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Genet Resour Crop Evol (2008) 55:33–43
DOI 10.1007/s10722-007-9212-y
R E S E A R C H A RT I C L E
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- cluster analysis also revealed that various acces-
nal plant of immense therapeutic value. The sions available in different eco-geographic regions
present study was aimed to elucidate its genetic might have originated from native places of wild
diversity based on morphochemical and RAPD abundance. Similarity matrices were generated
markers from 53 accessions belonging to 5 eco- for molecular markers and morphometric data to
geographic regions. Analysis of variance and D2 determine the degree of congruence between the
statistics revealed significant differences in all the two. A highly significant but low correlation
metric traits and sufficient inter-cluster distances (r = 0.547, P < 0.001) was obtained thus implying
indicating considerable diversity among the the correspondence between the two. The species
accessions. The complementary approach of is hermaphroditic and a habitual inbreeder. The
RAPD was used to evaluate the genetic dissim- present study yielded a typical triangular congru-
ilarities among all the accessions using 6 highly ence between its breeding system, morphometric
polymorphic primers. The average proportion of traits and RAPD markers thus elucidating the
polymorphic loci across primers was 96.28%. The usefulness of complementary approaches to make
molecular genetic diversity based on Shannon diversity analysis more explanatory and purpose-
index per primer averaged 5.585 with values ful for optimum genetic amelioration and effec-
ranging from 3.08 to 8.70 indicating towards wide tive conservation of its genotypic variability.
genetic base. RAPD based UPGMA and D2
Keywords Andrographis paniculata
Andrographolide Genetic diversity
S. K. Lattoo (&) S. Khan S. Bamotra
Mahalanobis D2 statistics Medicinal herb
M. K. Bhan A. K. Dhar
Department of Genetics and Plant Breeding, Shannon index
Regional Research Laboratory (CSIR), Canal Road,
Jammu Tawi 180 001, India
e-mail: surrinlattoo@rediffmail.com
Introduction
R. S. Dhar
Department of Biotechnology, Regional Research Andrographis paniculata (Acanthaceae) is a
Laboratory (CSIR), Jammu Tawi, India medicinal herb of immense therapeutic value
with wide geographic distribution from peninsular
K. K. Gupta
Division of Natural Products Chemistry, Regional India, Sri Lanka, south-east Asia, China, Amer-
Research Laboratory (CSIR), Jammu Tawi, India ica, West Indies and Christmas Island in Indian
123
34 Genet Resour Crop Evol (2008) 55:33–43
ocean (Anonymous 1985). The crude extract from Materials and methods
whole plant has shown anti HIV activity (Otake
et al. 1995). Its therapeutic value is ascribed to Morphometeric and chemical variation
various bioactive constituents synthesized and
accumulated in its leaves and roots. Some of the Present study was conducted at Regional Re-
major constituents include andrographolide, search Laboratory (CSIR), Jammu, India
deoxyandrographolide, 14-deoxy-11,12-didehy- (3244¢ N, 7555¢ E; 305 m in altitude) where
droandrographolide, neoandrographolide and the annual temperature fluctuates between 5C
andrographiside. Andrographolide protects liver and 45C and mean annual rainfall measures upto
and gallbladder and is more efficacious than 1,100 mm. The material for present study com-
silymarin, a known hepatoprotective drug (Sara- prised 53 accessions procured from different eco-
swat et al. 1995). Neoandrographolide has shown geographic regions of India (Table 1; Fig. 1) and
greater activity against malaria (Misra et al. 1992) maintained in the germplasm repository at RRLJ.
and is hepatoprotective against carbontetrachlo- For the purpose of studying variation, individual
ride induced hepatic damage (Kapil et al. 1993),
while 14-deoxyandrographolide produces a more
Table 1 List of Andrographis paniculata accessions from
potent hypotensive effect in anesthetized rats and different sources in India and their status
in isolated right atria (Zhang et al. 1998).
Accessions Origin Status
For optimum genetic amelioration and effec-
tive conservation of the allelic and genotypic APJ 001-APJ 017 Karnataka Cultivated
variability in a given species, it is imperative to APJ 018-APJ 027 Himachal Pradesh Cultivated
(H.P.)
evaluate and catalogue the variability. The
APJ 028-APJ 039 Delhi (NBPGR)* Composite seed
improvement in quantitative and quality traits collection
can be achieved through understanding the nature APJ 040-APJ 048 Madhya Pradesh Wild
and amount of variability present in the genotypes (M.P.)
APJ 049-APJ 053 Assam Cultivated
/ accessions for breeding purposes. Diversity
analysis based on morphological, chemical and *National Bureau of Plant Genetic Resources, New Delhi
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 1
123
Genet Resour Crop Evol (2008) 55:33–43 35
open-pollinated seed lots from 53 accessions were 94C, 1 min at 35C, 2 min at 72C and as a last
germinated in earthen pots containing mixture of step 10 min at 72C. 20 ll reaction mixture
soil and sand in the proportion of 4:1 under contained 1· PCR buffer (10 mM Tris–HCl,
outdoor conditions during the month of June pH 9.0; 50 mM KCl; 2.5 mM MgCl2; 200 lM
2002. The plants were transplanted after 45 days dNTP (Promega); 200 lM random primers; 20–
of germination into 4 · 2.5 m plots at spacing of 30 ng of DNA template and 0.5 U of Taq DNA
40 · 50 cm apart in a randomized block design polymerase (Banglore Genei, Banglore, India).
with 3 replications. Plants were raised in well- On the completion of the programme for the
drained sandy loam soil (pH 7.4) under uniform amplification of DNA samples, they were stored a
cultivation conditions. Data were recorded on 15 –20C. The separation of DNAs was performed
competitive plants for each accession for series of by electrophoresis in 1.5% (w/v) agarose gel. The
seven metric traits: plant height (cm), leaf length gel was documented using Image Master VDS
(cm), leaf width (cm), plant spread (cm), number (Amersham Biopharmacia, USA). All the PCR
of leaves per plant and herbage yield per plant reactions were repeated at least twice to check
(g). The data were subjected to analysis of the reproducibility.
variance and only those characters in which Amplification profiles were recorded and the
variation observed was significant were consid- size of each fragment was estimated using soft-
ered for multivariate analysis of D2 statistics ware (SEQUAID II [tm] version 2.2, 1987).
based on Mahalanobis (1936) and Rao (1952). Amplicons were scored as discrete variables,
The analysis was done using SYSTAT (version using 1 to indicate presence and 0 for absence.
10.2) statistical package and cluster formation was The binary matrix was used to generate pair-wise
confirmed by Tocher’s method. The relative similarities between the accessions based on the
contribution of each character towards genetic number of shared amplification products (Nei and
divergence was also worked out. All the acces- Lei 1979). Similarity matrix was subjected to
sions were evaluated for androgrpholide content cluster analysis to develop a dendrogram using a
after 120 days of transplantation. For chemical procedure of NTSYS-pc (Rohlf 1993), which uses
analysis fresh herbage was air dried under shade the unweighted pair group method with arithme-
and analyzed for andrographolides according to tic averages (UPGMA) (Sneath and Sokal 1973).
HPLC method of Saxena et al. (2000). Genetic diversity was estimated by Shannon
index (Lewontin 1972):
DNA fingerprinting: molecular analysis
X
k
H ¼ Pi ln Pi
Genomic DNA was extracted from leaves of 4-
i¼1
week old seedling from 53 accessions. DNA was
isolated following the modified procedure of where H denotes the diversity of RAPD markers
Doyle and Doyle (1990). The yield of DNA was in a population, k is the number of bands
40–60 lg g–1 tissue, the UV absorbance ratio at produced with the respective primer and pi is
260 nm/280 nm, were at least 1.9–2.1 respectively. the frequency of the ith fragment.
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- Morphometric analysis
iculata plants, six arbitrary decamer primers
(Operon Technologies, USA) were selected for For morphmetric traits, data (Table 2) were
polymerase chain reaction (PCR). These primers subjected to linear transformation by computing
produced distinct amplification profiles that were the mean and standard deviation of the states of
easily scorable. DNA amplifications were per- each trait and the values were expressed as the
formed in Master Cycler Gradient (Ependorf, matrix of the deviations from the mean in
Germany) and the PCR conditions were as standard deviation units (Bookistein 1991). A
follows: 3 min at 95C 40 cycles of: 1 min at pair-wise distance matrix was generated using the
123
36 Genet Resour Crop Evol (2008) 55:33–43
APJ 001 36.92 0.395 61.86 5.10 1.68 45.00 259.0 5.74
APJ 002 54.26 0.445 59.81 4.59 1.41 44.80 338.0 5.41
APJ 003 42.60 0.368 57.36 4.84 1.51 45.69 295.0 4.41
APJ 004 42.76 0.412 59.66 5.15 1.65 51.63 325.0 5.61
APJ 005 42.54 0.386 56.23 4.85 1.49 53.83 344.0 5.53
APJ 006 47.96 0.328 64.03 5.12 1.53 55.98 281.0 4.58
APJ 007 48.49 0.439 55.03 4.77 1.42 47.88 370.0 5.06
APJ 008 43.68 0.315 60.53 4.62 1.42 49.17 281.0 5.94
APJ 009 38.86 0.323 61.66 4.95 1.51 48.56 323.0 5.76
APJ 010 52.26 0.399 59.47 5.08 1.67 46.88 343.0 5.60
APJ 011 54.50 0.296 68.91 4.70 1.57 63.41 357.0 4.14
APJ 012 32.19 0.427 80.23 4.65 1.48 42.45 317.0 4.92
APJ 013 34.99 0.410 76.16 4.84 1.68 44.15 326.0 2.67
APJ 014 39.20 0.480 60.91 5.07 1.68 41.33 419.0 3.74
APJ 015 46.50 0.423 61.00 4.85 1.48 51.45 299.0 3.19
APJ 016 30.90 0.336 60.58 4.56 1.51 51.50 284.0 4.66
APJ 017 47.70 0.353 54.83 4.61 1.43 46.42 277.0 5.11
APJ 018 48.50 0.390 50.66 4.38 1.53 51.76 331.0 5.17
APJ 019 50.10 0.337 57.50 4.16 1.38 52.36 387.0 4.43
APJ 020 48.50 0.354 56.83 4.94 1.60 50.87 360.0 3.90
APJ 021 50.60 0.280 64.50 5.20 1.70 52.00 216.6 4.45
APJ 022 36.60 0.380 58.60 5.90 1.50 45.00 293.3 5.40
APJ 023 35.00 0.320 51.60 4.80 1.60 44.20 244.0 4.58
APJ 024 31.80 0.430 56.50 5.00 1.80 42.45 251.0 3.75
APJ 025 54.20 0.440 59.80 4.60 1.50 44.80 338.0 5.41
APJ 026 48.50 0.350 56.80 4.90 1.60 51.87 360.0 3.90
APJ 027 52.20 0.390 59.40 5.00 1.60 46.88 343.0 5.60
APJ 028 46.00 0.420 61.00 4.80 1.40 42.45 317.0 4.92
APJ 029 50.00 0.330 57.50 4.10 1.30 52.00 387.0 4.43
APJ 030 32.10 0.420 80.20 4.60 1.40 42.25 317.0 4.92
APJ 031 42.50 0.380 56.20 4.80 1.40 53.83 344.0 5.53
APJ 032 38.80 0.320 61.60 4.90 1.50 48.56 323.0 5.76
APJ 033 30.90 0.330 60.50 4.50 1.50 51.50 287.0 4.66
APJ 034 39.20 0.480 60.90 5.00 1.60 41.33 419.0 3.74
APJ 035 52.60 0.400 59.40 5.00 1.60 46.88 343.0 5.60
APJ 036 47.70 0.360 54.80 4.60 1.40 46.42 277.0 5.11
APJ 037 50.10 0.380 57.60 4.20 1.40 52.02 388.0 4.44
APJ 038 42.70 0.410 59.70 4.80 1.50 45.70 295.0 4.41
APJ 039 48.40 0.430 55.00 4.70 1.40 47.88 372.0 5.06
APJ 040 32.90 0.430 80.30 4.60 1.50 42.45 317.0 4.92
APJ 041 46.70 0.440 61.50 5.00 1.50 42.30 327.0 4.93
APJ 042 47.90 0.390 64.00 5.10 1.50 55.90 281.0 4.58
APJ 043 52.30 0.400 60.40 5.00 1.70 46.90 345.0 5.60
APJ 044 47.50 0.350 54.80 4.60 1.50 51.50 290.0 4.66
APJ 045 42.60 0.410 56.20 4.90 1.50 45.70 344.0 4.41
APJ 046 48.00 0.430 55.00 5.00 1.50 49.17 340.0 5.94
APJ 047 50.30 0.340 57.30 4.50 1.60 52.21 370.0 4.43
APJ 048 43.50 0.430 57.00 5.00 1.90 45.69 351.0 3.19
APJ 049 49.60 0.400 59.80 5.20 2.00 51.87 372.0 4.24
APJ 050 33.00 0.410 62.20 5.50 1.90 42.45 342.0 4.92
APJ 051 35.00 0.400 60.00 5.00 1.90 44.80 342.0 5.74
APJ 052 52.00 0.450 65.00 5.30 1.90 46.80 366.0 5.60
APJ 053 50.10 0.380 58.90 5.60 1.40 47.40 349.0 4.85
123
Genet Resour Crop Evol (2008) 55:33–43 37
Table 2 continued
Accession Dry herbage Leaf stem/ Plant Leaf Leaf Plant Number of Total andro-
No. yield/plant ratio height length width spread leaves/plant grapholides (%)
(g) (cm) (cm) (cm) (cm)
average taxonomic distance coefficient (Rohlf in separate clusters on the basis of either low,
1993) between a pair of accessions (j and k) for moderate or high mean values for different
n characters as characters (Table 6). Cluster means revealed
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi considerable differences among the clusters.
1X n Accessions of cluster V had highest dry herbage
djk ¼ ðjxij xjk jÞ2 per plant, maximum plant height and leaf length
n i¼1
with highest plant canopy. Assemblage of 9
The similarity matrix for morphmetric markers accessions representing cluster II, manifested
was generated by calculating 1–dij. moderate to low averages for all the traits but
interestingly revealed highest total andrographo-
lide content. 31 accessions belonging to cluster I
Results and discussion represented heterogeneous assemblage as they
presented moderate to low mean values for
Analysis of variance revealed highly significant different traits. Four of these accessions are
differences among all the accession metric traits cultivated and had incidentally less of total
under investigation (Table 3). However, the andrographolide.
chemical constituents were not significant. The Table 4 revealed that average intra-cluster
computed D2 values for all the n (n–1)/2 = 1,378 distance ranged from 0.34 to 4.06, while inter-
pairs of accessions ranged from 0.01 to 76.98 cluster distance ranged between 3.95 and 6.85.
thereby indicating considerable diversity in the Cluster III and V showed maximum inter-cluster
present material. Based on D2 values, all the 53
accessions could be grouped into five clusters such Table 4 Intra- and inter-cluster divergence (D2) among 5
that the accessions within a cluster have smaller clusters involving 53 accessions of Andrographis paniculata
D2 values among themselves than those belonging Cluster 1 2 3 4 5 D2
to different clusters (Table 4). The distribution of
1. 2.63 4.88 5.93 4.75 3.95 4.88
different groups revealed that there were 31 2. 2.77 4.25 5.71 5.57 5.10
accessions in cluster I, 9 in cluster II, 6 in cluster 3. 2.84 4.63 6.85 5.41
III, only 2 in cluster IV and 5 accessions in cluster 4. 0.34 6.81 5.47
V (Table 5). Accessions tended to group together 5. 4.06 5.79
123
38
123
Table 5 Cluster composition of 53 accessions of Andrographis paniculata based on morphometric traits and RAPD markers
Morphometric RAPD
Cluster Number of Serial number of accessions* Origin Number of Serial number of Origin
accessions accessions accessions*
1 31 002, 003, 004, 005, 007, 010, 015, Karnataka, H.P., 2 001, 023 Karnataka, H.P.
017, 018, 019, 020, 025, 026, 027, Delhi, M.P.,
028, 029, 031, 035, 036, 037, 038, Assam
039, 041, 043, 044, 045, 046, 047,
048, 049, 053
II 9 001, 008, 009, 016, 022, 023, Karnataka, H.P., 21 002, 003, 004, 005, 006, 007, 008, Karnataka, H.P.,
024, 032, 033 Delhi 009, 010, 011, 012 013, 014, 015, M.P.
016, 017, 018, 019, 020 046, 047
III 6 012, 013, 030, 040, 050, 051 Karnataka, Delhi, 20 021, 025, 026, 028, 029, 031, 032, H.P., M.P., Assam,
M.P., Assam, 034, 035, 036, 039, 040, 041, 042, Delhi
043, 044, 049, 050, 051, 053
IV 2 014, 034 Karnataka, 5 024, 037, 038, 045, 048 H.P., M.P., Assam,
Delhi Delhi
V 5 006, 011, 021, 042, 052 Karnataka, H.P., 1 033 Delhi
M.P., Assam
VI – – – 4 027, 052, 022, 030 H.P., Delhi
Assam
* APJ – accession prefix; H.P. – Himachal Pradesh; M.P. – Madhya Pradesh
Genet Resour Crop Evol (2008) 55:33–43
Genet Resour Crop Evol (2008) 55:33–43 39
divergence (6.85), thus indicating wide diversity three dimensional representation (Fig. 3), show-
between the accessions of these two clusters. The ing distance relationship among 53 accessions was
minimum inter-cluster distance occurred between found adequate (Table 7).
clusters I and V, suggestive of close relationship Of 48 decamer primers used to screen repre-
(Fig. 2). On the basis of average divergence (D2) sentative DNA samples, 6 (12.5%) detected
cluster V was the most divergent from the rest. scorable polymorphism in banding patterns
Inter-cluster divergence was also confirmed by among all the 53 accessions (Table 8). Six
canonical analysis. Since x1 + x2 + x3 = 84%; a selected primers generated a total of 185 bands
of which 177 were polymorphic. An example of
4 (0.34) the representative profiles of 20 accessions with
two primers is shown in Fig. 5. The number of
4 .6
1
3 bands per accession ranged from 4 to 16 and the
6.8
bands amplified ranged in size from 250 bp to
5
6.85
3 (2.84) 7,000 bp, although 68.9% (122 out of 177) ranged
(4.06) between 300 bp and 1,200 bp. The average num-
5.71
5
4.7
7
4.2
Fig. 3 Spatial
distribution of 53
accessions of
Andrographis paniculata
along three principle
coordinate axes based on
morphological characters
123
40 Genet Resour Crop Evol (2008) 55:33–43
Fig. 4 Dendrogram
generated using UPGMA
of 53 accessions of
Andrographis paniculata
based on RAPD markers
Table 8 Comparison of genetic diversity and dissimilarity coefficient among 53 accessions of Andrographis paniculata
Primer Sequence 5¢ fi 3¢ Total number of Polymorphic Percentage Shannon
number bands bands polymorphism index
exotic collections of A. paniculata. However, in exclusive RAPD loci, which account for substan-
the present material, there was comparatively tial portion of genetic diversity. Diversity based
higher proportion of genetic diversity as the on Shannon’s index averaged 5.585 with 6 primers
Jaccard’s similarity values ranged between 0.333 (Table 8), with values ranging between 3.08
and 0.812 with an average value of 0.61. Molec- (OPH-08) to 8.70 (OPB-02). This is indicative of
ular markers generated by RAPD displayed large differences among the accessions, conse-
adequate polymorphism and large proportion of quently wide genetic base in A. paniculata.
123
Genet Resour Crop Evol (2008) 55:33–43 41
123
42 Genet Resour Crop Evol (2008) 55:33–43
predominant, leading to mixture of highly Kantety RV, Zeng X, Bennetzen JL, Zehr BE (1995)
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is that there existed a typical triangular relation- Theor Appl Genet 101:1234–1241
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Acknowledgements The authors thank Dr. G.N. Qazi, detected by RFLPs, RAPDs, SSRs and AFLPs. Theor
Director Regional Research Laboratory (CSIR) for useful Appl Genet 97:1248–1255
discussions. One of us (S.B.) was in receipt of research Powell W, Morgnate M, Andre C, Hanafey M, Vogel J,
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