RESEARCH ARTICLE

Genetic Diversity in Punica granatum Revealed by Nuclear rRNA,Internal Transcribed Spacer and RAPD Polymorphism

S. K. Singh • P. R. Meghwal • Rakesh Pathak •

Ragini Gautam • Suresh Kumar

Received: 13 December 2012 / Revised: 27 December 2012 / Accepted: 4 March 2013 / Published online: 3 April 2013

� The National Academy of Sciences, India 2013

Abstract The morpho-physiological and molecular

markers were used to reveal genetic diversity among 13

putative varieties of Punica granatum grown in India. A

hybrid protocol of DNA isolation was developed to obtain

high molecular weight quality genomic DNA. Ten RAPD

primers generated 119 marker bands amounting to 92.44 %

polymorphism with 88 % polymorphic information con-

tent. The 5.8S rRNA gene region was found to be highly

conserved (99.34 %) followed by ITS-1 (96.58 %) and

ITS-2 (89.21 %) intron regions encompassing the gene.

ITS-2 region was recorded with the highest percentage of

parsimony informative sites with high divergence mainly

due to SNPs. The PCA accounted for 84.21 % of total

variance. The AMOVA of RAPD data revealed the maxi-

mum genetic variation within population rather than among

populations. Nei’s gene diversity (h) was 0.2684 and

Shannon information index (i) was 0.4135. The Nei’s

genetic distance ranged from 0.0620 to 0.0878. The coef-

ficient of gene differentiation between populations (Gst) of

0.2584 and gene flow (Nm) of 1.4347 validates that the

main proportion of genetic variation was within

populations. The present study validates the utility of ITS

rDNA region as a reliable indicator of phylogenetic inter-

relationships, especially ITS-2 as probable DNA barcode at

higher levels and can serve as an additional approach for

genetic cataloguing of pomegranate germplasm for crop

improvement.

Keywords Genetic diversity � Pomegranate �Punica granatum � RAPD � rRNA gene diversity

Introduction

Pomegranate (Punica granatum L.) belongs to Punicaceae

family and is an important horticultural crop of tropical and

subtropical regions of the world. The origin of pomegran-

ate is considered to be in central Asia [1] from where it has

spread to rest of the world [2]. It was introduced from the

Mediterranean region to Asia, North America, Europe and

into the Indian peninsula where it was first reported to be

grown [3]. Consumption of fresh and processing pome-

granate is considered to be functional product with health

benefits as it contains substances that are useful in pre-

vention of ageing and diseases [4]. The fruit rind is rich in

natural antioxidants belonging to the polyphenolic class

which includes tannins, polyphenols and flavonoids [5].

Different varieties/cultivars exist in the world vary

widely in their morphological, agronomical and post har-

vest characteristics [6–8]. The pomegranates have routinely

been characterized using morphological and or biochemi-

cal markers [9, 10]. These markers are of great significance

but they exhibit limited information in distinguishing dif-

ferent varieties due to environmental plasticity. DNA

markers reveal greater genetic diversity and relationships

of horticultural plants and have been used to reconstruct

S. K. Singh (&) � P. R. Meghwal � R. Pathak � R. Gautam �S. Kumar

Central Arid Zone Research Institute, Jodhpur 342003,

Rajasthan, India

e-mail: sksingh1111@hotmail.com

P. R. Meghwal

e-mail: prmeghwal@cazri.res.in

R. Pathak

e-mail: pathakjodhpur@gmail.com

R. Gautam

e-mail: tina_ragini@yahoo.com

S. Kumar

e-mail: skumar@cazri.res.in

123

Natl. Acad. Sci. Lett. (March–April 2013) 36(2):115–124

DOI 10.1007/s40009-013-0120-8

phylogenetic relationships. Such information is of great

importance for understanding the evolution and crop

improvement in pomegranate.

Among the various marker systems, the randomly

amplified polymorphic DNA (RAPD) is one of the most

popular DNA based approaches [11]. It allows the analysis

of individual and large number of markers in relatively

short time, as only a few primers allow the generation of

sufficient data to obtain a robust estimate of diversity index

and have allowed the resolution of complex taxonomic

relationships. This technique has recently been used in a

number of fruit trees Mangifera indica [12], Citrus species

[13] including P. granatum [14, 15]. In addition to this

AFLP [16], ISSR [17] and SSR [18] have also been used to

determine genetic diversity in pomegranate.

Currently, nuclear ribosomal internal transcribed spacer

(ITS) intron regions encompassing highly conserved 5.8S

rRNA gene is considered as one of the most useful phy-

logenetic marker due to less functional constrains and

comparatively higher evolutionary changes [19]. It evolves

relatively quickly and can be useful in determining inter-

specific [20] and sometimes intra-specific relationships

[21]. The rate and patterns of ITS sequence mutation are

typically appropriate for resolving relationships among

species and genera [22]. Although thousands of copies of

the ITS exist in Angiosperm genomes, but are generally

homogenized by concerted evolution, and thus can be

treated as a single locus [23]. The nuclear ribosomal RNA

(rRNA) gene complex is a tandem repeat unit of one to

several thousand copies. This complex has several domains

that evolve at varying rates [24] and thus have different

phylogenetic utilities.

The region of 5.8S rRNA gene was shown to contain

considerable phylogenetic information [25]. The ITS

polymorphism might occur at a genus, species or individual

levels, making it useful for phylogenetic, evolutionary and

bio-geographical diversity studies [26]. ITS sequences

have been widely used in several species [27, 28] for

phylogenetic inferences.

The present investigation is an attempt to explore the

extent of genetic diversity based on nuclear ribosomal

DNA internal transcribed spacer and RAPD polymorphism

in 13 varieties of P. granatum for crop improvement.

Materials and Methods

Field Experiment

Morpho-physiological and molecular characterization

studies were conducted in seven years old orchard of P.

granatum maintained in horticulture block at Central Arid

Zone Research Institute, Jodhpur. The composite samples

of fruits and leaves were collected from ten randomly

selected trees of each of the 13 varieties namely, P-23,

P-26, G-137, Arakta, Sinduri, Jodhpur red, Dholka, Gulsa

red, GKVK, Basein seedless, Ganesh, Jalore seedless,

Ganesh 9 Jalore seedless during January and February

2012. Morpho-physiological observations namely, plant

height, canopy area, fruit weight, fruit height, fruit width,

aril per cent, total soluble solids (TSS) were recorded. The

phenotypic correlation of all the morpho-physiological

traits studied with fruit yield was calculated to identify

possible phenotypic markers.

DNA Isolation and Quantification

The genomic DNA was extracted from the leaves of each

of the 13 varieties of P. granatum. A hybrid protocol for

genomic DNA isolation was developed using initial steps

of CTAB method [29] and subsequently columns and

solutions of the Plant Genomic DNA Purification spin kit

(Hi-media Company). One gram of composite fresh leaves

of each sample was crushed three times in liquid nitrogen,

transferred to a 30 ml tube containing pre-heated 3 %

CTAB and then incubated for 1 h in a water bath main-

tained at 65 �C with intermittent mixing. Subsequently,

5 ml of chloroform: iso-amyl-alcohol (24:1) was added,

mixed gently and centrifuged at 8,000 rpm for 10 min.

Then 500 ll of supernatant containing DNA was mixed

with 600 ll of precipitation buffer in a centrifuge tube and

placed on ice for 15 min. The contents were then passed

through red column and centrifuged at 14,000 rpm for

2 min. The flow through was added with 900 ll of binding

buffer and again passed through blue column and centri-

fuged at 8,000 rpm for 1 min. The DNA adhered to blue

column filter was centrifuged twice at 8,000 rpm for 1 min

with 500 ll of wash buffer containing ethanol. The geno-

mic DNA was finally eluted using 200 ll of Tris–EDTA

buffer at 10,000 rpm for 1 min to obtain high molecular

weight pure DNA for fingerprinting. DNA was quantified

with UV/VIS spectrophotometer by measuring OD260 and

OD280. The quantified DNA samples were diluted in TE

buffer to make a final concentration of 50 ng/ll for PCR

reactions.

RAPD Analysis

Eighteen decamer random primers of OPA, OPB and OPP

series (Operon Technologies) were used for initial

screening of 13 varieties of P. granatum. Based on the

reproducibility of scorable bands, the multi-locus geno-

typing by RAPD was performed using ten decamer arbi-

trary primers. Each amplification was performed in a total

volume of 25 ll containing: decamer primer, 1 ll

(50 pmol/ll); dNTP mix, 2 ll (2.5 mM/ll Bangalore

116 S. K. Singh et al.

123

Genei); Taq DNA polymerase, 0.4 ll (5U/ll, Sigma

Chem); MgCl2, 1 ll (25-mM, Sigma Chem); 109 PCR

buffer, 2.5 ll (100 mM, Tris–HCl, pH 8.3, 15 mM MgCl2,

250 mM KCl), 14.1 ll of dH2O and 4 ll of genomic DNA

(50 ng/ll). RAPD–PCR amplifications were performed in a

gradient thermal cycler (Corbett Research, USA) with

initial denaturation step of 94 �C for 3 min followed by 36

amplification cycles of 94 �C for 40 s, 50 �C for 40 s and

72 �C for 2 min and final elongation at 72 �C for 10 min.

Amplicons were separated on to a 1.4 % agarose gel

pre-stained with ethidium bromide in 19 TAE buffer. The

gel was run for 3 h at 50 V. The size of the amplified

fragments was determined using 100 bp plus ladder (MBI

Fermentas). Reproducibility of RAPD amplified DNA

fragments were examined by repeating PCR reactions as

well as running on gel for three times.

PCR Amplification of ITS Region

The universal primers ITS-1 and ITS-4 developed by

White et al. [30] were used to amplify the ITS-1 and ITS-2

regions of ribosomal DNA, encompassing the 5.8S rRNA

gene. Each PCR amplification was performed in a total

volume of 50 ll containing: 1U Taq DNA polymerase

(Bangalore Genei), 2.5 mM MgCl2, 160 lM dNTP mix

(MBI Fermentas, Germany), 50 pmol of each of ITS-1 and

ITS-4 primers (Bangalore Genei), 50 ng genomic DNA in

dH2O. The reactions were performed in a thermal cycler

(Corbett Research, USA) with following conditions: 1 min

denaturation at 95 �C, 30 s annealing at 50 �C, 80 s elon-

gation at 72 �C, for 34 cycles with a final elongation step of

72 �C for 10 min. Agarose gel was stained with ethidium

bromide and photographed under UV light using Syngene

gel documentation system.

Sequence Analysis

Amplified ITS regions were sequenced with an ABI Prism

DNA sequencer (Applied Biosystems, Carlsbad, CA, USA)

using ITS-1 and ITS-4 primers separately for labeling of

each DNA by the BigDye terminator method (Applied

Biosystems, Foster City, CA, USA). The sequenced data

obtained from the ITS-4 primer were inversed using Gene

Doc software [31] and clubbed with the sequence data

obtained with the ITS-1 primer, to obtain the complete

sequence of the ITS region. Comparison of nucleotide

sequences was performed using the basic local alignment

search tool (BLAST) network services of the National

Centre for Biotechnology Information (NCBI) database

(http://www.ncbi.nlm.nih.gov). Molecular characterization

of P. granatum varieties was done on the basis of similarity

with the best aligned sequence of BLAST search. The

phylogenetic relationships of P. granatum varieties were

established by multiple alignment of sequences using Clu-

stalX 2.0.11 and generating phylogram depicting bootstrap

values using NJ plot software [32] based on single nucleo-

tide polymorphisms (SNPs), insertions/deletions (INDELS),

and or length polymorphism in the ITS and 5.8S nuclear

rDNA regions. To assess the robustness of phylogenetic

relationships of pomegranate varieties, best aligned avail-

able reference sequences representing all the test genera

from GenBank database were downloaded in FASTA for-

mat. A composite phylogenetic tree with bootstrap values

showing grouping of 13 pomegranate varieties sequenced

with two reference sequences FM886997 and FM887006

was generated to measure phylogenetic accuracy.

Molecular Analysis of RAPD

Reproducible bands of each locus of RAPD were scored as

binary characters (present = 1, absent = 0). Combined

data matrix obtained from all the ten random primers was

used to determine Jaccard’s similarity coefficient with

NTSYS-pc software [33]. The polymorphic information

content (PIC) values for all the selected primers amplified

by a particular primer pair was calculated for the RAPD

markers to characterize the capacity of each primer to

detect polymorphic loci using the formula derived by

Smith et al. [34]. To perform analysis of molecular vari-

ance (AMOVA), 13 varieties were divided into three

populations based on the state from where they were

originally developed. Principal coordinate analysis (PCA)

via covariance matrix was calculated using GenALEx 6

software [35]. Whereas, diversity in the frequency of

fragment size of RAPD patterns was apportioned within

and among pomegranate varieties using Shannon’s infor-

mation index (i) [36] and gene diversity index (h) follow-

ing Nei [37], coefficient of genetic differentiation between

populations (GST) and gene flow (Nm) using PopGen 32

programme.

Results

Morpho-physiological Characterization

Data presented in Table 1 revealed that the plant height

ranged from 1.5 (Arakta) to 1.95 m (P-23), canopy area

from 2.21 (Ganesh) to 5 m2 (GKVK), fruit weight from

169.7 (Basein seedless) to 231.26 g (P-23), fruit height

from 4.58 (Sindhuri) to 7.07 cm (G-137), fruit width from

5.63 (Sindhuri) to 7.8 cm (G-137), aril per cent from 52.56

(P-23) to 70.15 % (Gulsa red) and TSS from 16.7 (Gulsa

red) to 19.1 % (Ganesh 9 Jalore seedless). However,

many varieties of pomegranate shared the similar ranges of

morpho-physiological values at 5 % level of significance.

Genetic Diversity in Punica granatum 117

123

The mean values of morpho-physiological characters

exhibited phenotypic diversity among and within the pop-

ulations. The population 1 (Maharashtra) exhibited the

maximum phenotypic diversity within the population i.e.

the variety Sinduri was recorded with the minimum fruit

height and fruit width and at the same time variety G-137

was recorded with the highest fruit height and fruit width

both representing Maharashtra. The phenotypic correlation

of morpho-physiological traits with fruit yield resulted in

highly significant and positive correlation with fruit weight

(0.58) followed by TSS (0.54), fruit width (0.18) and

canopy area (0.16), Table 1.

RAPD Analysis

Out of 18 decamer primers initially tested, ten primers

detected intra-specific variations generating scorable

amplicons reproducible patterns and generated 119 marker

bands in the range of 250 bp to 4 kb (Table 2). Out of 119

marker bands, 110 markers were polymorphic amounting

92.44 % polymorphism and exhibited 83.33–100 % poly-

morphism in banding patterns. The number of PCR

amplified products ranged from 8 (OPB-13) to 16 (OPA-02)

with an average of about 12 bands per primer. The RAPD

profile generated by OPB-04 exhibiting the maximum PIC

is shown in Fig. 1. The dendrogram obtained from cumu-

lative binary matrix analysis of ten RAPD primers scorable

fragments clearly delineated all the 13 varieties of

P. granatum into three main clusters (Fig. 2). Cluster I

contained four varieties i.e. P-23, G-137, Sindhuri and

Ganesh. Cluster II included three varieties i.e. Dholka,

Gulsa red and GKVK. Whereas, the variety P-26 can be

seen as distinct variety between cluster I and cluster II.

Cluster III included three varieties namely, Jalore seedless,

Ganesh 9 Jalore seedless and Basein seedless. Jodhpur red

and Arakta were delineated as the most distinct varieties of

pomegranate tested.

ITS Amplification and Sequencing

All the 13 P. granatum varieties generated a single identical

prominent band on the gel electrophoresis which included

partial sequences of 18S gene, complete sequence (ITS-1,

5.8S gene, ITS-2) and partial sequence of 28S gene upon

direct sequencing using ITS-1 and ITS-4 universal primers

(Fig. 3). All the gene sequences have been submitted to

National Centre for Biotechnological Information (NCBI),

USA and have been assigned Gen accession numbers from

JQ655163 to JQ65175 (Table 3). The conserved 5.8S rDNA

region was recorded with a uniform nucleotide length of 165

base pair in all the pomegranate varieties. Whereas, ITS-1

region exhibited base pair length diversity from 204 to

206 bp and ITS-2 region was recorded with a uniform base

pair length of 241 bp except in variety Arakta which was

recorded with ITS-2 length of 240 bp.

Upon multiple sequence alignment of all the 13 pome-

granate sequences, we detected not only SNPs at number of

places but also INDELS in both the ITS regions. ITS-1

region exhibited INDELS at three places and SNPs at four

places. Whereas, 5.8S gene region was recorded with SNPs

Table 1 Morpho-physiological characterization of Punica granatum varieties

Genotype State Plant

height (m)

Canopy

area (m2)

Fruit

weight (g)

Fruit height

(cm)

Fruit

breadth

(cm)

Aril

(%)

TSS

(%)

Fruit yield/

plant (kg)

P-23 Maharashtra 1.95 4.46 231.26 6.54 7.1 52.56 17.3 17.2

P-26 Maharashtra 1.90 5.32 203.38 6.3 6.4 64.20 17.8 16.72

G-137 Maharashtra 1.70 3.26 212.45 7.07 7.8 67.53 17.8 17.45

Arakta Maharashtra 1.50 3.76 187.78 4.97 6.2 68.77 16.8 14.2

Sinduri Maharashtra 1.70 3.79 198.26 4.58 5.63 66.10 17.2 18.2

Jodhpur red Rajasthan 1.84 3.07 182.42 6.83 7.2 66.12 17.0 12.26

Dholka Rajasthan 1.88 3.98 190.57 6.4 6.5 69.97 16.8 13.26

Gulsa red Maharashtra 1.78 3.48 170.40 5.8 6.1 70.15 16.7 15.36

GKVK Karnataka 1.80 5.00 201.40 6.3 6.8 67.52 18.0 16.5

Basein seedless Karnataka 1.94 4.83 169.70 6.10 6.8 72.02 18.8 13.62

Ganesh Maharashtra 1.75 2.21 183.42 6.12 6.28 68.49 17.6 14.32

Jalore seedless Rajasthan 1.88 4.26 194.9 6.7 6.87 72.0 18.2 19.2

Ganesh 9 Jalore

seedless

Maharashtra 9 Rajasthan 1.75 3.54 204.28 5.99 7.2 69.18 19.1 21.2

CD (5 %) 0.20 0.36 22.82 0.62 0.58 3.10 0.98 3.00

Phenotypic correlation with fruit yield -0.05 0.16 0.58 -0.06 0.18 -0.12 0.54 1.00

118 S. K. Singh et al.

123

at two places one each in Arakta and Sindhuri. However,

ITS-2 region exhibited INDELS at only one place

and SNPs at 25 places by way of replacement of single

nucleotide.

Table 2 Details of RAPD primers and their banding pattern

S.N. Primer code Primer sequence GC (%) Molecular

weight (bp)

No. of bands No. of polymorphic

bands

Polymorphism

(%)

PIC value

(%)

Min. Max.

1. OPA-02 TGC CGA GCT G 70 250 2,800 16 15 93.75 0.89

2. OPA-09 GGG TAA CGC C 70 275 2,000 9 9 100.00 0.86

3. OPA-13 CAG CAC CCA C 70 275 1,800 13 12 92.31 0.91

4. OPB-04 GGA CTG GAG T 60 400 3,500 13 12 92.31 0.92

5. OPB-05 TGC GCC CTT C 70 250 3,500 13 12 92.31 0.91

6. OPB-06 TGC TCT GCC C 70 350 1,800 12 10 83.33 0.88

7. OPB-13 TTC CCC CGC T 70 250 1,800 8 7 87.50 0.84

8. OPB-14 TCC GCT CTG G 70 300 4,000 13 12 92.31 0.88

9. OPP-09 GTG GTC CGC A 70 450 3,000 11 10 90.91 0.87

10. OPP-16 CCA AGC TGC C 70 350 2,400 11 11 100.00 0.86

Total 119 110

Average 92.44 0.88

Fig. 1 RAPD profiles of 13 varieties of Punica granatum amplified

by OPA-04 primer

Fig. 2 Dendrogram of 13

varieties of Punica granatumbased on ten random RAPD

informative primers

Fig. 3 PCR amplified products of r DNA of 13 varieties of Punicagranatum on gel electrophoresis

Genetic Diversity in Punica granatum 119

123

The phylogram generated based on multiple sequence

alignment showing delineation of pomegranate cultivars and

two best aligned reference sequences downloaded from

NCBI, Database is shown as Fig. 4. The pomegranate varie-

ties which were shown to have clustering in RAPD dendro-

gram and were further delineated from each other along with

their reference sequences with significant bootstrap values.

Genetic Analysis

The graphical representation of PCA of the three P.

granatum populations is shown in Fig. 5. The first three

principal coordinates accounted for 62.38, 15.08 and

6.75 %, respectively, amounting to total of 84.21 % of total

variance. The Eigen vector analysis indicated that the con-

tribution of the first three factors was 25.67, 6.20 and 2.78,

respectively, (explaining a total 34.65 % of total variability).

The population 1 comprising of seven varieties is seen as

genetically most distinct group of varieties as compared to

others. The AMOVA of RAPD data (Table 4) revealed that

100 % genetic variation existed within populations. The

results validate the existence of higher genetic diversity

among Indian pomegranate varieties studied.

The mean genetic variation statistics of all the three

populations and mean of all the loci is presented in Table 5.

The mean values of all the three populations together for

Nei’s gene diversity (h) was 0.2684 and Shannon Infor-

mation Index (i) was 0.4135. Results exhibited that the

genetic diversity of P. granatum varieties of population 1

was the richest among the three populations and in popu-

lation 3 it was the lowest. The greater genetic variability of

population 1 can also be seen as out group in both RAPD

dendrogram and rDNA phylogram.

Nei’s unbiased measure of genetic distances was eval-

uated to further elucidate the gene differentiation between

and among populations. The Nei’s genetic distance ranged

from 0.0620 to 0.0878 and genetic identity ranged from

0.9160 to 0.9398 based on RAPD analysis (Table 6). The

Table 3 Nucleotide base pair lengths of nuclear ribosomal RNA gene of 13 varieties of P. granatum

Genotype Gen accession number ITS-1 (bp) 5.8S (bp) ITS-2 (bp) Total (bp)

P-23 JQ655163 205 165 241 611

P-26 JQ655164 205 165 241 611

G-137 JQ655165 205 165 241 611

Arakta JQ655166 206 165 240 611

Sinduri JQ655167 205 165 241 611

Jodhpur red JQ655168 205 165 241 611

Dholka JQ655169 204 165 241 610

Gulsa red JQ655170 205 165 241 611

GKVK JQ655171 204 165 241 610

Basein seedless JQ655172 204 165 241 610

Ganesh JQ655173 205 165 241 611

Jalore seedless JQ655174 205 165 241 611

Ganesh 9 Jalore seedless JQ655175 205 165 241 611

Fig. 4 Phylogram based on

multiple sequence alignment

showing intra-specific

relationship among 13 varieties

of Punica granatum and with

two GenBank reference

sequences

120 S. K. Singh et al.

123

largest genetic distance occurred between population 1 and

3 and the least between population 1 and 2 and vice versa

for genetic identity. Coefficient of gene differentiation

between populations (Gst) was 0.2584 indicating that

mainly proportion of genetic variations was within popu-

lations than among populations. The gene flow (Nm) varied

from 0.2528 to 16.695 between pair wise populations and

was 1.4347 among all the populations.

Discussion

Initially attempts were made to isolate the genomic DNA

using CTAB method [29] but due to the presence of

polyphenols, mucilages, polysaccharides as glucans and

cuticular wax and subsequently by the Plant Genomic DNA

Purification spin kit ‘‘Hi Pura’’ of Hi-media Company due

to clogging and plugging of columns of the kit we ended up

with low molecular weight poor quality genomic DNA.

Therefore, a hybrid protocol of DNA isolation was devel-

oped not only to eliminate the interference of said com-

pounds and to subsequently feed precipitated DNA devoid

of impurities to red column of the kit. This resulted in

isolation of high molecular weight good quality genomic

DNA.

There is no consistency in grouping of pomegranate

varieties with similar morphological traits as phenotypi-

cally similar varieties were genetically catalogued into

different clusters of RAPD dendrogram. It is evident from

the fact that the pomegranate varieties belonging to dif-

ferent states and agro-climatic regions fall into same RAPD

phylogenetic cluster for example, Gulsa red (Maharashtra),

GKVK (Karnataka) and Dholka (Rajasthan) together

formed cluster II and similarly Jolore seedless (Rajasthan),

Ganesh 9 Jolore seedless (Maharashtra) and Basein seed-

less (Karnataka) formed cluster III of RAPD dendrogram.

This validates that P. granatum genetic diversity has wide

genetic distribution across agro-climatic regions of the

country. Previous studies on Iranian pomegranate using

RAPD [14, 38], using ISSR and RAPD [39], north eastern

Turkey using AFLP markers [40], Spain using 18–28S

rDNA [41], Tunisia using AFLP [16] and RAPD [42] could

not detect any correlation between geographic distribution

and genetic distances measured by DNA markers. Solei-

mani et al. [43] used sequence related amplified polymor-

phism (SRAP) markers to assess genetic diversity and

population of wild, cultivated and ornamental pomegran-

ates in different regions of Iran. They detected low dif-

ferentiation in allele frequencies among populations and

high gene flow indicating that the genetic diversity of

pomegranates is independent of their geographical origin.

This can be attributed to the fact that the exchange of plant

materials across the regions during the history of pome-

granate cultivation.

In general, the robustness of a molecular marker tech-

nique depends on the amount of polymorphism, it can

Fig. 5 The PCA of all the three populations using GenALEx

software (Peakall and Smouse [35]) for13 varieties of Punicagranatum

Table 4 Summary of AMOVA analysis

Source df SS MS Est. Var. %

Among pops 2 17.011 8.505 0.000 0

Within pops 10 137.143 13.714 13.714 100

Total 12 154.154 13.714 100

Table 5 Summary of genetic variation statistics for all loci

Locus (mean) Sample size na ne h i

Pop 1 7 1.8824 1.5623 0.3150 0.4660

Pop 2 4 1.3193 1.2605 0.1398 0.1998

Pop 3 2 1.1092 1.0772 0.0453 0.0661

Mean of all loci 13 1.9244 1.4408 0.2684 0.4135

na observed number of alleles, ne effective number of alleles, h Nei’s

gene diversity, i Shannon information index

Table 6 Matrix of unbiased genetic identity and genetic distance

according to Nei [37] among three populations of Punica granatumbased on 119 RAPD markers

Population Pop 1 Pop 2 Pop 3

Pop1 – 0.9398 0.9160

Pop2 0.0620 – 0.9189

Pop3 0.0878 0.0845 –

Nei’s genetic identity (above diagonal) and genetic distance (below

diagonal)

Genetic Diversity in Punica granatum 121

123

detect. Among the set of accessions investigated, the

cumulative analysis of all the ten informative RAPD

primers detected an average of 92.44 % polymorphism in

banding pattern with overall 88 % PIC values indicating its

efficiency in evaluating genetic diversity in pomegranate.

Earlier researchers have reported lower PIC values using

different molecular marker techniques [18, 40, 43, 44].

Under present study a uniform base pair length of 165 bp

in all the 13 sequenced genotypes validates the conserved

nature of 5.8S gene region. The length polymorphism in

intervening intron ITS regions indicates non-coding and

variable nature of these regions. The 5.8S region was found

to be highly conserved (99.34 %) followed by ITS-1

(96.58 %) and ITS-2 (89.21 %). ITS-2 region was recorded

with the highest percentage of parsimony informative sites

with high divergence mainly due to SNPs. ITS sequence

data determined the genetic diversity among putative Indian

pomegranate varieties studied with high boot strap values.

All the test varieties could be clearly distinguished by gen-

erating phylogram using NJ Plot.

The ITS length variants and polymorphism have been

reported in several plant species [25, 28, 45]. Saini et al. [22]

reported heterogeneity in nuclear rDNA ITS region in Vigna

radiata which did not cause any phylogenetic errors at

species level. Barkley et al. [46] observed SNPs and sug-

gested EcoTILLING as a powerful genetic analysis tool for

rapid identification of naturally occurring variation in plants.

The PCA analysis of RAPD data clearly delineated all

the 13 varieties of pomegranate and showed the first three

components contributed as much as 84.21 % of total var-

iance. The AMOVA of RAPD data revealed the cent per

cent genetic variation existed within population rather than

among populations. The result of AMOVA also supports

genetic relationship between pomegranate varieties by

cluster analysis, where varieties belonging to different

localities grouped together. Moslemi et al. [44] also did not

found significant differences between Iranian pomegranate

genotypes from different provinces by using AFLP mark-

ers. Similar results were obtained by Yuan et al. [47] and

Noormohammadi et al. [38] using molecular marker anal-

ysis. The high level of genetic diversity within groups

(populations) and low level of that among them is attrib-

uted to the clone propagation of pomegranate and the

excessive gene flow between different localities due to

germplasm exchange. The molecular analysis validates the

existence of total genetic diversity within population than

amongst population with higher h and i values because of

rich genetic diversity in pomegranate varieties studied. Our

results are in agreement with Narzary et al. [15] and Ercisli

et al. [40] who reported more than 90 % genetic diversity

among pomegranate genotypes. By contrast, earlier studies

using RAPD markers have reported inconsistent and lower

genetic variability ranging from 22 to 85 % among

cultivars of pomegranate from different countries [48–51].

The higher Gst of 0.2584 indicated that the main propor-

tion of genetic variations was within populations than

among populations which was also supported by the

AMOVA. Similarly the higher gene flow (Nm) ranging

from 0.2528 to 16.695 was recorded between pair wise

populations than amongst populations. Moslemi et al. [44]

reported 0.124 (Gst) and 0.969–10.404 (Nm) in Iranian,

and Yuan et al. [47] recorded 0.2018 (Gst) and an average

of 1.9027 (Nm) in Chinese populations to explain gene

differentiation within and amongst pomegranate popula-

tions, respectively.

The morpho-physiological characterization proved

insufficient to distinguish pomegranate varieties studied.

The primers identified in the present study are robust

RAPD markers that explained an average of 92.44 %

polymorphism in banding pattern and 88 % PIC and can be

used as powerful markers to reveal genetic diversity in

pomegranate. This study validates the utility of ITS rDNA

region as a reliable indicator of phylogenetic interrela-

tionships, especially ITS-2 as probable DNA barcode at

higher levels and can serve as an additional approach for

identification and genetic cataloguing of pomegranate

germplasm for crop improvement. The high genetic vari-

ability in this set of Indian pomegranate genotypes suggest

that they might have originated from genetically divergent

parents or has a long history of adaptation to their

respective micro-climatic regions and could be of signifi-

cance to contribute to pomegranate breeding programmes.

Acknowledgments The authors are thankful to Dr. M. M. Roy

Director, Central Arid Zone Research Institute, Jodhpur for providing

necessary laboratory and field facilities to carry out this study.

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