Euphytica 129: 15–23, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

15

Genetic relationships among annual and perennial wild species of Cicer

using inter simple sequence repeat (ISSR) polymorphism

P.N. Rajesh1 , V.J. Sant1 , V.S. Gupta1 , F.J. Muehlbauer2 & P.K. Ranjekar1,∗
1 Divisionof Biochemical Sciences, National Chemical Laboratory, Pune 411 008, Maharashtra State, India;
2 USDA-ARS, Grain Legume Genetics and Physiology Unit and Department of Crop and Soil Sciences, Washington
State University, Pullman, WA 99164, U.S.A.; (∗ author for correspondence, e-mail: [email protected])

Received 15 August 2001; accepted 31 May 2002

Key words: Cicer sections, crossability, genetic diversity, species

Summary
Wild Cicer germplasm is known to have genes for disease resistance, stress tolerance and other important traits, and
hence could be exploited for improving cultivated genotypes. However, only few Cicer species are interfertile and
it is essential to overcome crossability barriers to utilize the germplasm more effectively. Genetic diversity analysis
of Cicer species can give important clues in understanding species relationships and may assist in developing and
planning breeding strategies. We selected 6 annual and 7 perennial wild species, which were amplified using
15 ISSR primers and UPGMA and AMOVA were used to evaluate the genetic diversity. On an average, 6.6
polymorphic bands per primer were observed. Cluster analysis using the UPGMA algorithm indicated three major
groups of species at the similarity value of 0.60 with many subclusters. The clustering pattern was in agreement
with the data based on crossability, seed storage protein, isozyme, allozyme and RAPD marker analysis. The
among-population component of annual and perennial groups calculated using AMOVA accounted for 39.00%.
Our results suggested that wild annuals of Cicer were not monophyletic in nature.

Abbreviations: AMOVA – Analysis of Molecular Variance ISSR – Inter Simple Sequence Repeat; PHYLIP –
Phylogeny Inference Package; UPGMA – unweighted pair group method, arithmetic mean

Introduction have been responsible for the limited unconventional

exploitation of wide hybridization for introgression of
The genus Cicer contains 42 wild species including economically important traits such as sturdiness, high
9 annuals and 33 perennials with chromosome num- seed number per pod, disease resistance and efficient
ber of 2n=2x=16 in almost all the species (Labdi et plant type, shape in chickpea leading to reduction in
al., 1996). Cicer arietinum is an economically import- its yield (Saxena & Singh, 1987). Moreover, there
ant crop in India, the Middle East, North Africa and are constraints of strong post fertilization barrier to
Ethiopia, and is the third most important pulse crop in interspecific hybridization between many of the wild
the world next to Phaseolus vulgaris and Pisum sat- species in the genus Cicer and the lack of complete
ivum. In India, over 70% of the world’s chickpea crop crossability data. Considering the agro-economic im-
is produced. The oldest report concerning this species portance of chickpea, it becomes essential to study the
was 5450 BC (Helbaek, 1959) and it has been cultiv- genetic variability within and among the Cicer species
ated for at least 7000 years (Van der Maesen, 1972). It and this data may help the chickpea breeders to exploit
is a self-pollinated crop, with natural cross-pollination the genetic resources for hybridization programs.
ranging between 0–1% (Singh, 1987). This nature of Most of the interspecific relationship studies in
Cicer and its sexual incompatibility with most of the Cicer have been carried out using plant morphology
other wild genotypes in natural interspecific crosses (Robertson et al., 1997), karyotype (Ocampo et al.,
16

1992; Tayyar et al., 1994), crossability data (Ladizin- fusarium wilt resistant gene cluster in chickpea (Rat-
sky & Adler, 1976; Pundir & Van der Maesen, 1983; naparkhe et al., 1998). In this paper, I report the use
Ahmad et al., 1987), RFLP (Patil et al., 1995) as well of ISSRs for the assessment of genetic diversity in
as seed storage protein analyses (Vairinhos & Murray, perennial and annual wild species of Cicer.
1983; Ahmad & Slinkard, 1992), allozyme mark-
ers (Kazan & Muehlbauer, 1991) and more recently
RAPD markers (Ahmad, 1999). In addition, allelic Materials and methods
variation at a microsatellite locus [(TAA)n ] (Udupa et
al., 1999) and also in Ty1-copia like retrotransposon DNA procurement and isolation
sequence (Sant et al., 2000) have been utilized to study
DNA of 6 annual and 7 perennial wild species of
diversity in Cicer. Since annual and perennial wild
chickpea (Table 1) was procured from Washington
Cicer germplasm were partially represented in these
State University, USA. They were isolated from veget-
studies, it would be useful to evaluate additional ger-
ative buds and leaf tissues of the wild species using the
mplasm systematically, which would ultimately help
miniprep method of Doyle & Doyle (1987), with little
to gather more information about various important
modifications as described by Simon & Muehlbauer
traits (Van der Maesen, 1987).
(1997). DNAs of Cicer arietinum cultivars; Vijay, JG
Molecular markers like RFLP, PCR-based RAPD,
62 and ICC4958 were isolated by the same protocol at
AFLP, ISSR and SSLP have been extensively used
National Chemical Laboratory, Pune, India. One gram
to study the genetic variability in many crop species.
of young leaves of the cultivars was ground in liquid
Representative examples are pigeonpea (Ratnaparkhe
nitrogen to a fine powder, which was quickly trans-
et al., 1995), Amaranthus (Chan & Sun, 1997), wheat
ferred to a tube containing 7.5ml of ice–cold extraction
(Barret & Kidwell, 1998; Plaschke et al., 1995),
buffer (0.35 M sorbitol, 0.1 M Tris-HCl, 5 mM EDTA
soybean (Thomson et al., 1999) and rice (Aggarwal
pH 7.5). The tube was briefly shaken and 7.5 ml nuc-
et al., 1999; Joshi et al., 2000, Davierwala et al.,
lei lysis buffer (2 M NaCl, 0.2 M Tris-HCl, 50 mM
2001). In chickpea, RFLP was of less use in detect-
EDTA, 2% CTAB, pH 7.5) was then quickly added,
ing polymorphism because of the homogeneous nature
followed by 3ml of 5% sarkosyl solution. Sample sets
of Cicer (Simon & Muehlbauer, 1997). This narrow
were incubated in 65 ◦ C water bath for 20 min and
genetic basis of Cicer is due to the domestication of
these were allowed to cool and 18 ml of chloroform:
chickpea occurred at one place in the Old World and
isoamyl alcohol (24:1) was added to each tube. The
then spread much later to other areas. Some archaeolo-
tubes were centrifuged at 5000 g for 15 min, fur-
gical evidence supports this assumption (Serret et al.,
ther, the aqueous layer was removed and given another
1997). RAPDs, however, have revealed more diversity
chloroform/isoamyl alcohol treatment. The aqueous
among the annual wild species of Cicer (Ahmad,
layer was transferred to a new tube and the DNA was
1999). In our laboratory, we have demonstrated the use
precipitated with double volume of chilled ethanol.
of another class of molecular markers, the microsatel-
The DNA pellet was dried, suspended in 500µl TE
lite markers, for diversity analysis, which were highly
buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0)
polymorphic even within the cultivars of Cicer ariet-
and quantified by mini-gel method (Sambrook et al.,
inum (Sant et al., 1999). Other laboratories also have
1989)
shown the potential of these markers in chickpea gen-
ome analysis (Sharma et al., 1995; Gotz et al., 1998;
Oligonucleotide primers, PCR amplification and
Udupa et al., 1999). More recently, the microsatellite
electrophoresis
sequences have been used as primers in polymerase
chain reaction, where repeat motifs are anchored either A set of 100 primers representing di, tri, tetra and
at 5’ or 3’ end with one or few specific nucleotides and pentamer repeats (UBC set # 9) was procured from
amplify the sequences between the two microsatellite the Biotechnology Laboratory, University of British
loci referred to as inter simple sequence repeat (ISSR) Columbia, Canada. PCR amplifications were per-
markers. In addition, ISSRs can be targeted towards formed in 100 mM Tris-HCl, pH 8.3; 500 mM KCl;
particular sequences, which are reported to be abund- 15 mM MgCl2 ; 0.01% (w/v) gelatin, 200 mM dNTP,
ant in the genome and can overcome the technical 0.5 mM spermidine, 5 pM primer and 20 ng of ge-
difficulties of RFLP and RAPD. These primers have nomic DNA, per 20µl reaction volume and 0.8 unit
been successfully used in identifying markers linked to of AmpliTaq DNA polymerase (Perkin Elmer, USA),
 17
Table 1. Analysis of molecular variance (AMOVA) for 23 individuals from two groups
(annuals and perennials) using 115 ISSR bands. The data show the degrees of freedom
(d.f.), variance component estimates, the percentage of total variance contributed by
each component (% Total) and the probability of obtaining a more extreme component
estimate by chance alone (P-value)

Source of variation d.f. Variance component % Total P-value

Nested analysis
Among group 1 3.8 12.85% <0.001
Among populations 11 9.9 39% <0.001
within groups
Within populations 10 12.28 48% <0.001

Analysis among populations

Among populations 11.75 48.90% <0.001
Within populations 12.28 51.10% <0.001

Analysis among group

Among groups 4.32 16.95% <0.001
Within groups 21.17 83.05% <0.001

using a PTC-200 Peltier thermal cycler (MJ Research, 2000 replications in this program (Nelson & Yap,
USA). After initial denaturation at 94 ◦ C for 4 min, 40 1996). Analyses of Molecular Variance (AMOVA;
cycles of PCR were carried out where each cycle com- Excoffier et al., 1992) were used to partition the
prised 1min denaturation at 94 ◦ C, 45 sec annealing at ISSR variation among annual and perennial groups,
54 ◦ C and 2 min extension at 72 ◦ C for 5 min with among species within groups and within species us-
a final extension at 72 ◦ C for 5 min, at the end of 40 ing the program WINAMOVA 1.55 provided from L.
cycles. PCR products were separated on 2% agarose Excoffier (http://anthropologie.unige.ch/ftp/comp).
gels, stained with ethidium bromide and scored for the
presence or absence of bands.
Results
Data analysis Detection of DNA polymorphism in Cicer germplasm

The genetic relationships between 13 wild species of Using ISSRs, we screened 13 Cicer species consisting
Cicer were studied by means of scorable bands using of 6 annuals and 7 perennials with two accessions of
15 different ISSR primers. Since ISSRs are dom- each species wherever possible as listed in Table 1 and
inant, a locus was considered to be polymorphic if four cultivars of C. arietinum: Vijay, JG62, ICC4958
the band was present in one lane and absent in the and V65R. Among 100 ISSR primers representing di,
other. The presence or absence of bands was scored tri, tetra and penta repeats, 25 primers showed good
as binary code (1 and 0) and a simple coefficient amplification but no polymorphism while 15 primers
was used to calculate similarity for all the possible revealed reproducible polymorphic patterns. With 15
pairs of wild species. A dendrogram was construc- primers, a total of 115 bands were scored giving an
ted based on the similarity index matrix using the average of 7.6 bands amplified per primer. Among 115
arithmetic average of unweighted pair group method bands, 100 were polymorphic leading to an average of
(UPGMA) (Sneath & Sokal, 1973) to illustrate the 6.6 polymorphic bands per primer (Table 2). As seen
genetic relationships among the wild species using in Table 2, dinucleotide (GA)8 with single nucleotide
software package Taxan. Winboot, a software pro- anchor T/C/A at the 3’ end and (AC)8 , with dinuc-
gram developed at IRRI, was run for bootstrapping. leotide anchor YT at 3’ end, were found to be the most
PHYLIP (Phylogeny Inference Package) format (Fel- polymorphic (∼ 100%) compared to all others.
senstein, 1985) was used to determine the confidence Figure 1 shows a representative amplification pat-
limits of UPGMA based dendrogram by carrying out tern of Cicer species using [(GA)8C] primer with
18
Table 2. List of Cicer arietinum and related wild species used in the present study

Group Wild species Annual/perennial Section Origin

I C. acanthophyllum Boriss. Perennial Acanthocicer Tajikistan

W6-11515 & W6-14190
C. macracanthum M. Pop Perennial Acanthocicer Pakistan
W6-11177
C. pungens Boiss. Perennial Acanthocicer Afghanistan
W6-17172 & w6 19489
C. nuristanicum Kitam. Perennial Polycicer Pakistan
W6-15003
C. arietinum L. Annual Monocicer India
V65R, Vijay, ICC4958 & JG62
C. reticulatum Ladiz. Annual Monocicer Turkey
5667
C. echinospermum P.H. Davis Annual Monocicer Turkey
020689-0302 & 527932

II C. anatolicum Alef. Perennial Polycicer Turkey

PI 383626 & W6-17512
C. microphyllum Benth. Perennial Polycicer India
W6-015003 & W6 19489
C. oxyodon Boiss&Hoh Perennial Polycicer Turkey
CS-11

III C. yamashitae Kitam. Annual Monocicer Afghanistan

510657 & 510664
C. bijugum K.H. Rech Annual Monocicer Turkey
5781 & 458550
C. judaicum Boiss. Annual Monocicer Lebanon
510660

bands in the range of 2.5 kb to 0.5 kb. A total of 9 ilarity index values ranging from 0.84 to 0.54 with an
loci were amplified with this primer and not a single average value of 0.69. Three broad clusters of chick-
band was monomorphic in all the species of Cicer, pea genotypes were obtained in the dendrogram at a
thus revealing a high level of polymorphism in Cicer similarity value of 0.60. Cluster I included accessions
genome. Only one band of 0.8 kb shown by an arrow of C. acanthophyllum, C. macracanthum, C. pun-
was present in all the accessions of C. bijugum (lanes gens, C. nuristanicum, C. arietinum, C. reticulatum
14 & 15), C. arietinum (lanes 17, 23, 24 & 25), C. and C. echinospermum. Cluster II had C. anatolicum,
echinospermum (lanes 18 & 19), Cicer reticulatum C. microphyllum and C. oxyodon accessions while
(lanes 21) and Cicer judaicum (lane 22). Cluster III included C. yamashitae, C. bijugum and
C. judaicum. These three major clusters had many
Clustering pattern of chickpea genotypes subgroups at higher levels of similarity values.
C. arietinum cultivars, Vijay, ICC4958, JG62 and
Using the software package ‘Taxan’, similarity indices V65R and C. reticulatum and C. echinospermum,
were generated based on pairwise comparison of gen- the putative progenitors of cultivated C. arietinum,
otypes. This matrix was used as a basis to cluster the formed a closer cluster at a genetic distance (GD =
genotypes in the form of a dendrogram (Figure 2), 1-similarity) of 0.23 while the accessions of wild per-
which revealed the genetic relationships between the ennials C. acanthophyllum and C. pungens formed two
Cicer species at inter- and intra specific level with sim-
 19

Figure 1. ISSR profile obtained with primer (GA)8 C on a 2% agarose gel. Lanes 1, 16 Marker DNA (φX 174 Hae III digest); 2, 3 C.
acanthophyllum (W6-11515 & W6 11513); 4, 5 C. pungens (W6 17172 & W6 14190); 6, 7 C. anatolicum (PI 383626 & W6 19489); 8, C.
nuristanicum (W6 11190); 9, 10 C. microphyllum (W6 15003 & W6 19489); 11, C. macracanthum (W6 11177); 12, 13 C. yamashitae (510657
and 510664); 14, 15 C. bijugum (5781 & 458550) 17, C. arietinum (V65R); 18, 19 C. echinospermum (020689-0302 & 527932); 20, C.
oxyodon (CS-11); 21, C. reticulatum (5667); 22, C. judaicum (510660); 23, 24, 25 C. arietinum (Vijay, JG62 & ICC4958). A band of 0.8 kb
shown by an arrow was common to all accessions of C. bijugum (lanes 14&15), C. arietinum (lanes 17, 23, 24 & 25), C. echinospermum (lanes
18 & 19), C. reticulatum (lane 21) and C. judaicum (lane 22).

clusters at a genetic distance of 0.29 and 0.31 with C. Intraspecific similarities possessed higher bootstrap
macracanthum and C. nuristanicum, respectively. In values than the interspecific ones.
cluster II, C. bijugum and C. judaicum showed max- In a nested AMOVA analysis based on the two
imum similarity (0.80), which joined together with main groups (annual/perennial), 12.85% of the total
C. yamashitae at a genetic distance 0.37. In Cluster variation was found between the groups. In an
III, accessions of C. anatolicum showed higher sim- AMOVA analysis among groups, a total variance of
ilarity with C. microphyllum with genetic distance 83.05% was found within groups while in the analysis
value of 0.34 and later clustering with C. oxyodon among species, 51.10% of total variance was found
at a genetic distance 0.39. At the intraspecific level, within the species (Table 1).
different accessions of each species used wherever
possible revealed higher similarity. For example, two
different accessions of C. echinospermum and C. pun- Discussion
gens clustered at the similarity level of 0.84 and 0.83
respectively. But the accessions of C. anatolicum
There have been a number of efforts to transfer agro-
clustered at the similarity level of 0.66 only.
economically important genes from wild species into
Dendrogram is constructed based on the degrees
cultivated species through conventional breeding prac-
of similarity between a pair of individuals at a time.
tices. For example, interspecific hybridizations were
But bootstrapping is a way to obtain a nonparametric
performed to introgress perennial germplasm to cul-
estimate of these confidence limits. It involves re-
tivated one in alfalfa (McCoy & Echt, 1993) and
peated sampling with replacement (bootstrapping) of
nematode resistance gene in peanut (Garcia et al.,
the characters in a matrix of Operational Taxonomic
1996). However, similar attempts were not success-
Units (OTUs) × characters to create numerous boot-
ful to develop hybrids between C. arietinum and any
strap matrices of the same size as the original matrix
wild species of Cicer except C. reticulatum and C.
(Nelson & Yap, 1996). Comparing the bootstrap val-
echinospermum. A few efforts using plant tissue cul-
ues of all clusters, C. bijugum and C. judaicum showed
ture techniques such as embryo rescue to develop
the maximum value of 91%, while the remaining
hybrids between C. arietinum and C. pinnatifidum
interspecific clusters showed relatively lower values.
were reported (Anonymous, 1995). High hybrid vigor
was obtained in F1 and F2 generations, when C.
20

Figure 2. Dendrogram of the annual and perennial Cicer species based on the similarity matrix developed using ISSR markers.

arietinum was crossed with C. reticulatum and C. of chickpea have been used for diversity analysis at
echinospermum (Singh & Ocampo, 1997) ascertaining DNA level.
the potential of increasing the chickpea yield using In order to study inter and intraspecific relation-
interspecific hybridization. However, knowledge of ships in genus Cicer, we used 15 ISSR primers rep-
genetic relationships among various wild species is resenting twelve dinucleotide repeats anchored at the
necessary for successful and efficient exploitation of 3’ end, two unanchored trinucleotide repeats [(ATG)6
genetic diversity present in the wild species and such and (GAA)6 ] and one unanchored penta nucleotide re-
information is very poorly available in the genus Cicer, peat [(GGAGA)3 ]. We did not use the ISSR primers
especially using molecular markers. In all the previ- anchored at the 5’ end as they lack selective nucle-
ous studies, the genetic relationships were carried out otides at the critical 3’ end and impose selection for
either only among annuals (Ladizinsky & Adler, 1976; long simple sequence repeats that bind along the entire
Labdi et al., 1996; Ahmad, 1999) or among annuals length of the primers. Since such a selection pressure
with one (Kazan & Muehlbauer, 1991) or two peren- is not exerted by the 3’ anchored primers, they may
nials (Tayyar & Waines, 1996). Our report is the first detect microsatellites at a higher frequency than 5’
one where many perennial (7) and annual (6) species anchored primers (Blair et al., 1999).
 21
Table 3. List of ISSR primers used in genetic variability
analysis
& Adler, 1976; Ahmad et al., 1987; Ahmad, 1988).
These groups also resembled the clustering of an-
No. Sequence Number of Total number nual Cicer species based on their seed protein profiles
monomorphic of bands (Vairinhos & Murray, 1983). Interestingly, the cluster-
bands scored ing of annual species based on ISSR markers in our
studies is in agreement with the clustering based on
1 (GA)8 T 1 15
2 (GA)8 C – 9
crossability as well as seed storage protein data except
3 (GA)8 A – 9
that C. yamashitae was clustered with C. bijugum and
4 (GA)8 YT 1 4 C.judaicum (Figure 2).
5 (AG)8 YT 4 11
6 (CT)8 RC 2 8 Study on evolution of annuals
7 (CA)8 T – 5
8 (CA)8 RT 2 8 Various molecular markers including chloroplast
9 (AC)8 YT – 9 DNA, RFLP, RAPD, ISSR and STMS have been util-
10 (GT)8 A – 5 ized earlier to analyze the origin and evolution of
11 (GT)8 YT – 7 different plant species including the genus Oryza (Ishii
12 (TG)8 A – 5 et al., 1988; Wang et al., 1992; Ishii et al., 1996;
13 (ATG)6 2 8 Joshi et al., 2000; Davierwala et al., 2001). Allelic
14 (GAA)6 2 6
variation at a microsatellite locus like (GATA)n and
15 (GGAGA)3 1 6
(CAC)5 in rice (Gupta et al., 1994) and (TAA)n in
Total 15 115
chickpea (Udupa et al., 1999) has given direction
to understand the genetic relationship among various
species. Yang et al. (1994) have predicted that 28%
of allelic diversity has been lost during the process of
Since ISSR markers are dominant, the similarity cultivar development from landraces in rice. Retro-
at the sequence level of monomorphic bands can be transposons, specifically Ty1-copia rt domain, have
questioned. But numerous studies have now verified also been used to estimate the evolutionary relation-
that most co-migrating fragments are identical by des- ships between wheat, rice and maize (Matsuoka et al.,
cent, at least at the intraspecific level (Wu et al., 1999; 1999). In a recent study from our laboratory, Ty-1
Sales et al., 2001). Dominant markers have been util- copia like retrotransposon sequences have been used
ized to study the interspecific relationship based on to study the phylogenetic relationship among the Cicer
this assumption in various crops (Campos et al., 1994; species (Sant et al., 2000).
Brummer et al., 1995; Lanner et al., 1996; Laner- There are different theories regarding the transition
Herrera et al., 1996; Rieseberg, 1996; Fahima et al., of wild chickpeas from perenniality to annual state.
1999). Based on analysis of allozyme variation of 9 annuals
Our studies have shown relatively a high level of and one perennial, it is reported that annual species
polymorphism between wild species of chickpea and are monophyletic (Kazan & Muehlbauer, 1991). On
less polymorphism between the accessions of each the contrary, on the basis of isozyme studies using
wild species used in the study including the cultivated 9 annuals and 2 perennials, it is reported that the
ones except C. anatolicum. The observed heterogen- annual species are not monophyletic in origin (Tay-
eity in those accessions could be related to the mode yar & Waines, 1996). These contrasting conclusions
of collection of them. might be due to small size of the sample surveyed
and variation in the source of seeds, in the enzyme
Grouping of chickpea based on ISSR data system studied and in the procedure of sample ana-
lysis. Our results based on 7 perennials and 6 annuals
Earlier studies based on crossability data established probably support the view of Tayyar & Waines (1996)
4 groups for 9 annuals of Cicer. Group I consisted of in which annual species are not monophyletic. The
C. arietinum, C. reticulatum and C. echinospermum; seven perennials included in my study do not form a
group II included C. bijugum, C. judaicum and C. single cluster as is clear from Figure 2. Here two per-
pinnatifidum; group III comprised only C. cuneatum ennials of C. acanthophyllum and C. macracanthum
while group IV contained C. yamashitae (Ladizinsky form subgroup I; two perennials of C. pungens and
22

C. nuristanicum form subgroup II while three annuals Ahmad, F., 1999. Random amplified polymorphic DNA (RAPD)
namely C. arietinum, C. reticulatum and C. echino- analysis reveals genetic relationships among the annual Cicer
species. Theor Appl Genet 98: 657–663.
spermum form subgroup III in the Group I. Secondly, Ahmad, F. & A.E. Slinkard, 1992. Genetic relationships in the genus
C. anatolicum, C. microphyllum and C.oxyodon form Cicer L. as revealed by polyacrylamide gel electrophoresis of
group II all representing perennials while three more seed storage proteins. Theor Appl Genet 84: 688–692.
annuals namely C. yamashitae, C. bijugum and C. Ahmad, F., A.E. Slinkard & G.J. Scoler, 1987. The cytogenetic
relationship between Cicer judaicum Boiss. and Cicer chroas-
judaicum form group III. However, the annuals of sannicum (Bge) M. Pop. Genome 29: 883–886.
subgroup III cluster with perennials C. pungens and Anonymous, 1995. Kabuli Chickpea Improvement. Annu Rep
C. nuristanicum of subgroup II, while the three annu- Germplasm Program: Legumes. ICARDA, Aleppo, Syria pp.
als of group III cluster with perennials of subgroup I 59–132.
Barret, B.A. & K.K. Kidwell, 1998. AFLP based genetic diversity
and annuals of subgroup III and subgroup II of per- assessment among wheat cultivars from the pacific Northwest.
ennials. Thus, the dendrogram in Figure 2, suggests Crop Sci 38: 1261–1271.
that probably all the annual species might not have Blair, M.W., O. Panaud & S.R. McCouch, 1999. Inter simple
evolved simultaneously from the perennials. For the sequence repeat (ISSR) amplification for analysis of microsatel-
lite motif frequency and fingerprinting in rice (Oryza sativa L.)
precise phylogeny and evolutionary analysis of Cicer Theor Appl Genet 98: 780–792.
genus, studies with more perennial and annual spe- Brummer, E.C., J.H. Bouton & G. Kochert, 1995. Analysis of
cies and with a large number of accessions of each annual Medicago species using RAPD markers. Genome 38:
362–367.
species are required. However, the scarcity of seeds
Campos, L.P., J.V. Raelson & W.F. Grant, 1994. Genome re-
of perennial species and their poor growing ability at lationships among Lotus species based on random amplified
various locations are the major limitations to carry out polymorphic DNA (RAPD). Theor Appl Genet 88: 417–422.
this exercise. Chan, K.F. & M. Sun, 1997. Genetic diversity and relationships
detected by isozyme and RAPD analysis of crop and wild species
In summary, the evolutionary data generated us- of Amaranthus. Theor Appl Genet 95: 865–873.
ing ISSRs with 6 annuals and 7 perennials of Cicer Davierwala, A.P., K.V. Chowdari, A. Shivkumar, A.P.K. Reddy, P.K.
has supported the earlier report that the annuals are Ranjekar & V.S. Gupta, 2001. Genetic diversity evaluation of
not monophyletic. Since the genetic relationship ana- Indian elite rice varieties using molecular markers. Genetica (in
press).
lysis based on ISSRs supports the morphological and Doyle, J.J. & J.L. Doyle, 1987. A rapid DNA isolation procedure
crossability data, ISSRs prove to be an efficient marker for small amount of fresh leaf tissue. Phytochem Bull 19: 11–15.
system for such types of studies. Fahima, T., G.L. Sun, A. Beharav, T. Krugman, A. Beiles & E.
Nevo, 1999. RAPD polymorphism of wild emmer wheat pop-
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Acknowledgements proach using the bootstrap. Evolution 39: 783–791.
Garcia, G.M., H.T. Stalker, E. Shroeder & G. Kochert, 1996. Iden-
PNR and VJS acknowledge the Council of Scientific tification of RAPD, SCAR and RFLP markers tightly linked to
nematode resistance genes introgressed from Arachis cardenasii
and industrial Research for the grant of Senior Re- into Arachis hypogaea. Genome 39: 836–845.
search Fellowship. We thank Drs M.B. Ratnaparkhe Gotz, G., M. Nenno, K. Weising, D. Zink, W. Nagl & G. Kahl,
and D.K. Santra for offering valuable suggestions. 1998. Chromosomal localization and distribution of Simple se-
We also thank Rahul B and Gauri A for their help quence repeats and the Arabidopsis-type telomere sequence in
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