American Journal of Molecular Biology, 2013, 3, 187-197 AJMB Published Online October 2013 (
Characterization of winged bean (Psophocarpus
tetragonolobus (L.) DC.) based on molecular,
chemical and physiological parameters
Chandra Sekhar Mohanty1*, Sushma Verma1, Vinayak Singh1, Shahina Khan1, Priyanka Gaur2,
Priya Gupta1, M. Abdul Nizar3, Nilamani Dikshit3, Rojalin Pattanayak4, Alpika Shukla4,
Abhishekh Niranjan1, Nayan Sahu1, Soumit Kumar Behera1, Tikam Singh Rana1
1CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, Uttar Pradesh, India
2Sanjay Gandhi Institute of Post-Graduate Medical Sciences, Lucknow, Uttar Pradesh, India
3National Bureau of Plant Genetic Resources, Regional Station, Akola, Maharashtra, India
4University of Lucknow, Lucknow, Uttar Pradesh, India
Email: *, *
Received 9 July 2013; revised 11 August 2013; accepted 6 September 2013
Copyright © 2013 Chandra Sekhar Mohanty et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Winged bean (Psophocarpus tetragonolobus (L.) DC.)
is a potential legume crop of the tropics with high
protein and oil content in the seeds. Analysis of the
mutual genotypic relationships among twenty four
genotypes of P. tetragonolobus through Mantel test
found a significant correlation (r = 0.839) between
similarity matrices of the results obtained from the
use of the RAPD and ISSR molecular markers. The
UPGMA tree based on Jaccard’s similarity coeffi-
cient generated from their cumulative data showed
two distinct clusters and seven sub-clusters among
these accessions. Quantification of total polyphenols,
flavonoids and tannin revealed the highest percentage
of occurrence of kaempferol (1.07 - 790.5 μg/g) and
the lowest percentage of gallic acid (0.09 - 3.49 μg/g) in
the seeds. Phytochemical analysis of the winged bean
genotypes revealed that, some of the exotic lines are
distinct. Analysis of photosynthesis rate, photosyn-
thetic yield and stomatal conductance data also
showed two clusters and was in congruence with the
phytochemical affinities of the genotypes. The overall
high level of polymorphism and varied range of ge-
netic distances across the genotypes revealed a wide
range of genetic base of P. tetragonolobus. The pre-
sent investigation therefore, has provided significant
insights for further improvement of winged bean
germplasm for its qualitative and quantitative traits.
Keywords: Psophocarpus tetragonolobus; Polyphenols;
Flavonoid; RAPD; ISSR; Physiological Parameter
Psophocarpus tetragonolobus (L.) DC. (Fam. Fabaceae)
popularly known as Winged bean or Goa bean is a tropi-
cal legume found growing abundantly in hot, humid
equatorial countries, like India, Burma, Sri Lanka, Thai-
land and Philippines. It is also called a wonder legume as
it has the high protein content in the seeds and therefore
considered as a versatile legume [1]. It is a diploid (2n =
2x = 18), self-fertilizing leguminous crop with multifari-
ous usage [2]. It can be grown as a grain legume, green
vegetable, tuber-crop or a forage and cover-crop [3].
Seeds of winged bean contain some pharmacologically
active anti-nutrients such as trypsin and chymotrypsin
inhibitors [4], haematoglutins and amylase inhibitors.
There are other unfavorable compounds like tannins
(proanthocyanidins), which have been reported to be
present in the seeds of winged bean [5]. Tannins are
polyphenolic compounds and are either hydrolysable or
condensed. It can interact and precipitate with protein
and therefore, reduce food protein quality in monogas-
trics [6]. Knowledge of genetic diversity within and
among genotypes of any crop is fundamental to estimate
the potential of genetic gain in breeding programs and
for effective conservation and sustainable utilization of
available genetic resources [7]. All food legumes are
valuable sources of proteins, vitamins and minerals and
occupy an important place in human nutrition. Assess-
ment of genetic variations and relationships among these
leguminous crops may therefore, play a significant role
*Corresponding author.
C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
in breeding programs to improve grain yield, oil and pro-
tein content. Winged bean is of rapidly increasing inter-
est as a high-protein multipurpose crop. The breeding of
winged bean as a grain legume requires the development
of an improved ideotype with the highest nutritional
content and lowest anti-nutritional factors [8].
Over the years, the methods for detecting and assessing
genetic diversity have extended from analysis of discrete
morphological traits to advanced biochemical and mo-
lecular traits. Several DNA based molecular markers are
now currently used in diversity study of plants. Amongst
them RAPD and ISSR-PCR are commonly used markers
to characterize the genetic diversity of crop plants and
can be immensely helpful in identifying the potential
elite genotypes. The utility of these markers as a poten-
tial tool for documenting the genetic variations in several
legume crops like Chickpea [9-11]. Lens [12], Lentil [13],
Groundnut [14], Cajanas cajan [15], Cluster bean [16],
Mung bean and Black gram [17] etc., have been clearly
established over the period of time. However, no such
reports on genetic diversity assessment studies using mo-
lecular markers separately or in combination with other
biochemical markers are available in winged bean. The
efficiency of assessment of genetic diversity to be used
in breeding programme will be improved if combined
biochemical and molecular data are used [18,19]. Thus,
in the present study, RAPD and ISSR markers were used
to evaluate the extent of genetic diversity amongst 24
winged bean genotypes and their efficiency in analyzing
the genetic variations was compared. Furthermore, total
phenolic as well as flavonoid contents along with their
physiological responses to various conditions were enu-
merated. The use the biochemical and molecular markers
will lead to select the lines with high-levels of specific
polyphenol and flavonoid contents and suitable physio-
logical performances along with genetic variability.
There exists a considerable scope for incorporating pro-
mising winged bean genotypes in conventional breeding
program. Apart from identifying the genetically diverse
lines, attempts have been made to physiologically and
biochemically characterize these plants and to establish
effective correlation among these attributes.
2.1. Plant Material
The plant material was procured from National Bureau
of Plant Genetic Resources (NBPGR), Akola (Maharash-
tra), India that included genotypes from India, Thailand
and Nigeria. A total of 24 winged bean genotypes along
with Vigna radiata as an out-group were analyzed in the
present study. The details of plant materials are given in
(Table 1). The collected germplasm is now maintained in
the Botanic Garden of CSIR-National Botanical Re-
Table 1. List of genotypes of P. tetragonolobus L. (DC.) used
in the present study with their morphometric characters.
S. No.
Country Seed ColourShape of
1 IC 95232Pt1 India Light BrownSpherical
2 IC 95233Pt2 India Light BrownSpherical
3 IC 95234Pt3 India Dark BrownRound
4 IC 95235Pt4 India Light BrownSpherical
5 IC 95236Pt5 India Light BrownRound
6 IC 95237Pt6 India Light BrownSpherical
7 IC 95241Pt7 India Red BrownSpherical
8 IC 95242Pt8 India Light BrownSpherical
9 IC 112416Pt9 India Dark BrownSpherical
10EC 142660Pt10 N.A. Light BrownSpherical
11EC 142661Pt11 N.A. Light BrownRound
12EC 142665Pt12 N.A. Light BrownSpherical
13EC 142667Pt13 Nigeria Dark CreamSpherical
14EC 178267Pt14 Thailand Light BrownSpherical
15EC 178268Pt15 Thailand Light BrownSpherical
16EC 178274Pt16 Thailand Dark BrownSpherical
17EC 178279Pt17 Thailand Dark BrownSpherical
18EC 178283Pt18 Thailand Light BrownSpherical
19EC 178291Pt19 Thailand Dark BrownWrinkled
20EC 178292Pt20 Thailand Dark BrownSpherical
21EC 178293Pt21 Thailand Dark BrownSpherical
22EC 178296Pt22 Thailand Light BrownSpherical
23EC 178333Pt23 Thailand Light BrownRound
24EC 178334Pt24 Thailand Dark BrownSpherical
search Institute, Lucknow, India for further studies.
2.2. Genomic DNA Extraction
Total genomic DNA was isolated from the fresh leaves
using DNeasy Plant Mini Kit (Qiagen, USA), according
to the manufacturer’s protocol. Yield and purity of ge-
nomic DNA was estimated at OD260/280 on spectropho-
tometer the integrity of genomic DNA was determined
by running the DNA on 0.8% agarose gel.
2.3. RAPD and ISSR-PCR Amplification
A total of 13 random primers (Operon Technologies,
USA), that resulted in discrete, well-separated fragments
on agarose gel were selected for further amplification of
entire set of the winged bean genotypes. All RAPD reac-
tions were carried out in 25 µl volume containing 50 ng
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C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
Copyright © 2013 SciRes.
template DNA, 10 pm primer, 200 µm each dNTPs, 2.5
mM MgCl2 ion concentration in suitable 1X buffer and
0.5 unit of the thermostable Taq DNA polymerase (Ban-
galore Genei, India). DNA amplification was performed
in a thermal cycler (PTC 200, MJ Research Inc., USA),
programmed to include pre denaturation at 94˚C for 1
min, followed by 45 cycles of denaturation at 94˚C for 1
min, annealing at 35˚C for 1 min and extension at 72˚C
for 1 min, the final cycle allowed an additional 5 min
period of extension at 72˚C.
A total of 7, 16 - 18 mer anchored microsatellite prim-
ers from (University of British Columbia Canada) UBC
set were selected and used for ISSR analysis (Table 2).
PCR amplifications were performed in a volume of 25 μl
containing 50 ng of template DNA, 10 pm primer, 200
µm each dNTPs, 2.5 mM MgCl2 ion concentration in
suitable 1X buffer and 0.5 unit of the thermostable Taq
DNA polymerase enzyme (Bangalore Genei, India). The
thermal-cycler was programmed with an initial denatura-
tion for 5 min at 94˚C, followed by 44 cycles of denatu-
ration at 94˚C for 1 min, annealing at 55˚C for 45 sec and
extension at 72˚C for 1 min followed by 7 min of exten-
sion at 72˚C.
2.4. Gel Electrophoresis
The amplified products were electrophoresed on 1.5%
agarose gels containing 0.5X TBE buffer stained with 0.5
µg/ml ethidium bromide. The PCR product was electro-
phoresed at 72 V for 2 hrs. Low range DNA ruler of
known molecular weight was used for comparing the
band size of amplified products. After electrophoresis the
gel was visualized in a trans-illuminator using gel docu-
mentation system (UV Tech, UK).
2.5. Data Analysis
The amplified bands from each DNA accession were
transformed into a binary character matrix where the
presence of band on a gel is scored as “1” and the ab-
sence of band as “0”. The resulting data obtained by
Table 2. RAPD and ISSR primers used to analyze the genetic diversity in Psophocarpus tetragonolobus genotypes.
Primers Sequence 5’-3’ Loci amplified Polymorphic lociPercentage polymorphismPIC Approx. band size range (bp)
OPH 04 GGAAGTCGCC 7 3 42.8 0.124 250 - 2500
OPH 06 ACGCATCGCA 6 6 100 0.347 400 - 2000
OPH 09 TGTAGCTGGG 7 5 71.4 0.169 300 - 2000
OPH 14 ACCAGGTTGG 7 7 100 0.202 400 - 1500
OPH 15 AATGGCGCAG 6 3 50 0.190 200 - 2000
OPM 07 CCGTGACTCA 9 8 88.8 0.149 400 - 3000
OPM 10 TCTGGCGCAC 6 3 50 0.161 400 - 3000
OPM 11 GTCCACTGTG 5 3 60 0.154 200 - 2500
OPM 14 AGGGTCGTTC 3 1 33.3 0.110 500 - 1000
OPN 08 ACCTCAGCTC 6 2 33.3 0.091 300 - 3000
OPN 09 TGCCGGCTTG 5 3 60 0.077 280 - 1500
OPU 03 CTATGCCGAC 4 3 75 0.234 300 - 3000
OPU 10 ACCTCGGCAC 5 5 100 0.299 400 - 2000
76 52 68.4 0.170av 250 - 3000
UBC 810 (GA)8T 15 14 93.3 0.310 300 - 2000
UBC 811 (GA)8C 15 14 93.3 0.203 500 - 2500
UBC 825 (AC)8T 12 11 91.6 0.250 400 - 3000
UBC 827 (AC)8G 16 15 93.7 0.354 400 - 3000
UBC 840 (GA)8YT 06 05 83.3 0.225 300 - 2000
UBC 841 (GA)8YC 14 14 100 0.335 350 - 1900
UBC 855 (AC)8YT 13 13 100 0.291 400 - 3000
91 86 94.5 0.281av 300 - 3000
167 138 82.63 0.213av
av = Average.
C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
scoring the RAPD and ISSR profiles with selected prim-
ers were used individually as well as cumulatively to
construct a pair-wise matrix of similarities between ac-
cessions using Jaccard’s coefficient [20] for unweighted
pair group method with arithmetic averages, whereas the
distances were computed by using Jaccard’s coefficient
for NJ method in the FREE TREE program ver.
[21]. The pair-wise similarity data were used to generate
a UPGMA (Unweighted Pair Group Method with Arith-
metic Means) tree after allowing a 1000 replicate boot-
strap test using the same program. The tree was viewed,
annotated and printed using TREEVIEW ver. 1.6.5. The
data were further used to calculate different genetic di-
versity parameters such as total number of bands per
primer, polymorphic bands, genetic distances and poly-
morphic information content (PIC). Diversity index (DI)
and marker index (MI) [22] was calculated to determine
the utility of the two marker systems used in the present
study. Mantel test [23] was carried out in MXCOM mod-
ule of NTSYS pc software ver. 2.01e [24] to compute the
matrix correlation (r) between the similarity matrices
generated from different assays to test the goodness of fit
between RAPD and ISSR markers used in the present
2.6. Estimation of Biochemical Characters
Total Polyphenols
The finely powdered plant material (~25 mg) was ex-
tracted overnight with 50% methanol: water (3 × 10 ml).
The combined extracts were centrifuged at 6000 × g for
10 min, filtered and maintained at 30 ml. The total phe-
nolics content (TPC) in the extract was measured by us-
ing method of Ragazzi and Veronese [25] In 0.5 ml ex-
tract, 0.5 ml Folins reagent (1N) and 1 ml of sodium
carbonate (20%) were subsequently added. The test mix-
ture was mixed properly on a cyclomixer, left at room
temperature for 30 min and maintained at 12.5 ml with
water. The absorbance of test mixture was measured at
λmax 720 nm on a Varian Cary 50 spectrophotometer.
Further analysis of individual polyphenolic com-
pounds were performed through HPLC-UV (Shimadzu
LC-10A, Japan) equipped with dual pump LC-10AT bi-
nary system, UV detector SPD-10A at 254 nm, rheodyne
injection valve furnished with a 20 l loop on phenome-
nex Luna RP-C 18 column (4.6 × 250 mm, i.d., 5 m
pore size) preceded with guard column of same chemis-
try. Data was integrated by Shimadzu class VP series
software. HPLC analysis of the dried powered seeds was
used to generate metabolic profiles [26]. For the purpose
of our analysis we selected only major metabolic peaks
that showed identical retention times and uniform UV
spectra in samples. Separation followed by qualitative
and quantitative analysis of naturally occurring phenolics
like: gallic, protocatechuic, chlorogenic, caffeic, ferulic
acid and some plant flavonols like: rutin, quercetin and
kaempferol were carried out (Figure 1). The correlation
study among these polyphenols and flavonoid contents
were analyzed through hierarchical cluster software
SPSS ver. 16.0 and SAS ver. 9.2. This procedure at-
tempted to identify the relatively homogenous groups on
the basis of their chemical constituents (Figure 2). Prin-
cipal component analysis was used to summarize these
2.7. Estimation of Physiological Characters
2.7.1. Light Response
Photosynthetic light responses to photosynthetic photon
flux density (PPFD) were measured with Li-Cor 6400
XT portable photosynthesis system using artificial LED
light source (Li-Cor 6400-02B). Leaf gas exchange mea-
surements were made between 08:00 a.m. and 12:00 p.m.
on the abaxial surface of leaves of all the genotypes of P.
tetragonolobus. Six replicates of each genotype were
measured for each physiological attributes. The leaf area
under measurement was 2 cm2. The air flow rate, relative
humidity, air temperature (T air), vapour pressure deficit
(VPD) and reference CO2 concentration was kept con-
stant for all the genotypes at 500 µmols1, 55% - 65%,
28˚C, 1.5 KPa and 400 µmol·m2·sec1, respectively. The
light response was measured at a constant saturating
PPFD of 1200 µmol·m2·sec1. The leaves were dark
adapted by using dark adapting clips (Li-Cor 9964-091)
20 minutes prior to beginning of each measurement.
2.7.2. Chlorophyll Fluorescence
Leaf chlorophyll fluorescence was measured on intact,
fully expanded leaves of P. tetragonolobus with Li-Cor
6400 XT portable photosynthesis system using artificial
LED light source (Li-Cor 6400-02B). Five leaves were
measured per genotype and average fluorescence value
was obtained. Leaves were dark acclimated for 20 min-
ute before measurement of maximum quantum efficiency
of the photosystem II (Fv/Fm). Later, Fv’/Fm’ (light har-
vesting efficiency by oxidized PS II reaction centers in
light) was measured at saturating PPFD of 1200
µmol·m2·sec1. All the statistical analysis including the
cluster diagram for the physiological attributes were car-
ried out by Systat 13.0 (Systat Software Inc., Chicago,
3.1. RAPD Analysis
A total of thirteen decamer primers were used for RAPD
profiling of 24 genotypes of P. tetragonolobus. Repre-
sentative gel image for primer OPH-04 is shown in
(Figure 3(a)). The sizes of the amplified products ranged
rom 250 - 3000 bp. These thirteen primers amplified a f
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C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197 191
Figure 1. HPLC chromatogram of seed extract of selective genotypes of P. tetragonolobus at 254 nm.
Copyright © 2013 SciRes. OPEN ACCESS
C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
Figure 2. Principal component analysis (PCA) of polyphe-
nol and flavonoid content of seeds in different genotypes of
P. tetragonolobus.
total of 76 bands with an average of 5.8 bands per primer,
out of which 52 bands were polymorphic with an average
of 4 polymorphic bands per primer, revealing 68.4%
polymorphism. Minimum number (01) of polymorphic
band was amplified by primer OPM-14 and maximum
number (08) of polymorphic bands was amplified by
primer OPM-07. The PIC value ranged from 0.077 -
0.347 with an average value of 0.170 across the used
primers (Table 2). Genetic distances among the geno-
types ranged from 0.607 (between Pt4 and Pt15; Pt6 and
Pt16) to 0.974 (between Pt09 and Pt17).
3.2. ISSR Analysis
A total of seven ISSR primers that resulted into distinct
polymorphic bands were selected for ISSR profiling in
the present study. Representative gel image for ISSR
primer UBC 811 is shown in (Figure 3(b)). A total of 91
bands with an average of 13 bands per primer were ob-
tained with seven ISSR markers. Primers UBC 810 and
UBC 811 amplified the maximum number of bands (15),
whereas primer UBC 840 amplified a minimum of 5
bands. Out of 91 bands, 86 bands were polymorphic re-
vealing 94.5% polymorphism. Average number of poly-
morphic bands per primer was 12.2 with primer UBC
827 producing a maximum of 15 polymorphic bands,
whereas primer UBC 840 producing a minimum of 5
polymorphic bands. The PIC value obtained ranged from
0.203 - 0.354 with an average of 0.281 across all seven
ISSR primers (Table 2). Genetic distances ranged from
0.250 (between Pt4 and Pt24 pair of genotypes) to 0.887
(between Pt10 and Pt11 pair of genotypes) across all 24
genotypes of winged bean.
3.3. Cumulative Analysis Using RAPD and
ISSR Markers
Twenty RAPD and ISSR primers together amplified a
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6
Pt 7
Pt 8
Pt 9
Pt 10
Pt 11
Pt 12
Pt 13
Pt 14
Pt 15
Pt 16
Pt 17
Pt 18
Pt 19
Pt 20
Pt 21
Pt 22
Pt 23
Pt 24
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6
Pt 7
Pt 8
Pt 9
Pt 10
Pt 11
Pt 12
Pt 13
Pt 14
Pt 15
Pt 16
Pt 17
Pt 18
Pt 19
Pt 20
Pt 21
Pt 22
Pt 23
Pt 24
Figure 3. Representative gel images showing RAPD and
ISSR-PCR profiles of Psophocarpus tetragonolobus geno-
types using primers (a) OPH 4 and (b) UBC 811. Lanes
indicated by marker contains Low range DNA ruler.
total of 167 bands, out of which 138 were polymorphic
revealing 82.63% polymorphism across all winged bean
genotypes. The average PIC value was 0.242 with twenty
primers across all the genotypes (Table 3). Genetic dis-
tances varied from of 0.348 (between Pt6 and Pt16) to
0.883 (between Pt10 and Pt11) across different geno-
types of winged bean.
3.4. Comparison of Diversity Parameters Using
RAPD and ISSR Markers
Comparative study of different diversity parameters was
made on the basis of two marker systems used in the
present study. The average diversity index (DIa) value by
ISSR was higher (0.299) than RAPD (0.261), similarly
the marker index (MI) value in case of ISSR was higher
(3.47) in comparison to RAPD (0.717) (Table 3). ISSR
revealed higher polymorphism (94.5%) than the RAPD
(68.4%), indicating that ISSR are more suitable and reli-
able than RAPD markers to unravel the genetic variabil-
ity in winged bean genotypes. The correlation of genetic
distances was performed between 1) RAPD vs. ISSR; 2)
RAPD vs. cumulative; and 3) ISSR vs. cumulative. The
matrix correlation value (r) between RAPD vs. ISSR was
0.83, whereas it was 0.96 and 0.92 between RAPD vs.
cumulative and ISSR vs. cumulative respectively (Table
4). These values revealed that RAPD vs. cumulative data
have good correlation and are best fit to each other.
3.5. Quantification of Phenolic Acids, Flavonols
and Condensed Tannin
Plant phenolic acids namely: gallic acid, protocatechuic
acid, chlorogenic acid, caffeic acid, ferulic acid and fla-
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C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197 193
Table 3. Comparison of RAPD, ISSR and cumulative band
data analyses in Psophocarpus tetragonolobus.
No. of accessions 24 24 24
Total no of assays/primer 13 07 20
Band size range (bp) 200 - 3000 300 - 3000 200 - 3000
Total no. of bands amplified
(n) 76 91 167
Polymorphic bands (p) 52 86 138
Polymorphism (%) 68.4 94.5 82.6
Genetic distance range 0.607 - 0.974 0.250 - 0.887 0.348 - 0.883
Average PIC 0.170 0.281 0.242
Average Diversity Index
(DIav) 0.261 0.299 0.283
Effective Multiplex ratio
(EMR) 2.73 11.61 6.07
Marker Index (MI) 0.717 3.47 1.72
*combined data of RAPD and ISSR.
Table 4. Mantel correlation between the genetic distances
obtained from RAPD, ISSR and cumulative data analyses
in Psophocarpus tetragonolobus.
Marker pairs Correlation coefficient (r) (p) value
RAPD vs ISSR 0.83935 0.0020
RAPD vs *cumulative 0.96996 0.0020
ISSR vs *cumulative 0.92355 0.0020
*combined data of RAPD and ISSR.
vonols namely: rutin, quercitin and kaempferol were
quantified from the dried powdered seeds of P. tetra-
gonolobus through HPLC. The results were obtained by
comparison with standards (Figure 1). The values ob-
tained here are the mean values for three replicates of the
same sample.Elution was carried out at a flow rate of 0.6
ml/min with water: acetic acid (99.0:1.0 v/v) as solvent A
and acetonitrile as solvent B using a gradient elution in
0-14 min with 20% - 35% of solvent B, 14 - 40 min with
35% - 50% of solvent B.
3.6. Physiological Measurements of Light
Response and Chlorophyll Fluorescence
Light saturated maximum assimilation rate per unit leaf
area (Asat; µmol·m2·sec1) during light response meas-
urements (at PPFD of 1200 µmol·m2·sec1) was highest
in P. tetragonolobus genotype Pt2 from India (20.72 ±
1.41) while lowest assimilation rate was observed in
genotype Pt16 from Thailand (2.97 ± 0.91) (Figure 4).
The genotypes Pt13 (Nigeria), Pt4 (India), and Pt17
Figure 4. Vertical error bars of Photosynthesis (A; µmol·
m2·s1), Stomatal Conductance (G; mol H2O m2·s1) and
Leaf Transpiration (E; mmol H2O m2·s1) of 24 genotypes
of P. tetragonolobus.
(Thailand) showed similar assimilation rates (16.85 ±
0.19, 16.14 ± 0.7 and 14.78 ± 0.01, respectively). The
genotypes Pt25 (Thailand), Pt1 (India) showed assimila-
tion rate of 12.55 ± 0.62 and 12.31 ± 0.48 respectively,
while the genotypes Pt19 (Thailand), Pt11, and Pt24
(Thailand) showed slightly lower assimilation rates
(11.78 ± 0.06, 11.71 ± 0.93 and 11.08 ± 0.88 respec-
tively.) The Pt22 (Thailand) having an assimilation rate
of 7.57 ± 0.27 µmol·m2·sec1 had maximum instantane-
ous water use efficiency (WUE; 0.89 ± 0.05) (Figure 5)
and showed minimum leaf transpiration (E; 0.86 ± 0.08
mol H2O m2s1) (Figure 4). The photosynthetic yield
(0.08 ± 0.001) and electron transport rate (ETR; µmol
em2sec1; 41.35 ± 0.41) were also minimum in the
genotype Pt16 (Thailand) whereas maximum yield (0.30
± 0.001) and ETR (156.57 ± 0.57) was observed in
genotype Pt2 (India) (Figure 6). Stomatal conductance
was also maximum (0.28 ± 0.04) in genotype Pt2 (India)
(Figure 4). Intercellular CO2 conc. (Ci; µmol CO2 mol1)
values ranged between 158.13 ± 12.73 to 305.44 ± 29.18
among all genotypes (maximum in Pt2) (Figure 6).
Maximum quantum yield of photosystem II (Fv/Fm)
measured after dark acclimation ranged from 0.77 (low-
est) to 0.82 (highest) (Figure 5) whereas Fv’/Fm’ (Light
harvesting efficiency by oxidized PSII reaction centers in
light) ranged from 0.46 ± 0.01 to 0.57 ± 0.01 among the
global genotypes of winged bean (Figure 5).
The present study was intended to evaluate the level of
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C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
Figure 5. Vertical error bars of Water use efficiency (WUE),
Fv/Fm (variable to maximal Fluorescence ratio (dark)) and
ɸPSII (Photosynthetic Quantum Yield of PSII) of 24 geno-
types of P. tetragonolobus.
Figure 6. Vertical error bars of Fv’/Fm’ (variable to maxi-
mal Fluorescence ratio (light)), ETR (Electron transport
rate; µmol em2sec1), Ci (Intercellular CO2 conc.; µmol
CO2 mol1) of 24 genotypes of P. tetragonolobus.
genetic diversity and establish a genetic relationship
across the winged bean genotypes with respect to spe-
cific polyphenol and flavonoid content and their response
to various physiological conditions. The two DNA fin-
gerprinting methods (RAPD and ISSR) were used in this
study. Though differing in the underlying principle, were
very informative with regard to the amount of polymor-
phism detected with each method. However, ISSR tech-
nique was more efficient than RAPD in the present study
to unravel polymorphism in winged bean genotypes.
Utility and efficiency of ISSR markers over RAPD to
unravel the genetic polymorphism have been proved in
other legume crops such as chickpea [11], Arachis [27]
etc. Nagaoka and Ogihara [28] have also reported that
the ISSR primers produced several times more informa-
tion than RAPD markers in wheat. The number of poten-
tial ISSR markers depends on the frequency of microsa-
tellites, which changes with species and they are highly
polymorphic and have been used in innumerable crop
plants to study the genetic diversity, phylogeny, gene
tagging, and genomic mapping [29]. In order to achieve a
comprehensive genetic relationships, RAPD and ISSR
band data were pooled together and analyzed to generate
a cumulative UPGMA dendrogram, (Figure 7) thereby
screening much larger portion of the genome to ensure
its wider coverage and providing better reflection of the
genetic relatedness and affinities of the winged bean
genotypes in the present study. The UPGMA dendrogram
grouped all the 24 genotypes into two major groups (A
and B). These groups were again categorized into 7 clus-
ters. Cluster I consisted of four genotypes (Pt1, Pt2, Pt3
and Pt5) from India and two genotypes (Pt12 and Pt17)
from Nigeria and Thailand and three genotypes (Pt9-Pt11)
from unknown origin. Clusters II, III and IV clustered to-
gether all Thailand genotypes (Pt14-Pt24), except Pt17
which showed affinity to cluster I. Cluster V consisted of
genotypes Pt8 from India, showing affinity to Thailand
genotypes. Genotypes Pt4 and Pt5 from India were
grouped into Cluster VI whereas genotypes Pt6 separated
out in Cluster VII with 100 percent bootstrap support.
Furthermore, the out-group taxon clearly separated out
from rest of the studied genotypes. Clustering pattern in
UPGMA dendrogram revealed considerable genetic va-
riations among winged bean genotypes and are indica-
tive of the fact that the groupings were not in congruence
with their geographical affiliations. Principal component
analysis (PCA) performed on data matrices showed two
major significant groups (Figure 2). In the grouping,
Pt12 completely differs from other genotypes. Pt8, Pt11
and Pt14 also formed a separate stand in the groupings.
The grouping of Pt8 as a different genotype is agreeable
with the cumulative result of RAPD and ISSR.
The PCA analysis clearly separated tannin from the
polyphenols and flavonoids but grouped closely with
several other compounds like: rutin, ferulic acid and
chlorogenic acid. As tannins are generally defined as
naturally occurring polyphenolic compounds of high
molecular weight (500 - 3000 Da), which form com-
plexes with proteins [30]. They are classified into two
groups based on their structural type as hydrolysable
tannins composed of a polyhydroxyl alcohol esterified
with gallic or ellagic acid and the condensed tannins
which are flavonoid-based plymers. Total phenolics and o
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C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
Copyright © 2013 SciRes.
Figure 7. UPGMA dendrogram (generated for cumulative data of RAPD and ISSR) showing relationships of P. sophocarpus
tetragonolobus gentotypes.
tannin compounds play an important role in human nutri-
tion and the qualitative and quantitative data of these
compounds may help in identification of seed genotypes
with lower and higher content of polyphenols in the seed
which may be further utilized for genetic improvement
purposes. The range of total phenolics was between 0.09
to 3.49 µg/g. The genotypes (Pt8 and Pt16) from Thai-
land showed highest 1.07 and 790.5 µg/g Kaempferol
content in seeds.
Weighted clustering for plant performance upon three
key physiological parameters especially photosynthesis
rate (A), photosynthetic yield (Y) and stomatal conduc-
tance (Gs) were carried out among these 24 genotypes
(Figures 4-6) of winged bean. The above cluster analysis
showed two clusters (Figure 8). Cluster 1 again formed
two sub-clusters 1a and 1b. Cluster 1a consisted of 5
genotypes (Pt8, Pt12, Pt14, Pt16 and Pt18) having lowest
CO2 assimilation rate. Out of these 5 genotypes Pt14,
Pt16 and Pt18 all from Thailand, showed close affinity to
each other, while cluster 1b clustered together 8 closely
related genotypes (Pt6, Pt7, Pt9, Pt10 Pt15, Pt21, Pt22,
Pt23). Cluster 2 consisted of two sub-cluster 2a and 2b.
In cluster 2a genotypes Pt1, Pt3, Pt11, Pt17, Pt19, Pt25
Figure 8. Cluster analysis (weighted) of photosynthesis,
stomatal conductance and photosynthetic quantum yield
among 24 genotypes of P. tetragonolobus.
C. S. Mohanty et al. / American Journal of Molecular Biology 3 (2013) 187-197
and Pt4, Pt5 showed close relationship whereas cluster
2b had a single genotype (Pt2) showing maximum
physiological performance with high transpiration rates,
which is the most diverse among all the genotypes,
whereas, the genotype Pt16 showed least physiological
performance among all and occupied maximum cluster
distance from the best performing genotype Pt2. There
was little significant correlations between the physio-
logical clustering patterns and those obtained by cumula-
tive analysis of RAPD and ISSR data except for few
genotypes (Pt22, Pt23) in which the inter cluster distance
was least. The results obtained in the present study fur-
ther suggested that physiological diversity in the geno-
types of winged bean need not be necessarily related to
genetic diversity.
The present study provided significant insights on the
genetic diversity presenting in a global collection of
winged bean genotypes and confirmed the potential use
of RAPD and ISSR markers to unravel the extent of ge-
netic variability. ISSR was found to be more efficient in
comparison to RAPD markers revealing 94.5% poly-
morphism among the genotypes of winged bean. The
level of genetic variability detected in this study could be
further utilized in prospecting of highly diverse geno-
types which could be of paramount significance for im-
proving the food and nutrition quality of winged bean.
The plants from Thailand have the highest content of
polyphenols and tannin with hardiness towards improved
physiological efficiency and transpiration rate. The in-
formation generated on the biochemical and physiologi-
cal attributes of winged bean will certainly pave the ways
for prospecting of this important underutilized food crop
for its quality traits.
This work was supported under EMPOWER programme of Council of
Scientific and Industrial Research (CSIR), Government of India and
hence acknowledged. Due acknowledgment goes to N.B.P.G.R. for
providing genotypes of winged bean seeds. Director CSIR-NBRI is
duly acknowledged for providing basic infrastructure facilities to carry
out this research work.
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