Characterization of Three Maize Bacterial Artificial
Chromosome Libraries toward Anchoring of the Physical
Map to the Genetic Map Using High-Density Bacterial
Artificial Chromosome Filter Hybridization1
Young-Sun Yim, Georgia L. Davis*, Ngozi A. Duru, Theresa A. Musket, Eric W. Linton,
Joachim W. Messing, Michael D. McMullen, Carol A. Soderlund, Mary L. Polacco, Jack M. Gardiner, and
Edward H. Coe Jr.
Department of Agronomy, University of Missouri, 1–87 Agriculture, Columbia, Missouri 65211 (Y.-S.Y.,
G.L.D., N.A.D., T.A.M., M.D.M., M.L.P., J.M.G., E.H.C.); Waksman Institute, Rutgers, The State University of
New Jersey, Piscataway, New Jersey 08854 (E.W.L., J.W.M.); United States Department of AgricultureAgricultural Research Service, Plant Genetics Research Unit, 210 Curtis Hall, Columbia, Missouri 65211
(M.D.M., M.L.P., E.H.C.); and Plant Science Department, University of Arizona, Tucson, Arizona 85721
(C.A.S.)
Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density
filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were
evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat
sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage
␭. The results indicate that the libraries are of high quality with low contamination by organellar and ␭-sequences. The use
of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction
fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6⫻ coverage of
the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 ⫻ Mo17 genetic map
and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three
BACs. Twenty-two single-copy number core markers identified an average of 7.4 ⫾ 3.3 positive clones, consistent with the
expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute
to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.
Maize (Zea mays) has a relatively large genome size
of about 2,300 to 2,700 Mb (Arumuganathan and
Earle, 1991). The genome size of many plant species
differs as a result of variable amounts of repetitive
DNA. Repetitive sequences make up a significant
portion of the maize genome, estimated at approximately 50% to 73% (Bennetzen et al., 1998; Meyers et
al., 2001; Walbot and Petrov, 2001). In recent years,
rapid progress has been made in the rice (Oryza
sativa) and Arabidopsis genome projects, making
these organisms models for plant genome research
(Bevan et al., 1998; Mozo et al., 1999; Yuan et al.,
2000). Because of its large genome size, duplication of
genomic regions (Gaut, 2001), low percentage of
single-copy DNA, and high content of retroelements
(Meyers et al., 2001), maize is a challenging target for
genome analysis. However, the development of ge1
This work was supported by the National Science Foundation
(Plant Genome grant nos. DBI 9872655 and 9975618).
* Corresponding author; e-mail [email protected]; fax 573–
882–1469.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.013474.
1686
netic and physical map resources is making maize
genomics tractable.
Good genetic maps are an invaluable resource in
many aspects of genome research because they enable the mapping of QTL or genes without any prior
knowledge beyond phenotypic effects. They also provide a framework for anchoring the physical map.
Davis et al. (1999) constructed a maize linkage map
with 1,736 markers, including genomic and cDNA
clones, isozymes, and expressed sequence tagged
sites. A high-resolution genetic linkage map of maize
with 978 simple-sequence repeat markers was recently constructed (Sharopova et al., 2002). The intermated B73 ⫻ Mo17 (IBM) population used for the
simple-sequence repeat map has been used for additional mapping to produce a ⬎1,800-marker map that
is serving as the standard for ongoing genomics efforts (see http://www.maizemap/org). To facilitate
an understanding of the genome sequence, the gene
content, and the structure and function of the maize
genome, it is necessary to construct integrated genetic and physical maps at all levels of resolution.
Successful construction of an integrated genetic
and physical map in maize relies on the availability
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Maize Bacterial Artificial Chromosome Libraries
of deep-coverage large-insert genomic libraries.
Yeast artificial chromosome (YAC) libraries were
widely used in constructing physical maps in human
(Cohen et al., 1993) and in many other plant species,
including Arabidopsis (Grill and Somerville, 1991),
tomato (Lycopersicon esculentum; Martin et al., 1992),
and rice (Kurata et al., 1994). A maize YAC library
has been constructed and is publicly available for
genome research (Edwards et al., 1992). However,
the maize YAC library is limited in its general use
because of its high frequency of chimerism, low copy
number, and low stability of clones (Haldi et al.,
1994; Chumakov et al., 1995). Bacterial artificial chromosomes (BACs) have instead become the more popular choice for large-insert genomic libraries for
structural genome research in plants, including Arabidopsis (Choi et al., 1995; Mozo et al., 1998), rice
(Wang et al., 1995), sorghum (Sorghum bicolor; Woo et
al., 1994), tomato (Hamilton, 1997), potato (Solanum
tuberosum; Song et al., 2000), soybean (Glycine max;
Tomkins et al., 1999), and wheat (Triticum aestivum;
Lijavetzky et al., 1999). Compared with a YAC library, the BAC system has a low frequency of chimerism and a high stability of clones and is easy to
manipulate.
In the current study, we report the characterization
of three maize BAC libraries: the HindIII library
made at the Clemson University Genomics Institute
(South Carolina), and the EcoRI and the MboI libraries
made at the Children’s Hospital Oakland Research
Institute (Oakland, CA). These libraries are being
used to construct a physical map in maize and to
anchor the genetic map (Cone et al., 2002). A set of
complex repeat probes were hybridized to six highdensity BAC filters from each of the three BAC libraries, to provide information on chromosome organization and on organellar DNA content. In
addition, a second set of probes containing 90 maize
RFLP core markers was screened against the HindIII
library filters. These core markers function as bin
delimiters and provide a framework for anchoring
the BAC contigs to the IBM genetic map. The results
are integrated with maize BAC fingerprinting data
from the Arizona Genomics Institute (http://www.
genome.arizona.edu/fpc/maize) to anchor BAC contigs to the IBM genetic map. The goal of producing a
comprehensive, integrated genetic and physical map
(see http://www.maizemap.org/iMapDB/iMap.
html) is to facilitate map-based positional cloning,
application of comparative mapping across grass
species, and large-scale genome sequencing based on
a minimum tiling path.
RESULTS
BAC Library Screening with Complex Probes
Use of three libraries to make a physical map
should minimize the underrepresentation of certain
genomic regions arising from the use of a particular
Plant Physiol. Vol. 130, 2002
restriction enzyme (Frijters et al., 1997). To characterize and compare the quality of the libraries, highdensity filter sets from all three libraries were constructed. Each filter set contained six filters, and the
total of 18 filters collectively contained 331,776 BAC
clones with an overall average insert size of 154 kb.
The HindIII has an average insert size of 136 kb,
which is equivalent to 6⫻ haploid genome coverage
(Tomkins et al., 2002). The EcoRI has an average
insert size of 160 kb, and the MboI has an average
insert size of 167 kb, each resulting in 7⫻ haploid
genome coverage (K. Osoegawa and J. Messing, unpublished data). These BAC filters were evaluated
with a set of complex probes, which were aimed to
provide information on chromosome organization
and organellar DNA content. Probes in this set include the 185-bp knob repeat, ribosomal DNA, two
telomere-associated repeat sequences, four centromere repeat sequences, mitochondrial DNA, ␭, and a
chloroplast DNA cocktail. All of the raw data for the
high-density BAC filter hybridization are available
from MaizeDB (http://www.agron.missouri.edu/
bacs.html).
The percentages of positive clones in the three BAC
libraries that were hybridized with the set of complex
probes are shown in Figure 1A. In total, four centromeric repeat probes, CentA-ltr-11 (CentA-1), CentAint-7 (CentA-2), CentC, and Cent4, hit 8,409 BAC
clones (2.53%) of 331,776 clones tested. This number
includes some duplicate hits because of BACs that
cross-hybridized between four centromeric probes.
Hybridization results of centromere repeat probes
showed similar results for the percentage of BACs hit
in all three BAC libraries. The Cent4 probe is a maize
Figure 1. The HindIII, EcoRI, and MboI maize BAC libraries were
hybridized with 12 complex probes. A, Eight repetitive DNA elements were used to identify the distribution of the repeat elements
and chromosomal architecture. A set of probes including the four
centromere repeat probes (CentA-1, CentA-2, Cent4, and CentC),
two telomere-associated repeat sequences (Telo-1 and Telo-2), ribosomal DNA (pZMRI), and knob repeat (185 bp). B, Three organellar
DNA, a multifragment chloroplast DNA probe and two different
mitochondrial genome probes (Mito-A619 and Mito-Mo17, MitoMo17 were only hybridized to the maize HindIII BAC library), were
hybridized to measure the extent of organellar contamination, and
one ␭-DNA probe was also tested.
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1687
Yim et al.
chromosome 4-specific centromere sequence. The results show that Cent4 hybridized to only 70 BAC
clones (0.02%) in all three sets of filters, which is
much lower than the number of hits for the other
three centromeric repeat probes. The Cent4 sequence
has been reported to share high homologies to CentC
and the knob repeat and also to possess six copies of
the common telomeric motif CCTAAA (Page et al.,
2001), but our results show that BAC clones hybridized with Cent4 did not match those that hybridized
with CentC and telomere probes. However, under
low-stringency hybridization, Cent4 shows positive
hybridization signals, which coincide with the ones
to which the knob repeat probe hybridizes (results
not shown). The coincident location of these sequence elements may suggest a common functional
or structural role. By way of contrast, the number of
positives for telomere repeat sequences was 4-fold
higher in the EcoRI library. The hybridization results
of centromeric and telomeric repeat probes show
several contigs with multiple BAC hits, which can be
assigned to each maize chromosome with further
study. The contig data provide valuable information
that will be a key resource for future sequence and
structural analysis for those chromosomal regions.
The rDNA probe showed an even greater difference in hybridization results, the EcoRI and MboI
positives were increased 7- and 16-fold, respectively,
compared with the HindIII library. The rDNA of
maize contains approximately 10,000 tandem repeats
of a unit of 9.1 kb each. In a standard rDNA repeat,
there are no HindIII recognition sites and only a
single EcoRI site (McMullen et al., 1986). In contrast,
there are numerous MboI sites within the repeat. The
increased number of hits in MboI positives can be
explained by rDNA being cloned with higher frequencies into the MboI library because of an increased number of the clonable restriction recognition sites, compared with the HindIII and EcoRI
libraries.
The mitochondrial probe hybridization was performed using two different maize mitochondrial
genomic probes (mito-A619 and mito-Mo17). The results show mito-A619 and mito-Mo17 hybridized to
535 (0.5%) and 335 (0.3%) BAC clones, respectively,
from HindIII filters. Fauron et al. (1995) reported
physical maps of three mitochondrial genomes from
different maize cytotypes. These three maps show
differences in organization of the sequences attributable to rearrangements and size differences attributable to the location and amount of repeat sequences.
The differences in mitochondrial genome size and
sequences among different maize cytotypes could
explain the gap in hybridization results using two
different mitochondrial genomes. In addition, nDNA
contamination in the mito-A619 probe could account
for the difference in total hits.
Figure 1B shows that a very low percentage of the
HindIII, EcoRI, and MboI libraries consisted of chlo1688
roplast and mitochondrial clones, ⬍0.68% and
⬍1.46%, respectively. In total, 3,570 (1.08%) BAC
clones of 331,776 clones tested were derived from
organellar DNA. In all three libraries, fewer than
1,247 (0.5%) clones appeared to contain chloroplast
DNA. This is substantially lower than the previously
reported chloroplast contamination in sorghum and
tomato BAC libraries, which were 10.5% and 1.1%,
respectively (Budiman et al., 2000; Klein et al., 2000).
BAC filters were hybridized with ␭-DNA to check the
extent of random contamination during the lab procedures. Only 29 BAC clones positive to ␭-DNA were
detected, indicating an exceptionally low percentage
of ␭-DNA contamination.
BAC Library Screening with RFLP Core Markers
To anchor the bin boundaries and to begin to provide a framework for anchoring the genetic and
physical maps, probes representing the 90 core RFLP
markers were used to screen the HindIII (six filters,
representing 6⫻ haploid genome equivalents) library. The core markers are evenly spaced in the
genome and are the boundary markers for the chromosomal bin divisions on several maize genetic
maps, including the IBM (Gardiner et al., 1993; Davis
et al., 1999; see http://www.maizemap.org). All of
the data for the high-density BAC filter hybridization
with core markers are available from MaizeDB
(http://www.agron.missouri.edu/bacs.html). Figure 2 shows the relationship between the probe copy
number and the number of HindIII BACs identified
by each probe. The graph of the best linear fit demonstrates that the actual number of hits is very close
to the expected number of hits based on their copy
number. Correlation analysis on copy number of the
probe and number of BACs hit by each probe shows
a strong relationship (r ⫽ 0.910, P ⫽ 4.05E⫺26) between these two variables. RFLP probe copy number
Figure 2. Relationship between probe copy number and the number
of BACs identified per probe. Dot denotes number of positive HindIII
BAC clones for individual core markers. The best linear fit, Y ⫽
1.74 ⫹ 5.99X, is given by the solid line, whereas the dotted line is the
expected pattern.
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Plant Physiol. Vol. 130, 2002
Maize Bacterial Artificial Chromosome Libraries
was determined by the number of hybridized restriction fragments on Southern image. However, it is
possible that two homoeologous bands from wellconserved genomic regions could have the same
flanking restriction sites and therefore migrate
simultaneously.
About one-fourth of the core markers are single
copy in the genome, and the remainder are present in
two or more copies. Twenty-two of the core probes
including 20 probes reported by Tomkins et al. (2002)
and other two probes, p-asg8 (GenBank accession
nos. G10762 and G10763; identified 14 BACs) and
p-umc245 (GenBank accession nos. G13169 and
G13170; identified 11 BACs) that represent singlecopy sequences in the maize genome, identified an
average of 7.4 ⫾ 3.3 positive clones with a range of
three to 15 positive clones. Because the library represented 6⫻ genome equivalents, each single-copy
sequence should be represented six times, if all sequences are equally represented in the library. A
Student’s t test was performed to compare the mean
of actual positives, 7.4, versus expected number of
6.0. The statistical test (P ⫽ 0.120) indicates that the
difference between the mean and the expected number is not statistically significant. The variation in the
number of positive signals identified by the single-
copy probes may be indicative of the effects of preferential cloning obtained from the use of the HindIII
restriction enzyme or the effects of instability of certain sequences in the BAC vector.
The remaining core markers represent sequences
that are present in the genome in more than one
copy. Forty-nine markers with two to three copies
gave three to 28 positive BAC clones. At least three
positive clones were identified for all of the markers
analyzed (Tables I and II). This hybridization result
confirms that the HindIII library collectively provides
good representative coverage of 90 sequences that
are distributed throughout the maize genome.
Hybridization images of p-php20581 and p-umc64
show significant intensity differences even between
positives, and thus, the scanned images of these two
probes were scored with high stringency (highdensity filter reader threshold level set at 0.8, other
core markers were scored using the default threshold
of 0.5) to score the positives only with strong signals.
BAC data of these two probes scored at a highstringency setting were integrated into fingerprint
contig (FPC). The positive hits from the hybridization
filters of the other four probes with exceptionally
high numbers of positive BACs, p-bnl6.32, p-bnl7.08,
p-umc108, and p-umc124, were not scored because
Table I. Maize HindIII BAC library hybridization results using 30 probes with duplicated loci, out of 34 tested probes. Six high-density BAC
colony filter arrays were used for each probing allowing the screening of six haploid genome equivalents.
Probe
Bin
Probe Size
No. of Hits
GenBank Accession Nos.
11
19
21
14
12
20
12
17
13
19
15
10
20
14
18
7
8
4
17
9
3
13
15
15
14
21
6
18
21
13
G10822, G10823
G10865, G10866
G10864, G13173
G10812, G10813
G10776, G10777
G10851, G10852
G10855, G10856
G10816, G10817
G10834
G10847, G10848
G10837, G10838
G10752, G10753
G10801, G10802
G10828, G10829
G10857
G10748, G10749
G10820, G10821
G10862, G10863
G10797, G10798
G10870, G10871
G10810, G10811
G10841, G10842
G10818, G10819
G10760, G10761
G10754, G10755
G10832, G10833
G10778, G10779
T12667, T12668
G10782, G10783
–
bp
p-umc157
p-umc76
p-umc67
p-umc128
p-bnl8.45
p-umc53
p-umc6
p-umc131
p-umc255
p-umc5
p-umc32
p-asg24
p-umc102
p-umc17
p-umc63
p-agr r37
p-umc156
p-umc66
p-php20608
p-umc90
p-umc126
p-umc38
p-umc132
p-asg7
p-asg34
p-umc254
p-bnl9.11
p-csu31
p-npi268
p-umc259
Plant Physiol. Vol. 130, 2002
1.02
1.03
1.06
1.08
2.01
2.02
2.03
2.05
2.06
2.07
3.01
3.03
3.05
3.08
3.09
4.05
4.06
4.07
4.10
5.02
5.06
6.06
6.07
6.08
7.02
7.04
8.02
8.06
8.07
10.5
1,220
760
650
740
2,100
640
590
810
1,050
850
990
550
1,010
850
620
949
570
1,020
1,500
1,240
670
1,010
500
550
1,350
1,050
2,400
800
710
550
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1689
Yim et al.
Table II. Maize HindIII BAC library hybridization results using 29 probes with three or more copies. Six high-density BAC colony filter arrays were used for each probing allowing the screening of six haploid genome equivalents.
Probe
Bin
Probe Size
p-csu3
p-asg62
p-csu164
p-umc107
p-umc49
p-csu32
p-asg48
p-bnl5.37
p-bnl6.16
p-php20725
p-umc31
p-npi386
p-umc127
p-umc52
p-umc169
p-csu93
p-bnl5.24
p-umc59
p-umc21
p-umc168
p-npi220
p-umc192
p-umc95
p-csu54
p-csu61
p-npi285
p-umc64a
p-umc44
p-bnl7.49
1.05
1.07
1.09
1.10
2.09
3.02
3.04
3.06
3.07
4.02
4.03
4.04
4.08
4.09
4.11
5.05
5.08
6.02
6.05
7.06
8.01
9.02
9.05
9.08
9.06
10.2
10.4
10.6
10.7
1,200
500
700
1,090
630
500
1,600
2,300
2,450
1,650
1,200
550
1,210
780
670
800
2,500
930
1,050
1,080
400
1,750
680
1,400
500
1,250
710
800
2,100
Copy No.
No. of Hits
GenBank Accession No.
3
3
4
4
8
8
4
4
3
3
3
3
3
⬎8
⬎8
3
3
5
3
3
4
5
4
5
3
3
3
3
⬎8
23
16
25
20
53
55
28
34
6
26
8
28
17
48
63
13
24
42
22
8
25
31
29
29
16
28
18
19
66
T12525, T12526
G13181, G13182
T12748
G10803, G10804
G10845, G10846
T12669
G13183, G13184
G10766, G10767
G10768, G10769
–
G10835, G10836
G10786, G10787
G13175, G13176
G10849, G10850
G15653, G15654
T12714
G10764, G10765
G10853, G10854
G10830, G10831
G13171, G13172
G10780, G10781
X13502
G10872
T12684
T12691
G10784, G10785
G10858, G10859
G10843, G10844
G10774, G10775
bp
a
BAC filters scored at high-stringency reading.
there was no intensity difference between positives
and because each probe showed more than 300 positives, which suggests possible existence of repeat
elements within the probe sequences (data not
shown).
After the scoring of hybridized filter images, sequences of all 90 RFLP clones were retrieved from the
GenBank database and were used as queries in a
BLAST search against all maize sequences. These
analyses demonstrated that only eight probes contain
multiple regions homologous to either known genes
with repeat elements or a transposon. Six out of eight
probes had many more positive signals on hybridized filters than expected by chance. Core markers,
p-php20581, p-umc108, and p-bnl7.08, showed significant homology to a 22-kD zein sequence (AF031569).
These three markers also showed significant sequence similarities to the chloroplast ATPase
␤-subunit gene (X03396) and the ␣-tubulin gene
(AJ420857). Meyers et al. (2001) previously reported
the 22-kD zein region as a genomic region containing
repeat elements. The other three core markers also
show significant homology to multiple known genes
(Table III).
1690
Validation by Dot-Blot Hybridization and
Southern Analysis
To further validate the BAC-addressing method
and hybridization results, two types of DNA hybridization experiments were performed on selected
clones. Dot-blot hybridization was performed using
four hybridization probes, p-umc161, p-csu164,
Cent4, and pZmRI (rDNA). Among the BAC clones
scored as positive for these probes, two to four clones
were randomly selected for the dot-blot analysis.
Figure 3 shows all of the clones hybridized with the
expected probe except for the negative control. This
indicates that the addressing process was robust.
A second type of validation was focused on analyzing BACs identified by two probes, which were
thought to be single copy in the genome, p-tub1 and
p-tub4. These probes were selected because they hybridized to a higher number of BACs than expectations (15 for p-tub1 and 10 for p-tub4) and because
the BACs identified for each probe assemble into
multiple contigs, instead of a single contig (WebFPC,
October 24, 2001 version at 8⫻ coverage; http://
www.genome.arizona.edu/fpc/maize). To test the
robustness of the BAC hybridization and contig as-
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Plant Physiol. Vol. 130, 2002
Maize Bacterial Artificial Chromosome Libraries
Table III. Blast results from query of six core RFLP marker sequences with higher than expected total positive BACs against the GenBank
database
Significant hits (E ⬍ 1 ⫻ 10⫺5)a are listed.
Probe
Bin
GenBank Accession No.
p-bnl6.32
1.12
G10770, G10771
p-php20581
2.10
G10795, G10796
p-umc108
5.07
G10805
p-umc124
8.03
G10808, G10809
p-bnl7.08
8.04
G10772, G10773
p-umc64
10.04
a
G10858
Homology
Chromosome 9 bz genomic region (AF391808)
D3 mol H⫹-transporting ATPase (Mha1) gene (U09989)
See2a gene (AJ251453)
DWARF8 gene (AF413200)
Polyamine oxidase gene (AJ251018)
Repeat 22-kD zein region (AF031569)
Chloroplast ATPase ␤-subunit gene (X03396)
␣-Tubulin gene (AJ420857)
Repeat 22-kD zein region (AF031569)
Chloroplast ATPase ␤-subunit gene (X03396)
␣-Tubulin gene (AJ420857)
ADP-Glc pyrophosphorylase (shrunken-2) gene (M81603)
1-Acyl-glycerol-3-phosphate acyltransferase (Z29518)
Repeat 22-kD zein region (AF031569)
Chloroplast ATPase ␤-subunit gene (X03396)
␣-Tubulin gene (AJ420857)
Transposon frequent flyer (AF323026)
Cytosolic aldehyde dehydrogenase RF2C gene (AF348412)
Maize peroxidase 2 (pox2) gene (AJ401275)
E-Value
2E-11
7E-11
4E-06
5E-08
3E-06
7E-14
2E-08
7E-14
5E-18
2E-08
2E-14
7E-06
7E-06
2E-08
5E-09
2E-08
5E-30
1E-21
1E-09
E, Probability cutoff value.
sembly, several BACs associated with p-tub1 in five
contigs and several others associated with p-tub4 in
the other three contigs were digested with HindIII,
and the fragments were then separated on agarose
gels, blotted, and hybridized with the cognate
probes. Figure 4A shows the ethidium bromidestained gels and the corresponding blots.
All of the BACs associated with p-tub1 (lanes 1–19),
except one, share a common hybridizing band (Fig.
4B). The exceptional clone, 11P17 (lane 10), does not
appear to be contiguous with the other clones based
on three observations: 11P17 DNA hybridized poorly
to the p-tub1 probe on the Southern analysis, the
clone belonged to the contig by fingerprinting but
was not detected when the BAC filters were screened
by hybridization with p-tub1, and a recent version of
WebFPC BAC assembly (May 3, 2002) placed 11P17
in another contig.
Figure 4B shows that four of the p-tub1 hybridizing
clones (lanes 6, 7, 8, and 11) have an additional
hybridizing band, which suggests a possible complication that may require further investigation to assemble a robust physical map of a recently duplicated genome such as that of maize. One possibility
is that a tandem (or at least proximal) duplication of
the tub1 gene exists and that all of the BACs (except
11P17) do truly represent one contiguous stretch of a
single chromosome, which contains two p-tub1 loci.
However, additional information will be needed before we can rule out the possibility that two unlinked,
perhaps homoeologous, loci exist that have retained
a sufficient level of DNA sequence conservation so
that most restriction fragments are still the same, and
therefore FPC groups them into a single contig.
Plant Physiol. Vol. 130, 2002
Six of the BAC clones associated with p-tub4 (lanes
20–26) shared a hybridizing band of the same size.
These Southern-blot results further validate our hybridization results and BAC-addressing procedure.
Contig Assembly and Anchoring to the Genetic Map
FPC (Soderlund et al., 2000) is a program that
assembles clones into contigs based on fingerprints
and markers. WebFPC (Soderlund et al., 2002) is a
Java program that displays the results of FPC on the
Web. Of the 90 RFLP probes hybridized, 82 probes
hybridized to less than 50 BACs and thus were integrated into FPC contig assembly and contributed to
anchor contigs to the genetic map (see Arizona
Genomics Institute Web site, http://www.genome.
arizona.edu/fpc/maize/). Four probes (p-bnl7.49,
p-csu32, p-umc49, and p-umc169) hybridized with
more than 50 BAC clones but have high copy numbers (ⱖ8) based on Southern blots with genomic
DNA, meaning large numbers of positives are legitimate (Table II). These data were not integrated into
FPC to avoid ambiguity in assembly. The other four
probes, p-bnl6.32, p-bnl7.08, p-umc108, and
p-umc124, containing repetitive elements with over
300 positive BACs were also excluded for the FPC
assembly.
Thirteen single-copy probes hit a single contig and
thus could be unambiguously assigned to the IBM
genetic map. Single-copy probes should associate
with one contig, but nine single-copy RFLP markers
hit two or more contigs. Further evaluation of the
data will sort out these ambiguities, and manual
editing of the FPC could merge these multiple con-
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1691
Yim et al.
contigs to their respective genetic positions. Twentyone of 34 duplicate-copy probes hit three or more
contigs. Four of the duplicate-copy probes hit only a
single contig, indicating that one of the loci for these
probes may not be represented in the HindIII BAC
library or was not represented on this subset of filters. The possibility that these contigs could represent
assemblies of well-conserved duplicated genomic regions will require further investigation.
Finally, we also used a probe to screen the HindIII
library for BAC clones containing the R1 and B1 loci
on chromosomes 10 and 2, respectively. A total of 16
clones were obtained. These clones were subjected to
Southern-blot analysis (not shown) exhibiting clones
with two different restriction patterns, one that corresponded 100% to WebFPC contig 318 (R1) and the
other 100% to contig 65 (B1). From this example and
the asg24, it appears that FPC can distinguish duplicate factors in the allotetraploid maize genome.
DISCUSSION
Figure 3. Dot blot of positively scored BAC DNA probed with four
RFLP markers (A, umc161; B, Cent4; C, csu164; and D, pZmRI). BAC
clones are shown as follows: A1, 101F15; A2, negative control; A3,
244B02; B1, 18H07; B2, 14J07; B3, 132L19; B4, 132A03; C1,
124K13; C2, 124E13; C3, 200N03; C4, 200I12; D1, 163O18; D2,
165E02; D3, 233F11; and D4, 266C14. BAC clone 124E13 was used
for negative control for p-umc161 dot blot; this clone was not scored
as positive for hybridization with this probe.
tigs into one larger contig. Several probes with two
contig hits have been merged into a single contig
during the last few FPC updates (data not shown).
Probes hybridizing to two or more copies in the
genome should hit multiple contigs from duplicate
sequences in the genome. Among 34 duplicate-copy
probes, nine probes hit two contigs. BAC contigs for
maize core probe p-asg24 are shown in Figure 5.
RFLP hybridization data of p-asg24 with maize
genomic DNA indicates that this probe recognized
two loci in the genome. This is consistent with the
contig assembly data, which also indicates that there
are two copies of p-asg24. The FPC display also
shows that both contigs were not only hit by p-asg24
but also share an overgo CL743_1. This result shows
robustness of hybridization results and thus supports
the data generated by overgo hybridization and the
contigs assembled by fingerprinting. However, further evaluation of the data is needed to anchor the
1692
Maize genetic research has a long history of traditional breeding and mutant mapping studies. Recent
progress in maize high-resolution molecular genetic
mapping (Davis et al., 1999; Sharopova et al., 2002) is
helping to facilitate current molecular genomic research. It has also facilitated construction of comparative genetic maps among closely related grass species (Ahn et al., 1993; Zwick et al., 1998; Wilson et al.,
1999; Tikhonov et al., 2000; Gaut, 2001), and molecular dissection of quantitative trait loci (Thornsberry
et al., 2001).
The three BAC libraries reported in this paper have
a total genome coverage of 26-fold, providing an
accessible tool for genomic research. Tomkins et al.
(2002) reported construction and characterization of
the HindIII BAC library. Their results show that the
HindIII BAC library is of good quality and allows
⬎99% probability of recovery of any specific sequence of interest. Our hybridization results demonstrate that, despite its good quality, the HindIII library has a bias and that the bias is overcome by
using libraries made by several restriction enzymes.
The results for telomere and pZmRI (rDNA) probes
suggest that each library has a different bias because
of the use of a specific restriction enzyme for the
construction of the BAC library. The lower percentage of rDNA clones in the HindIII library compared
with the EcoRI and the MboI libraries suggests that
other regions of the maize genome devoid of HindIII
sites could also be underrepresented in the HindIII
library. These libraries will provide enough coverage
for future genome research, and they complement
each other to minimize the underrepresentation of
certain genomic regions caused by the use of a particular restriction enzyme for BAC library construction (Frijters et al., 1997). Our hybridization results
are significant in that BAC clones have been identi-
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Plant Physiol. Vol. 130, 2002
Maize Bacterial Artificial Chromosome Libraries
Figure 4. Analysis of 26 BAC clones digested with HindIII. A, Ethidium bromide-stained agarose gel. B, Southern blot of the
gel in A after hybridization with radioactively labeled p-tub1 and p-tub4 probes. BAC clones are shown as follows from lane
1 through 27: 188N16, 158C08, 49G01, 121N08, 21E04, 207P12, 344O08, 391H16, 32E08, 11P17, 129G10, 220J21,
155I03, 2K20, 190E08, 296P19, 187D08, 8K16, 172P22, 20F09, 339L08, 169O03, 232P15, 252D07, 32E08, 11P17, and
molecular marker (␭/HindIII). All of the clones except 11P17 (from EcoRI library) were from the HindIII library.
fied that correspond to key chromosome architectural features such as centromeres, telomeres, and the
rDNA. Further research will be necessary to anchor
these BAC contigs to their specific chromosomes.
Hybridization of BAC filters with chloroplast, mitochondrial, and ␭-DNA probes demonstrated that
all three maize BAC libraries exhibit a low percentage of contamination with these sequences. The suitability of a BAC library for positional cloning, physical mapping, and the ultimate goal of whole-maize
genome sequencing using a minimum tiling path
depends on the ability to recover clones from specific
regions by screening (Lijavetzky et al., 1999). Data
from the 90 RFLP core marker hybridization show at
least three positive BAC clones per probe, which
confirms adequate genome coverage of the maize
HindIII BAC library for the most of markers. However, this library can show biased representation because certain genomic regions lack HindIII restriction
sites. The extensive level of genome coverage and
low level of non-nuclear maize DNA contamination
Plant Physiol. Vol. 130, 2002
demonstrate the high quality of the three BAC libraries. The collective depth of these BAC libraries appears to be sufficient for retrieval of virtually any
maize sequence.
The primary purpose of the core marker hybridizations is to provide a framework for genome assembly
and sequencing. The data generally indicate that the
6⫻ subset of HindIII clones was sufficient to anchor
maize contig before manual editing. After identifying
the library addresses of each positive signal, results
were integrated into FPC analysis. Results from the
hybridization of 71 single- and low-copy number
probes generally confirmed BAC contigs assembled
by fingerprinting and positively demonstrated the
utility of single-copy sequences, such as single-copy
RFLP and gene probes, as anchor points for tying the
physical map to the genetic map. Ambiguities from
paralogous and/or homoeologous sequences may be
overcome by putting more genetically mapped markers onto the physical map, by screening BAC pools
using locus-specific PCR primers, by manual editing
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1693
Yim et al.
Figure 5. RFLP hybridization data for p-asg24 with maize genomic DNA indicates that this probe recognizes two loci in the
genome. This is consistent with the contig assembly data, which also indicates that there are two copies of p-asg24. In contig
36 (A) and contig 231 (B), BAC clones hit by p-asg24 are highlighted in green.
of the FPC analysis, and perhaps by finer resolution
fingerprinting approaches. Hybridization to additional subsets of the BAC libraries might also aid in
assigning paralogous sequences to their correct genetic location. The three BAC libraries can currently
be ordered from http://www.genome.clemson.edu/
orders (the b library is the HindIII; the c library refers
both EcoRI and MboI), and the hybridization data are
publicly available from http://www.agron.missouri.
edu/bacs.html. These public resources will be valuable for maize genome research, positional cloning,
and comparative research between cereal plants.
p-PMTY7SC (Telo-1; GenBank accession no. U39641) and p-PMTY9ER
(Telo-2; GenBank accession no. U39642) were provided by J.G. Both telomeric subclones contained CA-rich regions with sporadic occurrences of the
telomere repeat (Gardiner et al., 1996). Four centromere repeat clones,
CentA-1, CentA-2, CentC, and Cent4, were provided by James Birchler
(University of Missouri) and Kelly Dawe (University of Georgia, Athens).
CentA is a medium-copy number-dispersed retrotransposon (GenBank accession no. AF082532; Ananiev et al., 1998), CentC is a tandem repeat
element (GenBank accession nos. AF078922 and AF078923; Dong et al.,
1998), and Cent4 is a centromere 4-specific sequence (GenBank accession no.
AF242891; Page et al., 2001). Mitochondrial genome DNAs isolated from
maize inbred lines A619 and Mo17 were provided by Kathleen Newton
(University of Missouri).
BAC Library Screening
MATERIALS AND METHODS
Maize (Zea mays) Core RFLP and Complex Probes
Maize core RFLP probes used in this study included both cDNA and
genomic DNA clones. They included agr (Mycogen Plant Sciences, Des
Moines, IA), asg (Asgrow Seed, Galena, MD), bnl (Brookhaven National
Laboratory, Upton, NY), csu (Chris Baysdorfer, California State University,
Hayward), npi (Native Plants [Salt Lake City] and Pioneer-Hi-Bred International, Des Moines, IA), php (Pioneer-Hi-Bred International), and umc
(University of Missouri, Columbia) clones and several known genes (Davis
et al., 1999). Detailed information on these probes is available (see MaizeDB;
http://www.agron.missouri.edu/probes.html). GenBank accession numbers for the clones are given in Tables I and II.
Repetitive probes were obtained from various sources. The three
chloroplast-specific clones (pBHP20, pBPH134, and pBHE319; GenBank accession no. NC001666) were provided by Rod Wing (University of Arizona).
These three chloroplast genes are evenly distributed around the 133-kb
barley chloroplast genome. The 185-bp repeat (GenBank accession no.
M35408) was from M.D.M. (McMullen et al., 1986). Telomere probes
1694
The HindIII library made at the Clemson University Genomics Institute
has an average insert size of 136 kb with a genome coverage of 13.5⫻
(Tomkins et al., 2002). For this study, only six high-density BAC filters,
which provide genome coverage of 6⫻, were used. The EcoRI and the MboI
libraries made at the Children’s Hospital Oakland Research Institute have
an average insert size of 160 and 167 kb, respectively, with a genome
coverage of about 7⫻. High-density BAC filters were gridded in double
spots using a 4 ⫻ 4 pattern with six fields per nylon (Hybond NT) filter.
Each field consists of 16 ⫻ 24 boxes, and within each box, there are eight
independent clones in duplication. This allows each filter to represent 18,432
independent maize BAC clones.
BAC hybridization screening was performed using six filters equivalent
to 6⫻ (HindIII) and 7⫻ (EcoRI and MboI each) haploid genome coverage.
Colony filters were processed and hybridized using standard protocol (Sambrook et al., 1989). Films were digitized on a large-bed ScanMaker 6400XL
(Microtek Inc., Hsinchu, Taiwan), and the images were imported into the
semi-automated high-density filter reader 1.0 (Incogen Inc., Clemson, SC)
for the BAC scoring and addressing. Hybridization of the MboI library was
performed at the Waksman Institute under the same conditions.
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Plant Physiol. Vol. 130, 2002
Maize Bacterial Artificial Chromosome Libraries
Clone Verification by Dot-Blot Hybridization and
Southern-Blot Analyses
BAC clones giving positive signals upon hybridization were picked
individually from the 384-well microtiter plates of the maize BAC library
and inoculated into a starter culture of 5 mL of Luria-Bertani medium
containing 12.5 ␮g mL⫺1 chloramphenicol for initial culture. After 8 h of
incubation at 37°C, the starter culture was diluted into new Luria-Bertani
medium, 1:1,000 (v/v), and grown overnight at 37°C. BAC DNA was
isolated using the plasmid midi kit (Qiagen USA, Valencia, CA). Five
hundred nanograms of DNA was transferred to an Immobilon NY⫹ nylon
membrane (Millipore, Bedford, MA), and the DNA was fixed by UV crosslinking. Prehybridization and hybridization were done at 65°C in 50 mm
Tris, 10 mm EDTA, 5⫻ SSC, 1⫻ Denhardts solution, 0.2% (w/v) SDS, and
100 ␮g mL⫺1 denatured salmon sperm DNA. After hybridization, the membranes were washed three times with low-stringency wash (2⫻ SSC and
0.5% [w/v] SDS) and a final high-stringency wash (0.1⫻ SSC and 0.1%
[w/v] SDS) was done. Both high- and low-stringency washes were done at
65°C. The blots were exposed to x-ray films at ⫺80°C overnight.
Restriction digestions were performed using HindIII for Southern-blot
analysis. BAC DNA was electrophoresed in 0.8% (w/v) agarose gels at 50 V
for a distance of about 10 cm. Gels were denatured for 30 min in 0.4 n NaOH
and 0.6 m NaCl, followed by a 30-min neutralization in 0.5 m Tris, pH
7.5, and 1.5 m NaCl. DNA was transferred to Immobilon NY⫹ nylon
membrane, and the rest of procedures were done based on the procedure
described by Davis et al. (1999).
Homology Searching
All maize core RFLP markers were queried against public sequence
databases using the BLASTN search engine. A sequence was classified as
homologous to other sequences if a BLAST probability value (E-value) less
than 10⫺5.
Received August 23, 2002; returned for revision October 2, 2002; accepted
October 8, 2002.
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