Plant Breeding 123, 173—179 (2004)

2004 Blackwell Verlag, Berlin

ISSN 0179-9541

Genetic diversity estimates in Cicer using AFLP analysis

T . T . N g u y e n 1 , P . W . J . T a y l o r 1 , R . J . R e d d e n 2 and R . F o r d 1 , 3
1
BioMarka, Joint Centre for Crop Innovation, School of Agriculture and Food Systems, University of Melbourne, Melbourne,
Victoria 3010, Australia; 2 Australian Temperate Field Crops Collection, Department of Primary Industries, Private Bag 260,
Horsham, Victoria 3401, Australia; 3 Corresponding author, E-mail: rebeccaf@unimelb.edu.au
With 2 figures and 3 tables
Received May 22, 2003/Accepted October 22, 2003
Communicated by A. Graner

Abstract Accessions of wild Cicer species are known to possess genes

Amplified fragment length polymorphism (AFLP) analysis was used to for resistance to many biotic and abiotic stresses (Muehlbauer
evaluate the genetic variation among cultivated chickpea and wild et al. 1994). According to Singh et al. (1994), many Cicer
Cicer relatives. In total, 214 marker loci were assessed, of which 211 species contain sources of resistance to pathogens such as
were polymorphic (98.6%) across the 95 accessions that represented ascochyta blight, fusarium wilt, leaf miner, seed beetle and
17 species of Cicer. The genetic variation within a species was cereal cyst nematode. In particular, accessions of the annual
highest in C. pinnatifidum followed by C. reticulatum and lowest in wild Cicer species C. pinnatifidum, C. judaicum, C. bijugum and
C. macracanthum. Three main species groups were identified by C. echinospermum were shown to contain useful levels of
UPGMA clustering using Nei’s pair-wise distance calculations. Group
resistance to Ascochyta rabiei (Singh et al. 1981, Haware et al.
I included the cultivated species C. arietinum, C. reticulatum and
1992, Stagmina et al. 1998, Collard et al. 2003b). Success in
C. echinospermum. Within this group, C. reticulatum accessions were
clustered closest to the C. arietinum cultivars ÔLasseterÕ, ÔKanivaÕ and crossing cultivars with accessions of the two most closely
ÔBumperÕ, supporting the hypothesis that C. reticulatum is the most related wild species, C. reticulatum and C. echinospermum,
probable progenitor of the cultivated species. Cicer bijugum, indicated the potential to move valuable traits into elite
C. judaicum and C. pinnatifidum were clustered together creating cultivars (Singh and Ocampo 1997).
group II. Group III contained all nine perennial species assessed and Investigations into the genetic relatedness among Cicer
two annual species C. yamashitae and C. cuneatum. The genetic species were previously conducted by assessing similarities in
distance detected between group I and group III (0.13) was equivalent plant morphology (Ladizinsky and Adler 1976, Robertson
to the genetic distance detected between group I and group II (the et al. 1997), karyotype (Ocampo et al. 1992), crossability
primary and annual tertiary species, respectively; 0.14). This indicated
(Ladizinsky and Adler 1976, Ahmad et al. 1988, Singh and
that the perennial tertiary species may be as valuable for increasing
Ocampo 1993), seed storage proteins (Ahmad and Slinkard
variation to incorporate novel germplasm in the cultigen as the annual
tertiary species. 1992) and isozymes (Kazan and Muehlbauer 1991, Ahmad
et al. 1992, Labdi et al. 1996, Tayyar and Waines 1996).
Key words: Cicer arietinum — AFLP — genetic variation These studies revealed that C. reticulatum was likely to be
the progenitor of cultivated chickpea. However, analyses at
Chickpea (Cicer arietinum) is the only cultivated species these levels were limited due to the small numbers of loci
belonging to the Cicer genus, which is classified in the family analysed and the potential effect of environment and growth
Leguminoase, tribe Cicereae Alef (van der Maesen 1987). stage of the plant on expression of the genetic markers
Chickpea is grown as a crop in many geographical regions assessed.
including South Asia, West Asia, North and East Africa, Conversely, DNA-based molecular markers are tools that
Southern Europe, North America and Australia. However, enable plant breeders to directly evaluate genetic variation
the average annual yield world-wide (0.78 ton/ha) is con- between related individuals without any concern for environ-
sidered to be somewhat lower than its potential yield mental factors and effects on gene expression levels. In
(Singh et al. 1994, Sudupak et al. 2002). Therefore, many addition, DNA techniques allow for the assessment of a
chickpea breeding programmes are focused on improving the theoretically unlimited number of polymorphic marker loci.
genetic potential both to increase yield and to provide Previously, assessment of the genetic relationships within and
protection against abiotic and biotic stresses. In order to among Cicer species were conducted using DNA-based
enhance genetic potential, there must be a comprehensive molecular maker techniques such as restriction fragment
understanding of the amount and pattern of genetic variation length polymorphism (RFLP) (Serret et al. 1997), sequence-
that exists within and between the available cultivated and wild tagged microsatellite site (STMS) (Choumane et al. 2000),
Cicer accessions. World germplasm collections of cultivated random amplified polymorphic DNA (RAPD) (Ahmad 1999,
chickpea are lacking in diversity that may include traits needed Sudupak et al. 2002) and inter-simple-sequence-repeat (ISSR)
for effective improvement of the crop (Robertson et al. 1997, markers (Iruela et al. 2002).
Collard et al. 2003a). However, this may be overcome by The amplified fragment length polymorphism (AFLP)
looking to the wild relatives to widen the genetic bases of technique has emerged as a new powerful tool for genomic
breeding programmes through interspecific hybridization analysis (Vos et al. 1995). This technique has been applied to
(Singh and Ocampo 1997). determine genetic relationships among populations of legume

U. S. Copyright Clearance Center Code Statement: 0179–9541/2004/2302–0173 $ 15.00/0 www.blackwell-synergy.com

174 N g u y e n , T a y l o r , R e d d e n and F o r d

crops such as wild bean (Tohme et al. 1996), Lima bean for 30 s; and 72C for 1 min, until reaching an optimal annealing
(Caicedo et al. 1999), Azuki bean (Ru-Qiang et al. 2000) and temperature of 56C, followed by 24 more cycles of 94C for 30 s; 56C
peanut (Guohao and Channapatna 2001). The high frequency for 30 s and 72C for 1 min.
of identifiable polymorphic AFLP markers, coupled with their
reproducibility, make this technique an attractive tool for Polyacrylamide gel electrophoresis: Five microlitres of each final
detecting polymorphism and for determining genetic linkages AFLP amplification product was combined with 5 ll of AFLP gel
loading dye (98% formamide, 10 mM EDTA, 0.25% v/v each of
among individuals (Gupta et al. 1999).
bromophenol blue and xylene cyanol FF). Solutions were denatured at
The aims of this study were to use AFLP markers to 95C for 4 min, quenched on ice and 5 ll was then loaded on a 6%
determine the genetic diversity and relationships within and (w/v) polyacrylamide gel in 0.5 · TBE electrophoresis buffer. The
among unique accessions of the Cicer species. standard 30–330 bp AFLP DNA ladder (Invitrogen Life Technologies,
USA) was loaded in the flanking lanes. Gels were run at 50 V for
80 min, dried onto Whatman 3MM paper (Whatman International,
Materials and Methods USA), exposed to Kodax Bio-Image film (Kodak Ltd, USA) for at
Plant materials: Ninety-five accessions, representing 16 wild and one least 40 h and developed to produce an autoradiograph.
cultivated Cicer species, originating from the main Cicer centres of the
world diversity, were obtained from the Australian Temperate Field Data analysis: The AFLP bands were visually scored as either
Crops Collection (ATFCC) at the Victorian Institute for Dryland present (1) or absent (0) for each accession and each primer
Agriculture (VIDA), Horsham, Australia (Table 1). These are unique combination. The binary matrix was then used to measure pair-wise
accessions as described by Berger et al. (2003). genetic distance using Nei’s (1978) unbiased genetic distance within
POPGENE version 1.32 (Yeh et al. 1997). A dendrogram, showing
Genomic DNA extraction: Total genomic DNA was extracted using the genetic relationships among 95 accessions, was constructed from
the adapted CTAB method of Taylor et al. (1995) from young leaves the pair-wise genetic distance values based on the unweighted pair-
collected from plants grown in the quarantine glasshouse at VIDA. group method using arithmetic means (UPGMA) analysis in the
Molecular Evolutionary Genetic Analysis (MEGA) software version
AFLP procedure: Five hundred nanograms of genomic DNA of each 2.1 (Kumar et al. 2001). Unbiased measures of genetic distances were
accession was digested simultaneously with 10 units each of MseI and also calculated between each of the species and between phylogenetic
EcoRI (New England Biolabs, USA) at 37C for 4 h. Following groups of species using the algorithm of Nei (1978). A dendrogram of
digestion, EcoRI and MseI adapters (Invitrogen AFLP starter kit, Cicer species relationships was also constructed from the clustering of
USA) were ligated to restricted fragments at 20C for 2 h and digested the genetic distance values among Cicer species using the UPGMA
fragments were preamplified using 20 cycles of 94C for 30 s, 56C for method. Measures of genetic variation within species and among
1 min and 72C for 1 min according to the manufacturer’s instruc- species groups (number of polymorphic loci and gene diversity) were
tions. Selective amplification was then performed according to the also calculated (Weir 1990). No intraspecific variation was calculated
manufacturer’s instructions (Invitrogen, USA), with a 33P ATP for species represented by a single accession (C. multijugum,
labelled (Perkin-Elmer Life Sciences, USA) EcoRI primer and an C. canariense, C. flexuosum, C. nuristanicum, C. songaricum,
unlabelled MseI primer using a ÔTouchdownÕ cycle (Don et al. 1991) C. cuneatum and C. yamashitae).
programmed as follows: 12 cycles of 94C for 30 s; 65C ()0.7C/cycle)

Table 1: Details of Cicer species, number of accessions assessed and Results

their geographical origins AFLP marker profile
Number A total of 214 reproducible and clearly scorable bands,
Species of accessions1 Origins produced from five primer combinations, were assessed across
the entire collection of 95 Cicer accessions. Of these, three were
Group I
monomorphic and 211 were polymorphic. The average number
C. arietinum 3 Australia
C. echinospermum 7 Turkey of polymorphic bands detected per primer combination was
C. reticulatum 13 Turkey 42.2 and their molecular weights ranged from approximately
Group II 60 to 330 bp. Polymorphism varied remarkably among primer
C. bijugum 14 Iraq, Syria and Turkey combination. The most informative primer combination was
C. juidaicum 23 Israel, Jordan, Lebanon, the E-AAG/M-CTA pair, which produced the largest number
Syria and Turkey of polymorphic bands (59). This accounted for 28% of the
C. pinnatifidum 17 Lebanon, Lebanon,
total polymorphic bands examined in the study.
Syria and Turkey
Group III
C. anatolicum 2 Turkey Genetic variation among accessions
C. canariense 1 Spain
C. cuneatum 1 Ethiopia Nei’s coefficient of genetic distance between 95 accessions of 17
C. flexuosum 1 Russia species ranged from 0.01 (between accession ÔLasseterÕ and
C. macracanthum 2 Pakistan ÔKanivaÕ, both C. arietinum) to 0.66 (between accessions
C. microphyllum 5 India and Pakistan
ÔBumperÕ, from C. arietinum and ATC 46927, from C. bijugum)
C. multijugum 1 Uzbekistan
C. nuristanicum 1 Pakistan (data matrix not shown). The genetic distance between any two
C. oxyodon 2 Turkey accessions in the same species was generally quite low.
C. songaricum 1 Uzbekistan Conversely, the genetic distances were high between two single
C. yamashitae 1 Afghanistan accessions from different species. For instance, among 13
1
Unique accessions were provided by the Australian Temperate Field
accessions of C. reticulatum, the genetic distance index ranged
Crops Collection, Victorian Institute for Dryland Agriculture, from 0.04 to 0.23 while the distance to another species
Hosham (Berger et al. 2003). accession was as high as 0.66 (data not shown).
Genetic diversity estimates in Cicer using AFLP analysis 175

Table 2: Genetic variation detected

within Cicer species No. of No. of Gene
Species accessions polymorphic loci % Polymorphic loci diversity (h)

Group I
C. arietinum 3 20 9.3 0.036 ± 0.11061
C. reticulatum 13 79 36.9 0.104 ± 0.1714
C. echinospermum 7 30 14.0 0.038 ± 0.1099
Group II
C. pinnatifidum 17 102 47.6 0.126 ± 0.1746
C. judaicum 23 85 39.7 0.119 ± 0.1809
C. bijugum 14 66 30.8 0.080 ± 0.1522
Group III
C. anatolicum 2 12 5.6 0.023 ± 0.0955
C. oxyodon 2 29 13.5 0.023 ± 0.0955
C. macranthum 2 8 3.7 0.015 ± 0.0788
C. microphyllum 5 29 13.5 0.042 ± 0.1186
For all accessions 95 211 98.6 0.267 ± 0.1415
1
Standard deviation.

Genetic variation within Cicer species Genetic variation between species

Genetic variation was measured within each of the Cicer Pair-wise genetic distances between species ranged from 0.05
species assessed (Table 2). The most variable species was between C. multijugum and C. nuristanicum to 0.50 between
C. pinnatifidum, for which 102 polymorphic loci were detected C. arietinum and C. songaricum (Table 3). Cicer reticulatum
among 17 accessions with an overall gene diversity (h) of 0.126. was the closest species to the cultivated species (0.12) followed
The lowest genetic variation was detected within C. macra- by C. echinospermum (0.25). The most distant species to the
canthum among two accessions, with only eight polymorphic cultivated species was C. songaricum (0.50). In general, the
loci detected (h ¼ 0.015). For the cultivated species species in groups II and III possessed relatively higher genetic
(C. arietinum), 20 polymorphic loci were detected among three distances to the cultivated species, C. arietinum, than to the
accessions (h ¼ 0.036). wild species in group I (Table 3).

Dendrogram of genetic relationships among Cicer accessions Genetic variation between groups
and species Nei’s unbiased measures of genetic distance between groups I
The dendrogram constructed by the UPGMA method and II was 0.14, between groups II and III was 0.08 and
demonstrated that three main subgroupings existed in the between groups I and III was 0.13. This indicated a close
collection (Fig. 1). Group I (the primary and secondary relationship between groups II and III, both similarly distant
crossability groups documented by Ladizinsky and Adler to group I. The highest level of variation was detected within
(1976) clustered C. arietinum between C. echinospermum and group II, within which 73% of the detected loci were
C. reticulatum. Group II (the annual tertiary group) included polymorphic (h ¼ 0.19). Group III contained 65% polymor-
C. judaicum, C. pinnatifidum and C. bijugum. Group III phic loci (h ¼ 0.17), whereas, the least varied group, group I,
(mostly perennial tertiary) included C. anatolicum, contained 58% polymorphism (h ¼ 0.15).
C. oxyodon, C. canariense, C. flexuosum, C. macracanthum,
C. microphyllum, C. multijugum, C. nuristanicum and
C. songaricum as well as two annual species C. yamashitae Discussion
and C. cuneatum. The annual species C. cuneatum grouped The AFLP method was very useful for assessing the genetic
with C. canariense creating a separate subcluster within group relationships among a world collection of accessions that
III (Fig. 1), whilst another annual species C. yamashitae was represented species of the Cicer genus. The ability to determine
placed between the remaining perennial species in the other genetic variation among the accessions, species and species
subcluster. groups at the molecular level was directly related to the
Generally, all accessions were positioned into species clus- number of polymorphisms detected and their reproducibility.
ters with one exception: all accessions of C. reticulatum Previously, AFLP was considered to be a reliable technique for
clustered closely together with the three accessions of the the assessment of genetic variation among and between plant
cultivated species (ÔLasseterÕ, ÔKanivaÕ and ÔBumperÕ), except populations (Hill et al. 1996, Paul et al. 1997). This technique
for ATC 42326, which was placed further away. was more informative than previously used biochemical and
A dendrogram was also constructed to show relationships molecular methods to study variation and genetic relationships
among species. Within this dendrogram, three subgroups were in Cicer such as isozymes, storage proteins (Ahmad et al. 1992,
identified when a transect line was placed at approximately Labdi et al. 1996) and RAPD markers (Ahmad 1999, Sudupak
0.15 on the distance scale (Fig. 2). Cicer cuneatum and et al. 2002).
C. canariense fell outside the three main clusters. The dendro- In the dendrogram, the three accessions of C. arietinum
gram generally supported clustering observed previously were placed between C. reticulatum and C. echinospermum.
among the 95 accessions (Fig. 1). Together these species formed a single cluster within which
176 N g u y e n , T a y l o r , R e d d e n and F o r d

Fig. 1. Dendrogram of 95 Cicer accessions constructed based on UPGMA clustering. Accession names are provided as the ATC number
(R.J. Redden, ATFCC, VIDA, Horsham, Australia). Species symbols are the same as for Table 3
Genetic diversity estimates in Cicer using AFLP analysis 177

Fig. 2. Dendrogram of 17 Cicer

species constructed based on
UPGMA clustering

Table 3: Nei’s unbiased pair-wise measures of genetic distance among 17 Cicer species (Nei 1978)

Species CAR CRE CEC CPI CJU CBI CAN COX CCA CFL CMA CMI CMU CNU CSO CCU

CRE 0.12
CEC 0.25 0.15
CPI 0.35 0.22 0.23
CJU 0.39 0.27 0.27 0.12
CBI 0.49 0.34 0.36 0.18 0.25
CAN 0.46 0.34 0.35 0.29 0.31 0.34
COX 0.38 0.25 0.24 0.20 0.22 0.32 0.18
CCA 0.44 0.29 0.33 0.27 0.30 0.34 0.34 0.24
CFL 0.41 0.32 0.33 0.25 0.28 0.36 0.21 0.20 0.34
CMA 0.44 0.32 0.37 0.25 0.29 0.33 0.18 0.19 0.33 0.13
CMI 0.40 0.30 0.28 0.23 0.24 0.30 0.22 0.18 0.32 0.16 0.14
CMU 0.42 0.34 0.34 0.28 0.32 0.37 0.20 0.21 0.39 0.11 0.18 0.10
CNU 0.45 0.35 0.37 0.29 0.32 0.37 0.19 0.21 0.39 0.13 0.17 0.11 0.05
CSO 0.50 0.36 0.38 0.30 0.33 0.42 0.18 0.22 0.37 0.14 0.18 0.16 0.10 0.12
CCU 0.43 0.33 0.35 0.29 0.34 0.38 0.35 0.28 0.30 0.33 0.30 0.30 0.33 0.35 0.33
CYA 0.42 0.33 0.32 0.28 0.29 0.37 0.25 0.22 0.30 0.25 0.26 0.19 0.20 0.21 0.22 0.28

CAR, C. arietinum (cultivated species); CRE, C. reticulatum; CEC, C. echinospermum; CPI, C. pinnatifidum; CJU, C. judaicum; CBI, C. bijugum;
CAN, C. anatolicum; COX, C. oxyodon; CCA, C. canariense; CFL, C. flexosum; CMA, C. macrocanthum; CMI, C. microphyllum; CMU,
C. multijugum; CNU, C. nuristanicum; CSO, C. songaricum; CCU, C. cuneatum; CYA, C. yamashitae.

C. reticulatum was the least distant species to C. arietinum. demonstrated the close association of these three species
This agrees with the common hypothesis that C. reticulatum is (Ahmad and Slinkard 1992, Tayyar and Waines 1996). In the
the progenitor species of cultivated chickpea (Ladizinsky and species dendrogram this group also clustered closely with most
Adler 1976, Ohri and Pal 1991, Ahmad et al. 1992, Tayyar and of the perennial species. In addition, group II was closer to
Waines 1996, Iruela et al. 2002, Rajesh et al. 2002, Sudupak group III than group I suggesting that the annual species in
et al. 2002). Previous studies examining crossability, karyo- group II were more closely related to the perennial species than
type, isozyme polymorphism, seed protein and RAPD analy- the annual species in the primary crossability group.
sis, reported the close genetic relationship among C. arietinum, Several accessions of C. bijugum clustered with accessions of
C. reticulatum and C. echinospermum (Ahmad 1999). A low C. pinnatifidum. However, when assessed as species groups,
genetic distance was also detected between C. echinospermum C. bijugum was not clustered separately with C. pinnatifidum.
and C. arietinum, which may infer that C. echinospermum also This was due to the lower genetic distance detected between
played a role in the evolution of the cultivated species, as C. pinnatifidum and C. judaicum species than detected between
previously proposed by Iruela et al. (2002). C. pinnatifidum and C. bijugum species. This result was in
The clustering of C. judaicum, C. pinnatifidum and C. bijugum agreement with previous studies based on morphology analysis
in group II was in agreement with previous studies that by Ladizinsky and Adler (1976); seed storage protein analysis
178 N g u y e n , T a y l o r , R e d d e n and F o r d

by Ahmad and Slinkard (1992) and isozyme analysis by Labdi Within group II, C. judaicum, C. bijugum and particularly
et al. (1996) and Tayyar and Waines (1996) who demonstrated C. pinnatifidum possess very high levels of genetic diversity and
that C. pinnatifidum and C. judaicum were more closely related were reported as sources of resistance or tolerance to biotic
to each other than to C. bijugum. Conversely, this contradicted and abiotic stresses (Singh et al. 1998). Therefore, they offer
the previous finding of RAPD analysis by Sudupak et al. great potential sources for the future of chickpea breeding.
(2002) and RAPD and ISSR analysis by Iruela et al. (2002), Similar distances between group I and group II were detected
in which C. bijugum was clustered with and closer to as between groups I and III thus indicating that germplasm
C. pinnatifidum than C. judaicum. from groups II and III are both potential sources of novel
In the accession dendrogram, two annual species, C. yama- germplasm for improving chickpea breeding programmes.
shitae and C. cuneatum, were placed in the group III cluster However, before germplasm from groups II and III can be
together with all of the perennial species studied. However, in utilized in chickpea breeding, the barriers preventing interspe-
the species dendrogram, C. cuneatum was grouped with the cific hybridization need to be overcome.
perennial C. canariense outside the main group III cluster, as
similarly demonstrated in the dendrogram constructed from
RAPD and ISSR analysis by Iruela et al. (2002). The genetic Acknowledgements
distance of C. cuneatum to other annual species within the This research was funded by the Vietnamese Government, the
Cicer genus has previously been reported (Ahmad and University of Melbourne and the Department of Primary Industries,
Slinkard 1992, Labdi et al. 1996, Tayyar and Waines 1996, Victoria, Australia.
Ahmad 1999, Iruela et al. 2002). The close genetic relationship
of C. cuneatum and C. yamashitae with other perennial species
References
in this study raised questions regarding whether these species
have recently evolved from perennial species, or conversely Ahmad, F., 1999: Random amplified polymorphic DNA (RAPD)
analysis reveals genetic relationships among the annual Cicer
and more hypothetically, they have evolved to be annual
species. Theor. Appl. Genet. 98, 657—663.
candidates for perennial species. However, only single acces- Ahmad, F., and A. E. Slinkard, 1992: Genetic relationships in the
sions of both C. cuneatum and C. yamashitae were included in genus Cicer L. as revealed by polyacrylamide gel electrophoresis of
the study and much further research is required to fully seed storage proteins. Theor. Appl. Genet. 84, 688—692.
determine these possibilities. Ahmad, F., A. E. Slinkard, and G. J. Scoles, 1988: Investigations into
Although C. reticulatum is widely considered to be the barrier(s) to interspecific hybridization between Cicer arietinum L.
progenitor of the cultivated species, the question remains as to and eight other annual Cicer species. Plant Breeding 100, 193—198.
progenitor relationships between the annual and perennial Ahmad, F., P. M. Gaur, and A. E. Slinkard, 1992: Isozyme
species and which perennial species is most closely related to the polymorphism and phylogenetic interpretations in the genus Cicer
annual species gene pool. The closest perennial species to the L. Theor. Appl. Genet. 83, 620—627.
Badami, P. S., N. Nallikarjuna, and J. P. Moss, 1997: Interspecific
members of group I (annual) was C. oxyodon, which may
hybridization between Cicer arietinum and C. pinnatifidum. Plant
indicate that the C. oxyodon species was either involved in the Breeding 116, 393—395.
annual species evolution, or was derived from an annual species. Berger, J., J. Abbo, and N. C. Turner, 2003: Ecogeography of annual
However, insufficient numbers of accessions assessed may wild Cicer species: The poor state of the world collection. Crop
influence the results of evaluating genetic variation hence, Science 43, 1076—1090.
studies including more accessions with greater genome coverage Caicedo, A. L., E. Gaitan, M. C. Duque, O. T. Chica, D. G. Debouck,
are required before conclusions regarding the ancestry among and J. Tohme, 1999: AFLP fingerprinting of Phaseolus lunatus L. and
annual and perennial groups can be made. Nevertheless, Kazan related wild species from South America. Crop Sci. 39, 1497—1507.
and Muehlbauer (1991) used allozyme analysis and Choumane Choumane, W., P. Winter, F. Weigand, and G. Kahl, 2000: Conser-
et al. (2000) used STMS markers to demonstrate that vation and variability of sequence-tagged microsatellite sites
(STMSs) from chickpea (Cicer arietinum L.) within the genus Cicer.
C. anatolicum was closely related to the primary gene pool.
Theor. Appl. Genet. 101, 269—278.
However, this was not supported by the isozyme analysis of Collard, B. C. Y., E. C. K. Pang, and P. W. J. Taylor, 2003a:
Tayyar and Waines (1996), the RAPD analysis of Sudupak et al. Selection of wild Cicer accessions for the generation of mapping
(2002), or the RAPD and ISSR analysis of Iruela et al. (2002). populations segregating for resistance to ascochyta blight. Euphy-
The amount of genetic variation detected within C. arieti- tica 130, 1—9.
num was less than that detected within almost all of the wild Collard, B. C. Y., E. C. K. Pang, P. K. Ades, and P. W. J. Taylor,
Cicer species. This indicated that the wild Cicer species may 2003b: Preliminary investigation of QTLs associated with seedling
offer potential novel sources of genetic variation, which may be resistance to ascochyta blight from Cicer echinospermum, a wild
introduced into cultivars to broaden the genetic base of relative of chickpea. Theor. Appl. Genet. 107, 719—729.
chickpea through interspecific hybridization. Successful cros- Don, R. H., P. T. Cox, B. J. Wainwright, and J. S. Mattick, 1991:
Touchdown PCR to circumvent spurious priming during gene
ses have been made between the two most closely related wild
amplification. Nucl. Acids Res. 19, 4008.
species (C. reticulatum and C. echinospermum) and the Guohao, H., and P. Channapatna, 2001: Evaluation of genetic
cultivated species (Ladizinsky and Adler 1976, Singh and relationships among botanical varieties of cultivated peanut (Arachis
Ocampo 1997, Collard et al. 2003b). In addition, limited hypogaea L.) using AFLP markers. Genet. Res. Crop Evol. 48,
success has been reported in crossing the species in group I 347—252.
with group II (Singh et al. 1994, International Centre for Gupta, P. K., R. K. Varshney, P. C. Sharma, and B. Ramesh, 1999:
Agricultural Research in the Dryland Areas 1998). However, Molecular markers and their applications in wheat breeding. Plant
tissue culture methods such as embryo rescue techniques may Breeding 118, 369—390.
provide the means to negate crossability barriers to produce Haware, M. P., J. R. Narayana, and R. P. S. Pundir, 1992: Evaluation
wide and interspecific hybridizations in the future (Badami of wild Cicer species for resistance to four chickpea diseases.
International Chickpea Newsletter 27, 16—18.
et al. 1997).
Genetic diversity estimates in Cicer using AFLP analysis 179

Hill, M., H. Witsenboer, M. Zabeau, P. Vos, R. Kesseli, and Serret, M. D., S. M. Udupa, and F. Weigand, 1997: Assessment of
R. Michelmore, 1996: PCR-based fingerprinting using AFLP’s as genetic diversity of cultivated chickpea using microsatellite-derived
a tool for studying genetic relationships in Latuca spp. Theor. Appl. RFLP markers: implications for origin. Plant Breeding 116,
Genet. 93, 1202—1210. 573—578.
International Centre for Agricultural Research in the Dryland Areas, Singh, K. B., and B. Ocampo, 1993: Interspecific hybridization in
1998: Interspecific Hybridization in Chickpea. Annual Report 1998, annual Cicer species. J. Genet. Breed. 47, 199—204.
31. http://www.icarda.cgiar.org/Publications/AnnualReport/98/An- Singh, K.B, and B. Ocampo, 1997: Exploitation of wild Cicer species
Page31.HTML. for yield improvement in chickpea. Theor. Appl. Genet. 95,
Iruela, M., J. Rubio, J. I. Cubero, J. Gil, and T. Millan, 2002: 418—423.
Phylogenetic analysis in the genus Cicer and cultivated chickpea using Singh, K. B., G. C. Hawtin, Y. L. Nene, and M. V. Reddy, 1981:
RAPD and ISSR markers. Theor. Appl. Genet. 104, 643—651. Resistance in chickpeas to Ascochyta rabiei. Plant disease 65,
Kazan, K., and F. J. Muehlbauer, 1991: Allozyme variation and 586—587.
phylogeny in annual species of Cicer (Leguminosae). Plant Syst. Singh, K. B., R. S. Malhotra, M. H. Halila, E. J. Knights, and M. M.
Evol. 175, 11—21. Verma, 1994: Current status and future strategy in breeding
Kumar, S., K. Tamua, I. B. Jakobsen, and M. Nei, 2001: MEGA 2.1: chickpea for resistance to biotic and abiotic stresses. Euphytica 73,
Molecular Evolutionary Genetics Analysis. http://www.megasoft- 137—149.
ware.net/. Singh, K., B. Ocampo, and L. D. Robertson, 1998: Diversity for
Labdi, M., L. D. Robertson, K. B. Singh, and A. Charrier, 1996: abiotic and biotic stress resistance in the wild annual Cicer species.
Genetic diversity and phylogenetic relationships among the annual Genet. Res. Crop Evol. 45, 9—17.
Cicer species as revealed by isozyme polymorphisms. Euphytica Stamigna, C., R. Mancinelli, P. Crino, A. Infantino, A. Porta-Puglia,
88, 181—188. and F. Saccardo, 1998: Multiple resistances to diseases in wild
Ladizinsky, G., and A. Adler, 1976: Genetic relationships among the relatives of chickpea (Cicer arietinum L.). In: Proceedings of the 3rd
annual species of Cicer L. Theor. Appl. Genet. 48, 197—203. European Conference on Grain Legumes, Valladolid, Spain.
van der Maesen, L. J. G., 1987: Origin, history and taxonomy of Sudupak, M. A., M. S. Akkaya, and A. Kence, 2002: Analysis of
chickpea. In: M. C. Saxena, and K. B. Singh (eds), The Chickpea, genetic relationships among perennial and annual Cicer species
11—34. CAB International Publishers, UK. growing in Turkey using RAPD markers. Theor. Appl. Genet. 105,
Muehlbauer, F. J., W. J. Kaiser, and C. J. Simon, 1994: Potential for 1220—1228.
wild species in cool season food legume breeding. Euphytica 73, Taylor, P. W. J., T. A. Fraser, H. L. Ko, and R. J. Henry, 1995: RAPD
109—114. analysis of sugarcane during tissue culture. In: M. Terzi, R. Cella,
Nei, M., 1978: Estimation of average heterozygosity and genetic and A. Falavigna (eds), Current Issue in Plant Molecular and
distance from a small number of individuals. Genetics 89, 583—590. Cellular Biology, 241—246. Kluwer Academic International, Dord-
Ocampo, B., G. Venora, A. Errico, K. B. Singh, and F. Saccardo, 1992: recht.
Karyotype analysis of the Cicer genus. J. Genet. Breed. 46, 229—240. Tayyar, R. I., and J. G. Waines, 1996: Genetic relationships among
Ohri, D., and M. Pal, 1991: The origin of chickpea (Cicer arietinum L.) annual species of Cicer (Fabaceae) using isozyme variation. Theor.
karyotype and nuclear DNA amount. Heredity 66, 367—372. Appl. Genet. 92, 245—254.
Paul, S., F. N. Wachira, W. Powell, and R. Waugh, 1997: Diversity Tohme, J., D. O. Gonzalez, S. Beebe, and M. C. Duque, 1996: AFLP
and genetic differentiation among populations of Indian and analysis of the gene pool of a wild bean core collection. Crop Sci. 36,
Kenyan tea (Camellia sinensis (L) O.Kuntze) revealed by AFLP 1375—1384.
markers. Theor. Appl. Genet. 94, 255—263. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. V. D. Lee, M. Hornes,
Rajesh, P. N., V. J. Sant, V. S. Gupta, F. J. Muehlbauer, and P. K. A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau, 1995:
Ranjekar, 2002: Genetic relationships among annual and perennial AFLP: a new technique for DNA fingerprinting. Nucl. Acids Res.
wild species of Cicer using inter simple sequence repeat (ISSR) 23, 4407—4414.
polymorphism. Euphytica 129, 15—23. Weir, B.S., 1990: Intraspecific differentiation. In: D.M. Hillis, and C.
Robertson, L. D., B. Ocampo, and K. B. Singh, 1997: Morphological Moritz (eds), Molecular Systematics, 373—410. Sinauer Associates,
variation in wild annual Cicer species in comparison to the cultigen. Sunderland, MA, USA.
Euphytica 95, 309—319. Yeh, F.C., R.-C. Yang, T.B.J. Boyle, Z.-H. Ye, and J.X. Mao 1997:
Ru-Qiang, X., N. Tomooka, and D. A. Vaughan, 2000: AFLP POPGENE, the User-Friendly Shareware for Population Genetic
markers for characterizing the Azuki bean complex. Crop Sci. 40, Analysis. Molecular Biology and Biotechnology Centre, University
808—815. of Alberta, Canada. http://www.ualberta.ca/fyeh/.