RESEARCH ARTICLEOpen Access
A BAC/BIBAC-based physical map of chickpea,
Cicer arietinum L
Xiaojun Zhang1,6, Chantel F Scheuring1, Meiping Zhang1,2, Jennifer J Dong1, Yang Zhang1, James J Huang1,
Mi-Kyung Lee1, Shahal Abbo3, Amir Sherman4, Dani Shtienberg4, Weidong Chen5, Fred Muehlbauer5,
Background: Chickpea (Cicer arietinum L.) is the third most important pulse crop worldwide. Despite its
importance, relatively little is known about its genome. The availability of a genome-wide physical map allows
rapid fine mapping of QTL, development of high-density genome maps, and sequencing of the entire genome.
However, no such a physical map has been developed in chickpea.
Results: We present a genome-wide, BAC/BIBAC-based physical map of chickpea developed by fingerprint analysis.
Four chickpea BAC and BIBAC libraries, two of which were constructed in this study, were used. A total of 67,584
clones were fingerprinted, and 64,211 (~11.7 ×) of the fingerprints validated and used in the physical map
assembly. The physical map consists of 1,945 BAC/BIBAC contigs, with each containing an average of 28.3 clones
and having an average physical length of 559 kb. The contigs collectively span approximately 1,088 Mb. By using
the physical map, we identified the BAC/BIBAC contigs containing or closely linked to QTL4.1 for resistance to
Didymella rabiei (RDR) and QTL8 for days to first flower (DTF), thus further verifying the physical map and
confirming its utility in fine mapping and cloning of QTL.
Conclusion: The physical map represents the first genome-wide, BAC/BIBAC-based physical map of chickpea. This
map, along with other genomic resources previously developed in the species and the genome sequences of
related species (soybean, Medicago and Lotus), will provide a foundation necessary for many areas of advanced
genomics research in chickpea and other legume species. The inclusion of transformation-ready BIBACs in the map
greatly facilitates its utility in functional analysis of the legume genomes.
Chickpea (Cicer arietinum L., 2n = 2x = 16) is the third
most important pulse crop in the world [1,2]. It is often
used as a drought-tolerant crop grown in the drier
regions of India, East Africa, the Mediterranean basin,
and the Americas. In the cereal-based crop rotation sys-
tems, chickpea is used as a rotation crop to break dis-
ease cycles, fix atmospheric nitrogen and improve soil
fertility. It is a major source of high-quality proteins and
starch for a large Asian vegetarian population and a
health-conscious food in developed countries. Therefore,
understanding its genome is of significance not only
economically, but also for annotation, functional and
evolutionary analysis of legume genomes, particularly
the underlying mechanisms of plant drought tolerance
and nitrogen fixation. The most significant constraints
to its production are Ascochyta blight caused by Dydi-
mella rabiei and cultivar adaptation to terminal drought
and high temperature during pod set [3,4]. Ascochyta
blight is a destructive, devastating disease of chickpea
and may result in total crop loss [3,5]. Hence, combin-
ing Ascochyta blight resistance with early flowering is
crucial to increasing grain yield and quality of chickpea
in various rain-fed environments [6,7]. However, it has
been proven difficult, without assistance of modern
molecular tools, to breed for high grain yield and quality
combining both Ascochyta blight resistance and early
To facilitate complex trait breeding, and to clone and
characterize the genes and loci of agronomic importance
* Correspondence: email@example.com
1Department of Soil and Crop Sciences, Texas A&M University, College
Station, Texas 77843-2474, USA
Full list of author information is available at the end of the article
Zhang et al. BMC Genomics 2010, 11:501
© 2010 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
in chickpea, some molecular tools have been developed.
These include several molecular genetic maps [8-13],
identification of quantitative trait loci (QTL) for a num-
ber of agronomic traits [4,9,13-19], several large-insert
bacterial artificial chromosome (BAC) and plant-
transformation-competent binary BAC (BIBAC) libraries
[20,21], and limited tissue- or treatment-specific
expressed sequence tags (ESTs) [22-24]. Nevertheless,
significant efforts will be needed to make the tools suita-
ble for chickpea breeding. QTL for controlling resis-
tance to Didymella rabiei (RDR) were mapped to five of
its eight chromosomes and QTL for days to first flower
(DTF) to four chromosomes [9,18]. Unfortunately, the
markers for most of the QTL are not sufficiently close
to the loci for their effective use in marker-assisted
selection for the traits.
Genome-wide integrative physical mapping has been
used in several species to effectively integrate genomic
tools for marker-assisted breeding, high-resolution map-
ping and positional cloning of genes and QTL [25,26].
Simultaneously, physical maps will also provide desirable
platforms for advanced EST analysis, genome sequen-
cing [27,28] and comparative genomics. Despite these
advantages, a genome-wide physical map has not been
developed for chickpea. Lack of such genomic tools and
infrastructure for the species has not only limited the
deeper analysis of its agronomic genes and QTL, but
also prevented genomic information flow to chickpea
from the model or related legumes such as Medicago,
Lotus and soybean whose genomes have been sequenced
[29-32]. Therefore, a comprehensive platform is needed
to rapidly access the QTL for RDR, DTF and many
other agronomic traits, and advance chickpea and
related legume genomics research. In comparison with
other major crops such as wheat, maize, soybean, cotton
and tomato, chickpea has a relatively smaller genome
size (740 Mb/1C). The size of the chickpea genome is
comparable to those of the non-cultivated legume mod-
els, Medicgo [540 Mb/1C, ] and Lotus [480 Mb/1C,
]. The relatively small genome size of chickpea facili-
tates physical mapping and high-throughput sequencing
of its genome, thus facilitating annotation, functional
analysis and evolutionary investigation of the legume
In this study, we constructed one new BAC library
and one new BIBAC library from the chickpea cultivar,
Hadas, using different restriction enzymes and vectors
from those used in the existing chickpea libraries. From
the two new BAC and BIBAC libraries, and the two
BAC and BIBAC libraries of the cultivar previously
reported , we constructed a genome-wide, BAC/
BIBAC-based physical map of the chickpea genome.
Using the physical map, we identified the BAC/BIBAC
contigs containing or closely linked to two QTL, one
controlling RDR and the other controlling DTF. These
results, especially the genome-wide BAC/BIBAC physical
map, will provide a framework for many aspects of geno-
mics and genetics research of the species and finally, for
sequencing its genome by using the next-generation high-
throughput genome sequencing technology.
New BAC and BIBAC library construction
In a previous study , we constructed one BAC
library (Chickpea-CHI) and one BIBAC library (Chick-
pea-CBV) for chickpea cv. Hadas using Hind III and
BamH I, respectively (Table 1). To facilitate quality phy-
sical map development of the genome, we constructed
one new BAC library (Chickpea-CME) and one new
BIBAC library (Chickpea-CHV) from the nuclear DNA
of the same genotype partially digested with MboI and
HindIII, respectively (Table 1). The chickpea-CME BAC
library contains 22,272 clones. Analysis of 84 random
clones showed that it has an average insert size of 130
kb and provides a 4.0-fold coverage of the chickpea hap-
loid genome. The chickpea-CHV BIBAC library contains
38,400 clones. Analysis of 100 random clones showed
that it has an average insert size of 142 kb and provides
a 7.5-fold coverage of the chickpea genome (Figure 1).
For both libraries, less than 5% of their clones contain
no inserts of chickpea DNA. We screened 36,864 clones
of the chickpea-CHV BIBAC library using the chloro-
plast genes, ndhA, rbcL and psbA, as probes. A total of
49 positive clones were obtained, suggesting that
approximately 0.13% (49/36,864) of the new libraries
clones were derived from chloroplast DNA. This num-
ber is close to the chickpea BAC and BIBAC libraries
that we constructed previously, 0.3% of whose clones
were derived from chloroplast DNA . Therefore, the
two new libraries (11.5 ×), along with the two BAC
(Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries
(6.1 ×) constructed previously , provide comprehen-
sive and high-quality source libraries for construction of
the chickpea physical map [25,35].
Our previous studies showed that BAC and BIBAC fin-
gerprints generated with different restriction enzyme
combinations may result in different quality BAC/
BIBAC physical maps [36,37]. Therefore, we first tested
twenty-four 3-, 4- and 5-enzyme combinations of Hind
III, BamH I, EcoR I, Xba I, Xho I, and Hae III using 32
BACs randomly selected from the Chickpea-CHI library.
In the combinations, only the ends produced by Hind
III, BamH I, EcoR I, Xba I or Xho I digestion are labeled
with a fluorescent dye (NED-ddATP or HEX-ddATP).
Hae III is used to further digest the labeled fragments to
sizes that allow separation on a capillary sequencer.
Zhang et al. BMC Genomics 2010, 11:501
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According to the criteria that there are no partial diges-
tion, no star activity, an average of 35 - 70 bands per
clone and a relatively even size distribution of the bands
in a window ranging from 35 - 500 bases in a single-
tube single-step digestion-labeling system, the enzyme
combination of Hind III/Xba I/Xho I/Hae III was
selected for generation of BAC and BIBAC fingerprints
for the chickpea genome physical mapping.
A total of 67,584 (12.4-fold) BAC and BIBAC clones
were fingerprinted from the four chickpea BAC and
BIBAC libraries (Table 1), with 1.4-fold to 5.6-fold
clones from each library, or 5.4-fold coverage BACs and
7.0-fold coverage BIBACs. Of the clones, the fingerprints
of 64,211 (95.0%) clones were validated and used in the
physical map assembly. The validated fingerprints repre-
sented approximately 11.7-fold of the chickpea genome
(Table 2). Each clone had an average of 39.2 restriction
fragment bands in the window of 35 - 500 bases, with a
range from 5 to 192 bands per clone. According to our
previous studies [36,38], the genome coverage of 11.7
fold should be sufficient to allow the assembly of a
high-quality genome-wide physical map of the chickpea
Determination of tolerance and cutoff values
The FingerPrinted Contig (FPC) program was used to
assemble the contig map from the BAC and BIBAC fin-
gerprints, by which two parameters, tolerance and cut-
off, are crucial to quality contig assembly. Tolerance is
the window size in which two restriction fragments are
considered as the same or equivalent bands. To deter-
mine the tolerance value to be used for the contig
assembly, we selected the four pECBAC1 vector frag-
ments generated with the enzyme combination (Hind
III/Xba I/Xho I/Hae III) of sizes 60, 161, 230 and 375
bases in the range from 35 to 500 bases released from
200 BACs randomly selected from the fingerprint data-
set. We calculated the mean size deviation of each of
the fragments. At a 95% confidence interval, the mean
deviations of the four vector fragments, 60, 161, 230 and
375 bases, were 0.56, 0.48, 0.43 and 0.55 base, respec-
tively, with an average of 0.505 bases. Therefore, a toler-
ance value of 5 (0.5 × 10) was chosen as the candidate
tolerance value for FPC contig assembly, with all frag-
ment sizes of each fingerprint multiplied by 10 .
Furthermore, the tolerances of 1 - 7 were tested using
the entire fingerprint dataset to determine the para-
meters suitable for contig assembly. On the basis of
these results, a tolerance of 5 was finally selected for the
The cutoff value is a threshold of the probability that
fingerprint bands match by coincidence. Lowering its
value would increase contig assembly stringency, and
therefore, increase the likelihood that overlapping BAC
clones are truly overlapping. To determine the cutoff
value for the chickpea physical map assembly, we tested a
series of cutoff values ranging from 1e-4 to 1e-30 and a
tolerance of 5 for automatic contig assembly. The resul-
tant numbers of contigs, singletons, and Q (question-
able)-clones were analyzed. At higher stringencies (1e-14
to 1e-30), “chimeric” contigs were split and Q-clones
were reduced, but the number of singletons increased
drastically. At lower stringencies (1e-4 - 1e-8), a smaller
number of or larger contigs were obtained, but a larger
number of clones were fallen in the category of Q-clones.
The relationship among the three factors is plotted in
Figure 2, from which it was apparent that a cutoff value
Table 1 BAC and BIBAC libraries of chickpea cv. Hadas constructed and used in chickpea genome physical mapping
No. of clones
Libraries No. of clones Genome coverage Insert size (kb)VectorType Enzyme
* The average insert size of fingerprinted clones was estimated to 135 kb.
Figure 1 BIBACs randomly selected from the chickpea-CHV
BIBAC library (see Table 1). BIBAC DNA was isolated, digested
with Not I and electrophoresed on a pulsed-field gel.
Zhang et al. BMC Genomics 2010, 11:501
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of approximately 1e-12 resulted in reasonable low num-
bers of all three factors, suggesting the desirable quality
contig assembly. While the number of Qs was 18.5%
(11868/64211) when automatic contigs was initially
assembled at the cutoff of 1e-12, it was reduced to 3.0%
(1092/64211) after subsequent Dqer at lower cutoff
values (1e-13, 1e-14 and 1e-15) that allows re-analysis of
the initial assembly step-wisely to reduce the Q-clones in
the contigs. We, therefore, chose 1e-12 as the cutoff
value for the initial assembly of the physical map.
Contig map assembly
Contigs were assembled from the fingerprint data of the
clones using the computer program FPC version 8.9.
A total of 1,945 contigs were assembled from the
validated 64,211 BAC and BIBAC fingerprints at a cutoff
value of 1e-12 and a tolerance of 5, followed by Dqer,
end-to-end merging and end-to-singleton merging at
progressively lower stringencies (Additional files 1, 2, 3,
4 and 5; for example, see Figure 3). The 1,945 contigs
contained a total of 55,029 clones whereas the remain-
ing 9,182 clones remained as singletons.
Table 2 summarizes the resultant BAC/BIBAC contig
map of the chickpea genome (Additional files 1, 2, 3, 4
and 5). The contigs each contained from 2 to over 199
clones, with the largest 225 (11.6%) contigs containing
56.0% (31,102 clones) of the total 55,029 clones. Each
contig contained an average of 28.3 clones and spanned
an average length of 559 kb, with a range of 200 - 6,107
kb. The 1,945 contigs contained a total of 265,614 con-
sensus bands (CBs), thus representing a total length of
approximately 1,087,954 kb. On average, each clone
contributed 4.8 unique CBs to the assembly, or approxi-
mately 19.7 kb (14.6% of the 135-kb average clone insert
size) to the physical length of the contig assembly.
Assessment of the contig map and identification of the
contigs containing or closely linked to RDR and DTF QTL
Several approaches were used to assess the reliability of
the chickpea contig map. First, we assembled automatic
contigs from the fingerprints using two different contig
building strategies and compared the resultant contigs,
as described by Wu et al. . A sample of 100 random
contigs from the two assemblies was analyzed compara-
tively. The result showed that 99.0% of the automated
contigs resulted from the two strategies was completely
consistent in both clone content and order.
In the second approach, we assembled contigs from
the clones of the Chickpea-CME and, Chickpea-CHV
Table 2 Statistics of the BAC/BIBAC physical map of the chickpea genome
Total number of BAC clones fingerprinted
Valid fingerprints for FPC assembly
Average number of bands per BAC
Total number of contigs assembled
No. of contigs containing >199 clones
No. of contigs containing 100-199 clones
No. of contigs containing 50-99 clones
No. of contigs containing 25-49 clones
No. of contigs containing 10-24 clones
No. of contigs containing 3-9 clones
No. of contigs containing 2clones
Clones contained in the 1,945 contigs
Average BAC clones per contig
Average estimated size per contig (kb)
Number of singletons
Total number of CB bands included in the contigs
Total physical length of assembled contigs (kb)
12.4 × genome coverage
11.7 × genome coverage
10.0 × genome coverage
Figure 2 Plot of cutoff values versus the numbers of contigs,
singletons, and Q clones for physical map assembly. The cutoff
value that yielded the lowest numbers for all three factors, contigs,
singletons and Q clones, was selected for the chickpea genome
physical map assembly.
Zhang et al. BMC Genomics 2010, 11:501
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libraries, separately. We randomly selected 100 contigs
from the contigs assembled from each library and com-
pared them with their corresponding contigs in the phy-
sical map. We found that 96% and 95% of the contigs
were shown to be in complete agreement with the con-
tigs of the chickpea physical map in terms of both clone
content and order.
In the third approach, we randomly selected five con-
tigs from the chickpea physical map, fingerprinted their
BACs and BIBACs with a different enzyme combination,
BamHI/HindIII/XhoI/HaeIII, and then reassembled
them into the contigs. As a result, the same contigs as
those selected from the physical map were reassembled.
Finally, we verified the accuracy of the contig map by
screening the source BIBAC clones of the chickpea-
CHV library with SSR primers flanking the RDR and
DTF QTL previously mapped to Linkage Groups LG4
and LG8 (Figure 4) and to other regions of the chickpea
genetic maps [9,18,21]. If the physical map contigs were
assembled properly, the positive clones of a single-copy
SSR primer pair should be located to a single BAC/
BIBAC contig of the map. We screened two high-
density filters of the chickpea-CHV library containing a
total of 36,864 double-spotted clones, of which 27,724
clones were used in the map assembly. A total of 16
SSR primer pairs were used as probes for the library
screening (Additional file 6). Of the 16 SSR primer
pairs, seven, H1G20, H1C092, TA3, H1A19, H1H22,
H1C9-2 and H2J2, were clearly derived from multiple-
copy sequences, as indicated by Southern analysis and
the library screening result that they hybridized to 47 -
672 positive clones, respectively. The remaining nine
SSR primer pairs were shown to be single- or low-copy
by Southern analysis, with each hybridizing to 2 - 8
positive clones, being within the range of expected 6.5
positive clones per single-copy probe. The positive
clones of each of all nine single- or low-copy SSR pri-
mer pairs were found to be assembled into a single con-
tig, further suggesting that the physical map contigs are
assembled properly. Of the nine single- or low-copy
Figure 3 Example of the chickpea physical map contigs. The contig3270 (ctg3270) contains 54 clones and spans 335 CB units, thus having a
physical length of 955 kb. The two highlighted clones, V066I24 and V053O04, are the positive clones of the SSR marker TA2 flanking the RDR QTL4.1.
Zhang et al. BMC Genomics 2010, 11:501
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marker-containing contigs, contig71 (ctg71) was located
to the region of DTF QTL8 and spanned a physical
length of 2,939 kb, and contig3270 (ctg3270) and con-
tig2831 (ctg2831) were located to the region of RDR
QTL4.1 and spanned a physical length of 955 kb and
1,036 kb, respectively.
We have developed a genome-wide, BAC/BIBAC-based
physical map of the cultivated chickpea, C. arietinum,
from cultivar Hadas. The map consists of 55,029 BAC
and BIBAC clones assembled into 1,945 contigs. Each
contig contains 2 to >199 clones with an average of 28.3
clones per contig, spanning from 200 to 6,107 kb, with
an average physical length of 559 kb (Table 2). These
contigs collectively span approximately 1,088 Mb, larger
than the 740-Mb estimated genome size of chickpea by
approximately 47%. The longer physical length of the
physical map contigs than the estimated genome size
may be attributed to one or more of the following fac-
tors. Overlaps existed between contigs, but they were
too small to be detected or merged unambiguously by
using the FPC program alone; the average insert size
of the source clones was over-estimated; and/or the gen-
ome size of chickpea was underestimated. According to
previous studies [40-43], the undetected overlaps among
Figure 4 BAC/BIBAC contigs containing the positive clones of the SSR primer pairs flanking the QTL for resistance to Didymella rabiei
(RDR) and days to first flower (DTF). The clones highlighted in bold face were located to contigs and the remaining clones are not analyzed
in this study. The QTL were genetically mapped using the phenotypic data collected at one or two locations (Gilat and Massuot, Israel) .
Zhang et al. BMC Genomics 2010, 11:501
Page 6 of 10
the contigs likely account for much of the discrepancy.
As described above, the current physical map has an
average overlap of 85.4% (1 - 14.6%) of clone average
insert size (135 kb), indicating that the clones having an
overlap of <85.4% insert size might be assembled into
separate contigs in this study. The existence of overlaps,
even though they were undetected by the PFC program
under the conditions used in this study, will allow the
contigs to be further merged into larger ones. However,
further unambiguously merging these contigs will
require additional supporting evidence in addition to
their fingerprint similarities, such as anchoring of the
contigs to the genetic map by mapped DNA markers
and to the related genome sequences using the end
sequences of the source BACs and BIBACs as anchors.
The quality of the chickpea physical map is sufficient
for its use in different aspects of chickpea genomics
research. The clones equivalent to 10.0 x of the chickpea
genome (55,029 clones) were assembled into the physical
map contigs (Table 2). According to our previous studies
[36,38,40-44], this number of clones is adequate for con-
struction of a high-quality physical map of the chickpea
genome, though it is much smaller than those used for
the physical maps of human and bovine developed pre-
viously [45,46]. Moreover, the source clones of the map
were randomly selected from four BAC and BIBAC
libraries constructed with three restriction enzymes
(Hind III, BamH I and Mbo I) in three vectors (pIndigo-
BAC5, pECBAC1 and pCLD04541) (Table 1). Although
the distribution of the restriction sites of any particular
restriction enzyme is uneven throughout a genome, the
use of the clones constructed with three different restric-
tion enzymes in three vectors would further increase the
actual coverage of the resultant map for the whole gen-
ome of chickpea . The accuracy of the contig physical
map was confirmed using different approaches, including
independent contig building methods, different finger-
printing methods and SSR marker hybridization. While
the number of the DNA makers used in the verification
of the physical map contigs was limited, the employment
of multiple approaches that were previously proven
[40,43] to the research purpose provided evidence on the
reliability of the map assembly. The results of all of the
approaches together have verified that the physical map
contigs were assembled properly.
The physical map provides a powerful foundation for
many aspects of advanced genome research of chickpea
and related legume species. Using the physical map, we
have identified three contigs, ctg3270, ctg2831 and
ctg71, that likely contain or are closely linked to the
RDR QTL4.1 contributing to 14.4% of Ascochyta blight
resistance and the DTF QTL8 contributing approxi-
mately 4 days to earlier flowering (Figure 4). The
ctg3270 spans 955 kb, the ctg2831 spans 1,036 kb and
the ctg71 spans 2,939 kb in physical length. Assuming
that the average physical/genetic ratio of the chickpea
genome is from 300 kb/cM [9,12] to 630 kb/cM ,
the ctg3270 may span approximately 1.5 - 3.2 cM, the
ctg2831 may span 1.6 - 3.4 cM, and the ctg71 may span
4.7 - 9.8 cM. Since the RDR QTL4.1 region spans
2.1 cM and the DTF QTL8 region spans within 2.6 cM
, these genetic distances may cover the entire
regions of the RDR QTL4.1 and DTF QTL8, respec-
tively. Therefore, the contigs have provided powerful
tools for fine mapping and cloning of the QTL. Further-
more, since the physical map was constructed from
both BACs and Agrobacterium-mediated plant transfor-
mation-ready BIBACs, the inclusion of the BIBACs will
further facilitate cloning and applications of the QTL
and other agronomic genes of interest, and promote
functional analysis of the chickpea genome by genetic
transformation at the whole genome level [[47-49];
Chang Y-L, Chuang H-W, Meksem K, Wu F-C, Chang
C-Y, Zhang M and Zhang H-B, submitted].
The physical map represents the first generation of the
physical map of chickpea. Although its utility in isola-
tion of BAC/BIBAC contigs containing or closely linked
to QTL has been preliminarily demonstrated in this
study, further efforts remain to optimize its effectiveness
for whole genome sequencing and sequence assembly,
high-density mapping of the genome (e.g., SNP map-
ping), and comparative genome analysis with related
genome sequences such as those of soybean, Medicago
and Lotus. Efforts will be also needed to integrate the
contig map with the developed genetic maps of the spe-
cies and to locate all mapped genes and QTL to the
BAC/BIBAC contigs, as was done for RDR QTL4.1 and
DTF QTL8 in this study. These include, but are not lim-
ited to, sequencing of the source BAC and BIBAC ends,
alignment of the BAC/BIBAC contigs along the
sequenced soybean, Medicago and/or Lotus genomes,
targeted (e.g., the physical map BAC/BIBAC end
sequences are used as the targeted sites) development of
DNA markers and high-density genome maps, and
screening of the source BACs and BIBACs using
mapped DNA markers, especially those flanking the
genes and QTL mapped (e.g., Figure 4). These experi-
ments will not only allow facilitating subsequent sequen-
cing, sequence assembly and sequence annotation of the
chickpea and other legume genomes, and deciphering
the evolution of the legume genomes as a whole, but
also further merging the contigs, thus significantly
enhancing the physical map.
It has been documented that a genome-wide physical
map is of significance for advanced genome research.
We have developed a genome-wide quality BAC/BIBAC
Zhang et al. BMC Genomics 2010, 11:501
Page 7 of 10
physical map of chickpea and using the map, identified
three large contigs containing or closely linked to QTL
contributing to Ascochyta blight resistance and earlier
flowering in chickpea. This map, along further improve-
ments as discussed above, will greatly advance the geno-
mics and genetics research of chickpea and related
legumes. The inclusion of plant transformation-ready
BIBACs in the physical map will further promote its uti-
lity in cloning of genes and QTL of interest and func-
tional analysis of the chickpea and related genomes.
Source BAC/BIBAC libraries
Two BAC and two plant transformation-ready BIBAC
libraries of chickpea cv. Hadas, named Chickpea-CHI
(BAC), Chickpea-CBV (BIBAC), Chickpea-CME (BAC)
and Chickpea-CHV (BIBAC), were used to construct the
chickpea physical map. The Chickpea-CHI and Chick-
pea-CBV libraries were previously constructed from the
nuclear DNA of chickpea cv. Hadas with Hind III and
BamH I in the BAC vector pIndigoBAC5 and BIBAC
vector pCLD04541, respectively, and have average insert
sizes of 121 kb and 145 kb . The two new libraries,
Chickpea-CME and Chickpea-CHV, were constructed
from the nuclear DNA of chickpea cv. Hadas partially
digested with Mbo I and Hind III in the BAC vector
pECBAC1 and BIBAC vector pCLD04541, respectively.
The megabase-sized nuclear DNA isolation and library
construction were described previously [35,50-52].
BAC or BIBAC clones arrayed in 384-well microplates
were inoculated into 96-deep well plates containing
1.0 ml TB (Terrific Broth) medium with proper antibio-
tics using a 96-pin replicator (BOEKEL, Feasterville, PA,
USA). Thus, the clones of a 384-well plate were inocu-
lated into four 96-deep well plates. To ensure clone
tracking, the clones were always inoculated with the
A01 pin of the 96-pin replicator aligned with the A01
position of the 384-well plate as the 96-deep well plate
A, followed by the A01 pin of the replicator aligned
with the B01, A02 and B02 of the 384-well plate as the
96-deep well plates B, C and D, respectively. The 96-
deep well plates were covered with air-permeable seals
(Excel Scientific, Wrightwood, CA, USA) and incubated
in an orbital shaker at 320 rpm, 37°C for 20-22 h.
The overnight cultures were centrifuged at 2,500 g for
10 min in a Beckman bench-top centrifuge to harvest the
bacterial cells. BAC or BIBAC DNA was isolated using a
modified alkaline lysis method , dissolved in 15 μl TE
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) with
16 μg/ml RNase (Ambion, USA) and stored at -20°C
before use. The DNA was digested and end-labeled in a
reaction containing reaction buffer (50 mM NaCl, 10 mM
Tris-HCl, 10 mM MgCl2, 1.0 mM dithiothreitol, pH 8.0),
6.0 μM each dTTP, dCTP and dGTP, 1.0 μg/μl BSA, 1 U
each of Hind III, Xba I, Xho I, and HaeIII (New England
Biolabs, Ipswich, MA, USA), 0.3 U Taq FS and 6.0 μM
HEX-ddATP or NED-ddATP. The reaction was incubated
at 37°C for 2 h, followed by further incubation at 65°C for
45 min. The clone DNAs labeled with different fluorescent
dyes (HEX-ddATP or NED-ddATP) were combined, pel-
leted, washed, dried and dissolved in a mixture of 9.8 μl of
Hi-Di formamide and 0.2 μl of the internal GeneScan-500
Rox size standard (Applied Biosystems, Foster City, CA,
USA). The DNA was denatured at 95°C for 3 min, cooled
on ice and then subjected to analysis on the ABI 3100
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA) using the default GeneScan module (36-cm array,
The fragment sizes in each BAC fingerprint profile were
collected by the ABI Data Collection program. The data
of the ABI 3100 Genetic Analyzer were processed using
the software package ABI-ExportTabularData  and
SeqDisplayer (unpublished). The data were transformed
using an automatic algorithm contained in the Seq-
Displyer program into “bands” files. Several quality
checks were applied to the fingerprints, with sample-
empty wells being removed, fingerprints with fewer than
5 band peaks removed, the background peaks identified
and removed, the off-scale bands with peak heights
greater than 6,000 removed, and the vector band peaks
removed. Only the band peaks falling between 35 and
500 bases were used for the contig assembly.
Physical map contig assembly and manual editing
The program FPC version 8.9  was used to assemble
the clone fingerprint data into contigs. A series of tests
were conducted, in which the fingerprints of a set of
overlapping clones were analyzed using different toler-
ances (from 1 to 7) and cutoffs (1e-4 to 1e-30). On the
basis of these tests, a fixed tolerance of 5 and a cutoff of
1e-12 were selected for automatic contig assembly.
Library screening and assessment of physical map quality
Four sets of high-density filters of the Chickpea-CHV
BIBAC library, with each set containing 36,864 double-
spotted clones, were prepared according to Lee et al.
. Of these clones, 27,724 were used in the physical
map contig assembly. Sixteen pairs of SSR primers were
selected from published chickpea linkage maps [9,18,21]
and then purchased from Sigma Genosys (Woodlands,
TX, USA). The SSR primer oligos were end-labeled with
32P-ATP (Amersham, Piscataway, NJ, USA) at 37°C for
1 h, the unincorporated nucleotides removed using a
Sephadex G50 column, the probes denatured at 95°C for
Zhang et al. BMC Genomics 2010, 11:501
Page 8 of 10
10 min, and then added to the hybridization buffer con-
taining the high-density BIBAC filters. Hybridization was
performed at 40°C for 18 h, and the filters were washed
three times in 0.1% SDS, 0.5× SSC at 40°C, 30 min each
wash, and exposed to X-ray film at -80°C for 2-5 days.
Additional file 1: Shows the contigs constituting the physical map
of the chickpea genome 1 (ctg5-904).
Additional file 2: Shows the contigs constituting the physical map
of the chickpea genome 2 (ctg904-1902).
Additional file 3: Shows the contigs constituting the physical map
of the chickpea genome 3 (ctg1903-2845).
Additional file 4: Shows the contigs constituting the physical map
of the chickpea genome 4 (ctg2849-3727).
Additional file 5: Shows the contigs constituting the physical map
of the chickpea genome 5 (ctg3731-5052).
Additional file 6: Shows the positive clones and associated contigs
of SSR primers identified from 36,864 clones of the chickpea-CHV
BIBAC library (384-well microplates 1 - 96).
BAC: bacterial artificial chromosome; BIBAC: plant-transformation-competent
binary BAC; QTL: quantitative trait locus; RDR: resistance to Didymella rabiei;
DTF: days to first flower; SSR: simple sequence repeat; CB: consensus band;
EST: expressed sequence tag.
Authors thank Yen-Hsuan Wu and Yun-Hua Liu for their kind assistance; and
Guenter Kahl, Peter Winter, Paul Taylor and David A. Hoisington for their
kind support for the project development. This research was supported by
Research Grant Award No.: US-3870-60C from BARD, the United States-Israel
Binational Agricultural Research and Development Fund.
1Department of Soil and Crop Sciences, Texas A&M University, College
Station, Texas 77843-2474, USA.2College of Life Science, Jilin Agricultural
University, Changchun, Jilin 130118, China.3Institute of Plant Science and
Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, 76100,
Israel.4The Volcani Center, P.O. Box 6, Bet-Dagan, 50250, Israel.5USDA-ARS
and Department of Crop and Soil Sciences, Washington State University,
Pullman, WA 99164-6434, USA.6The Key Laboratory of Experimental Marine
Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao
XZ conducted Chickpea-CHV library construction, fingerprinting, data
processing, contig assembly, library screening, and manuscript writing; CFS
constructed the Chickpea-CME BAC library; JJD arrayed the Chickpea-CHV
library and provided assistance in sequencer operation; MZ and YZ
conducted library hybridization; JJH provided software assistance; M-KL
provided assistance for fingerprinting and FPC; SA, AS, DS, WC and FM
participated in the experimental design and manuscript preparation; H-BZ
designed the project, supervised its execution and wrote the manuscript. All
authors read and approved the final manuscript.
Received: 7 January 2010 Accepted: 17 September 2010
Published: 17 September 2010
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Cite this article as: Zhang et al.: A BAC/BIBAC-based physical map of
chickpea, Cicer arietinum L. BMC Genomics 2010 11:501.
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