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RESEARCH ARTICLE
Allelic diversity between and within three wild annual Cicerspecies
Zvi Peleg • Alon Shabtay • Shahal Abbo
Received: 23 March 2014 / Accepted: 2 June 2014 / Published online: 26 June 2014
� Springer Science+Business Media Dordrecht 2014
Abstract Most wild Cicer species have narrow eco-
geographic amplitude. Likewise, domesticated chick-
pea suffers from severe adaptive limitations due to its
unique evolutionary history. The wild progenitor may
offer only limited adaptive allelic variation for
improving the chickpea crop. Therefore, there is a
need to explore allelic diversity between and within
annual Cicer sp. that span diverse natural habitats.
Here we characterized the allelic diversity between
and within wild populations of C. pinnatifidum, C.
judaicum and C. cuneatum spanning most of their
documented native range in Turkey, Israel and Ethi-
opia. Eco-geographical analysis resulted in clear
separation between the collection sites of C. cuneatum
in east Africa and the other two east Mediterranean
species. Analysis of molecular variance shows that
only 18 % of the allelic variation accounts for
differences between the three species, while 34 %
was contributed from difference between populations.
Interestingly, most (48 %) of the allelic variation was
detected among accessions within populations. PCoA
analysis confirmed the independent taxonomic and
indeed the genetic integrity of the two east Mediter-
ranean sister species C. pinnatifidum and C. judaicum.
Conservation of large rich populations seems a more
effective strategy than the preservation of small thin
populations of annual Cicer sp. Given the relatively
narrow geographic range of most annual Cicer sp.,
accessing germplasm lines from ecologically distinct
habitats emerges as the most promising strategy for the
identification of useful adaptive allelic variation.
Keywords Chickpea � Cicer cuneatum � Cicer
judaicum � Cicer pinnatifidum � Eco-geographic
adaptation � Genetic diversity � Microsatellites
Introduction
Chickpea (Cicer arietinum L.) is an important grain
legume crop across the Mediterranean basin, East
Africa, the Indian sub-continent and in certain New
World regions. In rotation with cereals chickpea has
an indispensable role in breaking disease cycles and
fixing atmospheric nitrogen (e.g. Singh 1997; Kumar
and Abbo 2001). In recent years chickpea production
has increased, placing it now as the second in
importance grain legume in global terms (http://
faostat.fao.org/site/567/default.aspx#ancor). Tradi-
tionally, chickpea was of relatively lesser significant in
the economy of highly industrialized nations, and as a
Electronic supplementary material The online version ofthis article (doi:10.1007/s10722-014-0141-2) contains supple-mentary material, which is available to authorized users.
Z. Peleg (&) � A. Shabtay � S. Abbo
The Levi Eshkol School of Agriculture, The Robert H.
Smith Faculty of Agriculture, Food and Environment,
The Hebrew University of Jerusalem, P.O. Box 12,
7610001 Rehovot, Israel
e-mail: zvi.peleg@mail.huji.ac.il
123
Genet Resour Crop Evol (2015) 62:177–188
DOI 10.1007/s10722-014-0141-2
consequence our understanding of the biological and
the ecological determinants of its yield potential was
limited as compared with better studied grain crops
like maize (Zea mays L.), rice (Oryza sativa L.), wheat
(Triticum aestivum L.), and soybean (Glycine max (L.)
Merr.) (e.g. Kumar and Abbo 2001).
Massive efforts conducted in ICRISAT and else-
where in recent years have brought chickpea genetics
and genomics to the forefront thereby providing new
opportunities for identification of the genetic systems
that control grain yield (e.g. Lichtenzveig et al. 2005;
Hiremath et al. 2012; Nayak et al. 2010; Varshney
et al. 2012, 2013). However, while sequence and
genomic data are no longer likely to limit crop
physiology studies or molecular breeding efforts, it
becomes clear that phenotyping of diverse domesti-
cated and wild germplasm are likely to be the weakest
link. While domesticated chickpea germplasm collec-
tions are rich and diverse (Pundir et al. 1988), and have
been analysed by large scale field experiments (e.g.
Berger et al. 2006), wild Cicer germplasm collections
are still quite small relative to the number of acces-
sions from the wild progenitors of the temperate
cereals (wheat and barley) deposited in genebanks
(Berger et al. 2003). Efforts to increase the number of
Cicer accessions are currently being done by various
groups and this is likely to create an impact in the near
future.
Although the genus Cicer as a whole spans a vast
geographic range, from the Canary Islands in the west
to the Himalaya foothills in the east and from
Uzbekistan in the north to Ethiopia in the south, most
of the annual and perennial Cicer taxa have a rather
limited geographic distribution attesting to their strict
ecological affinities and indeed narrow adaptive range
(e.g. Abbo et al. 2003; Berger et al. 2003; van der
Maesen 1972). More specifically, the relatively nar-
row range of the wild progenitor of domesticated
chickpea, C. reticulatum Ladiz., suggests that the
prospects for improving the adaptive range of domes-
ticated chickpea by direct crossing and recruiting
environmental adaptation alleles from this species into
modern high yielding cultivars are quite limited. As a
consequence, an alternative strategy involving more
remote taxa and employing comparative genetic
approaches was proposed (e.g. Ben-David and Abbo
2005; Abbo and Mallikarjuna 2008). Regrettably
however, recent attempts to apply the latter approach
faced difficulties and progress was limited due to
crossability barriers between closely related wild taxa
(e.g. Abbo et al. 2011).
As part of our interest in chickpea and its wild
relatives we have assembled a working collection of
several wild annual Cicer species (e.g. Ben-David and
Abbo 2005; Ben-David et al. 2010, 2006; Abbo et al.
2011). When taken together, the latitudinal range of
the three species involved in our long-term studies
spans almost the entire range of the genus as a whole,
these are C. pinnatifidum Jaub. et Spach native to
Turkey, C. judaicum Boiss. native to Israel and Jordan,
and C. cuneatum Hochst. ex A, Rich. native to Eritrea
and Ethiopia. In some of the cases collections were
made while sampling more than one individual from
certain habitats thereby providing an opportunity to
explore between- and within-populations diversity in
both genetic and phenotypic terms (Ben-David and
Abbo 2005; Frenkel et al. 2008). Measures of genetic
diversity between and within populations are impor-
tant parameters in devising germplasm collection and
conservation strategies, but thus far such information
for wild Cicer species is rather scarce.
The availability of such geographically diverse
wild Cicer material enables to address questions
concerning between-species phenotypic and genetic
comparisons including the structure of genetic diver-
sity within the genus as a function of eco-geographic
variables. Accordingly, our objectives in this study
were as follows: (1) to estimate the overall genetic
variation in the collection by using a limited set of
microsatellite DNA markers; (2) to evaluate the
microsatellite allelic diversity between and within
annual Cicer taxa spanning a wide latitudinal gradient;
(3) to reassess the independent taxonomic status of the
closely related species C. judaicum and C. pinnatifidum;
(4) to try and associate between the microsatellite
variability, phenological variation and response to
the ascochyta pathogen Didymella rabiei in Israeli
C. judaicum.
Materials and methods
Population sampling
One hundred and twenty two accessions of wild Cicer
from tree species: C. pinnatifidum (33 accessions), C.
judaicum (61 accessions), and C. cuneatum (28
accessions) were employed in this study. The
178 Genet Resour Crop Evol (2015) 62:177–188
123
accessions are maintained in Rehovot, Israel, as pure
lines and were propagated inside insect-proof screen
houses to minimize the scope for cross-pollination.
Full description of these accessions is given in Fig. 1
and Table S1.
Sample preparation and genotyping
Fresh leaf tissue (*200 mg) from 2 months old
greenhouse-grown plants was used for DNA extrac-
tion by CTaB method. A NanoDrop� ND1000 Spec-
trophotometer (NanoDrop Technologies, Inc.,
Wilmington, DE, USA) was used to measure the
DNA concentration. Thirty-two microsatellites (SSR)
primers (Lichtenzveig et al. 2005; Winter et al. 2000)
were tested for polymorphism using 3-5 accessions
from each species and the reference line cv. Hadas.
Eight SSR markers showed polymorphism within and/
or between species and were used for the analysis (see
Table S2). Polymerase chain reaction (PCR) protocols
and primers used in this study were as previously
described by (Lichtenzveig et al. 2005) while adjust-
ing the annealing temperature for each species to
improve amplification efficiency (Table S2). Lengths
of the amplified segments were detected with an
automated laser fluorescence (A.L.F.) sequencer and
analysed using the computer program Fragment
Analyzer Ver. 1.02 (Amersham Pharmacia Biotech)
by comparing with internal size standards. The
chickpea cultivar Hadas was used as a reference in
each run to ensure size accuracy and to minimize run-
to-run and gel-to-gel variations. The list of SSR
markers and their chromosomal location is given in
Table S2.
Genetic analysis
PowerMarker software (Liu and Muse 2005) was used
to calculate allelic frequencies, allele numbers, and
diversity indices (He) for each marker. PowerMarker
was also used to calculate polymorphism information
content (PIC) values according to Anderson et al.
(1993). For marker i, the PIC was calculated as:
PICi ¼ 1�Xn
j¼1
p2ij
where pij is the frequency of the jth allele for marker
i and summed across n alleles. This value provides an
estimate for the discriminatory power of a given
microsatellite locus by taking into account not only the
number of alleles per locus, but also its relative
frequency in the population genetics studied.
Fig. 1 A Geographic distribution of the three wild Cicer
species. B Principle component analysis (based on correlation
matrix) of eco-geographical parameters characterizing the wild
Cicer habitats. Lt, latitude; Ln, longitude; Alt, altitude above sea
level; Tmin coldest period, minimum temperature in January;
Tmax warmest period, maximum temperature in August. Biplot
vectors are trait factor loadings for principal component (PC)1
and PC2
Genet Resour Crop Evol (2015) 62:177–188 179
123
Individual pairwise genetic distances (Peakall and
Smouse 2006) were calculated for all markers. A
principal coordinate analysis (PCoA) was performed
on markers data set with GENEALEX 6.0 (Genetic
Analysis in Excel) software (Peakall and Smouse
2006). Analysis of molecular variance (AMOVA) was
employed to estimate the variance between popula-
tions and among accessions within populations with
1,000 bootstrap replicates.
The correlation between pairwise genetic distances
and geographic (measured in kilometres) distances
among populations was investigated by the Mantel test
(Mantel 1967) of matrix correspondence. All Mantel
tests were performed using the GENEALEX 6.0
software.
The STRUCTURE program was used to analyse
and cluster the studied genotypes. This program
implements a model-based clustering method assign-
ing individuals to clusters and identifying migrants
and individuals resulting from admixture (Pritchard
et al. 2000). The number of clusters (K) was set from 1
to 12. Each K was replicated 10 times for 10,000
iterations after a burn-in period of 100,000. An
admixture model was employed in which the fraction
of ancestry from each cluster is estimated for each
individual without prior information on the population
of origin.
Results
Eco-geographic characterization of the wild
chickpea sampling sites
Principle component analysis of the eco-geographical
variables, alongside a general distribution map, for the
collection sites of the three wild Cicer species is
presented in Fig. 1. The PCA extracted two major
principle components (Eigenvalue [ 1) that collec-
tively accounted for 80.9 % of the eco-geographical
variance. Principle component 1 (PC1, X-axis)
explained 58.2 % of the dataset variance, and was
loaded positively with minimum temperature (Tmin)
in the coldest period and annual precipitation, and
negatively with maximal temperature (Tmax) in the
warmest period and latitude (Lt). PC2 (Y-axis)
explained 22.7 % of the variance and was loaded
positively with longitude (Ln) and negatively with
altitude above sea level (Alt). The PCA resulted in
clear separation between the collection sites of
C. cuneatum in east Africa (depicted in red) and the
other two east Mediterranean species along PC1. The
separation between C. judaicum (green) and C.
pinnatifidum (blue) is apparent along PC2, presumably
influenced by the latitudinal differences (Fig. 1).
In C. pinnatifidum, the PCA exposed two major
components explaining 78.9 % of the total variance in
the dataset. PC1 explained 45.8 % and was loaded
positively with Tmax, Lt, Ln, and annual precipitation.
PC2 explained 33.1 % and was loaded positively with
Tmin and negatively with Alt (Fig. S1a). In C.
judaicum, the PCA exposed two major components
explaining 89.9 % of the variation in the data set. PC1
accounted for 56.8 % of the variation and was
positively loaded with Ln, Alt and Tmax and nega-
tively with Ln and Tmin. PC2 explained 34 % of the
variation and was positively loaded with the annual
precipitation and longitude (Fig. S1b). The PCA
created a clear separation between the northern
(Kerem Maharal, Nahal Milek, Mei-Ami, Nahal
Narbeta) and the southern (Kiryat Sefer, Nahal Anava,
Nahal Meara, Menora) populations in Israel. In
C. cuneatum, the PCA accounted for 94.8 % of the
variation in the data set. PC1 explained 72.8 % of the
variation and was loaded positively with annual
precipitation, Alt and Ln and negatively with Tmax
and Lt. PC2 accounted for 22 % of the variation and
was loaded positively with Tmin (Fig. S1c). The
effectiveness of the PCA is apparent from the distinct
position of the eastern most population near Harar
relative to the other highlands sites of Ethiopia and
Eritrea.
Allelic variation of microsatellite markers
in the three wild chickpea species
Cicer pinnatifidum—Total of 88 amplified fragments
were detected among the 33 C. pinnatifidum acces-
sions over the 12 tested microsatellite loci (Table 1).
The number of detected alleles per locus varied
between 3 and 11, with an average of 7.1 alleles per
locus. The genetic diversity ranged between 0.88
(XH3F8) to 0.41 (XH1D24a) with average diversity of
0.71. The PIC value was highest for XH3F8 (0.87) and
lowest for XH1D24a (0.38). When analysed according to
the source populations, the highest PIC value 0.41 was
detected among Kahramanmarash–Adiyaman road
population, and the lowest 0.15 in Burc (Table 2).
180 Genet Resour Crop Evol (2015) 62:177–188
123
Cicer judaicum—Total of 46 amplified fragments
were detected among the 27 C. judaicum accessions
over 10 microsatellite loci (Table 1). The number of
alleles ranged from two to nine with an average of 4.6
alleles per locus. The mean observed heterozygosity
was highest for XH5A4a (0.11). The PIC values ranged
between 0.81 in XH1B13b and 0.26 in XH1D24a, with an
average of 0.56. When analysed according to the
source population, the highest genetic diversity
(Table 2) was detected in Nahal Anava (0.43) and
the lowest in Nahal Milek (0.1). Heterozygosity was
detected only in the Kerem Maharal population (0.04).
Cicer cuneatum—Total of 50 amplified fragments
were detected in 28 accessions of C. cuneatum over 10
microsatellite loci (Table 1). Frequency of the major
allele ranged between 0.84 for XH1D24a to 0.25 for
XH1B13b, with an average of 0.5. The genetic diversity
ranged between 0.84 (XH3F8) and 0.28 (XH1D24a) with
average of 0.61. The PIC values ranged between 0.82
(XH3F8) and 0.26 (XH1D24a). When analysed based on
the source populations, heterozygosity was detected in
two populations, Mendefera and in the Mendefera-
Barantu road, both with value of 0.06. The PIC value
ranged between 0.3 in the Mendefera population and
0.21 in the Bilbala population (Table 2).
Table 1 Linkage group (LG), number of alleles detected,
major allele frequency, gene diversity (He) and polymorphism
information contents (PIC) of microsatellite markers in three
wild Cicer species from Turkey, Israel, Ethiopia and Eritrea
Marker LG A Major allele
frequency
He PIC
Cicer pinnatifidum
TA3 8 3 0.65 0.51 0.44
STMS21 1 4 0.45 0.63 0.55
H1B13b 7 10 0.25 0.85 0.83
H1D24a 7 6 0.76 0.41 0.38
H5A4a 6 7 0.27 0.80 0.77
H3F8 3 11 0.22 0.88 0.87
H1B13a 3 8 0.33 0.77 0.73
TR1 6 11 0.32 0.83 0.81
H1D24b 7 7 0.63 0.57 0.55
H1D24c 7 6 0.46 0.72 0.69
H5A4b 6 7 0.25 0.83 0.80
H5A8b 2 8 0.37 0.79 0.77
Average 7.1 0.42 0.71 0.68
Cicer judaicum
TA3 8 2 0.96 0.08 0.07
H1B13b 7 9 0.26 0.83 0.81
H1D24a 7 3 0.83 0.28 0.26
H5A4a 6 4 0.61 0.53 0.46
H3F8 3 5 0.37 0.73 0.68
H1B13a 3 4 0.55 0.57 0.49
TR1 6 4 0.55 0.60 0.53
H1D24b 7 5 0.47 0.68 0.63
H5A4b 6 3 0.65 0.51 0.45
H5A8b 2 7 0.42 0.75 0.72
Average 4.6 0.57 0.56 0.56
Cicer cuneatum
TA3 8 4 0.63 0.51 0.43
STMS21 1 4 0.59 0.51 0.41
H1B13b 7 7 0.25 0.83 0.81
H1D24a 7 3 0.84 0.28 0.26
H5A4a 6 3 0.52 0.57 0.47
H3F8 3 9 0.26 0.84 0.82
H1B13a 3 8 0.26 0.81 0.78
H1D24b 7 5 0.58 0.61 0.58
H1D24c 7 3 0.54 0.56 0.47
H5A4b 6 4 0.53 0.63 0.58
Average 5 0.50 0.61 0.56
Table 2 Mean umber of alleles detected per locus (A), allelic
diversity (He), heterozygosity per population (Ho), and poly-
morphism information contents (PIC) among the populations
of three wild Cicer species from Turkey, Israel, Ethiopia and
Eritrea
Population A He Ho PIC
Cicer pinnatifidum
Araban 1.7 – – 0.28
Besni 1.6 0.26 0.10 0.20
Burc 1.5 0.19 0.05 0.15
Kahramanmaras 3.0 0.46 0.08 0.41
Sof Mt. 2.3 0.37 0.01 0.32
Yolagzı 0.9 – – 0.28
Cicer judaicum
Nahal A’naba 2.3 0.43 – 0.36
West Carmel 1.6 0.21 – 0.17
Kerem Maharal 1.7 0.26 0.04 0.21
Mey A’mi 1.6 0.23 – 0.18
Nahal Mylek 1.2 0.10 – 0.08
Cicer cuneatum
Adi-Quala 2.4 0.30 0.06 0.27
Mendefera 1.6 – – 0.30
Barantu rd. 2.2 0.33 0.06 0.29
Bilbala 1.3 – 0.06 0.21
Full eco-geographical information can be found in
supplementary Table S1
Genet Resour Crop Evol (2015) 62:177–188 181
123
The pattern of allelic variation
between and within populations
Cicer pinnatifidum—According to our analysis of
microsatellite allelic diversity among the six C.
pinnatifidum populations 45 % of the variation was
detected between population and the remaining 55 %
within populations (Fig. 2A). The PCoA plot shows
two major components explaining 51.2 % of the
allelic diversity between accessions. PCo1 accounted
for 27.1 % of the allelic variance and PCo2 explained
24.1 %. The scatter plot shows a clear separation
between the sampled populations (Fig. 2B), and the
effectiveness of the PCoA is apparent from placing of
the eastern most population of Yolagzı distal to the rest
westerly and more adjacent populations (Fig. 2B).
Cicer judaicum—Most of the allelic variance
(69 %) was detected within populations (among
accessions) and the remaining 31 % between the
populations (Fig. 2C). The two major PCo axes
account for 50.4 % of the allelic variation, with
PCo1 contributing 31.2 % and PCo2 19.2 %. The
PCoA created a clear separation between the northern
and southern Israeli population, however no clear
distinction is apparent between accessions belonging
to the different populations of these two population
groups. That is, no separation was obtained between
the different northern populations, and likewise
between the different southern populations (Fig. 2D).
Additional 34 C. judaicum accessions, obtained
from three geographical regions using a three-levelled
sampling hierarchy (population, sub-population,
accession; see Frenkel et al. 2010; Ozkilinc et al.
2010) were analysed separately (Fig. 3). In each
region, three populations were identified at a minimum
distance of 1.5 km, while subpopulations were defined
as plant clusters with a distance of 50–100 meters.
Five individual plants were sampled at a minimum
Fig. 2 Analysis of
molecular variance
(AMOVA) of SSR markers
haplotypes of three wild
Cicer species. A principal
coordinate (PCo) plot of
pairwise individual genetic
distances. C. pinnatifidum
(A, B), C. judaicum (C,
D) and C. cuneatum (E, F)
182 Genet Resour Crop Evol (2015) 62:177–188
123
distance of 10 m within each sub-population (Frenkel
et al. 2008, 2010; Ozkilinc et al. 2010). The popula-
tions from central Israel (Nataf and Haela Valley)
clustered separately from the northern ones (Menashe
Hills). Interestingly, an almost perfect separation was
obtained between the three populations of the northern
sampling region, while a lesser degree of separation is
apparent for the southern populations and their sub-
populations (Fig. 3).
Cicer cuneatum—The AMOVA have shown that
31 % of the allelic variation resides between popula-
tions while 69 % of the variation was observed within
populations (Fig. 2E). The PCoA showed two major
components explaining 65 % of the allelic diversity
between accessions. Most of the variance was
explained by PCo1 (50.8 %), creating a separation
between accessions sampled in Eritrea and those
sampled in Ethiopia (Fig. 2F). However, similar to the
situation observed for C. judaicum, here also the
PCoA failed to separate the different populations in
the two countries from each other (Fig. 2F).
Species relationship among the studied taxa
Analysis of molecular variance (AMOVA) shows that
only 18 % of the allelic variation is accounted for by
differences between the three studied species
(Table 3). Additional 34 % of the allelic variation
was contributed from difference between populations
(nested within species), and 48 % of the allelic
variation was detected among accessions nested
within populations.
The probabilities of the K number of clusters
showed the best solution for K = 3 which was
considerably better than K = 2, while K C 4 gave
only a small probability improvement. Low level of
admixture was found between populations and like-
wise between species (Fig. 4). The PCoA (Fig. 5A)
confirmed the independent taxonomic and indeed the
genetic integrity of the two east Mediterranean sister
species C. pinnatifidum (blue) and C. judaicum
(green). The correlation (Rm) between geographic
distance and genetic distance of the two Mediterra-
nean species (Mantel test, Fig. 5B) was significant
(Rm = 0.61, P \ 0.001).
Discussion
The Earth biosphere errantly undergoes a rapid decline
in biodiversity (i.e. biological diversity; Ricketts et al.
2005). Preservation of both inter- and intra-specific
biodiversity is of growing concern, particularly in
relation to global climatic change and urbanization.
Thus, better understanding of the relationships
between genetic diversity in natural populations and
ecological factors is fundamental for planning effec-
tive conservation strategies. In the current study, we
used microsatellites markers as a tool for the
Fig. 3 Estimated population structure based on allele fre-
quency variation of microsatellite markers. Each individual is
represented by a vertical bar, which is partitioned into K-colored
components representing the ancestry fractions in K = 3
clusters. Individual accessions are ordered by species (labeled
above)
Fig. 4 A principal coordinate (PCo) plot of pairwise individual
genetic distances among C. judaicum populations in three
regions
Table 3 Summary of analysis of molecular variance (AM-
OVA) of microsatellite haplotypes of the three wild Cicer
species
Source of variance df SS % P
Between species 2 180 18 \0.001
Between populations 12 343 34 \0.001
Within populations 48 356 48 \0.001
Total 62 879 100
Genet Resour Crop Evol (2015) 62:177–188 183
123
characterization of allelic diversity between and
within wild populations of three annual Cicer species.
Ecogeography of wild Cicer
The different annual species in the genus Cicer are
characterized by relatively narrow geographic range
(van der Maesen 1972; Berger et al. 2003), which is
thought to reflect narrow eco-geographic adaptation
(Abbo et al. 2003; Berger et al. 2003). Likewise, most of
the less studied perennial Cicer taxa also span narrow
geographic range (van der Maesen 1972). This may
suggest that narrow eco-geographic adaptation is a more
general phenomenon and probably of a deep evolution-
ary origin in this genus. In this work we have analysed
only a single Cicer species from each of three regions,
Turkey, Israel and East Africa. In eastern Turkey
however, three additional wild annual Cicer species
are known namely, C. echinospermum, C. bijugum and
C. reticulatum. In this region, and in certain ecological
settings, individuals from more than one species can be
found in close proximity, e.g. C. pinnatifidum and C.
reticulatum, in calcareous habitats or C. echinospermum
and C. bijugum on basaltic soils. Therefore, it would be
of great interest to identify diagnostic combinations of
eco-geographical parameters capable of differentiating
between the unique niches of these sympatric but
genetically distinct and reproductively isolated annual
Cicer taxa (Ladizinsky and Adler 1976). Such differen-
tiation may improve our understanding of the environ-
mental determinants of chickpea distribution in the wild
and the agro-ecological adaptation of the cultigen under
domestication.
Given the relatively large distance between the
Turkish, Israeli and the east African sampling sites and
their different ecological and geobotanical features,
the clear PCA separation between the three respective
regions and the sampling sites of studied Cicer taxa is
not surprising (Fig. 1). Still, using a relatively small
number of eco-geographic parameters it was possible
to obtain a good separation between the different
sampled populations within each of the studied species
(Fig. S1). Moreover, even within the relatively small
range of C. judaicum in Israel, with ca. 120 km
between the northern- and the southern-most sites, a
clear separation was obtained between southern and
northern populations. A separation that was also
reflected in the pattern of allelic diversity and other
genetically controlled plant traits (below).
Within and between population allelic diversity
Patterns of genetic variability between and within
natural plant populations and their driving forces are
of great interest in evolutionary biology, as well as in
ecological- and population- genetics studies (e.g.
Futuyma 1998). Genetic variation within and between
plant populations is generated and maintained by
evolutionary forces such as mating system, meiotic
paring patterns, genetic drift (e.g. founder effect), gene
flow (e.g. pollen/seed dispersal), mutation and natural
selection (e.g. Hedrick 2006).
Fig. 5 A A principal coordinate (PCo) plot of pairwise
individual genetic distances between C. judaicum populations
(green) and C. pinnatifidum (blue). B Pair-wise geographical
versus genetic distances between Israeli wild C. judaicum
populations and Turkish C. pinnatifidum populations. C Repre-
hensive photo of leaf morphology of the two species. (Color
figure online)
184 Genet Resour Crop Evol (2015) 62:177–188
123
Ben-David and Abbo (2005) documented pheno-
logical variation between and within C. judaicum
populations in Israel. These authors later demonstrated
that this variation reflects heritable ecotypic variation
that is presumably selected for by environmental
determinants (Ben-David et al. 2010). The present
study, however, is the first report of DNA allelic
variation between and within wild annual Cicer
populations. The microsatellite markers used in this
study were originally developed for domesticated
chickpea C. arietinum. Consequently, we had to
screen more than 80 microsatellites before identifying
six markers capable of exposing polymorphism in all
three wild species. Still, the PCoA based on the allelic
variation in the studied materials resulted in clear
separation between the three Cicer species. Indeed,
low level of admixture was apparent based on the
STRUCTURE analysis (Fig. 4). This pattern was
observed despite the fact that only 18 % of the overall
allelic variation in the data set was accounted for by
differences between the three studied species
(Table 3).
Considering the unique ecology and morphology of
C. cuneaum compared with all other wild annual Cicer
taxa its genetic distinctness in terms of microsatellite
allelic variation is of no surprise. However, the clear-
cut separation obtained between C. pinnatifidum and
C. judaicum is very important. Over the years, the
independent taxonomic status of C. judaicum was
questioned. For example, Zohary (1972) considered C.
judaicum as a variant of C. pinnatifidum and com-
mented that individuals showing intermediate mor-
phology can be observed. In addition, van der Maesen
(1972) reported that the two species were found in
close proximity in the Damascus basin in Syria,
thereby attesting to (at least partial) overlap in their
eco-geographic affinities.
Our work experience with these two Cicer species
is entirely different. These two species have distinct
foliar morphological features that enable easy classi-
fication of both living and herbarium material
(Fig. 5C). The number of leaflets is larger in C.
judaicum while leaflet dentation is more pronounced
in C. pinnatifidum (Fig. 5C). In addition, C. judaicum
flowers are smaller and have a dull pink coloration as
compared with the larger and purple C. pinnatifidum
flowers. Over the years we have followed hundreds of
C. judaicum populations in Israel, about two dozens of
C. judaicum populations in Jordan, and several dozens
of C. pinnatifidum populations in southeastern Turkey.
Never have we observed any intermediate types
growing in any of the visited populations, neither did
we see any such types among several dozens of Cicer
specimen deposited in the herbarium of the Hebrew
University of Jerusalem. Moreover, our field observa-
tions and allelic variation analyses are in full accord
with the hybridization experiments conducted by
Ladizinsky and Adler (1976) and Abbo et al. (2011).
Namely, in both studies several reproductive barriers
were observed between C. judaicum and C. pinnatifidum
including abnormal floral morphology in the F1
hybrids and high degree of sterility among F1BC1
progeny (therein). Hence, the reproductive barriers
that maintain the distinct genetic integrity of C.
judaicum and C. pinnatifidum as separate biological
species are in full accord and indeed reflected in the
distinct microsatellite allelic pattern documented in
these two Cicer species. Unfortunately, we are unable
to inspect the Cicer populations in the Damascus
basin, however, the strong reproductive barriers
between these two species suggest that even if they
grow in close proximity the likelihood of successful
hybridization and introgression are slim.
Domesticated chickpea is a predominately self-
fertilizing species (Abbo et al. 2009). We are unaware
of studies documenting the reproductive biology of
wild Cicer, however, the size of the flowers in some
species and the observations of insects visits in wild
populations would suggest that the pattern observed
for the cultigen may not apply to all wild taxa. In the
current study, despite the small number of analysed
loci, we were able to pick up and identify heterozy-
gosity in certain accessions at certain loci.
Many authors consider microsatellite loci to be
environmentally neutral (e.g. Ellegren 2004). How-
ever, despite the relatively small number of studied
microsatellite loci, our PCoA was capable to differ-
entiate between Turkish C. pinnatifidum populations,
and likewise between Israeli C. judaicum populations
and between east African C. cuneatum populations.
Moreover, even on a relatively small geographic range
within Israel, the observed allelic variation enabled to
classify the accessions sampled from the different
sites. The observed north–south separation of the
Israeli C. judaicum population is of significance for
two reasons. First, it corroborates our observations and
interpretations despite the seemingly limited number
of analysed loci. Second and more important, it may
Genet Resour Crop Evol (2015) 62:177–188 185
123
suggest that these loci are not neutral but rather are
subject to a range of selection pressures. Specifically,
our results are in line with a previous work by (Frenkel
et al. 2008) that have shown that the northern Cicer
Israeli populations have a different pattern of response
to the ascochyta blight fungal pathogen Didymella
rabiei compared with the southern populations. More-
over, D. rabiei isolates sampled from the northern C.
judaicum populations have shown a distinct allelic
pattern compared with D. rabiei isolates samples from
southern Israeli Cicer populations, thereby reflecting
their geographic origin and indeed co-evolutionary
history with their wild C. judaicum host (Frenkel et al.
2010).
Ben-David and Abbo (2005) studied the vernaliza-
tion response of Israeli C. judaicum populations but
observed no clear geographic pattern, and relatively
low (ca. 10 %) phenotypic variance component within
populations. This is in contrast to the considerable
(above 50 %) within population microsatellite allelic
variation observed in the present study. One possible
explanation to this discrepancy is the fact that there is
hardly any adaptive advantage for a wide phenological
response within any single habitat. All individuals at a
certain location are by definition exposed to the same
day-length and to similar seasonal temperature pro-
files, and hence are expected to be relatively similar in
terms of their life history traits, as observed by Ben-
David and Abbo (2005). This of course is quite
different with respect to selection pressures associated
with incidental disease infection and the resulting
allelic variation in disease response loci (Frenkel et al.
2008, 2010) or with microsatellite markers that are not
necessarily linked to such phenology loci.
Implications for germplasm exploration
and conservation
An important issue concerning germplasm exploration
revolves around what could be framed as finding the
desired balance between number of sampling sites
(habitats) and the number of sampled individuals
within any such site. Considering data obtained in
previous studies on wild chickpea (Ben-David and
Abbo 2005; Ben-David et al. 2006, 2010), sampling a
large number of ecologically and geographically
diverse habitats with relatively small number of
individuals per sites seems as the preferred strategy.
However, the large within-population allelic diversity
documented in this study calls for a reconsideration of
this view. At present, we have no clue about the
adaptive value of the local within-population allelic
variation in wild annual chickpea, and this is an
important issue worth pursuing. It should be borne in
mind that the relatively narrow geographic range of
the different annual Cicer species is a challenge, and
requires that different unique habitats of each such
species be explored in order to obtain a true estimate
for the potential of wild Cicer populations as a source
for agro-eco-ecologic adaptive genetic variation. At
present stage we would cautiously suggest that similar
to the finding with wild emmer wheat (Peleg et al.
2005, 2008) conservation of large rich populations
might be more rewarding a strategy than preservation
of small thin populations of annual Cicer. Clearly,
further work is required before more firm conclusions
could be drawn in this context.
In conclusion, the patchy distribution of wild Cicer
species (e.g. Ben-David et al. 2006), the relatively
small number of seeds produced per plant in the wild,
and the relatively high allelic variation within popu-
lations is a unique but highly vulnerable situation in
terms of germplasm conservation. This is because any
land development or construction within the natural
range of these species may wipe out unique local
genetic combinations. When taken together with the
extensive civil construction projects and agricultural
development that currently take place in many Turkish
provinces this situation calls for urgent deployment of
strict knowledge based conservation strategies (Abbo
et al. 2008).
Acknowledgments The authors are grateful to Prof. Tzion
Fahima, University of Haifa, Israel for providing the
infrastructure required for the microsatellite analyses. We
acknowledge the assistance of Dr. Roi Ben-David, Dr. Omer
Frenkel, Dr. Judith Lichtenzveig and Dr. Ruth vanOss.
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