RESEARCH ARTICLE

Comparative analysis of genetic diversity using molecularand morphometric markers in Andrographis paniculata(Burm. f.) Nees

Surrinder K. Lattoo Æ Rekha S. Dhar Æ Shabnam Khan Æ S. Bamotra ÆMaharaj K. Bhan Æ Autar K. Dhar Æ Kuldip K. Gupta

Received: 7 August 2006 / Accepted: 11 January 2007 / Published online: 12 May 2007� Springer Science+Business Media B.V. 2007

Abstract Andrographis paniculata is a medici-

nal plant of immense therapeutic value. The

present study was aimed to elucidate its genetic

diversity based on morphochemical and RAPD

markers from 53 accessions belonging to 5 eco-

geographic regions. Analysis of variance and D2

statistics revealed significant differences in all the

metric traits and sufficient inter-cluster distances

indicating considerable diversity among the

accessions. The complementary approach of

RAPD was used to evaluate the genetic dissim-

ilarities among all the accessions using 6 highly

polymorphic primers. The average proportion of

polymorphic loci across primers was 96.28%. The

molecular genetic diversity based on Shannon

index per primer averaged 5.585 with values

ranging from 3.08 to 8.70 indicating towards wide

genetic base. RAPD based UPGMA and D2

cluster analysis also revealed that various acces-

sions available in different eco-geographic regions

might have originated from native places of wild

abundance. Similarity matrices were generated

for molecular markers and morphometric data to

determine the degree of congruence between the

two. A highly significant but low correlation

(r = 0.547, P < 0.001) was obtained thus implying

the correspondence between the two. The species

is hermaphroditic and a habitual inbreeder. The

present study yielded a typical triangular congru-

ence between its breeding system, morphometric

traits and RAPD markers thus elucidating the

usefulness of complementary approaches to make

diversity analysis more explanatory and purpose-

ful for optimum genetic amelioration and effec-

tive conservation of its genotypic variability.

Keywords Andrographis paniculata �Andrographolide � Genetic diversity �Mahalanobis D2 statistics � Medicinal herb �Shannon index

Introduction

Andrographis paniculata (Acanthaceae) is a

medicinal herb of immense therapeutic value

with wide geographic distribution from peninsular

India, Sri Lanka, south-east Asia, China, Amer-

ica, West Indies and Christmas Island in Indian

S. K. Lattoo (&) � S. Khan � S. Bamotra �M. K. Bhan � A. K. DharDepartment of Genetics and Plant Breeding,Regional Research Laboratory (CSIR), Canal Road,Jammu Tawi 180 001, Indiae-mail: surrinlattoo@rediffmail.com

R. S. DharDepartment of Biotechnology, Regional ResearchLaboratory (CSIR), Jammu Tawi, India

K. K. GuptaDivision of Natural Products Chemistry, RegionalResearch Laboratory (CSIR), Jammu Tawi, India

123

Genet Resour Crop Evol (2008) 55:33–43

DOI 10.1007/s10722-007-9212-y

ocean (Anonymous 1985). The crude extract from

whole plant has shown anti HIV activity (Otake

et al. 1995). Its therapeutic value is ascribed to

various bioactive constituents synthesized and

accumulated in its leaves and roots. Some of the

major constituents include andrographolide,

deoxyandrographolide, 14-deoxy-11,12-didehy-

droandrographolide, neoandrographolide and

andrographiside. Andrographolide protects liver

and gallbladder and is more efficacious than

silymarin, a known hepatoprotective drug (Sara-

swat et al. 1995). Neoandrographolide has shown

greater activity against malaria (Misra et al. 1992)

and is hepatoprotective against carbontetrachlo-

ride induced hepatic damage (Kapil et al. 1993),

while 14-deoxyandrographolide produces a more

potent hypotensive effect in anesthetized rats and

in isolated right atria (Zhang et al. 1998).

For optimum genetic amelioration and effec-

tive conservation of the allelic and genotypic

variability in a given species, it is imperative to

evaluate and catalogue the variability. The

improvement in quantitative and quality traits

can be achieved through understanding the nature

and amount of variability present in the genotypes

/ accessions for breeding purposes. Diversity

analysis based on morphological, chemical and

biochemical traits is most rampant and extremely

useful. To complement and supplement the char-

acterization on the basis of morphochemical

descriptors and geographic origin with the appli-

cation of molecular methods has the potential to

accelerate the accumulation of this information.

In the backdrop of immense therapeutic value

of A. paniculata and scarce data regarding its

genetic diversity, the information on intraspecific

relatedness is important for selection of divergent

genotypes for crosses and effective conservation

and management of its germplasm resources.

Furthermore, it would be interesting, both con-

ceptually and empirically to find if there is any

agreement between the morphophysiological

traits and molecular markers. The present inves-

tigation was thus aimed to (1) characterize the

morphochemical diversity in the germplasm of

A. paniculata (2) evaluate the molecular diversity

employing RAPD and (3) also to compare and

understand the degree of congruence between the

morphometeric and RAPD markers.

Materials and methods

Morphometeric and chemical variation

Present study was conducted at Regional Re-

search Laboratory (CSIR), Jammu, India

(32�44¢ N, 75�55¢ E; 305 m in altitude) where

the annual temperature fluctuates between 5�C

and 45�C and mean annual rainfall measures upto

1,100 mm. The material for present study com-

prised 53 accessions procured from different eco-

geographic regions of India (Table 1; Fig. 1) and

maintained in the germplasm repository at RRLJ.

For the purpose of studying variation, individual

1

3

4

5

2

1. Himachal Pradesh (H.P.) 2. Delhi (NBPGR) 3. Madhya Pradesh (M.P.) 4. Karnataka 5. Assam

Fig. 1 Map of India showing geographical locations fromwhere Andrographis paniculata accessions were procured/collected. Map is only representative and distances are notto the scale

Table 1 List of Andrographis paniculata accessions fromdifferent sources in India and their status

Accessions Origin Status

APJ 001-APJ 017 Karnataka CultivatedAPJ 018-APJ 027 Himachal Pradesh

(H.P.)Cultivated

APJ 028-APJ 039 Delhi (NBPGR)* Composite seedcollection

APJ 040-APJ 048 Madhya Pradesh(M.P.)

Wild

APJ 049-APJ 053 Assam Cultivated

*National Bureau of Plant Genetic Resources, New Delhi

34 Genet Resour Crop Evol (2008) 55:33–43

123

open-pollinated seed lots from 53 accessions were

germinated in earthen pots containing mixture of

soil and sand in the proportion of 4:1 under

outdoor conditions during the month of June

2002. The plants were transplanted after 45 days

of germination into 4 · 2.5 m plots at spacing of

40 · 50 cm apart in a randomized block design

with 3 replications. Plants were raised in well-

drained sandy loam soil (pH 7.4) under uniform

cultivation conditions. Data were recorded on 15

competitive plants for each accession for series of

seven metric traits: plant height (cm), leaf length

(cm), leaf width (cm), plant spread (cm), number

of leaves per plant and herbage yield per plant

(g). The data were subjected to analysis of

variance and only those characters in which

variation observed was significant were consid-

ered for multivariate analysis of D2 statistics

based on Mahalanobis (1936) and Rao (1952).

The analysis was done using SYSTAT (version

10.2) statistical package and cluster formation was

confirmed by Tocher’s method. The relative

contribution of each character towards genetic

divergence was also worked out. All the acces-

sions were evaluated for androgrpholide content

after 120 days of transplantation. For chemical

analysis fresh herbage was air dried under shade

and analyzed for andrographolides according to

HPLC method of Saxena et al. (2000).

DNA fingerprinting: molecular analysis

Genomic DNA was extracted from leaves of 4-

week old seedling from 53 accessions. DNA was

isolated following the modified procedure of

Doyle and Doyle (1990). The yield of DNA was

40–60 lg g–1 tissue, the UV absorbance ratio at

260 nm/280 nm, were at least 1.9–2.1 respectively.

An aliquot of 3 ll of preparations was checked on

0.5% agarose gel for purity. In a pre-screen with

48 primers based on the amplification of A. pan-

iculata plants, six arbitrary decamer primers

(Operon Technologies, USA) were selected for

polymerase chain reaction (PCR). These primers

produced distinct amplification profiles that were

easily scorable. DNA amplifications were per-

formed in Master Cycler Gradient (Ependorf,

Germany) and the PCR conditions were as

follows: 3 min at 95�C 40 cycles of: 1 min at

94�C, 1 min at 35�C, 2 min at 72�C and as a last

step 10 min at 72�C. 20 ll reaction mixture

contained 1· PCR buffer (10 mM Tris–HCl,

pH 9.0; 50 mM KCl; 2.5 mM MgCl2; 200 lM

dNTP (Promega); 200 lM random primers; 20–

30 ng of DNA template and 0.5 U of Taq DNA

polymerase (Banglore Genei, Banglore, India).

On the completion of the programme for the

amplification of DNA samples, they were stored a

–20�C. The separation of DNAs was performed

by electrophoresis in 1.5% (w/v) agarose gel. The

gel was documented using Image Master VDS

(Amersham Biopharmacia, USA). All the PCR

reactions were repeated at least twice to check

the reproducibility.

Amplification profiles were recorded and the

size of each fragment was estimated using soft-

ware (SEQUAID II [tm] version 2.2, 1987).

Amplicons were scored as discrete variables,

using 1 to indicate presence and 0 for absence.

The binary matrix was used to generate pair-wise

similarities between the accessions based on the

number of shared amplification products (Nei and

Lei 1979). Similarity matrix was subjected to

cluster analysis to develop a dendrogram using a

procedure of NTSYS-pc (Rohlf 1993), which uses

the unweighted pair group method with arithme-

tic averages (UPGMA) (Sneath and Sokal 1973).

Genetic diversity was estimated by Shannon

index (Lewontin 1972):

H ¼ �Xk

i¼1

Pi ln Pi

where H denotes the diversity of RAPD markers

in a population, k is the number of bands

produced with the respective primer and pi is

the frequency of the ith fragment.

Morphometric analysis

For morphmetric traits, data (Table 2) were

subjected to linear transformation by computing

the mean and standard deviation of the states of

each trait and the values were expressed as the

matrix of the deviations from the mean in

standard deviation units (Bookistein 1991). A

pair-wise distance matrix was generated using the

Genet Resour Crop Evol (2008) 55:33–43 35

123

Table 2 Mean performance of 53 accessions of Andrographis paniculata

AccessionNo.

Dry herbageyield/plant(g)

Leaf stem/ratio

Plantheight(cm)

Leaflength(cm)

Leafwidth(cm)

Plantspread(cm)

Number ofleaves/plant

Total andro-grapholides (%)

APJ 001 36.92 0.395 61.86 5.10 1.68 45.00 259.0 5.74APJ 002 54.26 0.445 59.81 4.59 1.41 44.80 338.0 5.41APJ 003 42.60 0.368 57.36 4.84 1.51 45.69 295.0 4.41APJ 004 42.76 0.412 59.66 5.15 1.65 51.63 325.0 5.61APJ 005 42.54 0.386 56.23 4.85 1.49 53.83 344.0 5.53APJ 006 47.96 0.328 64.03 5.12 1.53 55.98 281.0 4.58APJ 007 48.49 0.439 55.03 4.77 1.42 47.88 370.0 5.06APJ 008 43.68 0.315 60.53 4.62 1.42 49.17 281.0 5.94APJ 009 38.86 0.323 61.66 4.95 1.51 48.56 323.0 5.76APJ 010 52.26 0.399 59.47 5.08 1.67 46.88 343.0 5.60APJ 011 54.50 0.296 68.91 4.70 1.57 63.41 357.0 4.14APJ 012 32.19 0.427 80.23 4.65 1.48 42.45 317.0 4.92APJ 013 34.99 0.410 76.16 4.84 1.68 44.15 326.0 2.67APJ 014 39.20 0.480 60.91 5.07 1.68 41.33 419.0 3.74APJ 015 46.50 0.423 61.00 4.85 1.48 51.45 299.0 3.19APJ 016 30.90 0.336 60.58 4.56 1.51 51.50 284.0 4.66APJ 017 47.70 0.353 54.83 4.61 1.43 46.42 277.0 5.11APJ 018 48.50 0.390 50.66 4.38 1.53 51.76 331.0 5.17APJ 019 50.10 0.337 57.50 4.16 1.38 52.36 387.0 4.43APJ 020 48.50 0.354 56.83 4.94 1.60 50.87 360.0 3.90APJ 021 50.60 0.280 64.50 5.20 1.70 52.00 216.6 4.45APJ 022 36.60 0.380 58.60 5.90 1.50 45.00 293.3 5.40APJ 023 35.00 0.320 51.60 4.80 1.60 44.20 244.0 4.58APJ 024 31.80 0.430 56.50 5.00 1.80 42.45 251.0 3.75APJ 025 54.20 0.440 59.80 4.60 1.50 44.80 338.0 5.41APJ 026 48.50 0.350 56.80 4.90 1.60 51.87 360.0 3.90APJ 027 52.20 0.390 59.40 5.00 1.60 46.88 343.0 5.60APJ 028 46.00 0.420 61.00 4.80 1.40 42.45 317.0 4.92APJ 029 50.00 0.330 57.50 4.10 1.30 52.00 387.0 4.43APJ 030 32.10 0.420 80.20 4.60 1.40 42.25 317.0 4.92APJ 031 42.50 0.380 56.20 4.80 1.40 53.83 344.0 5.53APJ 032 38.80 0.320 61.60 4.90 1.50 48.56 323.0 5.76APJ 033 30.90 0.330 60.50 4.50 1.50 51.50 287.0 4.66APJ 034 39.20 0.480 60.90 5.00 1.60 41.33 419.0 3.74APJ 035 52.60 0.400 59.40 5.00 1.60 46.88 343.0 5.60APJ 036 47.70 0.360 54.80 4.60 1.40 46.42 277.0 5.11APJ 037 50.10 0.380 57.60 4.20 1.40 52.02 388.0 4.44APJ 038 42.70 0.410 59.70 4.80 1.50 45.70 295.0 4.41APJ 039 48.40 0.430 55.00 4.70 1.40 47.88 372.0 5.06APJ 040 32.90 0.430 80.30 4.60 1.50 42.45 317.0 4.92APJ 041 46.70 0.440 61.50 5.00 1.50 42.30 327.0 4.93APJ 042 47.90 0.390 64.00 5.10 1.50 55.90 281.0 4.58APJ 043 52.30 0.400 60.40 5.00 1.70 46.90 345.0 5.60APJ 044 47.50 0.350 54.80 4.60 1.50 51.50 290.0 4.66APJ 045 42.60 0.410 56.20 4.90 1.50 45.70 344.0 4.41APJ 046 48.00 0.430 55.00 5.00 1.50 49.17 340.0 5.94APJ 047 50.30 0.340 57.30 4.50 1.60 52.21 370.0 4.43APJ 048 43.50 0.430 57.00 5.00 1.90 45.69 351.0 3.19APJ 049 49.60 0.400 59.80 5.20 2.00 51.87 372.0 4.24APJ 050 33.00 0.410 62.20 5.50 1.90 42.45 342.0 4.92APJ 051 35.00 0.400 60.00 5.00 1.90 44.80 342.0 5.74APJ 052 52.00 0.450 65.00 5.30 1.90 46.80 366.0 5.60APJ 053 50.10 0.380 58.90 5.60 1.40 47.40 349.0 4.85

36 Genet Resour Crop Evol (2008) 55:33–43

123

average taxonomic distance coefficient (Rohlf

1993) between a pair of accessions (j and k) for

n characters as

djk ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1

n

Xn

i¼1

ðjxij � xjkjÞ2s

The similarity matrix for morphmetric markers

was generated by calculating 1–dij.

Results and discussion

Analysis of variance revealed highly significant

differences among all the accession metric traits

under investigation (Table 3). However, the

chemical constituents were not significant. The

computed D2 values for all the n (n–1)/2 = 1,378

pairs of accessions ranged from 0.01 to 76.98

thereby indicating considerable diversity in the

present material. Based on D2 values, all the 53

accessions could be grouped into five clusters such

that the accessions within a cluster have smaller

D2 values among themselves than those belonging

to different clusters (Table 4). The distribution of

different groups revealed that there were 31

accessions in cluster I, 9 in cluster II, 6 in cluster

III, only 2 in cluster IV and 5 accessions in cluster

V (Table 5). Accessions tended to group together

in separate clusters on the basis of either low,

moderate or high mean values for different

characters (Table 6). Cluster means revealed

considerable differences among the clusters.

Accessions of cluster V had highest dry herbage

per plant, maximum plant height and leaf length

with highest plant canopy. Assemblage of 9

accessions representing cluster II, manifested

moderate to low averages for all the traits but

interestingly revealed highest total andrographo-

lide content. 31 accessions belonging to cluster I

represented heterogeneous assemblage as they

presented moderate to low mean values for

different traits. Four of these accessions are

cultivated and had incidentally less of total

andrographolide.

Table 4 revealed that average intra-cluster

distance ranged from 0.34 to 4.06, while inter-

cluster distance ranged between 3.95 and 6.85.

Cluster III and V showed maximum inter-cluster

Table 2 continued

AccessionNo.

Dry herbageyield/plant(g)

Leaf stem/ratio

Plantheight(cm)

Leaflength(cm)

Leafwidth(cm)

Plantspread(cm)

Number ofleaves/plant

Total andro-grapholides (%)

Mean 44.24 0.387 60.514 4.86 1.56 48.08 327.68 4.816Range 30.90–54.500 0.28–0.48 50.66–80.33 4.10–5.90 1.30–2.0 41.33–63.41 216.6–419.0 2.67–5.94SE 0.972 0.006 0.871 0.046 0.021 0.618 5.844 0.103CD 5.586 0.082 10.388 0.5508 0.1695 8.647 49.763 N.S.

Table 3 Analysis of variance in Andrographis paniculata

Source ofvariation

Degree offreedom(df)

Mean square

Herbage yield /plant (g)

Leaf/stemratio

Plantheight(cm)

Leaflength(cm)

Leafwidth(cm)

Plantspread(cm)

Number ofleaves/plant

Replication 2 3.1963 0.0038 19.9920 0.2253 0.0084 436.3455 179.6385Treatments 52 150.2645* 0.0062* 116.7330* 0.3140 0.0641* 60.7037* 5504.8287*Error 104 11.9088 0.0025 40.4793 0.1157 0.0109 28.5158 944.5890

*Significant P £ 0.01

Table 4 Intra- and inter-cluster divergence (D2) among 5clusters involving 53 accessions of Andrographis paniculata

Cluster 1 2 3 4 5 D2

1. 2.63 4.88 5.93 4.75 3.95 4.882. 2.77 4.25 5.71 5.57 5.103. 2.84 4.63 6.85 5.414. 0.34 6.81 5.475. 4.06 5.79

Genet Resour Crop Evol (2008) 55:33–43 37

123

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38 Genet Resour Crop Evol (2008) 55:33–43

123

divergence (6.85), thus indicating wide diversity

between the accessions of these two clusters. The

minimum inter-cluster distance occurred between

clusters I and V, suggestive of close relationship

(Fig. 2). On the basis of average divergence (D2)

cluster V was the most divergent from the rest.

Inter-cluster divergence was also confirmed by

canonical analysis. Since x1 + x2 + x3 = 84%; a

three dimensional representation (Fig. 3), show-

ing distance relationship among 53 accessions was

found adequate (Table 7).

Of 48 decamer primers used to screen repre-

sentative DNA samples, 6 (12.5%) detected

scorable polymorphism in banding patterns

among all the 53 accessions (Table 8). Six

selected primers generated a total of 185 bands

of which 177 were polymorphic. An example of

the representative profiles of 20 accessions with

two primers is shown in Fig. 5. The number of

bands per accession ranged from 4 to 16 and the

bands amplified ranged in size from 250 bp to

7,000 bp, although 68.9% (122 out of 177) ranged

between 300 bp and 1,200 bp. The average num-

ber of bands per primer ranged between 19

(OPB-02) and 42 (OPW-05) with a mean of

29.5. The proportion of polymorphic markers

across the primers ranged between 92.85% and

100% with an average of 96.28% (Table 8).

Padmesh et al. (1999) have also reported Jac-

card’s similarity range of 38–89% in 15 native and

Table 6 Cluster mean for 53 accessions in Andrographis paniculata

Clusterlevels

Number ofaccessions

Dry herbageyield/plant (g)

Leaf/stemratio

Plantheight(cm)

Leaflength(cm)

Leafwidth(cm)

Plantspread(cm)

Number ofleaves/plant

1. 31 48.04 0.39 57.99 4.79 1.52 48.58 3392. 9 35.94 0.35 58.53 4.93 1.55 47.34 2833. 6 33.36 0.42 73.18 4.86 1.64 43.09 3324. 2 39.20 0.48 60.91 5.04 1.64 41.33 4195. 5 50.59 0.35 64.11 5.09 1.64 54.90 300

1 (2.63)

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(0.34)

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6.85

2 (2.77)

Fig. 2 Mutual relationship among 53 accessions of And-rographis paniculata

Fig. 3 Spatialdistribution of 53accessions ofAndrographis paniculataalong three principlecoordinate axes based onmorphological characters

Genet Resour Crop Evol (2008) 55:33–43 39

123

exotic collections of A. paniculata. However, in

the present material, there was comparatively

higher proportion of genetic diversity as the

Jaccard’s similarity values ranged between 0.333

and 0.812 with an average value of 0.61. Molec-

ular markers generated by RAPD displayed

adequate polymorphism and large proportion of

exclusive RAPD loci, which account for substan-

tial portion of genetic diversity. Diversity based

on Shannon’s index averaged 5.585 with 6 primers

(Table 8), with values ranging between 3.08

(OPH-08) to 8.70 (OPB-02). This is indicative of

large differences among the accessions, conse-

quently wide genetic base in A. paniculata.

Table 7 Relative contribution of characters towards divergence in Andrographis paniculata

Character x1 x2 x3 Character contributing % Rank

Dry herbage yield/plant (g) 0.907 0.112 0.275 48.40 ILeaf stem/ratio 0.163 –0.412 0.427 8.56 IIIPlant height (cm) –0.318 –0.176 0.773 7.04 IVLeaf length (cm) 0.079 0.071 –0.049 1.60 VIILeaf width (cm) –0.069 –0.078 0.032 4.86 VIPlant spread (cm) 0.136 0.139 –0.131 5.01 VNumber of leaves/plant 0.139 –0.869 –0.352 24.53 II

Fig. 4 Dendrogramgenerated using UPGMAof 53 accessions ofAndrographis paniculatabased on RAPD markers

Table 8 Comparison of genetic diversity and dissimilarity coefficient among 53 accessions of Andrographis paniculata

Primernumber

Sequence 5¢ fi 3¢ Total number ofbands

Polymorphicbands

Percentagepolymorphism

Shannonindex

1. OPB-02 TGATCCCTGG 19 19 100 8.702. OPH-08 AGACGTCCAC 33 31 93.94 3.083. OPM-06 CTGGGCAACT 38 36 94.74 4.964. OPZ-04 AGGCTTCCTC 26 25 96.15 6.365. OPW-05 CTGCTTCGAG 42 39 92.85 4.386. OPH-17 CACTCTCCTC 27 27 100 6.03Total score 185 177 - -Mean per primer 30.83 29.5 96.28 5.586

40 Genet Resour Crop Evol (2008) 55:33–43

123

Molecular genetic variability and morphomet-

ric divergence revealed by UPGMA dendrogram

(Fig. 4) and D2 statistics respectively showed

loose parallelism in the clustering pattern (Ta-

ble 5). This is understandable as the genome

surveyed by the primers is random and may

involve the non-coding regions of the genome and

show little conformity with the functional gen-

ome. A perusal of dispersion of accessions in

UPGMA dendrogram and also the clustering

pattern based on D2 values revealed that there

was no correspondence between genetic diver-

gence and eco-geographic origin as the accessions

from one origin dispersed randomly into more

than one cluster. Tendency to yield such cluster-

ing pattern implies that the regional isolation may

not always dilute the genetic make up of the

introductions that contribute towards diversity in

naturalized populations. Hence clustering seems

to be influenced more by genetic constitution of

the accessions rather than the eco-geographic

origin. This is possibly due to free exchange of

seed material from the native places of wild

abundance to other places for cultivation.

UPGMA dendrogram revealed that one of the

accessions APJ 033 from Delhi (NBPGR) was

monotypic and displayed the maximum similarity

coefficient of 0.62 with APJ 029 and minimum of

0.33 with APJ 010. Accessions APJ 001 and APJ

022 (Karnataka and H.P.) branched out together

with minimum similarity coefficients of 0.33 and

0.35 with APJ 027 and APJ 016 (H.P. and

Karnataka) respectively. Molecular genetic dis-

tinctness of APJ 033, APJ 001 or APJ 022 and

their maximum dissimilarities with APJ 010, APJ

027 and APJ 016 respectively, makes them highly

suitable for breeding programme.

Characterization of diversity in majority of

plant species is primarily based on morphometric

evaluation, which is vulnerable to intrinsic

(ontogeny) and extrinsic (environment) factors.

It is desirable to compare any molecular charac-

terization with morphological markers to under-

stand the degree of correspondence between the

two. In the present study, there existed a positive

correlation between the RAPD and the morpho-

metric markers. Similarity matrices based on

RAPD and morphometric markers had mean

values of 0.61 and 0.627 respectively. Correlation

between the similarity coefficients for both sets of

data was highly significant (r = 0.547, P < 0.001),

implying the ‘congruence of the two.’ The extent

of genetic diversity observed in the present

material is in conformity with breeding behaviour

of the species. A. paniculata is hermaphroditic,

self-compatible and a habitual inbreeder. Inti-

mate proximity of adpressed stigma with the

anthers and synchronization of anther dehiscence

and stigma receptivity, provides for obligate

autonomous selfing in the species (Lattoo et al.

2006). The genetic differences existing among its

genotypes are maintained by repeated selfing.

The majority of reports where correlation be-

tween morphometric and molecular markers has

been found to exist are either autogamous or

inbred lines (Kantety et al. 1995; Powell et al.

1996; Russel et al. 1997; Pejic et al. 1998; Martin

and Sanchez-Yelamo 2000). The correlation

between morphometric and RAPD markers in

present study may be attributed to diverse genetic

make up of the accessions on account of obligate

selfing and limited gene flow in the natural

populations and / or under cultivation. This

situation is characteristic for populations when

self-pollination or vegetative propagation is

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

a

b

Fig. 5 DNA fingerprints of 20 accessions obtained withprimers OPH-17 (a) and OPW-05 (b): lanes 1-20 representaccessions from APJ 001-APJ 020 and M, molecularweight markers (10 kb DNA ladder)

Genet Resour Crop Evol (2008) 55:33–43 41

123

predominant, leading to mixture of highly

distinctive groups of individuals. In case of more

predominant cross-pollination a more uniform

population would be expected for progenies of

wild-grown plants (Arnholdt-Schmitt 2000)

To recapitulate our findings on the diversity

analysis of A. paniculata, the Mahalanbios D2

statistics provided a powerful conventional tool to

quantify the variation in metric traits and also

to measure the intra and inter group distances to

isolate divergent genotypes. To complement and

supplement the morphometric characterization,

RAPD analysis proved to be an effective and

efficacious technique to measure the magnitude

of diversity and discriminate between genotypes.

The point worth making about the present results

is that there existed a typical triangular relation-

ship between the breeding system, morphometric

traits and RAPD markers, and thus elucidating

the usefulness of complementary approaches to

make diversity analysis more explanatory and

purposeful. Furthermore, the congruence of

RAPD markers with the morphological descrip-

tors provides a viable alternative to characterize

the germplasm of A. paniculata for optimum

genetic improvement and effective conservation

of its genetic resources. The present investigation

also identifies that various accessions available in

different eco-geographic regions of India may

have actually originated from the native places of

wild abundance.

Acknowledgements The authors thank Dr. G.N. Qazi,Director Regional Research Laboratory (CSIR) for usefuldiscussions. One of us (S.B.) was in receipt of researchfellowship from GBPIHED, Almora, India, whilst thework was being carried out.

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