Asian Journal of Agricultural Sciences 2(3): 89-93, 2010
ISSN: 2041-3890
© M axwell Scientific Organization, 2010
Submitted Date: March 30, 2010
Accepted Date: April 13, 2010
Published Date: July 05, 2010
Comparative Proteomic Studies in Leguminous Species
B. Yasmin, A. Habib, S. Sazia, G. Sajid and A.K. Imtiaz
Department of Genetics, Hazara University, Mansehra, Pakistan
Abstract: During present work, comparative Proteomics were employed for elaborating genetic diversity in
27 accessions of 6 genera of family Pap ilionaceae (legume s family). Sod ium D odecyl Su lphate Poly
Acrylamide Gel Electrophoresis (SDS-PAGE) was employed for analyzing seed storage protein of the species.
The Proteomic assay comprised a total of 296 reliably score able protein alleles identified in the twenty-seven
accessions of legumes. The genetic distance estimates ranged from 0-100%. Phylogenetic relationship among
the legume based upon cluster analysis assorted all the accessions in to 4 groups A, B C and D comprising 15,
5, 4 and 3 accessio ns, resp ectively.
Key words: Dendrogram , genetic distances, legumes, phylogenetic analysis, SDS-PAGE, seed storage proteins
INTRODUCTION
All the cultivated legum es belong to family
Papilionaceae (Ali, 1977). They are the important sources
of proteins both for human and animals. In Pakistan two
major types of legumes viz; rabi season legu mes e.g
Chickpea (Cicer arietinum L.) and lentil (Lens culina ris)
and kharif season legumes e.g mung bean (Vigna
radiata), French beans(Phaseolus vulgaris) and mash
(Vigna mungo) are grown on large area. In Pak istan
legumes are grown as both rabi and Kharif crops on an
area of app roxim ately 100,000 ha with a total production
of more than 600,100 metric to nes giving an ave rage y ield
of 1.61 tones/ha (Anon ymou s, 2009). Legumes are used
as green vegetables, boiled or are ground and used as
soup, dhal, and to mak e bread. Seed of legumes have 3859% carbohydrates, 3% fiber, 4.8-5.5% oil, 3% ash, 0.2%
Calcium and 0 .3% Phospho rus.
Earlier work with legumes contributed a lot to
the developm ent of major ideas in biology (Haecker
et al., 2004). They have also been used as model
organism for the better understanding of genetic system of
plants (Weber et al., 2005 ). Their study has w idely
contributed to understanding the processes of seed and
embryo developm ent, including the cellular events that
occur before and after fertilization, and differentiation of
endosp erm (Wang et al., 2003; Zhu et al., 2005). During
present study, gene tic diversity in various legume species
com mon ly grown in Pakistan was studied using Sodium
Dodecyl Sulphate P oly Acrylamide G el Electrophoresis
(SDS -PAG E). The results were used to compa re gen etic
structure of various sp ecies o f legum es.
MATERIALS AND METHODS
Twenty-nine accessions of legume s we re obtained
from Plant G enetic Resource Institute, NARC, Islamabad.
The experiments were conducted at Department of
Genetics, Hazara University, Mansehra, during 2008.
Details of the accessions are presented in Table 1. For
SDS-PAGE analysis, single seed from each accession was
grounded to a fine powder with mortar and pestle. Four
hundred ml of Protein Extraction Buffer (PEB) was added
to 0.01 g of seed flour and vortexes (using G yro mixer
vortex machine) thoroughly to homogenize. The proteins
were extracted at room temperature for 20 min. In order
to purify, the homogenate samples were centrifuged
(using Eppendorf centrifuge model No 0021586) at
12,000 rpm for 10 m in at room temperature. The extracted
crude proteins were recovered as clear supernatant and
were transferred to a new 1.5 mL eppendorf tube
and stored at 4ºC until they were run on the
polya crylam ide gel.
The electrophoretic procedure was carried out using
slab type SDS-PAGE Mode l: MGV-202, with 12.5%
polyacrylamide gel. A 12.5% resolving gel (3.0M
Tris-HCL (Sigma) pH9, 0.4% SDS (W ako) and 4.5%
stacking gel (0.4M Tris-HCL pH 7.0, 0.4% SD S) was
prepared and polymerized chemically by addition of 17
ml of N, N’, N ’, N’ tetra ethylene diamine (Wako) and
10% Ammonium persulphate (Circa reagent). Electrode
buffer solution comprised 0.025 M Tris, 1.29 M Glycine
(Sigma), 0.125% SDS. Fifteen ml of the extracted protein
was loaded with the micropip ette into the wells of the gel.
The apparatus was connected with constant electric
supp ly (75V) till the tracking dye “Bromophenol Blue”
(BPB) reaches the bottom of the gel. After electrophoresis
the gels w ere stained w ith staining solution comprising
0.2% (W/V) Comassie Brilliant Blue (CBB) R 250
dissolved in 10% (V/V) acetic acid (Circa reagents), 40%
(V/V) methanol (BD H) for abo ut an hour at room
temperature. Gels were destained in a solution containing
5% (V/V) acetic acid and 2 0% (V/V) m ethanol. G els were
shacked using Double Shaker Mixer DH - 10 gently until
Corresponding Author: Imtiaz Ahmed Khan, Department of Genetics, University of Karachi, Pakistan
89
Asian J. Agr. Sci., 2(3): 89-93, 2010
Table 1: Various accessions used to study comparative proteomics in legumes
S.No.
Genus
Sp ecie
A c ce ss io n No
1
Vigna
Ra diate
013986
2
Vigna
Ra diate
014222
3
Vigna
Ra diate
014234
4
Vigna
Ra diate
014430
5
Vigna
Ra diate
014438
6
Vigna
Mungo
013861
7
Vigna
Mungo
013824
8
Vigna
Mungo
013938
9
Vicia
Erv ilia
013220
10
Vicia
Hir sute
013288
11
Vicia
Faba
013244
12
Vicia
Erv ilia
013221
13
Phaseo lus
Vu lgar is
018288
14
Phaseo lus
Vu lgar is
018287
15
Phaseo lus
Vu lgar is
018286
16
Cicer
Arietinum
017080
17
Pisum
Sativum
018372
18
Vigna
Mungo
01
19
Vigna
Mungo
02
20
Vigna
Mungo
03
21
Lens
Ca ulina ris
01
22
Lens
Ca ulina ris
02
23
Lens
Ca ulina ris
03
24
Lens
Ca ulina ris
04
25
Lens
Ca ulina ris
05
26
Lens
Ca ulina ris
06
27
Lens
Ca ulina ris
07
28
Lens
Ca ulina ris
08
29
Lens
Ca ulina ris
09
Source
PGRI
PGRI
PGRI
PGRI
PGRI
PGRI
PGRI
PGRI
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses program I
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Pulses Program
Orig in
Punjab, Pak
Balochistan, Pak
Sindh, Pak
NW FP, Pak
Pak
Punjab, Pak
Punjab, Pak
Punjab, Pak
Syria
Punjab, Pak
Balochistan, Pak
Syria
NW FP, Pak
NW FP, Pak
NW FP, Pak
Pakistan
Au stralia
NIAB Faisalabad
ARI D.I khan
NIAB Faisalabad
NIAB Tandojam
ARI Faisalabad
ARI Faisalabad
ARI D .I khan
AR S M anseh ra
AR C Q uetta
the background of the gel became clear and protein bands
were clearly visible. The excess CBB was removed by
addition of piece of tissue paper Kim wipe in the
distaining solution. After destaining the gels were
photographed using gel documentation system ”Uvitech”.
For statistical analysis, each individual band was
considered as a single locus/allele. Alleles/loci (bands)
were scored as present (1) or absent (0). G enetic diversity
was estimated using following formula (Nei and
Li, 1979).
GD = 1 - d x y /d x + d y -d x y
W here GD = G enetic distance between two
genotypes, d xy = number of common bands in 2 genotypes,
d x = total numb er of bands in genotype 1 and dy = total
number of ban ds in genotype 2 . The b i-variant 1-0 data
matrix was also u sed to construct a dendrogram using
computer program “Popgene 3.2”
Fig. 1: Seed storage protein profile of eight legumes accessions
using SDS-PAGE
11= Vicia faba Acc # 013244, 12= Vicia ervilia Acc #
013221, 13= Phaseolus vulgaris Acc # 018288, 14=
Phaseolus vulgaris Acc # 018287, 15= Phaseolus
vulgaris Acc # 018286, 16= Cicer arietinum Acc #
017080, 17= Pisum sativum Acc # 018372, 18= Vigna
mungo Acc # 01,
RESULTS AND DISCUSSION
Only clearly score able bands were included in the
analyses.
Minor bands, which could not be scored
reliably, were not included in the analyses. In two
accessions (Vigna mungo-02 and Vigna mung- 03)
proteins alleles (bands) could not b e scored reliably and
hence data from these 2 accessions is not included in the
analysis. An example of SDS-PA GE is presented in
Fig. 1.
Genetic distances (GD) among the genotypes were
calculated using U PG M A procedure outlined by Nei and
Li, (1979) and are presented in Table 2. Range of genetic
distance observed among the accessions was 0-100%.
Minimum genetic distances (0%) was observed
among 84 co mpa risons while maximum genetic distance
90
Asian J. Agr. Sci., 2(3): 89-93, 2010
Table 2: Genetic distances among 27 legume accessions used during present study
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23 24
25
26
2
0.1
3
0.1
0.0
4
0,1
0.0
0.0
5
0.2
0.1
0.0
0.1
6
0.5
0.6
0.6
0.6
0.5
7
0.5
0.6
0.6
0.6
0.5
0.2
8
0.5
0.5
0.5
0.5
0.6
0.1
0.4
9
0.7
0.6
0.5
0.6
0.6
0.3
0.6
0.6
10 0.6
0.5
0.5
0.5
0.6
0.6
0.6
0.7
0.6
11 0.6
0.5
0.5
0.5
0.6
0.2
0.5
0.7
0.6
0.7
12 0.8
0.5
0.7
0.7
0.3
0.7
0.7
0.7
0.6
0.6
0.4
13 0.7
0.9
0.7
0.7
0.6
0.7
0.7
0.7
0.6
0.7
0.2
0.3
14 0.6
0.5
0.3
0.5
0.5
0.7
0.7
0.7
0.6
0.7
0.2
0.4
0.5
15 0.6
0.5
0.6
0.5
0.5
0.7
0.7
0.7
0.6
0.7
0.2
0.4
0.5
0.1
16 0.7
0.6
0.6
0.6
0.6
0.7
0.8
0.7
0.6
0.6
0.5
0.4
0.3
0.3
0.4
17 0.7
0.7
0.7
0.7
0.6
0.6
0.7
0.7
0.6
0.8
0.4
0.4
0.5
0.4
0.4
0.5
18 0.4
0.4
0.4
0.3
0.6
0.7
0.2
0.4
0.5
0.7
0.4
0.5
0.5
0.4
0.5
0.5
0.5
19 0.9
0.8
0.8
0.8
0.8
1.0
1.0
0.8
0.6
0.7
0.8
0.7
0.5
0.8
0.8
0.7
0.9
0.8
20 0.5
0.4
0.5
0.3
0.4
0.6
1.0
0.5
0.7
0.6
0.3
0.6
0.7
0.4
0.5
0.8
0.6
0.1
0.9
21 0.3
0.2
0.2
0.2
0.3
0.6
1.0
0.6
0.6
0.6
0.7
0.7
0.8
0.6
0.6
0.7
0.7
0.5
1.0
0.5
22 0.3
0.2
0.2
0.2
0.3
0.5
0.5
0.5
0.6
0.6
0.6
0.7
0.8
0.6
0.6
0.7
0.7
0.5
1.0
0.4
0.3
23 0.2
0.1
0.2
0.1
0.2
0.5
0.5
0.5
0.6
0.6
0.6
0.8
0.8
0.6
0.6
0.7
0.6
0.5
0.9
0.4
0.1
0.1
24 0.2
0.1
0.1
0.1
0.2
0.5
0.5
0.5
0.6
0.6
0.6
0.8
0.7
0.6
0.6
0.7
0.6
0.4
0.9
0.4
0.1
0.1
0.0
25 0.2
0.1
0.1
0.1
0.2
0.5
0.5
0.5
0.6
0.6
0.6
0.8
0.7
0.6
0.6
0.7
0.6
0.4
0.9
0.4
0.1
0.1
0.0 0.0
26 0.2
0.1
0.1
0.1
0.2
0.5
0.5
0.5
0.6
0.6
0.6
0.8
0.7
0.6
0.6
0.7
0.6
0.4
0.9
0.4
0.1
0.1
0.0 0.0
0.0
27 0.2
0.1
0.1
0.1
0.2
0.5
0.5
0.5
0.6
0.6
0.6
0.8
0.7
0.6
0.6
0.7
0.6
0.4
0.9
0.4
0.1
0.1
0.0 0.0
0.0
0.0
1= Vigna radiata Acc # 013 986 , 2 = Vigna radiata Acc # 014222, 3 = Vigna radiata Acc # 014234, 4 = Vigna radiata Acc # 014430, 5 = Vigna radiata Acc # 014 438 , 6
= Vigna mungo Acc # 013861, 7 = Vigna mungo Acc # 013824 , 8 = Vigna mu ngo Acc # 013938, 9 = Vicia ervilia Acc # 013220, 10 =Vicia hirsute Acc #013288, 11 = Vicia
faba Acc # 013244, 12 = Vicia ervilia Acc #013221, 13 = Phaseolus vulgaris Acc #018288, 14 = Phaseolus vulgaris Acc # 018287, 15 = Phaseolus vulgaris Acc # 018286,
16 = Cicer arietinum Acc # 017080, 17 = Pisum sativum Acc # 018372, 18 = Vigna mungo Acc # 01, 19 = Lens caulinaris Acc # 01, 20 = Lens caulinaris Acc # 02, 21 =
Lens caulinaris Acc # 03, 22 = Lens caulinaris Acc # 04, 23 = Lens caulinaris Acc # 05, 24 = Lens caulinaris Acc # 06, 25 = Lens caulinaris Acc # 07, 26 = Len s cau linaris
Acc # 08, 27 = Lens caulinaris Acc # 09
(GD = 100%) was observ ed for 6 com parisons. G enetic
distances of 10, 20, 30, 40, 50, 60, 70, 89 and 90% were
observed for 31, 24, 14, 29, 61, 85, 56, 21, 9 comparisons,
respectively.
Cluster analysis of twenty-seven accessions of
legumes was carried out using computer program
“Popgene ver 3.2”. The dendrogram of 27 legume
accessions based on SDS-PAGE data is presented in
Fig.2. Twenty-seven genotypes were clustered in 4
groups “A”, ”B”, “C” and “D”. Group “A” wa s largest
group comprised 15 accessions. Groups “B”, “C” and
“D”, comprised 5, 4 and 3 accessions, respectively.
Accessions 1 (Vigna rad iata Acc# 013986) and 17 (Pisum
sativum Acc# 01837 2) were found most distantly related
to each othe r.
Previously morphological or cytological assay
procedures were used to estimate existing gene tic
variability in the cro ps of comm ercial importance
including legumes (Islam and Shepherd, 1991). These
assay procedures through were successful in many cases
but were not considered suitable for large scale screening
mainly because of limited number of such markers and
time consume d for the assay proce dures. More recently
protein mark ers (esp ecially seed storage proteins) are
being used for better and more reliable estimation of
genetic distances among species/lines/populations (Weber
et al., 2005 ). The technique o f Sodium D odecyl Su lphate
Poly Acryl am ide Gel E lectrophoresis (SDS-PAG E) is a
com mon ly used technique for separating different
molecules of proteins on the ba sis of their size. This
technique is commonly used fro the estimation of genetic
distances mainly because of reliability and simplicity of
the assay procedure. Comparative Proteiomics is a
recently developed branch of Proteom ics which d eals w ith
the study of relationships between the polypeptides of
different genera/species/strains. In legumes a great deal
of comparative proteomics studies have been carried out
during last decade which has resulted in better
understanding of
genome structure of legumes
(W eber et al., 2005).
The results of present studies further strengthened
previous findings by G hafoor et al. (2003), Asghar
et al. (2003), Ferreira et al. (2000) and Dasgup ta and
Singh (2003) who reported high genetic diversity in
various legume species.
t is suggested that breeding program aimed at
increasing genetic diversity with in legumes should be
launched and those programs may involve hybridization
among most distant accessions with in a species. It is also
suggested that more studies of similar nature should be
conducted for better understanding of the genome
structure of legumes, which will ultimately help designing
better strategies fo r legum e improvemen t in Pakistan.
CONCLUSION
It is conclude d that the legum e species co mm only
grown in pakistan have high amount of gen etic dive rsity
(GD ranging from 0-100%) which can be used for the
imporvement of legume crops in the country through
hybridization.
91
Asian J. Agr. Sci., 2(3): 89-93, 2010
Fig. 2: Dendrogram constructed for 27 accessions of legumes using 1-0 bivariate data matrix generated from SDS-PAGE analysis
1 = Vigna radiata Acc # 013986, 2 = Vigna radiata Acc # 014222, 3 = Vigna radiata Acc # 014234, 4 = Vigna radiata Acc #
014430, 5 = Vigna radiata Acc # 014438, 6 = Vigna mungo Acc # 013861, 7 = Vigna mungo Acc # 013824, 8 = Vigna mungo
Acc # 013938, 9 = Vicia ervilia Acc # 013220, 10 = Vicia hirsute Acc #013288, 11 = Vicia faba Acc # 013244, 12 = Vicia
ervilia Acc #013221, 13 = Phaseolus vulgaris Acc #018288, 14 = Phaseolus vulgaris Acc # 018287, 15 = Phaseolus vulgaris
Acc # 018286, 16 = Cicer arietinum Acc # 017080, 17 = Pisum sativum Acc # 018372, 18 = Vigna mungo Acc # 01, 19 = Lens
caulinaris Acc # 01, 20 = Lens caulinaris Acc # 02, 21 = Lens caulinaris Acc # 03, 22 = Lens caulinaris Acc # 04, 23 = Lens
caulinaris Acc # 05, 24 = Lens caulinaris Acc # 06, 25 = Lens caulinaris Acc # 07, 26 = Lens caulinaris Acc # 08, 27 = Lens
caulinaris Acc # 09
Dasgupta, T. and M. Singh, 2003. Diversity in advanced
breedin g lines of chickpea. Int. Chickpea Pigeon pea
Newslett., 10: 38-41.
Ferreira, J.J., E.A. lvarrez, M.A. Roca and R. Giraldez,
2000. Determination of the out crossing rate of
Phaseolus vulgaris L. using seed protein markers.
Euphytica, 113: 259-263.
Gha foor, A., F.N. Gulbazz, M. Afzal, M. Ashraf and
M . Arshad. 2003. Inter-relationship between SDSPAGE mark ers and agro nom ic characters in chickpea
(Cicer arietinum L.). Pak. J. Bot., 35: 613-624.
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