Abstract: Comparative Genomics is relatively a new discipline of Genetics, which deals with the relationships between the genomes of genera, species, varieties or strains. For elaborating comparative genomics of legumes; fourteen accessions belonging to seven species viz; Vigna radiate, Vigna mungo, Vicia ervilia, Vicia faba, Phaseolus vulgaris, Cicer arietinum and Lens culinaris, were analyzed using Randomly Amplified Polymorphic DNA (RAPD). Alleles of various sizes ranging from 500-1400 bp were amplified. On an average 3.5 allele per genotype were amplified. The average genetic distance ranged from 4-91%. Phylogenetic relationship among the legumes based upon DNA analysis was studied through dendrogram analysis. All the accessions were clustered in 6 groups. It was found that Vigna radiata and Vigna mungo accessions were most distantly related to each other

A Habib

A K Imtiaz

B Yasmin

Imtiaz Ahmad Khan

S Ghafoor

S Shazia

CiteSeerX

Current Research Journal of Biological Sciences 2(3): 178-182, 2010
ISSN: 2041-0778
© Maxwell Scientific Organization, 2010
Submitted Date: March 30, 2010 Accepted Date: April 13, 2010 Published Date: May 20, 2010
Corresponding Author: Imtiaz Ahmad Khan, Department of Genetics, University of Karachi, Pakistan
178
Comparative Genomic Studies in Leguminous Species
S. Shazia, A. Habib, B. Yasmin, S. Ghafoor and A.K. Imtiaz
Department of Genetics, Hazara University, Mansehra, Pakistan
Abstract: Comparative Genomics is relatively a new discipline of Genetics, which deals with the relationships
between the genomes of genera , species, varieties or strains.  For elaborating comparative genomics of legumes;
fourteen accessions belonging  to seven species v iz; Vigna radiate, Vigna mungo, Vicia ervilia , Vicia faba,
Phaseolus vulgaris, Cicer arietinum and Lens culinaris, were analyzed using Randomly Amplified
Polymorphic DNA (RAPD). Alleles of various sizes ranging from 500-1400 bp were amplified.  On an average
3.5 allele per genotype were amplified. The average genetic distance ranged from 4-91% . Phylogenetic
relationship among the legumes based upon DNA analysis was studied through dendrogram analysis. All the
accessions were clustered in 6 groups.  It was found that Vigna radiata and Vigna mungo accessions were most
distantly related to each other.  
Key words: Dendrogram, gene tic distances, legumes, phylogenetic relationship, randomly amplified
polymorphic DNA
INTRODUCTION
Papilionaceae (legumes family) is a very important
family of commercial crops all over the world. In Pakistan
various kinds of legumes viz; chickpea (Cicer arietinum
L.), Lentil (Lens culinaris), mung beam (Vigna mungo),
mash (Vigna radiata), beans (Phaseolus vulgaris) and
peas (Pisum sativum) are grown.  Although area under
legumes cultivation has increased in the past few years,
for example, area under cultivation of green peas in
Pakistan has increased from 2880 thousand ha during
200-2003 to 31031 thousand ha during 2005-2006
(Anonymous, 2007), over all yield of legume crops did
not increased substantially over the same period.  The
national average yield of major legume crops in Pakistan
is approximately 0.5 tonnes per ha as compared to the
average yield of legumes in the developed countries, for
example Australia, which is a lmost 8 times (5496.7
Kg.haG1) higher than national average of Pakistan  (Ali and
Randles, 1997).
A prerequisite for increasing yield of legumes is the
Comparative Genomics studies which help in better
understanding of genome structure (Alonso et al., 2003;
Adjaye et al., 2004; Zhu et al., 2005).  Previously various
kinds of molecular markers including Restriction
Fragment Length Polymorphism (RFLP), Polymerase
Chain Reaction (PCR), Amplified Fragment Length
Polymorphism (AFLP) and Single Nucleotide
Polymorphism (SNiPs) have been used for estimation of
genetic diversity in various crop species (Rafalski et al.,
1996; Demeke and Adams, 1994). Among these assays
procedures, Polymerase Chain Reaction are relatively
easier, cheaper and faster than other assay systems and
have been used extensively for the estimation of genetic
diversity. Among PCR based assays, Simple Sequence
Repeat Primers (SSR) Allele Specific Amplifications
(ASA ), Sequence Tag Site amplification (STS) etc have
been developed and used in breeding programs (Rafalski
et al., 1996).   Relatively recent introduction of Randomly
Amplified Polymorphic DNA (RAPD) is more user’s
friendly as it does not require any sequence information
(Williams et al., 1990).   Present study was undertaken to
study comparative genomics of legumes species using
RAPD based assays.  
MATERIALS AND METHODS
The study was conducted at the Department of
Genetics, Hazara University during 2008-2009. During
present study, DNA of 14 accessions belonging to seven
species of legumes were analyzed using Randomly
Amplified Polymorphic DNA (RAPD ).  Seeds were
obtained from Plant Genetic Resource Institute, NARC,
and Food Legumes (Pulses) Program, NARC, Islamabad
(Table 1). 
Total Genomic  DNA was isolated using modified
small scale D NA isolation procedure (Czaplicki et al.,
2000; Weining and Langridge, 1991; Doyle and Doyle,
1987). Quality and quantity of the DNA was checked on
1% agarose/TBE gel.  Gels were run at constant voltage
of 70 volts for approximately one hour and observed
under UV light using “Uvitech” gel documentation
system.  Polymerase Chain Reaction (PCR) was carried
using protocol described by M ukhtar et al., (2003) and
Devos and Gale (1992). Components of PCR reaction
were the genomic DNA used as template, dNTPs (dATP,
Curr. Res. J. Biol. Sci., 2(3): 178-182, 2010
179
Table 1: Species used to study comparative Genomics in Legumes
S.No. Genus Species Accession Nos Source Orig in
1 Vigna Radiata 013986 PGRI Punjab Pak
2 Vigna Radiata 014222 PGRI Balochistan Pak
3 Vigna Radiata 014234 PGRI Sindh Pak
4 Vigna Mungo 013861 PGRI Punjab Pak
5 Vigna mungo 013824 PGRI Punjab Pak
6 Vigna mungo 013938 PGRI Punjab Pak
7 Vicia ervilia 013220 PP Syria 
8 Vicia Faba 013244 PP Balochistan Pak 
9 Phaseo lus vulg aris 018288 PP NW FP Pak
10 Phaseo lus vulg aris 018287 PP NW FP Pak
11 Cicer arietinum 017080 PP Pakistan
12 Lens culin aris 01 PP NIAB Faisalabad (Desi) 2006
13 Lens culin aris 02 PP ARI  D.I khan 2004
14 Lens culin aris 03 PP NIAB Faisalabad 2002
PGR I = Plant Genetic Resource Institute Islamabad, PP = Pulses program, NAR C. Islamabad
Table 2: List of RAPD primers used for estimation of genetic diversity in 14 accessions of legumes used during present study
S. No Oligo name Sequ ence (5 ’-3’) M o l. W t %GC
1 GLA-14 TCTGTGCTGG 3050 60
2 GLA-16 AGCCAGCGAA 3040 60
3 GLB-16 TTTGCCCGGA 3108 60
4 GLD-03 CTCGCCGTCA 3003 70
dCTP, dGTP and dTTP), Random primer, Taq
Polymerase buffer, MgCl2 and Taq DNA Polymerase. 
Four Randomly Amplified Polymorphic DNA (RAPD)
primers viz; GLA-14, GLA-16, GLB-16 and GLD-03
(obtained from Gene-Link, Inc, USA) were used to study
genetic differences at DNA level in the material.
Sequence and molecular weight of the primers are
presented in Table 2. 
For statistical analysis, every band was considered as
single locus/allele.  Alleles/loci were scored as present (1)
or absent (0).  Bivariate 1-0 data matrix was generated
and genetic distances (GD) among the genotypes were
estimated using Unweighted Pair Group of Arithmetic
Means (UPGMA) as described by N ei and Li  (1979).
The formula used to calculate GD is given below
GD=1-dxy/dx+dy-dxy
Where GD = Genetic distance, dxy = Total number of
common bands in two genotypes, dx = Total number of
bands in genotypes # 1 and dy = Total number of bands in
genotype #2.  Computer program “Pop gene 32” was used
to construct Dendrogram using bivariate 1-0 data matrix.
RESULTS AND DISCUSSION
Total genomic DN A from 14 accessions of the six
legumes species v iz; Vigna radiata (Acc Nos
013986,014222,014234i), Vigna mungo (Acc Nos
013861,013824,013938i), Vicia ervilia  (Acc No 013220i),
Vicia faba (Acc No 013244i), Phaseolus vulgaris  (Acc
No 018288), Cicer arietinum (Acc No 017080), Lens
culinaris (Acc Nos 01,02,03) was isolated using small
scale DNA isolation procedure. An example of PCR
amplification of Legumes accessions using RAPD primer
GLB-16 are presented in Fig. 1.   
Fig. 1:  PCR amplification profile of 7 legume accessions using
RAPD primer GLB-16.  M= Molecular weight marker (in
bp) Is presented on left.
1 = Vicia faba Acc No 013244, 2 = Phaseolus vulgaris
Acc No 018288, 3 = Phaseolus vulgaris Acc No 018287,
4 = Cicer arietinum Acc No 017080, 5 = Lens culinaris
Acc No 01, 6 = Lens culinaris Acc No 02, 7 = Lens
culinaris Acc No 03
Average genetic distance estimates among the 14 legume
accessions ranged from 4-91% (Table 3).  Maximum
average genetic distance (GD = 91%) was estimated for
1 comparison viz; Vigna radiata Acc No 013986 - Cicer
arietinum Acc No 017080. One comparison (Vigna
mungo  Acc No 013861 - Vigna mungo Acc No 013938)
showed almost complete homozygosity (average GD =
4%).  Rest of the comparisons showed varying range of
Curr. Res. J. Biol. Sci., 2(3): 178-182, 2010
180
Tab le 3: A verage e stima tes of  Ge netic  distances among 14 legume accessions using four RAPD primers (GLA-14, GLA-16, GLB-16 and  GLD -03)
1 2 3 4 5 6 7 8 9 10 11 12 13
1
2 0.25
3 0.14 0.12
4 0.81 0.39 0.49
5 0.74 0.55 0.49 0.11
6 0.71 0.35 0.49 0.04 0.14
7 0.72 0.46 0.39 0.25 0.20 0.24
8 0.58 0.59 0.48 0.55 0.58 0.54 0.56
9 0.44 0.53 0.44 0.60 0.58 0.56 0.67 0.37
10 0.44 0.52 0.44 0.57 0.64 0.52 0.61 0.43 0.09
11 0.91 0.59 0.61 0.68 0.61 0.64 0.56 0.39 0.37 0.41
12 0.88 0.61 0.56 0.52 0.68 0.69 0.53 0.61 0.40 0.42 0.35
13 0.51 0.61 0.58 0.68 0.66 0.67 0.50 0.47 0.47 0.49 0.35 0.14
14 0.65 0.63 0.61 0.56 0.56 0.56 0.36 0.58 0.48 0.51 0.58 0.25 0.23
1 = Vigna r adia ta  Acc No 013986, 2 = Vigna r adia ta  Ac c N o 014222, 3  = Vigna r adia ta  Acc No 014234, 4 = Vigna mungo  Acc No 013861, 5 = Vigna
mungo Acc No  013824, 6 = Vigna mungo  Acc No 013938, 7 = Vicia  ervilia  Acc No 013220, 8 = Vicia faba Acc No 013244, 9 = Phaseolus  vulg aris
Acc No 018288, 10 = Phaseolus  vulg aris  Acc No 018287,  11 = Cicer arietinum Acc No 017080, 12 = Lens culinaris  Acc No 01, 13 = Len s cu linar is
Acc No 02, 14 = Lens culinaris  Acc No 03.
average genetic distances but in most of the comparisons
(63 comparisons) average genetic distance distances were
moderate (average GD ranging  from 40-70%). 
The data obtained during present study using four
RAPD primers were used to analyze phylogenetic
relationship among the legume accessions.  Computer
program “Popgene ver. 32” was used to construct the
dendrogram (Fig. 2). The fourteen  acccessions were
clustered in 6 groups “A”, “B” “C’ “D” “E” and “F”
comprising 3, 2, 2, 1, 3 and 3 accessions, respectively.
Groups A, B, C, D, E and F comprised Vigna radiata,
Vicia (vicia ervilia and Vicia faba), Phaseolus vulgaris,
Cicer arietinum, Lens Culinaris  and Vigna mungo,
respectively. It was found that Vigna radiata and Vigna
mungo  were most distantly related to each other on the
basis of data obtained using Randomly Amplified
Polymorphic DNA primers. It was also observed that
differences within Vigna mungo accessions were less than
that observed for accessions within Vigna radiata (Fig. 2).
Similarly differences within the two accessions of
Phaseolus vulgaris were minimum among all the
comparisons studied during present research work.
During present study PCR, alleles of various size
(ranging from 100-1400 bp) were amplified.  On an
average 3.5 allele  per genotype were amplified.  Similar
results  were reported by M ukhtar et al., (2003), Hamza
et al., (2004) and Fu et al., (2006) using PCR based
assays. Although a wide level of genetic diversity was
observed during present study (average genetic distance
estimates using the four RPAD primers ranged from 4-
91%), but most of the comparisons (63 comparisons)
showed moderate average genetic distance estimates
(average GD ranging from 40-70%).  These results further
strengthened previous findings by Ghafoor et al. (2005),
Asghar et al. (2003), Choi et al. (2004), Ferreira et al.
(2000) and Dasgupta and Singh (2003) who reported low
to medium level of genetic diversity in various legume
species.
Fig. 2: Dendrogram constructed for 14 legume accessions
based on data obtained using four Randomly Amplified
Polymorphic DNA primers. 
1 = Vigna radiata Acc No 013986, 2 = Vigna radiata
Acc No 014222, 3 = Vigna radiata Acc No 014234, 4
= Vigna mungo Acc No 013861, 5 = Vigna mungo Acc
No  013824, 6 = Vigna mungo Acc No 013938, 7 =
Vicia ervilia Acc No 013220, 8 = Vicia faba Acc No
013244, 9 = Phaseolus vulgaris Acc No 018288, 10 =
Phaseolus vulgaris Acc No 018287, 11 = Cicer
arietinum Acc No 017080, 12 = Lens culinaris Acc No
01, 13 = Lens culinaris Acc No 02, 14 = Lens culinaris
No 03.
Curr. Res. J. Biol. Sci., 2(3): 178-182, 2010
181
Phylogenetic relationship observed during present studies
revealed that Vigna radiata and V. mungo though
belonging to the same genera, were more distantly related
in terms of phylogenetics as compared to other genera.
Differences within Vigna mungo accessions were less
than that observed for accessions within Vigna radiata .
Similarly differences within the two accessions of
Phaseolus vulgaris were least among all the comparisons.
As very low level of genetic variability was observed
within  Vigna sp. (which are important legumes of
Pakistan), it is suggested that breeding program aimed at
increasing genetic diversity with in legumes should be
launched and those programs may involve wide
hybridization (for example crossing between Vigna
radiata and Vigna mungo accessions). 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 for legume improvement in Pakistan . 
CONCLUSION
It is concluded that the legume species commonly
grown in pakistan have sufficient amount of genetic
diversity which can be used for the imporvement of
legume crops in the country through hybridization. It has
also ben conlcuded that Vigna radiata and V. mungo
though belonging to the same genera were parted more
distantly in terms of phylogenetics as compared to other
genera.
REFERENCES
Adjaye, J., R. Herwig, D. Herrmann, W . Wruck, A.
Benkahla, T. Brink, M. Nowak and H. Niemann.
2004. Cross-species hybridisation of human and
bovine orthologous genes on high density cDNA
microarrays. BMC Genomics, 5: 83.
Ali, A. and J.W. Randles, 1997. Early season survey of
pea viruses in Pakistan and the detection of two new
pathotypes of pea seed born mosaic potyvirus. Plant
Diseases, 81: 343-347.
Alonso, J.M., A.N. Stepanova, T.J. Leisse,  C .J. Kim,
H.M.      Chen,      P.     Shinn,     D.K.     Stevenson,
J. Zimmerman, P. Barajas and R. Cheuk, 2003.
Genome-wide insertional mutagenesis of Arabidopsis
thaliana. Science, 301: 653-657.
Anonymous, 2007. Agriculture Statistics of Pakistan.
Ministry of Food Agriculture and Live Stock,
Islamabad. Pakistan.
Asghar, R., T. Siddique and M. Afzal, 2003. Inter and
i n t r a - s p e c i f i c  v a r ia t io n  i n  S D S - P A G E
electrophoregrams of total seed protein in chickpea
(Cicer arietinum L.) germplasm. Pak. J . Biol. Sci.,
6(24): 1991-1995.
Choi,   H.K.,  J.H.  Mun,  D.J.  Kim,  H. Zhu, J.M. Baek,
J. Mudge, B. Roe, N. Ellis, J. Doyle and G.B. Kiss,
2004. Estimating genome conservation between crop
and model legume species. Proc. Natl. Acad. Sci.
USA., 101: 15289-15294.
Czaplicki, A., P. Borsuk and I. Moraczewsk, 2000.
Molecular methods of identification of wheat
varieties. J. Biomol. Struc. Dyname. 17(6): 2.
Dasgupta, T. and M. Singh, 2003. Diversity in advanced
breeding lines of chickpea. Int. Chickpea Pigeonpea
Newslett., 10: 38-41.
Demeke, T. and R.P. Adams, 1994. The Use of PCR-
RAPD Analysis in Plant Taxonomy and Evolution.
In: Griffin, H.G. and A.M. Griffin, (Eds.), PCR
Technology: Current Innovations. CRC Press, Boca
Raton, Florida, pp: 179-191.
Devos, K.M. and M.D. Gale, 1992. The use of Randomly
Amplified DNA markers in wheat. Theor. Appl.
Genet., 101: 107-118.
Doyle, J.J. and J.L. Doyle, 1987. A rapid DNA isolation
procedure for Small quantities of fresh leaf tissue.
Photochem. Bull., 19: 11-15.
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. 
Fu, J., M.F. Zhang and X .H. Qi. 2006. Genetic diversity
of traditional Chinese mustard crops Brassica juncea
as revealed by phenotypic differences and RAPD
markers. Genet. Res., 53: 1513-1519.
Ghafoor,  A. and  Z. Ahmad, 2005. Diversity of
agronomic traits and total seed protein in blackgram
(Vigna mungo L. Hepper). Acta Biol. Cracov. Bot.,
47(2): 1-7.
Hamza, S., W.B. Hamida, A. Rebai and M . Harrabi,
2004. SSR based genetic diversity assessment among
Tunisian winter barley and relationship with
morphological traits. Euphytica, 135: 107-118.
Mukhtar, M.S., M. Rahmanand and Y. Zafar, 2003.
Assessment   of   genetic   diversity  among  wheat
(T. aestivum) cultivars from arrange of localities
across Pakistan  using  Rando m A mplifie d
Polymorphic DNA (RAPD) analysis. Euphytica, 128:
417-425.
Nei, N. and W. Li, 1979. Mathematical model for
studying genetic variation in terms of restriction
endonucleases.   Proc.   Natl.   Acad.   Sci.,  76:
5269-5273.
Rafalski, J.A., J.M . Vogel, M. Morgante , W . Powell and
C.S. Tingey,  1996. Generating and Using DNA
Markers in Plants. In: 1. Birren, B. and E. Lai, (Eds.),
In Non Mammalian Genomic Analysis: A Practical
Guide. Academic Press Inc., New York, pp: 73-134.
Curr. Res. J. Biol. Sci., 2(3): 178-182, 2010
182
Weining, S. and P. Langridge, 1991. Identification and
mapping of polymorphism in cereals base on
polymerase chain reaction. Theor. Appl. Genet., 82:
209-216.
Williams, J.G.K., A.R. Kubelik, K.I. Livak, J.A. Rafalski
and S.V. Tingey, 1990. DNA polymorphisms
amplified by arbitrary primers are useful as genetic
markers. Nucleic Acids Res., 18: 6531-6535.
Zhu, H., H.K. Choi, D.R. Cook and R.C. Shoemaker,
2005. Bridging model and crop legumes through
comparative   genomics.   Plant   Physiol.,  137:
1189-1196.


Comparative Genomic Studies in Leguminous Species

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=98991034C1748363A0EB1DB1466BA544?doi=10.1.1.1051.5541&rep=rep1&type=pdf

Comparative Genomic Studies in Leguminous Species

Abstract

Similar works

Full text

Available Versions

CiteSeerX