apple genotypes were
polymorphic and grouped into two different clusters (Table 2; Figs. 3 and 4). Cluster I
comprised of 9 genotypes, while cluster II comprised of remaining two genotypes viz.
AKCa 05 and AKCa 07.
The isozymes have proved useful to study the genetic diversity in a variety of
horticultural crops as they are reliable, uniform and genetically based and can frequently
be assayed in a variety of tissues both, from old and young plants (Ellstrand and Lee,
1987). Pascual et al. (1993) also successfully utilized the polymorphism in isozyme
banding patterns of Spanish and California cherimoya (Annona cherimola Mill.) cu1tivars
and differentiated both the groups from each other. Similar findings were obtained by
Perfectti and Pascual (1998) in Annona atemoya.

Diversity Based on RAPD Data

Out of 11 oligonucleotide primers used, only five could produce good, scorable
and reproducible bands (Table 3; Fig. 5) of these five primers, primer OPB 17 was
monomorphic while other produced distinct polymorphic banding patterns. A total of 26
markers were generated of which 19 were polymorphic. The size of fragments ranged
from 208 bp to 1354 bp, with 4 to 9 markers per primer. The banding pattern obtained
using different RAPD primers showed that the genotype AKCa 05 was different than the
rest of the genotypes. This may be because of the non availability of the primer binding
site at the corresponding locus in genotype AKCa 05.

617
 The cumulative data as obtained using polymorphic RAPD markers was utilized to
produce a dendrogram suggesting the extent of diversity. The 11 genotypes were grouped
into three distinct groups (Table 3; Fig. 6) which indicated that there exists variability
amongst the genotypes used in the present study.

Comparison Using Morphological, Biochemical and RAPD Data

The results obtained using morphological characterization revealed that the
genotypes had shown relatively wider variability, indicating that most of the genotypes
were divergent from each other. For example, the genotypes, AKCa 08, AKCa 09 and
AKCa 06 individually formed the separate cluster while, the remaining eight genotypes
were grouped in the same cluster (Table 2). However, the molecular characterization
based on RAPD data clearly showed that only three genotypes viz., AKCa 05, AKCa 07
and AKCa 10 were divergent and the remaining eight genotypes were almost similar
including the cultivar ‘Balanagar’ which was used as check in the present study. The
results obtained using peroxidase profile however, could distinguish only two genotypes
(AKCa 05 and AKCa 07) from the remaining nine genotypes. The wide morphological
variation observed may have been due to the environmental and managemental effects as
well as microclimate effect. Although the number of RAPD primers used in the present
study was very small, the results as obtained using isozyme and RAPD markers clearly
demonstrates that the diversity based on molecular markers was more reliable. Ronning et
al. (1995) used a set of 15 RAPD markers for the fingerprinting genotypes of atemoya,
sugar apple and cherimoya and for study of genetic variability among these selections and
observed highly distinct and polymorphic patterns for the genotypes used. Rahman et al.
(1997) studied the genetic relationship among A. cherimola, A. squamosa, A. reticulata,
A. glabra, A. muricata, A. montana and A. cherimola × A. squamosa by PCR-RFLP. The
results indicated that the cultivated and wild species were grouped separately. Rahman et
al. (1998) and Naravaez-Trujilla et al. (2007) studied genetic diversity of cherimoya
cultivars and observed that these genotypes could be clearly differentiated using AFLP
markers. Similarly, Kwapata et al. (2007) utilizing SSR markers studied the differences
among Annona senegalensis.
A number of similar studies was carried out by different workers in various fruit
crops using molecular markers and emphasized the importance and efficiency of
molecular characterization in assessing the genetic diversity. As the present study using
RAPD markers was planned on a pilot scale, it needs to be substantiated using additional
markers in the future.

CONCLUSIONS
The results as obtained using biochemical and RAPD markers have shown that
there was considerable diversity amongst the genotypes collected from different locations.
RAPD markers have proved to be suitable for characterizing sugar apple (Annona
squamosa) genotypes. Thus the molecular characterization is a very efficient, effective
and reliable technique to study diversity amongst sugar apple genotypes and the
genotypes AKCa 05, AKCa 07 and AKCa 10 can be conserved based on true genetic
diversity.
ACKNOWLEDGEMENTS
Financial assistance from Central Assistance-ICAR, New Delhi to SGB to attend
the BIOTECHFRUIT 2008 is acknowledged. PLK acknowledge with thanks ICAR New
Delhi for research grants for the project Pigeonpea Genomics Initiative.

Literature Cited
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(Annona cherimola Mill.). Scientia Hort. 105:491-497.
Escribano, P., Viruel, M.A. and Hormaza, J.I. 2004. Characterization and cross species
amplification of microsatellite markers in cherimoya (Annona cherimola Mill.

618
 Annonaceae). Mol. Ecol. Notes 4:746-748.
Ellstrand, N.C. and Lee, J.M. 1987. Cultivar identification of cherimoya (Annona
cherimola Mill.) using isozyme markers. Sci. Hort. 32:25-31.
Kumar, R.N. and Sailaja, B. 2006. Role of information and communication technology
for improving input efficiency of horticultural production. Ind. J. Arid Hort. 1:63-68.
Kwapata, K., Mwase, W.F., Bokosi, J.M., Kwapata, M.B. and Munyenyembe, P. 2007.
Genetic diversity of Annona senegalensis Pers. Populations as revealed by simple
sequence repeats (SSRs). African J. Biotech. 6(10):1239-1247.
Mahalnobis, P.C. 1936. On the generalized distance in statistics. Proc. Natl. Inst. Sci.
India 2:49-50.
Maniatis, T., Fritsch, E.F. and Sambrook, J. 1989. Molecular Cloning. A Laboratory
Manual, Second Edition, Cold Spring Harbour Laboratory Press, USA.
Narvaez-Trujilla, A., Barreiro, J.M. and Astudillo, R.M. 2007. Tracing the genetic base of
cherimoya (Annona cherimola Mill.) commercial cultivars through AFLP analysis of
diversity at the species putative centre of origin. Acta Hort. 738:459-465.
Pascual, L., Perfectti, F., Gutierrez, M. and Vargas, A.M. 1993. Characterizing isozymes
of Spanish cherimoya cultivars. Hort. Sci. 28:845-847.
Perfectti, F. and Pascual, L. 2004. Genetic diversity in a worldwide collection of
cherimoya cultivars. Genet. Res. Crop Evol. 52:959-966.
Popenoe, W. 1974. The Cerimoya. p.161-189. In: Manual of tropical and subtropical
fruits, facsmile of the 1920 (ed.), Hafner press, a division of Macmillan publishing Co.
Inc, New York, collier Macmillan publishers, London, chapter 5, (c.f. Pinto, 2005).
Rahman, M., Shimada, T., Yamamoto, T., Yonemoto, J.Y. and Yoshida, M. 1998.
Genetical diversity of cherimoya cultivars revealed by amplified fragment length
polymorphism (AFLP) analysis. Breeding Sci. 48:5-10.
Rahman, M., Yamada, M. and Yoshida, M. 1997. Relationship of Annona species as
revealed by PCR-RLP analysis. Breeding Sci. 47:335-339.
Rao, C.R. 1952. Advanced statistical methods in biometric research. John Wiley and
Sons, New York.
Rathore, D.S. and Sanyal, D. 2002. Annonas. p.293-328. In: T.K. Bose, S.K. Mitra and D.
Sanyal (eds.), Fruits:Tropical and subtropical, Vol.2, Naya Udyog, Kolkatta.
Ronning, C.M. and Schnell, R.J. 1995. Using randomly amplified polymorphic DNA
(RAPD) markers to identify Annona Cultivars. J. Amer. Soc. Hort. Sci. 120:726-729.
SCUC. 2000. Annona: Annona cherimola, A. muricata, A. reticulata, A. senegalensis and
A. squamosa, Field Manual for Extension Workers and Farmers, University of
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Tables

Table 1. The primers and their sequence screened during the study.

Sr. No. The RAPD primer Base sequence (5’-3’)

1 OPA 02 TGCCGAGCTG
2 OPA 09 GGGTAACGCC
3 OPA 11 CAATCGCCGT
4 OPB 01 GTTTCGCTCC
5 OPB 05 GGTCGCAGTT
6 OPB 07 GGTGACGCAG
7 OPB 08 GTCCACACGG
8 OPB 10 CTGCTGGGAC
9 OPB 12 CCTTGACGCA
10 OPB 15 GGAGGGTGTT
11 OPB 17 AGGGAACGAG

Table 2. Grouping of the genotypes on the basis of different parameters.

Cluster Biometric data Isozyme data RAPD data

No.
I AKCa 01, AKCa 02, AKCa 01, AKCa 02, AKCa 01, AKCa 02,
AKCa 03, AKCa 04, AKCa03, AKCa 04, AKCa 03, AKCa 04,
AKCa 05, AKCa 07, AKCa 06, AKCa 08, AKCa 06, AKCa 08,
AKCa 10, Balanagar AKCa 09, AKCa 10, AKCa 09, Balanagar
Balanagar
II AKCa 08 AKCa 05, AKCa 07 AKCa 07, AKCa 10
III AKCa 09 -- AKCa 05
IV AKCa 06 -- --

Table 3. Results as obtained using polymorphic primers.

No. Primer Total bands Polymorphic bands % polymorphism

1 OPB -01 6 5 83.33
2 OPB-07 9 5 55.56
3 OPB-08 4 3 75.00
4 OPB-10 7 6 85.71
Total 26 19 74.90

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Figures

Fig. 1. Map of Vidarbha region of Maharashtra State showing the collection sites of
Annona squamosa L.

Fig. 2. Dendrogram of sugar apple genotypes based on biometric data.

621
Fig. 3. Isozyme (peroxidase) banding profile of sugar apples genotypes (arrow indicates
polymorphism).

Fig. 4. Dendrogram of sugar apple genotypes based on isozyme polymorphism.

622
Fig. 5. Representative RAPD banding profile of sugar apple genotypes as obtained using
primer OPB 01.

Fig. 6. Dendrogram of sugar apple genotypes based on RAPD polymorphism.

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