Inter Specific Genetic Diversity within the Ethnomedicinally Important Ipomoea L. Species through Morphological and Biochemical Profiling
Noor Muhammad1,2*, Nisar Uddin2, M Khalil Ullah Khan2 and Niaz Ali2
1College of Horticulture, Hebei Agricultural University, China
2Department of Botany, Hazara University Mansehra, KP
Submission: April 15, 2019; May 17, 2019
*Corresponding author: Noor Muhammad, College of Horticulture, Hebei Agricultural University, Baoding, China & Department of Botany, Hazara University Mansehra, KP, Pakistan
How to cite this article: Noor Muhammad, Nisar Uddin, M Khalil Ullah Khan, Niaz Ali. Inter Specific Genetic Diversity within the Ethnomedicinally Important
Ipomoea L. Species through Morphological and Biochemical Profiling. Int J Environ Sci Nat Res. 2019; 19(3): 556012. DOI:10.19080/IJESNR.2019.19.556012
The present study has attempted to highlight the medicinal importance as well as genetic relationships among of Ipomoea species growing in Swat Valley, KP, Pakistan. There was a general agreement among the informers regarding the medicinal uses of plants and Informants Consensus Factor (ICF) value ranged from 0.991 to 0.965. The study shown that most of the informants agreed in the application of I. hederacea to use as blood purifier (ICF 0.991) that exhibited the highest fidelity level (100%). The intra and inter specific dissimilarity among the 28 genotypes of Ipomoea species viz., 14 genotypes of I. hedracea, 14 of I. purpurea, were tested using morphometric and biochemical profiling. Twenty-four morphological characters were counted for the assessment of intra and inter genetic disagreement through traits similarity index and cluster analysis. Total seed protein profiling was carried out on 12% slab gel electrophoresis; 6 reproducible bands were detected in both I. Purpurea and I hedracea with molecular weight ranging from 10KDa to 180KDa. Intra locus contribution toward the genetic disagreement was 33.33% in I. hedracea and 66.66% in I. purpurea. In the same way, inter species locus contribution toward genetic diversity was 83.33%. Interestingly, locus 2 (L-2) was monomorphic in the collected germplasms and may be shown as generic specific locus for Ipomoea species. SDS-PAGE profiling based on two-way cluster plotting successfully determined the two species into separate clusters and the diversity found between and within Ipomoea species may offer unique avenues for novel drug discovery.
The Ipomoea is an important genus belongs to family convolovulaceae; with many species of Ipomoea have wide therapeutic uses. Species are broadly dispersed throughout temperate and tropical regions and mostly found in tropical America and tropical Asia , used to treat blood disease, sterility in women, urinary infection, constipation and gynecological disorders . The plant is also having laxative, psychedelic  anticarcinogenic, hepatoprotectivity, oxytocic and antioxidant properties  as well as in rheumatism and fungal infection .
Ipomoea species are used for its medicinal contents and psychoactive compounds mostly alkaloids. The genus Ipomoea comprises of food crops such as the tubers of sweet potatoes and the leaves of water spinachare are commercially important food stuffs. The most important species Ipomoea including I.hederacea and I. purpurea are growing in the hilly areas of Swat, Pakistan. The I. purpurea and I. hederacea are distinguishable by sepal and leaf characteristics. The I. purpurea sepals are ovate-lanceolate with an apex that is acute to abruptly acuminate while I. hederacea’s
sepals are lanceolate, longattenuate, and caudate. Leaves are ovate and deeply 3-lobed with acuminate tips and cordate bases. Stem twine with their apex in a dextral fashion [6-8].
Morphological characterization has important role in the exploration of genetic diversity in crop plants but effected by environmental changes . On the other hand, genetic diversity evaluation through molecular methods such as biochemical assessment at protein level and DNA based techniques have a number of advantages over the classical morphology  but compared to biochemical assessment at protein level, molecular investigation of DNA level is highly robust but too expensive (Win et al., 2011). Among biochemical techniques, Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method is a simple, reliable, cheap and free of environmental fluxes . SDS-PAGE is now broadly used biochemical practice to designate the genetic structure of crop germplasm (Hameed et al., 2009). Enormous attention has been focused on the use of SDS-PAGE over the last two decades for estimation of genetic diversity, reliable judgment and identification of plant varieties. Seed storage protein markers have been successfully used to resolve
taxonomic relationships and characterize cultivated varieties
in a number of crop plant species; An attempt for estimation of
genetic relationships among the nine Ipomoea species using the
molecular technique of sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS-PAGE) of seed proteins was carried out
by Pragati et al. .
Pakistan is rich in naturally distributed species of Ipomea but
there are no reports about the ethnomedicinal uses or genetic
relationship of Ipomoea species based on total seed protein
profiling available. In this article, we report on ethnomedicinal
uses and SDS-PAGE analyses of 28 genotypes of Pakistani Ipomea
species (I. hedracea and I. purpurea) growing in hilly areas of Swat.
Both these species are locally adapted and are of widespread
use as ornamental and medicinal purposes. The objective of the
study to document the ethnomedicinal uses and level of genetic
polymorphism and relationships among Pakistani Ipomoea
species (I. hedracea and I. purpurea) growing in hilly areas of Swat
using morphological and SDS-PAGE characterization.
In the present study several exploratory trips were arranged
to different agro-ecological zones of Swat in 2017 – 2018. During
expedition, of different zones i.e. Swegalai, Kohay, Ziarat, Jawand,
Gadi, Sharfabad, Rangila, Gharai, Khazana, Aboha, Barikot,
Shingardar Manyar, Nawagai, Shahdeari, Sarsinai, Khawazakhela,
Mangultan, Manglor, Chamtalai, Chaliyar, Baidara, Sherpalam,
Kanju, Bandai, Derai and Hazara were selected for collection of
samples for Ethno medicinal uses, morphological characterization
and estimation of genetic diversity in seed storage protein profile.
Specimens for each specie have been collected and processed
using standard herbarium techniques . The data was collected
through semi structured interviews with inhabitants involved
in the plant’s management . A total of 120 residents were
interviewed for information on uses of plants to cure various
diseases of human being, parts used, of medicine have been
collected. Based on the information obtained from the informants
in the study area, all the reported diseases have been classified
into 9 groups. The use value (UV) demonstrates the relative
importance of locally known plants . It was calculated using
the formula: UV = ΣUi / N , where Ui is the number of uses
mentioned by each information for a given species and N is
the total number of informants. The level of similarity among
information delivered by various informants was calculated by
the Informants’ Consensus Factor or ICF  using the formula:
ICF = Nur – Nt / (Nur – 1) Where, Nur = number of use
reports from informants for a specific plant-use category; Nt =
number of taxa or species that are used for that plant use category
for all informants.
ICF Values range between 0 and 1, where ‘1’ indicates the
highest level of informant agreement. The fidelity level (FL), the
percentage of informants demanding the application of a certain
plant species for the same major purpose, was counted for the
most frequently reported diseases as: FL (%) = (Np / N) × 100
Where, Np = number of informants that claim a use of a plant
species to treat a particular disease; N = number of informants
that use the plants as a medicine to treat any given disease .
Qualitative and quantitative descriptors were taken.
Qualitative traits were recorded on the general visualization
(phenotypic observations). Nine qualitative traits i.e. Leaf Shape,
Leaf Color, Leaf Pubescent, Flower color, Seed texture, Hilum
color, Seed coat color, Seed shape, Spots on the seed coat while
the quantitative traits were Petiole length, Leaf length, Leaf width,
Seed length, Seed thickness, Seed width, Pod length, Seed per pod,
Pod per plant, Flower length, Flower width, Seed weight, Branches
per plant, Plant height, Biomass, Yield per plant. A total of 24 traits
were recorded for morphological description.
To assess genetic diversity and genetic relationships, SDSPAGE
was conducted (Lameli, 1970). For seed storage protein
profile, single seed of each genotypes was crushed into a powder.
400μl of Protein Extraction Buffer PEB (0.5M Tris-HCL, 0.2%SDS,
5M Urea, 1%B-mercaptoethanol under 8-pH) was added to 0.01g
of seed powder. Then mixture in E-tube was Vortexed thoroughly
to homogenize it. The Comassive Brilliant Blue (CBB) was added
to the E-tube as tracking dye to see the movement of PEB-FP on
the separation PAG. The homogenated samples were centrifuged
at 13,000 rpm for 10 minutes under room temperature. The
electrophoretic process was carried out using 12% polyacrylamide
gel (composition of resolution gel: 3.0M Tris-HCl pH9.0, 0.4% SDS
and staking gel 0.4M Tris-HCl pH 7.0, 0.4% SDS). The electrode
buffer containing 0.025M Tris, 129M Glycine and 0.125% SDS was
poured in the Electrophoresis tank. Similarly, 15μl PEB-FP was
loaded in each well of 12% PAG. The electrophoresis was run at
100V until the blue line passed through the bottom of gel plates.
The PAG were than stained and destained for data scoring of seed
storage protein profile.
In this research work 2 plant species of Ipomoea (I. hederacea
and I. purpurea) belonging to Convolovulaceae family in Swat
Valley, have been in listed for curing of 12 categories of ailments.
For each species botanical name, family, local name, ailments to be
treated, and part(s) used were noted (Table1).
Residents’ perspectives about this plant evaluated in this
category of results were very interesting. The responses of the people about the medicinal uses of Ipomoea L. are summarized
(Table 1). In the common domain, the plant was used for the
treatment of Blood purifier, Infertility, Rheumatism, Fungal
infection, Liver protection, Acne, Urinary infection, Diarrhea,
Constipation (Table 1). About 100% of people used it as Blood
purifier, 91.666% people used it for Infertility, 72.5% for
Rheumatism, 75% for reducing Fungal infection , 75% used it for
Liver protection , 62.5% it for Acne, 58.333 for Urinary infection,
41.666% for Diarrhea 25% used it for Constipation (Table 2).
Responses to the status about the survival of the species
suggest that over 74.166% of the respondents agree that the plant
has survived due to ornamental value, 65% people relate it to
medicinal value, 60% relate it to presence on fertile soil, 72.5%
of respondents believed its survival was due to its good water
absorption. Similarly, 55.833% of the informants suggested that
it has survived because of its strong secondary growth system
In the present study, reasons for the serious decline of
Ipomoea in the study area were also investigated. The most
important reason for its decline recorded was overuse of the
seed and leaves. Other reasons included overgrazing or use as
forage, lack of protection, water logging and salinity, less resistant
to drought condition etc. (Table 3). Major source of its decline
(73.333%) was regarded due to its overuse of the seed and leaves,
70.833% of respondents regarded its decline to the non-existence
of any conservation measures, 65% mentioned water logging
and salinity as important reason of its decline while, 55.83% of
respondents regarded the lack of drought resistance as the major
reason of its decline (Table 3).
Use of plant parts as medicine among the informants
demonstrated variations. Leaves and seeds are mostly used part
for majority, followed by bark (Table 1). Similar trend of harvesting
leaves for medicinal use has also been reported from Lawachara
National Park . In the present study area threat to the species
is marginal as leaves are the leading plant part used for medicinal Use of plant parts as medicine among the informants
demonstrated variations. Leaves and seeds are mostly used part
for majority, followed by bark (Table 1). Similar trend of harvesting
leaves for medicinal use has also been reported from Lawachara
National Park . In the present study area threat to the species
is marginal as leaves are the leading plant part used for medicinal
ICF values were calculated to know the settlement among
the informants of Swat valley for consumption of plants to treat
certain ailment categories. The ICF values are given in the Table 2
and varied from 0.991 to 0.965 with an average ICF value of 0.978.
Blood purifier has the highest ICF value 0.991 with 120 use-reports
for 1 plant species. The specie accountable for this high consensus
was I. hederacea. With 120 of the 120 defined events, linked by
Infertility (ICF = 0.99; 110 use-reports, 2 species), Rheumatism
(ICF = 0.988; 87 use-reports, 2 species), Fungal infection (ICF=
0.988, 90 use reports, 2 species) and so on. Medicinal plants
thought to be effective in treating specific illness have high ICF
values. The high ICF value for Blood purifier perhaps disclosed
that this ailment is common in the study area High ICF values
also designate that the specie conventionally used to treat these
ailments are worth searching for bioactive compounds. The least
agreement (ICF=0.985 and ICF= 0.965) between the informants
was observed for Urinary infection, Diarrhea and Constipation
respectively. The low ICF value as noted in our study could be due
to a lack of communication among people in different areas (Table
4 & Figure 1).
To find conventionally medicinal significance of the Ipomoea
species in the area, Fidelity Level (FL) of plants has been predicted
based on use reports, which have been cited by 50 or more
informants for being used against a given illness. The FL values
are shown in Table 5. The investigation displayed that the highest
FL value found in I. hederacea followed by I. purpurea. The least
FL value was found in the case of I. purpurea. FIC and FL studies
presented that the most commonly used species in the study area
is I. hederacea (ICF = 0.991) with 120 use-reports and FL value
(100%). When choosing the most ideal plant species for each
ailment category, we took the high-Fidelity Level (%) in each
category of ailment.
To determine the intra and inter specific variation among the
Ipomea Species, morphological and biochemical characterizations
(SDS-PAGE) were carried out. The germplasm for this study were
collected from different areas of Swat.
SDS-PAGE is one of the most extensively used techniques
to separate total seed protein sub-units and to understand
phylogenetic relationship of plant species. The protein in SDSPAGE
is separated according to the change in their molecular
weight. In this study 28 genotypes of two species i.e. I. hedracea
and I. purpurea, 14 of each were carried out. Each sample was
run on the gel in order to investigate the intra and inter specific
genetic diversity among them.
For morphological data analysis both the qualitative and
quantitative traits were taken. Quantitative traits which were
measured with the help of vernier calipers are: petiole length
(PL), leaf length (LL), leaf width (LW), seed length (SL), seed
width (SW), seed thickness (ST), and seed weight (SWt), pod
length (PodL), No. of seed per pod (SP), No. of pod per plant (PP),
inflorescence length (IL), inflorescence width (IW), 100 seed
weight, No. of branches per plant, plant height (Ph), Biomass.
Characters mean was found out after measuring of 3 different
samples (small, medium, large) of each quantitative character.
The observed qualitative characters are leaf type (LTY), leaf
color (LC), seed texture (St), Hilum color (Hc), seed coat color
(SC), seed shape (SS), leaf pubescent, leaf stipule presence, flower
color. The data of both quantitative and qualitative characters of
28 genotypes (total 24 characters) was recorded and the binary
matrix data was subjected to computer software the PCORD and
the cluster analysis was presented as in Figure 2.
By using the Pearson correlation coefficient, the result for
the association coefficient among the various traits for the two
species of Ipomoea (I. hedracea and V. I. purpurea) was performed
(Table 6). In correlation study the petiole length in the I. hedracea,
is negatively correlated with leaf length in the while positively
correlated with leaf length in I. purpurea. Leaf width is negatively
correlated with the leaf length in I. hedracea while positively
correlated in the I. purpurea. The flower length in I. hedracea and
I. purpurea is negatively correlated with the leaf length and leaf
width. Seed length seed width, seed thickness, No. of the Pods/
Plant, No. of seed/ Plant and 100 seed weight are negatively
correlated with petiole length, leaf length, leaf width, inflorescence
length, flower length and pod length in both the species.
The double data matrix of 28 genotypes based on morphology
was analyzed for the construction of phylogenetic tree to
represents the similarity of various species or genera and the two
species of the Ipomoea were investigated for similarities and the
phylogenetic tree was constructed (Figure 2). The phylogenetic tree
divided the two species in two lineages L1 and L2 at a linkage
distance 37.5 (Figure 1). L1 consisted of two clusters; (C1) and
(C2). The C1 and C2 comprised of all genotypes of I.heracea. While
the L2 was further divided into two clusters; C3, C4 composed of
all genotypes of I. hedracea.
The similarity indexes were performed for all the genotype of
2 species that was 33.33 for I. hedracea and I. purpurea (Table 7).
Six reproducible bands were observed in the both the species,
(Figure 3). The phylogenetic relationship among the 2 species
through phylogenetic tree has been shown in the (Figure 4).
The phylogenetic tree was splitted into eight groups. Groups I
comprised of only one genotype of I. purpurea collected from
Swegalai (IP16). Group I was 25% genetically similar with II which
was composed of 5 genotypes of I. purpurea collected from Kohay
(IP24), Ziarat (IP25), Jawand (IP26), Gadi (IP27) and Sharfabad
(IP28). Group III has 37.7 similarity with the genotypes of Group
II while 50% similar with the genotypes of Group IV. These
genotypes were collected from Rangila (IP17), Gharai (IP18), and
Khazana (IP19). Group V has 81.25% similarity with the genotypes
of Group VI. The genotype of Group IV was collected from Aboha
(IP15. Group V comprised of 4 genotypes collected from Barikot
(IP20), Shingardar (IP21), Manyar (IP22), Nawagai (IP23). Group
VI and Group II only one genotypes of I. hedracea collected from
Shahdeari (IH14) and Sarsinai (IH9) respectively. I. hedracea
has separatd from I. purpurea at a linkage distance 81.25%. I.
hedrarcea and I. purpurea has 81.25% genetic similarity. The
genotypes of Group VII and Group VIII 93.75% similar which were
collected from Khawazakhela (IH4), Mangultan (IH6), Manglor
(IH10), Chamtalai (IH1), Chaliyar (IH2), Baidara (IH3), Sherpalam
(IH5), Kanju (IH7), Bandai (IH8), Derai (IH11) and Hazara (IH).
Table 8 and Figure 5 show interspecific variation among 28
genotypes of the Ipomea species. Among all the genotypes, six
loci (L1-L6) were noted out these L2 was monomorphic and
were marked as generic specific which is used to discriminate the Ipomea species. Moreover, the loci L-1, L-4 and L-6, marked
as polymorphic with 32.14, 7.142, 3.571, 32.14 and35.71 percent
genetic diversity, respectively. The inter species comparative locus
contribution toward genetic disagreement (CLCTGD) was 83.33%
in the two species of 28 Ipomoea genotypes (Table 8 & Figure 5).
Intraspecific locus variation among 14 genotypes of I.
hedracea is represented in Table 9 Notably, L-1, L-2. L-3, L-4 was
monomorphic in I. hedracea. While L-5 and L-6 was polymorphic.
L-5 and L-6 shows 28.571 and 92.85, 17.24, 100 percent variation
and the locus contribution toward genetic disagreement (LCTGD)
of I. hedracea was 33.33% table 9.
The table 9 represents the intraspecific variation among the
14 genotypes of I. purpurea, exhibited high intra-specific locus
variation. Among six loci, out of which L-2 was monomorphic,
while L-1, L-3, L-4, L-5 and L-6 were polymorphic showed 64.28,
7.14, 37.7 and 164.28 percent variation. The locus contribution
toward genetic disagreement (LCTGD) of I. purpurea was 22.22%.
A vast majority of the public living in this area depends directly
on medicinal plant to cure an extensive range of diseases. However,
the disappearance of these plant species is steadily reported
chiefly due to fluctuations in the environment, land degradation
and unsustainable use of these plants; moreover, the expansion
of invasive species has donated a lot to their disappearance
. According to the local inhabitants the reason for its decline
recorded was overuse of the seed and leaves. Other reasons
included overgrazing or use as forage, lack of protection, water logging and salinity as well as less resistant to drought condition
. The present work has been subsequently initiated with an
objective to file the knowledge and practices on the Ipomoea
medicinal uses by the local inhabitants and its genetic diversity.
The present study is supposed to add up the communities’
knowledge in the country’s database of traditional knowledge
and will provide a baseline data for future pharmacological
and phytochemical studies. Genetic diversity suits more vital in
viewpoint of climatic change and associated unexpected events as
it may serve as the source of novel traits considering tolerance to
different biotic and abiotic stresses .
Though, the indigenous knowledge that has been recognized
was previously eroded, if not in all, formerly in most of the
parts studied . Contributions concerning conservation of
traditional knowledge remain scarce . Therefore, the study
focused on identifying local factors that promote the knowledge
on introduced and native medicinal plant species, and to describe
medicinal uses of plant species.
The present work is one of the first efforts to count the
ethnomedicinal information and interspecies genetic diversity in
Ipomoea species (I. hederacea and I. purpurea) from Swat which
offer better choice for the selection of broadly used medicinal
plants looking for bioactive compounds to cure illnesses. The
effectiveness and safety of the reported ethnomedicinal plants
need to be assessed by phytochemical and pharmacological
studies. Plants with high informant consensus factor use report
and fidelity level should be given priority to carry out bioassay
and toxicity studies. From this study we suggest I. hederacea for
further ethno pharmacological studies, since this species has the
high ICF and FL values. The results showed that this species may
be castoff for the improvement of new, cheap, effective, and ecofriendly
herbal formulations for healthcare organization. Further
use of these herbal formulations for healthcare management will
require safety and effectiveness testing. According to inhabitants
and our observations in the field, I. hederacea is now a very
infrequent plant in the area. Illegal and unsustainable collection
of leaves from this plant by the local crude drug traders is one of
the major causes of depletion of this species from nature. There
is crucial want to formulate suitable conservation strategies
for naturally growing ethno medicinal plants to overcome their
depletion from natural resources and to make these practices
more ecofriendly .
The study of genetic diversity and phylogenetic relationship
in medicinal plant species is helpful in their conservation.
Molecular markers are the most effective markers for genetic
polymorphism studies in many species . However, the use of
biochemical markers based on total seed protein and enzyme by
SDS-PAGE method has been a reliable, yet inexpensive method.
Genetic markers for identification and genetic analyses of several
plant species, as they reveal differences between storage proteins
or enzymes encoded by different alleles at a single (allozymes) or
more gene loci (isozymes) .
However, up to date, the present study for the estimation of
inter species phylogeny among the selected species (I. hedracea
and I. purpurea) through morphological and SDS-PAGE is the first
documented report from this area. As the SDS-PAGE has been
used as a practical and reliable method for species Phylogenetic
relationship and identification , therefore, the present study
was conducted that displayed positive results with low inraspecific
and high inter-specific divergence that could enable us to
distinguish two species through SDS-PAGE.
The two plant species under the genus Ipomea study revealed
that no two plants have similar protein banding patterns which
demonstrates the presence of genetic diversity between these
species. The presence of common bands/locus (L-2) among these
two Ipomea species suggests their close genetic similarity and
common ancestry. These loci coded for by a gene that has become
fixed in different species under genus Ipomea over evolutionary
time that is an agreement with work of  that the presence
of common bands in Lycopersicum and Trichosanthes species
describes their common evolutionary origin. Also,  accredited
the appearance of a common locus/band in all individual in
a population to the fact that the gene coding for the enzyme or
protein does not differ.
Due to High inter-species locus contribution toward genetic
disagreement SDS-PAGE could be a reliable technique for
identification of these two species, while intra-specie locus
contribution toward genetic discrepancy was high in genotypes
of I. purpurea (66.66%) as compare to I. hedracea (33.33%) .
In our present work, phylogenetic tree based on seed storage
protein analyses of selected species of Ipomea presented that the
two species of Ipomea had close similarity to one another. The
result showed that the I. purpurea was clustered adjacent to I.
hedracea at linkage distance 81.25% genetic similarity and has
revealed genetic relatedness 81.25% to I. hedracea. The results
obtained after SDS-PAGE electrophoresis disclosed that the method
provided a powerful tool for reliable germplasm discrimination
based on genetic differences in seed storage protein compared to
selected germplasm of Ipomoea. Thus, the present study explores
the existing polymorphism of total proteins through SDS PAGE to
facilitate characterization of selected germplasm of Ipomoea .
The current work provides evidence that medicinal plants
have an important role in the healthcare system of Swat area.
They continue to depend on medicinal plants for the treatment
of healthcare problems. The current paper documented a
significant ethnobotanical information on medical plants which
provides baseline data for future pharmacological studies and
genetic diversity is helpful in selection elite genotypes for future
conservation as well as may allow identification of important
therapeutic compounds that may be used for the discovery of new
NM collected plants carried out experimental work analyzed
the data, NA and NU reviewed the paper and helped in writing the
paper and MK helped in in providing chemicals for experiments.
All authors have read and approved the final manuscript.