International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(5) pp. 119-128, May, 2011
Available online http://www.interesjournals.org/IRJPS
Copyright © 2011 International Research Journals
Full length Research Paper
Phylogenetic relationships of Crotalaria species based
on seed protein polymorphism revealed by SDS-PAGE
L. Joelri Michael Raj1*, S.John Britto1, S.Prabhu1, and S.R.Senthilkumar2.
1
The Rapinat Herbarium and Center for Molecular Systematics, St. Joseph’s College,
Tiruchirappalli – 620 002, South India.
2
Department of Plant Biology and Plant Biotechnology, St. Joseph’s College,
Tiruchirappalli – 620 002, South India.
Accepted 13 May 2011
Seed protein profile of 12 accessions belonging to 9 species of genus Crotalaria were investigated
through SDS-PAGE. Intra and inter specific relationship was estimated using Nei and lei’s genetic
distances. A dendrogram based on UPGMA revealed the inter relationship of species within Crotalaria.
Most species had distinctly different protein patterns but close association was found between
C.pallida and C.laburnifolia. The Morphological variation observed between the two specimens of C.
grahamiana collected from two different geographical regions showed good variations in their protein
profiles also, that are enough to give it the rank of sub species of C. grahamiana. PCA had grouped
C.verrucosa and C.beddomeana together where both are specifically characterized by the presence of
well developed stipules indicating it as a phylogenetically important character. The present study
provides useful markers for the identification of the taxa, their relationship and the delimitation of their
taxonomic status both at interspecific and intraspecific levels.The phylogenetic tree obtained by
DAMBE predicts sequence of evolution within the genus.
Keywords: Crotalaria; Seed proteins; SDS-PAGE; UPGMA; PCA; Phylogenetic tree.
INTRODUCTION
Crotalaria is a species complex genus and is considered
to be the third largest genus of Papilionoideae, with
around 600 species distributed throughout the tropics and
subtropics (Polhill 1982). They are used as a source of
fibers, silage and green manure which are its agronomic
traits (Cook and White, 1996; Ramos et al., 2001; Sakala
et al., 2006). Crotalaria species can be used as forage for
horses and cattle owing to the large amounts of water
soluble gums and proteins in their seeds(Purseglove,
1981; Pandey and Srinivatsva, 1990). Their ability to fix
nitrogen enables these plants to be used for enhancing
soil fertility (Samba et al., 2002). Moreover these
leguminous plants develop a high resistance to main
species of root knot nematodes which cause severe
*Corresponding author Email: joelriraj@gmail.com
damage to crops (Antonio and Neumaier, 1986).
Crotalaria species are used as food plants for larvae of
Lepidoptera species (Eisner et al., 2000). The toxic
alkaloids produced by some members of this genus are
known to be incorporated by these larvae and used to
secure their defense from predators (Bernays et al.,
2003). Alkaloid monocrotaline, a pyrrolizidine alkaloid is
the main toxic principle of genus Crotalaria which is used
to induce experimental pulmonary hypertension in
laboratory animals (Werchan et al., 1989). Thus there
arises a great need to unravel the phylogenetic
relationship of such an agronomic ethnomedicinal and
horticulturally important genus. Moreover morphological
markers used in the past are insufficient for their correct
and proper identification. Sodium Dodecyl Sulphate
PolyAcrylamide Gel Electrophoresis (SDS-PAGE)
technology is widely used to study plant taxonomy,
120 Int. Res. J. Plant Sci.
affinities and genetic diversity. SDS-PAGE of total seed
proteins has found wide application in resolving
systematic relationships and for inter and intra specific
studies (Karihaloo et al., 2002). When the plants are
highly variable and contain large number of hybrids,
identification based on morphological characters is quite
difficult.
SDS-PAGE have extensively been used to study
genome structure in various crops of agronomic
importance like wheat, maize, soybean, sunflower, etc
(Weber et al., 2005, Zhu et al., 2005). Seed protein
profile obtained by electrophoresis has been successfully
used to resolve the taxonomic problems of Solanum and
Capsicum species (Edmonds and Glidewell ,1977;
Zubaida et al., 2006). SDS-PAGE is most widely used
due to its validity and simplicity for describing genetic
structures of group of plants (Sher et al., 2010) rather
relying on DNA based markers which are expensive and
time consuming (Wang et al., 2006) . However the
potentials of DNA markers to resolve inter and
intraspecific variations is well known and hence could be
used to study variations that could not be revealed by
protein markers. Moreover proteins are expressed form
of genome which can be biomarkers for identification of
botanic drugs (Muhammad et al, 2010). The mature seed
provides a stable and convenient system for biochemical
analysis to establish relationship in parents and hybrids
(Gangwar and Bajpa, 2006). The genetic similarity
information could make decisions regarding the choice
for selecting superior genotypes for improvement or to be
used as parents for the development of future cultivars
through hybridization. Legumes have played a primary
role in the search for vegetable sources of proteins owing
to the high protein content of the seed (Valizadeh, 2001).
The comparative study of protein variation in these
species is not well demonstrated. Hence, it is desirable to
increase our knowledge of the genetic resemblance
among the most important genus Crotalaria by employing
variations in seed storage proteins, which are their main
common characteristics. The present study was initiated
to find genetic similarity on the basis of seed protein
profile and its relationship with agronomic traits in genus
Crotalaria. It was also used to study the Intra specific
variations to identify the elite variety. For example wild
species of C.grahamiana RHT56077 has specific
features such as drought tolerance, high seed protein,
high pod set and viable large seeds whereas
C.grahamiana RHT56085 lacks these features. Since
genetic differences are reflected in shifts of seed protein
patterns, the present study was under taken on
biochemical characterization of seed storage proteins in
order to see variation for seed protein in the segregating
populations due to environmental pressures. Boatwright
et al (2008) has did a systematic study of generic
relationships in the tribe Crotalarieae based on ITS and
rbcL sequences but the inter and intra specific relations
among one of the genus Crotalaria in the tribe has not
specifically dealt with. Thus the present investigation
aims to determine the taxonomic relationship by using
SDS-PAGE of seed proteins of 12 taxa at interspecific
and intraspecific levels.
MATERIALS AND METHODS
Collection of samples
Two accessions of C.verrucosa, C.retusa and
C.grahamiana from widely separated geographical
regions were chosen for study to measure the range of
divergence that had occurred in the protein banding
patterns within the species.RHT (Rapinat Herbarium,
Tiruchirappalli) 56077, C.grahamiana and RHT56060,
C.beddomeana were collected from the Western Ghats
of,SouthIndia at high altitudes. Similarly C. pallida
RHT56081 and RHT56083 were collected from foothills
of kollihills and Puliyancholai hills respectively. The other
accessions were from plains of Central Tamil Nadu south
India (Table 1). The voucher specimens were identified at
the Rapinat Herbarium and Center for Molecular
Systematics, St. Joseph’s College Tiruchirappalli, Tamil
Nadu, India. The morphological identification and
comparison was done based on The Flora of the
Tamilnadu Carnatic (Matthew, 1981) and The Flora of the
Palni hills (Matthew, 1999). Mature seeds only were
collected and stored at room temperature for protein
analysis.
Sample extraction
Seed proteins were extracted by modified procedure of
Miller et al (1972). Total seed proteins were extracted
from 2.0 g of seed flour using 10ml of extraction buffer
that contained 125mM Tris-HCl pH 7.2, 0.2% SDS, 0.5M
NaCl, and 1% Mercaptoethanol. Seed flour was
thoroughly mixed with buffer by vortexing. The extracted
protein was separated by centrifuging the sample at the
rate of 15000rpm for 10 minutes. The supernatant was
the protein source.
Protein estimation and sample preparation
Total soluble protein in extracted samples was estimated
using
Perkin
Elmer
spectrum
lambda35
UV
spectrophotometer at 280nm. The protein solution was
Raj et al. 121
Table 1. List of species within the Genus Crotalaria taken for study
S.No
1
Accession No.
RHT56077
Botanical Name (Leaf Morphology)
C.grahamiana ( 5- 7 foliate)
Latitude
10.27083° N
Longitude
77.56944°E
Location and altitude
Kodaikanal Ghat road, 1835m.
2
3
RHT56060
RHT56075
C. beddomeana ( Unifoliate)
C. retusa ( Unifoliate)
10.2372 ° N
10.85444° N
77.4714 °E
78.66417°E
Kodaikanal S.H college, 1327m.
Kambarasampettai, 68m.
4
5
RHT56078
RHT56079
C. verrucosa( Unifoliate)
C. juncea( Unifoliate)
10.835° N
10.85545° N
78.73222°E
78.66518°E
Keelmulaikudi, 71m.
Kambarasampettai, 72m.
6
RHT56080
C.paniculata ( Unifoliate)
10.85570° N
78.66558°E
Kambarasampettai, 73m.
7
8
RHT56081
RHT56082
C. pallida ( Trifoliate)
C. laburnifolia ( Trifoliate)
10.75354° N
10.85345° N
78.43291°E
78.66398°E
Kolli hills, 754m.
Kambarasampettai, 73m.
9
10
11
12
RHT56083
RHT56084
RHT56076
RHT56085
C. pallida ( Trifoliate)
C. retusa ( Unifoliate)
C. verrucosa ( Unifoliate)
C.grahamiana ( 5- 7 foliate)
10.73543° N
10.83543° N
10.85450° N
10.85491° N
78.77219°E
78.73291°E
78.66554°E
78.66468°E
Puliancholai hills, 350m.
Cauvery river banks 65m.
Kambarasampettai, 73m.
Kambarasampettai, 73m.
diluted two-fold with 125mM Tris buffer pH 7.2 so that the
concentration is well within the accurate range of the
instrument. Accuracy is greatest, approximately at 0.3
absorbance. (Aitken and learmonth, 2002)
This gives an accurate estimate of the protein content by
removing the contribution to absorbance by nucleotides
at 280 nm, by measuring the A-260 which is largely
owing to the latter (Aitken and learmonth, 2002).
Protein (mg/mL) = 1.55 A280 -0.76 A260
All the samples were further diluted according to the yield
and brought to 1mg/ml concentration. The samples were
then mixed with equal volumes of sample buffer (Jha and
Ohri, 2002) and equal amount 50µl (25µg) of protein was
loaded in each well.
Protein profiling using SDS PAGE
Electrophoresis was carried out in a discontinuous SDSPAGE system of Laemmli (1970) using 12% acrylamide
gel. A Protein Molecular Weight Marker Medium range
(Genei, Bangalore) was also run along with protein
samples as standard (M.wt 97.4 - 14.3 KDa). SDS PAGE
was performed at constant voltage (100 V). Gels were
stained using Coomassie brilliant blue dye R-250 (Hames
and Rickwood, 1990) and documented by UVidoc Gel
Documentation System. The banding patterns were
analysed in GEL DOC software (UVItec, UK). The
experiment was repeated thrice to check the consistency
of the data.
Band scoring and analysis
Only the unambiguous bands were coded for presence /
absence (1/0) and analysed in POPGENE ver.3.5
software. The UPGMA dendrogram was generated based
on the Nei and lei’s genetic distances (Nei, 1978). The
tree was viewed in TREE VIEW software and later
imported in MEGA ver. 4.0 for branch swapping and
modification. The data was also analysed using MVSP
3.1 (Multi Variate Statstical Package) (Kovah, 2005) for
calculating Jaccards similarity coefficient, Squared
Euclidean distances and PCA (Principal Component
122 Int. Res. J. Plant Sci.
Analysis) was done based on the Euclidean distances.
The distance matrix was also subjected to analysis in
DAMBE 5.1.2. (Data Analysis for Molecular Biology and
Evolution) to trace the phylogenetic relationship (Xia,
2001).
RESULTS AND DISCUSSION
Protein markers for Crotalaria
The SDS PAGE analysis had generated 32 protein
markers for these species (Figure 1, Table 2).These
banding patterns clearly segregated the simple leafed
species from compound leafed species with one
exception where C.grahamiana, with compound leaves
(RHT56077, RHT56085) shared specific bands at
124kDa, 73kda, 63kDa, 60kDa and 42kDa with the
simple leafed ones (Table 2). Some specific markers at
120kDa, 92kDa and 46kDa were identified for the
trifoliate species C.pallida (RHT56081, RHT56083) and
C.laburnifolia (RHT56082). Another important marker
was observed at 30kDa common to all the species of
Crotalaria studied. Except for C.retusa (RHT56075,
RHT56084) the intensity of this band was observed to be
prominent for all the species studied (Figure 1). These
are suspected as lectin proteins that has a molecular
weight of 30kDa. The results obtained for the lectins of
C. juncea (Ersson et al., 1973, Ersson 1977), C. striata
(Khang et al., 1990), and C. pallida (Rego et al., 2002)
shows that most Leguminosae lectins consist of four
subunits of 30 kDa each, which are held together by noncovalent interactions to yield a tetramer of 110 kDa
(Sharon and Lis, 1990). The lectin shows a single band of
30 kDa in SDS-PAGE (Luzia et al., 2004). Moreover
previous studies indicated that there are many unique
features identified for each lectin proteins especially
lectins obtained for C.paulina seeds. These are observed
to have a peculiar feature capable of agglutinating
erythrocytes in the absence of divalent ions ( Luzia et al.,
2004) unlike the lectins obtained from C. striata (Khang et
al., 1990) and C. pallida (Rego et al., 2002). Our work
gives a scope to analyse the variations in the activity of
lectins from each species that can be specifically
identified by the protein markers identified in this study
(Table 2)
Genetic Similarity
The Jaccard’s similarity coefficient ranged between 0.063
-1.00 (Table 3). The minimum similarity value of 0.063
was observed between C.retusa (RHT56075) and
C.laburnifolia (RHT56082) and also between C.retusa
(RHT56084) and C.pallida (RHT56081, RHT56082). The
maximum similarity value was 1.00 showing no allelic
variations between the two accessions of C.pallida
collected from two different geographical regions (Table
1). Both the species did not show much variation in their
morphology too. This shows the genetic stability of seed
storage proteins that could be used to derive a
meaningful phylogeny. But on the contrast similarity value
of the two accessions of C.grahamiana was 0.75. But this
accession had many morphologically variant features
which was well depicted by the protein banding patterns
showing allelic variations at 143kDa, 99kDa and 60kDa
(Table 2). RHT56085 also has some morphologically
dominant characters like large seeds and fleshy leaves
with silky pubescent hairs. This may be due to the
difference in geographical regions that these two
accessions grow. Moreover the intensity of the shared
bands of RHT56077 with RHT56085 is much higher
indicating richness of the seed proteins in RHT56085
which is considered to be an elite variety. Another fact
about this species is that its occurrence is reported only
in the higher altitudes of palni hills (e.g. C.grahamiana
RHT56077. But RHT56085 was found in the plains of
Kambarasampettai which is the first report on its
occurrence at the hay fields (Table 1). These differences
are enough to give it the rank of sub species of
C.grahamiana.
Moreover specific markers for
identification of C.grahamiana was also identified at
90kDa and 40 KDa which was common to both these
accessions indicating that it was the variant of the elite
one in higher altitudes which could have been introduced
by native farmers a decade back to improve the fertility of
their lands. So it is emphasized that more work should be
done to identify the agronomic potentials of the elite
variety of C.grahamiana (RHT56077). Baijukya et al.
(2006) reports that the percentage fertiliser equivalency
(%FE) of legumes ranged between 25 and 59% with
higher values recorded with C. grahamiana.
Cluster analysis
The cluster analysis of the taxa based on Nei and lei’s
genetic distances was represented by an UPGMA
dendrogram in Figure 2. The protein patterns divided the
twelve species of Crotalaria into two broad clusters. All
the species in the cluster II were trifoliate ones. The
genetic relatedness of unifoliate ones like C.juncea with
C.retusa was much closer than that with trifoliate ones
like C.pallida and C.laburnifolia is precisely depicted by
the data generated by protein markers.Our studies are in
congruence with the study on four species of Crotalaria
based on EST SSR markers (Wang et al ., 2006). Thus
Raj et al. 123
124 kDa
120 kDa
114 kDa
99 kDa
92 kDa
90 kDa
86 kDa
81 kDa
76 kDa
73 kDa
68 kDa
66 kDa
63 kDa
60 kDa
56 kDa
53 kDa
50 kDa
48 kDa
46 kDa
42 kDa
40 kDa
38 kDa
37 kDa
35 kDa
34 kDa
33 kDa
31 kDa
30 kDa
1
0
0
0
0
0
0
0
0
0
0
0
134 kDa
RHT56077
RHT56060
RHT56075
RHT56078
RHT56079
RHT56080
RHT56081
RHT56082
RHT56083
RHT56084
RHT56076
RHT56085
136 kDa
Acc.No.
139 kDa
Mol.wt.
143 kDa
Table 2. Molecular weight values for various bands electrophoresed in seed proteins of Crotalaria species.
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
1
0
1
1
1
0
0
0
0
1
0
1
0
0
0
0
0
0
1
0
0
1
0
1
1
0
1
0
1
0
1
1
0
0
0
0
0
0
1
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
1
1
0
1
0
1
1
1
1
1
1
1
1
1
1
0
0
0
1
1
1
1
0
0
0
1
0
1
1
1
0
1
1
0
0
0
0
1
1
1
1
1
0
0
0
1
1
1
1
1
0
0
0
0
1
1
1
1
1
0
1
0
0
0
0
0
0
1
0
0
1
0
1
1
0
1
1
1
0
1
0
0
0
0
0
0
1
1
1
1
0
0
0
1
1
0
1
1
0
1
1
1
0
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
1
0
1
0
1
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
1
1
1
1
0
0
0
0
0
1
1
1
1
0
0
0
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
protein markers are simple and effective in
revealing the inter specific relationships.The
interesting fact revealed by protein markers is that
it had clustered C.grahamiana; a 5-7 foliate
species along with the simple leafed species
whereas the classical taxonomic key clusters
C.grahamiana with the compound leafed species
which is not supported by the protein data. Since
seed storage proteins are the products of gene
expression with genetic stability and not been
affected much by environment, the protein
banding patterns
poses a much validated
phylogenetic relationship than relying on few
morphological characters (Ghafoor et al., 2002;
Panigrahi et al., 2007). Within the Cluster I,
C.grahamiana is clustered within Cluster IA along
with C.juncea, C.retusa and C.paniculata. So
C.grahamiana is expected to have evolved from
any one of these these simple leafed species and
also is expected to share many agronomic traits
with C. juncea which is one of much exploited
species agronomically as a potential green
manure worldwide, particularly in the Asian
countries. C. grahamiana is already exploited as a
green manure in many African countries
(Tumuhairwe et al., 2007; Niang et al., 2002).
Phylogenetic tree obtained by DAMBE analysis
clearly predicts the sequence of evolution in
morphology that could have occurred within this
genus. The relationships revealed by protein
markers could be well explained by the presence
or absence of stipules which is declared to be an
evolutionarily important character in genus
Crotalaria where it had clustered the prominent
stipulated
species
C.verrucosa
and
C.beddomeana together.
The morphological
features are added to the tree for better
understanding of phylogenetic relationships.
(Figure 3).
PCA analysis
The Principal component analysis (PCA) of the
protein data represented as Figure 4, had also
grouped C verrucosa and C.beddomeana in the
same axis. This result is in congruence with the
classical taxonomic key, where both these
124 Int. Res. J. Plant Sci.
Figure 1. Seed protein profiles of 12 Crotalaria species showing inter and intra specific relationships.
M= Marker (medium range). Numbers 1-12 as in Table - 1.The 30kDa marker which was observed common to all the 12
species is indicated below the white line
0.000
RHT56081 C.pallida
8.495
0.000
24.408
RHT56083 C.pallida
8.495
11.362
4.567
9.775
15.929
6.845
RHT56080 C.paniculata
1.587
3.824
1.587
4.922
13.861
1.587
11.151
15.448
RHT56084 C.retusa
Cluster I A
1.587
6.304
RHT56075 C.retusa
RHT56079 C.juncea
4.922
14.832
Cluster II
RHT56082 C.laburnifolia
RHT56077 C.grahamiana
Cluster I
RHT56085 C.grahamiana
RHT56078 C.verrucosa
RHT56076 C.verrucosa
Cluster I B
RHT56060 C.beddomeana
5
Figure 2. Dendrogram for Protein markers based on Nei's (1978) Genetic distance: Method = UPGMA . Modified from
NEIGHBOR procedure of PHYLIP Version 3.5
Tree Caption. The tree with sum of branch lengths = 164.4436 is shown. The branch length in which the associated taxa
are clustered together in the bootstrap test (1000) replicates are shown next to the branches. The tree is drawn to scale
with branch lengths in same units as those of evolutionary distances used to infer the phylogenetic tree.
Raj et al. 125
Table 3. Similarity matrix based on Jaccard’s coefficient.
S.No.
RHT56077
(C.grahamiana)
RHT56060
(C.beddomeana)
RHT56075
(C.retusa)
RHT56078
(C.verrucosa)
RHT56079
(C.juncea)
RHT56080
(C.paniculata)
RHT56081
(C.pallida)
RHT56082
(C.laburnifolia)
RHT56083
(C.pallida)(
RHT56084
(C.retusa)
RHT56076
(C.verrucosa)
RHT56085
C.grahamiana)
RHT560
77
(C.graha
miana)
RHT560
60
(C.bedd
omeana)
RHT560
75
(C.retus
a)
RHT560
78
(C.verru
cosa)
RHT560
79
(C.junce
a)
RHT560
80
(C.panic
ulata)
RHT560
81
(C.pallid
a)
RHT560
82
(C.labur
nifolia)
RHT560
83
(C.pallid
a)
RHT560
84
(C.retus
a)
RHT560
76
(C.verru
cosa)
RHT560
85
(C.graha
miana)
1
0.2
1
0.308
0.118
1
0.333
0.529
0.267
1
0.333
0.238
0.462
0.368
1
0.063
0.056
0.333
0.118
0.267
1
0.15
0.136
0.125
0.19
0.471
0.286
1
0.1
0.091
0.063
0.091
0.333
0.308
0.643
1
0.15
0.136
0.125
0.19
0.471
0.286
1
0.643
1
0.333
0.125
0.833
0.286
0.385
0.222
0.063
0.05
0.063
1
0.389
0.5
0.25
0.929
0.421
0.111
0.238
0.136
0.238
0.267
1
0.75
0.211
0.333
0.353
0.438
0.067
0.222
0.105
0.222
0.364
0.412
species also share some specific morphological
features such as presence of angular branchlets,
prominent and foliaceous, semi-lunate stipules.
(Mathew, 1999). These two species shares many
protein markers at 114, 99, 73, 63, 60, 56, 50 and
38kDa. The 38kDa marker was very specific to
these two species and were absent in other
species of Crotalaria studied. Two markers at 136
and 134 kDa were specific in identifying
C.beddomeana exclusively from other species.
Similarity values of C.beddomeana with other
species were also very low indicating it to be a
unique species. The PCA has its advantage in
delimiting some in precise manner. For example,
it had grouped C.juncea and C.paniculata
together which gives some scope to test the
agronomic potentials of C.paniculata a hitherto
under utilized species. PCA has also pictured the
1
close association of C.pallida with C.laburnifolia,
where the latter is expected to share similar
chemical profiles to the former which is much
reported for its specific pyrrolizidine alkaloids like
retrorsine and a senecionine associated with
hepatotoxicity (Artz and mount, 1999).
126 Int. Res. J. Plant Sci.
RHT56079 C.juncea
Rooted ( stipules 0)
RHT56082 C.laburnifolia
RHT56081 C.pallida
Trifoliate species ( stipules 0)
RHT56083 C.pallida
RHT56080 C. paniculata
Stipules linear subulate
RHT56075 C.retusa
Stipules linear subulate
RHT56084 C.retusa
RHT56077 C.grahamiana
5- 7 foliate species ( linear stipules)
RHT56085 C.grahamiana
RHT56078 C.verrucosa
RHT56076 C.verrucosa
Presence of foliaceous semi-lunate Stipules
RHT56060 C beddomeana
0.05
Figure 3. Phylogenetic reconstruction with DAMBE 5.1.2 using Euclidean distance matrix.
Tree caption: Phylogenetic tree from a user-supplied distance matrix, based on the FastME algorithm (Desper and Gascuel
2002, 2004), with branch evaluation by the balanced method and with the initial tree generated by the GME method. The
final tree results from evaluating candidate trees generated by extensive NNI (nearest neighbor interchange). The tree
length (sum of branch lengths) is 1.65201. The tree is rooted by outgroup RHT56079. Phylogenetic reconstruction is done
with DAMBE 5.1.2(Xia 2001; Xia and Xie 2001).
Figure 4. Principle Component analysis (PCA) using Euclidean distances for 12 accessions based on 32 protein
Raj et al. 127
CONCLUSION
SDS-PAGE was found effective for phylogenetic studies
in Crotalaria species. It had proved its advantage over
morphological identification and had also detected
specific proteins of which lectins are tools of medical and
biological research which can be further analysed by 2D
gel electrophoresis. Although this study added new
findings to the literature, it is somewhat limited to the
known species and subspecies distributed in the central
Tamilnadu. Moreover the overall similarity values seemed
to be very low showing wide range of divergence
between the species (Table 3). A comprehensive study
covering a few more economically important species of
Crotalaria from the Western Ghats seems to be
necessary to construct a more satisfactory classification
and interpretation of evolutionary relationships. The
intraspecific variations between the two accessions of
C.pallida could be resolved only by DNA based
techniques like RAPD and ISSR markers. Sequence
analysis of chloroplast genes could obtain a much more
resolved phylogeny for genus Crotalaria.
REFERENCES
Aitken A, Learmonth M (2002). The Protein Protocols Handbook,
Second Edition: Protein Determination by UV Absorption. Humana
Press., New jersey, pp. 3-6.
Antonio H,
Neumaier N (1986). Reaction of soil improving plant
species to the root Knot nematode Meloidogyne iavanica.
Nematologia Brasileria. 10:207-215.
Arzt J, Mount ME (1999). Hepatotoxicity associated with pyrrolizidine
alkaloid (Crotalaria spp) ingestion in a horse on Easter Island. Vet
Hum Toxicol. 41(2):96-99.
Baijukya FP, Ridder N, Giller KE (2006). Nitrogen release from
decomposing residues of leguminous cover crops and their effect on
maize yield on depleted soils of Bukoba District, Tanzania. Plant and
Soil. 279:1-2
Bernays EA, Chapman RF, Lamunyon CW (2003).Taste receptors for
pyrrolizidine alkaloidsIn a monophagous caterpillar. Journal of
Chemical Ecology 29(7):1709-1722.
Boatwright JS, Roux L, Marianne M, Michael W, Tatiana M, Ben-Erik V
(2008) Phylogenetic Relationships of Tribe Crotalarieae (Fabaceae)
Inferred from DNA Sequences and Morphology. Systematic botany
33(4):752-761.
Cook CG and White GA (1996). Crotalaria juncea: A potential multipurpose fiber crop. pp. 389-394. In: J. Janick (ed.), Progress in new
crops. ASHS Press., Arlington, VA. Desper R, Gascuel O (2002).
Fast and accurate phylogeny reconstruction algorithms based on the
minimum-evolution principle. Journal of Computational Biology.
9:687-705.
Desper R , Gascuel O (2004). Theoretical foundation of the balanced
minimum evolution method of phylogenetic inference and its
relationship to weighted least-squares tree fitting. Mol. Biol. Evol.
21:587-598.
Edmonds JW, Glidewell SM (1977). Acrylamide gel electrophoresis of
seed proteins from some Solanum (section Solanum) species. Plant
Syst. Evol. 127: 277-291.
Eisner T, Eisner M, Rossini C, Iyengar VK , Roach BL (2000).
Chemical defense against predation in an insect egg.Proceedings of
the National Academy of Sciences of the USA 97: 1634–1639.
Ersson B (1977) Phytohemagglutinin from sunn hemp seeds (Crotalaria
juncea) II Purification by a high capacity biospecific affinity adsorbent
and its physicochemical properties. Biochim Biophys Acta. 494: 5160.
Ersson B, Aspberg K, Porath I (1973). Phytohemagglutinin from sunn
hemp seeds (Crotalaria juncea) Purification by biospecific affinity
Chromatography Biochim Biophys Acta. 310: 446-452.
Gangwar LK, Bajpa GC (2006). Seed protein variation in interspecific
hybrids of Pigeonpea. Indian J. Crop Sci. 1(1-2): 125-128
Ghafoor A, Ahmad Z, Qureshi AS, Muhammad B. (2002) Genetic
relationship in Vigna mungo (L.) Hepper and V. radiata (L.) R.
Wilczek based on morphological traits and SDS-PAGE.
Euphytica.123(3): 367-378.
Hames BO, Rickwood D (1990). “Gel Electrophoresis of Proteins, a
Practical Approach” (2nd Ed.) Oxford University Press, USA pp. 1147
Jha SS, Ohri D (2002) Comparative study of seed protein profiles in
the genus Pisum. Biologia Plantarum. 45 (4): 529-532.
Karihaloo JM, Kaur M, Singh S (2002) Seed protein diversity in
Solanum melongena L. and its wild and weedy relatives. Genetic
Resources and Crop Evolution. 49(6): 533-539.
Khang, NQ, Jean-Luc G, Johan H (1990). A blood group a specific
lectin from the seeds of Crotalaria striata.Biophys. Biochem. Acta.
33(2): 210-213
Kovach WL (2005). MVSP - A MultiVariate Statistical Package for
Windows, ver. 3.1. Kovach Computing Services, Pentraeth, Wales,
U.K.
Laemmli UK (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227 (5259): 680-685.
Luzia AP, Daniela DC,Marcos HT, Luciana DC, Jose C, Novello SF,
Pascholatti and Sergio M (2004) Purification and Characterization of
a Lectin from Crotalaria paulina Seeds. The Protein Journal. 23(7):
437- 444.
Matthew KM (1981). The Flora of Tamilnadu Carnatic part-I, Diocesan
press., India, pp 352-380
Matthew KM (1999). The Flora of Palni Hills part-I, Offset press., India,
pp 293- 309.
Miller MK, Schonhorst MH, McDaniel RG(1972). Identification of
Hybrids from Alfalfa Crosses by Electrophoresis of Single Seed
Proteins. Crop Sci. 12: 535- 537.
Muhammad IC, Abdul SM, Wang Y, Cheng Y, Tariq M, Muhammad A
(2010). Proteins as Biomarkers for Taxonomic Identification of
Traditional Chinese Medicines (TCMs) from Subsection Rectae
Genus Clematis from China. World Appl. Sci. 8: 62-70.
Nei M (1978). Estimation of average heterozygosity and genetic
distance from a small number of individuals. Genetics 89: 583-590
Niang A, Amadalo BA, Dewolf J, Gathumbi SM (2002). Species
screening for short term planted fallows in the highlands of western
Kenya. Agrofor Syst. 56:145–154
Pandey VN, Srinivastava AK (1990). Seed protein yield from some
Crotalaria spp. and in vitro nutritional quality of that from C. juncea..
Plant Foods Human Nutr. 40: 195-200.
Panigrahi J, Kumar DR, Mishra M, Mishra RP, Jena P (2007). Genomic
relationships among 11 species in the genus Cajanus as revealed by
seed protein (albumin and globulin) polymorphisms. Plant Biotechnol.
Reports. 1(2): 109-116.
Polhill RM (1982). Crotalaria in Africa and Madagascar. Royal Botanic
Gardens, Kew, pp. 389.
Purseglove, JW (1981). Leguminosae, pp. 250-254. In: Purseglove JW.
Tropical crops: dicotyledons. Longman Group Ltd., Essex, UK.
Ramos MG, Villatoro MAA, Urguiaga S, Alves BJR, Boddey RM (2001).
Quantification of contribution of biological nitrogen fixation to tropical
green manure crops and residual benefit a subsequent maize crop
using N isotope techniques. J. Biotechnol. 91: 105-115.
Rego EJL, DeCarvalho DD, Marangoni S, DeOliveira B, Novello JC
(2002). Lectins from seeds of Crotalaria pallida (smooth rattlebox).
Phytochemistry. 60(5): 441–446.
Sakala W, Aldish G, Giller KE(2000). Interactions between residues of
128 Int. Res. J. Plant Sci.
maize and pigeon pea and mineral N fertilizer during decomposition and
N mineralization Soil. Boil. Biochem. 32: 679-688.
Samba RT, Sylla SN, Neyra M, Gueye M, Dreyfus B, Ndoye I (2002).
Biological nitrogen fixation in Crotalaria species estimated using
15
the N isotope dilution method. Afr. J. Biotechnol. Vol. 1 (1): 17-22.
Sharon N, Lis H (1990). Legume lectins - a large family of homologous
proteins. FASEB J.4 (14): 3198-3208
Sher AK, Habib A, Shah MK, Ayub K, Sardar A, Muhammad S (2010).
Confirmation of sunflower F1 hybrids using SDS-PAGE analysis.
African Journal of Biotechnology. 9 (29): 4516-4520.
Tumuhairwe JB, Rwakaikara-Silver MC, Muwanga S, Natigo S (2007).
Screening Legume Green Manure for Climatic Adaptability and
Farmer Acceptance in the Semi-Arid Agro-ecological Zone of Uganda
pp. 255-259 In: Andre B, Boas W, Job K, Joseph K (eds.), Advances
in integrated soil fertility management in sub-saharan Africa:
challenges and opportunities, Springer publications, Netherlands.
Valizadeh M (2001). Seed storage protein profile of grain legumes
grown in iran, using SDS-PAGE. J. agric. sci. Technol. (3): 287-292.
Wang ML, Mosjidis JA, Morris JB, Dean RE, Jenkins TM, Pederson GA
(2006). Genetic diversity of Crotalaria germplasm assessed through
phylogenetic analysis of EST-SSR markers. Genome 49:707-715.
Weber H, Borisjuk L, Wobus U (2005). Molecular physiology of legume
seed development. Ann. Rev. Plant Biol. 56: 253-279.
Werchan PM, Summer WR, Gerdes AM, McDonough KH (1989). Right
ventricular performance after monocrotaline-induced pulmonary
hypertension. Am. J. Physiol. Heart. Circ. Physiol. 256:1328-1336.
Xia X (2001). Data analysis in molecular biology and evolution. Kluwer
Academic Publishers, Boston.
Xia X and Xie Z (2001). DAMBE: Software package for data analysis in
molecular biology and evolution. J. Hered. 92:371-373.
Zhu H, Choi HK, Cook DR, Shoemaker RC (2005). Bridging model and
crop legumes through comparative genomics. Plant Physiol. 137:
1189-1196.
Zubaida Y, Shahid M, Zabta KS, Mirajab K and Ashiq R (2006).
Evaluation of taxonomic status of medicinal species of the genus
solanum and capsicum based on poly acrylamide gel electrophoresis
Pak. J. Bot. 38(1): 99-106.