全 文 ：Received 7 Jan. 2003 Accepted 8 Jun. 2004
Supported by the National Natural Science Foundation of China (30270708, 39880024), Knowledge Innovation Program of The Chinese
Academy of Sciences (KZCX1-SW-19) and the National Special Program for Research and Industrialization of Transgenic Plants (Z2002-B-004).
* Author for correspondence. Tel: +86 (0)10 64889356; Fax: +86 (0)10 64889783; E-mail: .
** Coordinate first author.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1357-1365
Microdissection and Construction of Region-specific DNA Libraries
of Wheat Chromosome 6B
HU Zan-Min*, WANG Huai**, SHI Rui, DANG Ben-Yuan, HU Jun, YIN Wei-Bo, CHEN Yu-Hong,
JIANG Shu-Mei, CHEN Zheng-Hua
(Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China)
Abstract: Chromosome 6B of wheat (Triticum aestivum L.) in mitotic metaphase spreads was
micro-dissected with Nd:YAG laser microbeam into four segments and then each segment was collected
by glass needles. The DNAs of the isolated chromosomal segments were separately amplified by Sau3A
linker adaptor-mediated polymerase chain reaction (LA-PCR). The presence of region-specific DNA from
each of four segments was verified by Southern hybridization. The second round PCR products from four
segments of chromosome 6B were cloned into a pGEM T-vector to create four chromosome region-
specific libraries, named R1, R2, R3 and R4, which included 2.1×105; 2.74×105; 2.45×105 and 2.93×105
recombinant clones, respectively. A total of 150 randomly selected clones from each library were
characterized by mini plasmid DNA preparation and enzyme restriction. Results showed that the size of
inserts ranged from 300 to 1 800 bp with an average of 820 to 870 bp, of which 43%-48% were low/unique
copy and 42%-47% were medium/high copy sequences. A set of microsatellite sequences located on
chromosome 6B and other chromosomes of wheat were used for the verification of PCR products from
micro-dissected chromosomal segments. The results reported here should facilitate the molecular
genetics analysis of different fragments from single chromosomes of a plant.
Key words: wheat; chromosome microdissection; LA-PCR; chromosome region-specific DNA library
Wheat (Triticum aestivum) is one of the most important
crops in the world, and a great deal of research work in
general genetics, cytogenetics and molecular genetics of
wheat have been done. Many genes of economic interests
are located on different chromosomes (Dhitaphichit et al.,
1989; Dedryver et al., 1996; Procunier et al., 1997). Chromo-
some 6B has been shown to carry genes for resistance to
diseases, including Pm11 to powdery mildew (Tosa and
Tsujimoto, 1994); Lr9 to leaf rust (Schachermayr et al., 1994);
and Lr3 to leaf rust (Sacco et al., 1998). However, because
of the large size (approximately 1.6×1010 bp), the polyp-
loid complexity of wheat genome (2n=6X=42, AABBDD)
and low number of molecular markers, a high-resolution
linkage map of wheat has not been established to date.
Therefore, development of a high-density linkage map is
still needed in wheat genome research for marker-assisted
selection of agronomical important traits and isolation of
genes of economic interest. A potential strategy for clon-
ing an important gene located on a known chromosome is
to construct region-specific high-density linkage maps.
Chromosome microdissection and microcloning is a
direct way to construct chromosome region-specific DNA
libraries and to get probes for a high-density region-spe-
cific linkage map. These techniques were developed in 1981
in Drosophila (Scalenghe et al., 1981) and refined into an
efficient tool to generate chromosomal or chromosome-re-
gion-specific DNA libraries in human (Ludecke et al., 1989;
Hadano et al., 1991; Guan et al., 1992) and several animals
(Greenfield et al., 1987; Zimmer et al., 1997). However, this
technique has lagged far behind in plants than in human
and animals. To date some chromosome-specific DNA li-
braries have been constructed in a few plants, including
Lilium regale (Dang et al., 1998), wild beet (Jung et al.,
1992), barley (Schondelmaier et al., 1993), oat (Chen and
Armstrong, 1995), soybean (Zhou et al., 2001), rye (Zhou
et al., 1999), and wheat (Albani et al., 1993; Liu et al., 1997;
Liu et al., 1999). No defined chromosome regional specific
DNA libraries have been reported except maize (Stein et al.,
1998). There are now two distinct methods to dissect target
chromosomes; the glass needle method (Scalenghe et al.,
1981; Ludecke et al., 1989) and the laser microbeam method
(Monajembashi et al., 1986; Ponelies et al., 1989; Fukui et
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041358
al., 1992). For the latter, UV laser (Monajembashi et al.,
1986), argon-ion laser (Hadano et al., 1991; Kamisugi et al.,
1993) and Nd:YAG laser microbeam (Wang et al., 1998) have
been used. The microdissected chromosomal segments were
subsequently cloned by direct cloning method (Fisher et
al., 1985; Sandery et al., 1991), vector or adaptor-mediated
PCR with specific primers (Garza et al., 1989; Ludecke et al.,
1989; Chen and Armstrong, 1995) and degenerate oligo-
nucleotide-primed PCR using random primers (Wesley et
al., 1989, Telenius et al., 1992).
We report here microdissection of chromosome 6B into
four segments by Nd: YAG laser microbeam, collection by
glass needles from mitotic metaphase spreads of common
wheat “Chinese Spring” and construction of four DNA li-
braries from four segments of dissected chromosome 6B.
The four libraries were characterized in detail. Amplifica-
tion of a set of microsatellite sequences located on chro-
mosome 6B and other chromosomes of wheat confirmed
that the second round PCR products did originate from
specific regions of chromosome 6B, indicating the region-
specific sequences from four microdissected segments of
chromosome 6B, i.e. R1, R2, R3 and R4, were present in the
corresponding libraries. This provides a strategy for the
research of plant chromosome region-specific fragments.
1 Materials and Methods
1.1 Plant materials
The cultivar “Chinese Spring” of common wheat
(Triticum aestivum L. 2n=6x=42, AABBDD), which was
kindly provided by Dr. LI Yi-Wen, Institute of Genetics and
Developmental Biology, The Chinese Academy of Sciences,
was used in this research.
1.2 Chromosome preparation and microdissection
After being immersed in water for 5-8 h, “Chinese
Spring” seeds were germinated on moist filter paper in petri
dishes at 25 ℃ in dark. Root tips (0.5-1.0 cm) were har-
vested and treated in ice water (0 ℃) for 24-36 h to accu-
mulate metaphases, fixed in 3:1 ethanol:acetic acid for 5
min, then stored in 70% ethanol at –20 ℃ for at least 1 d.
Meristems were digested with an enzyme mixture of 2%
cellulase and 1.5% Pectolyase at 37 ℃ for 40 min, then
rinsed in ddH2O and stored at 4 ℃ for 15-20 min. After the
enzyme digested root meristems were stained by Carbo
Fuchsin solution for 1-2 min and squashed, they were im-
mediately used for microdissection by an Nd:YAG laser
microbeam system under a microscope (designed and made
by Institute of Genetics, The Chinese Academy of Sciences,
Beijing, and Laser Biological Institute of Chongqing, China).
The target chromosome 6B, which was distinguishable by
its large satellite on the short arm, was dissected into four
segments by laser microbeams with 1 µm diameter, 530 nm
wave length and 10 mj export energy. After the target chro-
mosome was dissected, the cover slip was removed and
each dissected segment was collected with glass needle
tips of 1-2 µm in diameter, respectively. Single chromo-
some segment was transferred into a 0.5-mL Eppendorf tube
containing 20 µL Proteinase K (5 ng/mL) in 1×T4 DNA
ligase buffer (Promega), according to the protocol devel-
oped by Hu et al. (1998).
1.3 Amplification of dissected chromosomal segments
After microdissection, the isolated chromosome seg-
ments were amplified using Sau3A linker adaptor-mediated
PCR (LA-PCR) following protocols described by Chen and
Armstrong (1995), Dang et al. (1998) and Zhou et al. (1999).
I n b r i e f , t h e 2 3 m e r D N A s e q u e n c e 5 -
GATCCTGAGCTCGAATTCGACCC-3 and the 19 mer DNA
sequence 5-GGGTCGAATTCGAATTCGAGCTCAG-3
were synthesized (Cybersyn Company) and Sau3A linker
adaptors were prepared as described by Chen and
Armstrong (1995). After the isolated target chromosome
segment was treated at 37 ℃ for 2 h in a Proteinase K
solution, the chromosomal DNA was digested with 0.02 U
Sau3A at 37 ℃ for 2 h. The digested chromosomal DNA
was linked with Sau3A adaptor (2 µL, 5 ng/µL) using T4
DNA 1igase (0.5 µL, 3 U/µL) in a total volume of 24.5 µL at
16 ℃ overnight. All the enzymes used in above mentioned
procedures were inactivated at 70 ℃ for 20 min after the
reactions. Two rounds of PCR were performed. The first
round of PCR was carried out in the same tube by adding 10
µL of 10×Taq buffer, 6 µL of 25 mmol/L MgCl2, 2 µL of 10
mmol/L dNTPs, 1 µL of 19 mer primer (50 ng/µL), 2 U Taq
DNA polymerase (Promega) and double distilled water in
100 µL total volume. After denaturing at 94 ℃ for 5 min,
amplification was performed with 35 cycles of 1 min at 94
℃, 1.5 min at 50 ℃, 3 min at 72 ℃, followed by a final 15
min extension at 72 ℃. The second round of PCR was done
under the same conditions described above except that
only a 2-µL product from the first round of PCR was used
as the template. In all the procedures, strict positive and
negative control experiments were carried out using the
same conditions but the templates. In the positive control
experiment, 10 pg of “Chinese Spring” genomic DNA was
used as template, whereas in the negative control no DNA
template was used.
1.4 Southern hybridization
Genomic wheat DNA was extracted from leaves by us-
ing CTAB protocol (Doyle and Doyle, 1990), and 2.5 µg
genomic DNA was digested with EcoRⅠand labeled with
Hu Zan-Min et al.: Microdissection and Construction of Region-specific DNA Libraries of Wheat Chromosome 6B 1359
digoxygenin (DIG)-11-dUTP as probes. The second round
PCR products from segments of chromosome 6B and con-
trols were separated on 1.5% agarose gel and transferred
onto nylon membranes (Hybond+, Amersham). Southern
hybridization, probe labeling and detection were carried
out according to the manufacturer’s instruction (Roche).
1.5 Verifiction of PCR products with wheat microsatellite
sequences
PCR product from microdissected chromosome seg-
ments was verified with 42 pairs of primers of wheat
microsatellite markers (kindly provided by professor Michael
Denis Gale, John Innes Centre) for different chromosomes
(Stehpenson et al., 1998), by a PCR procedure supplied by
Prof. Michael Denis Gale (personal communication). Briefly,
the second round PCR product was firstly denatured for 5
min at 94 ℃, then 30 cycles of 1 min at 94 ℃, 1 min at varied
temperature depending on primer sequences, 1 min at 72 ℃
and final extension for 5 min at 72 ℃. The products were
separated on 2.5% agarose gel for detection.
1.6 Library construction and characterization
The products of the second round PCR were purified
solved in 10 µL TE buffer with a DNA purification kit, and 2
µL of purified DNA (100 ng) was ligated into pGEM T-vec-
tor (Promega) in 10 µL volume at 4 ℃ for 16 h. One µL of
ligation mixture was used for transformation of DH5a com-
petent Escherichia coli cells by heat shock. One-tenth vol-
ume (100 µL) of transformed DH5a cells was spread on the
LB plate containing 100 mg/L ampicillin, X-gal and IPTG.
One hundred and fifty randomly selected recombinants
(white clones) from each of four DNA libraries were charac-
terized by plasmid mini preparation and restriction analysis.
Recombinant plasmids were extracted by alkaline lysis. The
inserts were released with EcoRⅠ and separated in a
1.2% agarose gel. DIG-labeled genomic wheat DNA was
hybridized to Hybond+ filters containing recombinant clones
to estimate the copy number of the inserts.
2 Results
2.1 Microdissection of chromosome 6B and amplifica-
tion of its segments
In a well spread mitotic metaphase (Fig.1A), chromo-
some 6B can be distinguished from other chromosomes by
its large satellite. It was more or less evenly dissected into
four segments (R1, R2, R3 and R4) with laser microbeams
(Fig.1B). The segments were separately transferred into a
0.5-mL Eppendorf tube with the tip of a glass needle under
an inverted microscope using a micromanipulator. The re-
sult demonstrated that Nd:YAG laser microbeam could dis-
sect chromosomes and generate a gap of about 1 mm in size
between chromosome segments. The DNA of isolated chro-
mosomal segments was amplified by two rounds of PCR.
The second round PCR products of these chromosome
segments ranged in size from 200-2 000 bp (Fig.2A), which
were similar to those of positive control (10 pg genomic
DNA as template). The negative control, to monitor pos-
sible contamination of DNA, was conducted during all
stages of the microdissection and amplification procedures.
No product was amplified from the negative control. Wheat
genomic DNAs were labeled with DIG and used as probes
for the hybridization with the second round PCR products
of microdissected chromosome segments. The result indi-
cated that DNAs of microdissected chromosome segments
were successfully amplified and they did originate from
wheat genome (Fig.2B).
2.2 Characterization of amplified DNA by microsatellite
markers
A total of 42 pairs of primers of microsatellite markers,
which were located on different parts of wheat
Fig.1. Chromosomes of Chinese Spring stained with Carbo Fuchsin solution. One of chromosome 6B indicated in A was microdissected
into R1, R2, R3 and R4 sections (B). Bar represents 10 mm.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041360
chromosomes, were used to detect the origin of the PCR
products from microdissected chromosomal segments.
When primer of Xpsp3009, a microsatellite sequence lo-
cated on the middle part of chromosome 6BS (Stephenson
et al., 1998), was used for PCR amplification, a 221 bp band
resulted from PCR reactions in which either wheat genomic
DNA, or amplified DNA from R2 of chromosome 6B, or PCR
products of positive control was used as template. No PCR
product was obtained in PCR reactions using amplified
DNAs from the segment R1, R3 or R4 as template as well as
in the negative control (Fig.3A). When primers of Xpsp3139,
another microsatellite marker also located on the middle
part of chromosome 6BS (Stephenson et al., 1998), was
used for the PCR amplification, the result was similar to that
of Xpsp3009 primers except the size of band 170 bp
(Fig. 3B). These results indicted that the DNA of R2 seg-
ment was amplified from a part of chromosome 6B on which
Xpsp3139 and Xpsp3009 were located and no cross con-
tamination from different segments occurred.
PCR amplification using the primers of a micosatellite of
Xpsp3079, which is located on distal region of chromo-
some 6BS, chromosome 4D and chromosome 7D
(Stephenson et al., 1998), yielded 298 bp, 271 bp, 178 bp
products in the reaction with wheat genomic DNA as
template; 198 bp and 178 bp bands in the positive control in
which the template was the PCR product from the second
round PCR using wheat genomic DNA as template; and a
198 bp band in the reaction with DNA from R1 segment as
the template. Nothing was produced in other reactions us-
ing DNAs from R2, R3 and R4 segments as templates
(Fig.3C). This showed that the second round PCR prod-
ucts of microdissected R1 segment was indeed from a cer-
tain part of chromosome 6B, and no contamination from
amplified DNAs of R2, R3 and R4 segments occurred. When
primers of Xpsp3131 (a microsatellite located on centro-
meric region of chromosome 6B) was used, a 144-bp band
was amplified only in the PCR reaction using wheat ge-
nomic DNA as the template (Fig.3D). Because of the un-
availability of sufficient microsatellite makers for chromo-
some 6B, the origin of amplified DNAs from R3 and R4
segments could not be demonstrated by amplification of
microsatellite sequences. Using other 38 pairs of primers of
microsatellite markers, located on other chromosomes than
chromosome 6B, all reactions using wheat genomic DNA
as template yielded an expected PCR product. Most of re-
actions using PCR products of positive control as template
yielded the same product, whereas reactions using ampli-
fied DNAs from R1, R2, R3 and R4 as template yielded no
product. Figure 3E and F show the results using the prim-
ers of Xpsp3034 and Xpsp3114, which were located on chro-
mosomes 2B and 7A, respectively.
2.3 Library analysis
The second round PCR products from microdissected
segments R1, R2, R3 and R4 were cloned into PGEM-T
vectors. In total, 420, 547, 490 and 586 recombinant clones
were obtained from a small fraction (1/500) of transformed
DH5a cells of the second round PCR products of segments
R1, R2, R3, and R4, respectively. It was estimated that ap-
proximately 2.10×105, 2.74×105, 2.45×105 and 2.93×
105 recombinant clones would be in the DNA libraries of
segments R1, R2, R3, and R4, respectively, if the clone re-
dundancy was not considered. One hundred and fifty ran-
domly selected recombinant clones were analyzed by plas-
mid extraction and restriction from each library. The length
of inserts ranged mainly from 300 to 1 800 bp, with an aver-
age of 830 bp estimated by agrose gel separation of inserts
Fig.2. Products of the second round PCR using microdissected chromosomal segments of chromosome 6B as templates and linker
adaptors as primers (A), and characterization of the product by Southern hybridization using DIG-labeled wheat genomic DNA as the
probe (B). G, wheat genomic DNA digested with HindⅢ; M, lDNA digested with HindⅢ and EcoRⅠ; N, negative control; P, positive
control (10 pg wheat genomic DNA as the template); R1, R2, R3 and R4, four microdissected chromosomal segments.
Hu Zan-Min et al.: Microdissection and Construction of Region-specific DNA Libraries of Wheat Chromosome 6B 1361
(Fig.4A). Wheat genomic DNA labeled with DIG was hy-
bridized to the filter containing inserts (Fig.4B). To estimate
the copy number of inserts, plasmid DNA of 128 recombi-
nant clones from each library were transferred onto
Hybond+ filters and hybridized with wheat genomic DNA
labeled with DIG (Fig.5). According to the intensity of hy-
bridization signals, inserts were estimated to be low/unique
copy (weak or no signals) and medium/high copy (strong
signals) sequences in wheat genome. The characterization
data obtained from each library are listed in Table 1.
3 Discussion
3.1 Microdissection of plant chromosomes by Nd:YAG
laser microbeam
It is difficult to study the molecular structure of large
plant genomes. Chromosome microdissection and
microcloning provide an efficient tool for this problem, by
which researchers can focus on a chromosome or chromo-
some segment. One of the key steps in chromosome micro-
dissection and microcloning is to dissect the target chro-
mosome and collected chromosome segments. In human
and some animals, procedures for chromosome microdis-
section and segment collecting have been well developed
using glass needles (Ludecke et al., 1989; Guan et al.,
1995). To dissect chromosomes in plant is more difficult
than in human, because chromosome preparation is more
difficult in plant. To date there is no report that a plant
chromosome has been dissected into several segments and
the segments were collected separately for subsequent DNA
cloning. In this paper, we report, for the first time, a suc-
cessful method to dissect plant chromosome using Nd:YAG
microbeam laser and to collect chromosomal segments with
glass needles and constructed four chromosome region-
specific DNA libraries from a single chromosome.
Fig.3. Verification of DNA amplified from microdissected chromosomal segments of chromosome 6B using microsatellite markers
located on chromosome 6B (Xpsp3009, Xpsp3139, Xpsp3079 and Xpsp3131), chromosome 2B (Xpsp3034) and chromosome 7A
(Xpsp3114). A, B, C, D, E and F show DNA amplification products using primers of Xpsp3009, Xpsp3139, Xpsp3079, Xpsp3131,
Xpsp3034 and Xpsp3114, respectively. Lane M, pUC19 DNA digested with MspⅠ (HpaⅡ); lane N, wheat genomic DNA as the
template; lane O, negative control (no template); lane P, positive control (using PCR product from positive control of LA-PCR); lanes
1, 2, 3 and 4, using the second round PCR product from the segment R1, R2, R3 and R4, respectively, as the template.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041362
Chromosome-specific DNA libraries were prepared from
flow-sorted chromosomes in wheat (Wang et al., 1992).
Improved methods for chromosome and chromosome arm
sorting have been developed (Vrana et al., 2000; Kubalakova
et al., 2002), and may be used to obtain large quantities of
chromosomes. DNA of sorted chromosome was reported
to be of high molecular weight (Simkova et al., 2003) and
thus suitable for construction of chromosome- and chro-
mosome arm-specific large insert DNA libraries cloned in a
BAC vector. However the flow cytogenetics methods can
not be used to constructed fine chromosome region-spe-
cific DNA libraries. Up to now, most of chromosomes of
plant can not be sorted by using flow cytometry. The ap-
proach we reported here should facilitate the analysis of
different fragments from single chromosome of plant.
3.2 Characterization of DNA from microdissected chro-
mosomal segments
Another important step in chromosome microdissection
and microcloning is the characterization of amplified DNA
from chromosomal segments to ascertain the origin of am-
plified DNA. It must be free from any DNA contamination
from other chromosomal segments, chromosomes and other
species. Amplified DNA from the target chromosome was
usually characterized by Southern hybridization using ge-
nomic DNA and/or PCR products from microdissected chro-
mosomes as probes (Albani et al., 1995; Chen and Armstrong
et al., 1996; Zhou et al., 2001; Stein et al., 1998). However,
the amplified DNAs can only trace to the genome of spe-
cies used in the experiment, not the dissected chromosome
and chromosomal segment. It cannot exclude DNA con-
tamination from other chromosomal segments or other
chromosomes.
Table 1 Characterization data of DNA libraries from segment
R1, R2, R3 and R4 of chromosome 6B
Characterization items
DNA libraries
R1 R2 R3 R4
Recombinant clone No. (×105) 2.10 2.74 2.45 2.93
Insert size (bp) 300- 300- 300- 300-
1 800 1 800 1 800 1 800
Average size of inserts 850 820 870 860
Low/unique copy clones (%) 42 44 43 47
Medium/high copy clones (%) 48 46 47 43
Clones without inserts (%) 10 10 10 10
Fig.4. Characterization of a part of recombinant clones from the DNA library of segment R1 of chromosome 6B by EcoRⅠ restriction
(A) and Southern hybridization using DIG-labeled wheat genomic DNA as probe (B).
Fig.5. Characterization of some recombinant clones from the
DNA library of segment R1 of chromosome 6B by dot hybridiza-
tion using DIG-labeled wheat genomic DNA as probe.
Hu Zan-Min et al.: Microdissection and Construction of Region-specific DNA Libraries of Wheat Chromosome 6B 1363
We used a set of microsatellite markers, located in dif-
ferent parts of wheat chromosomes (Stephenson et al.,
1998), to characterize the origin of amplified DNA. Accord-
ing to the location of Xpsp3009, Xpsp3139 and Xpsp3079,
it is estimated Xpsp3009 and Xpsp3139 might be located
on R2 region and Xpsp3079 on R1 region. Results showed
that PCR products were exactly amplified from correspond-
ing microdissected segments and had no contamination
from other segments. Due to the unavailability of
microsatellite makers on R3 and R4 regions, the origin of
amplified DNAs from R3 and R4 segments could not be
demonstrated by amplification of microsatellite sequences.
Therefore, microsatellite markers located on target chromo-
somal segment could be used to characterize the origin of
the amplified DNA. However, microsatellite markers located
in the centromere region of chromosome 6B could not be
amplified from the amplified DNA of that region.
Additionally, Xpsp3027, Xpsp7030, Xpsp3058, Xpsp3071
and Xpsp3114, which are also located in the centromere
region of other wheat chromosomes, could neither be am-
plified from any PCR products of microdissected chromo-
somal segments nor from the positive control. Probably
most of the highly repetitive DNAs within centromere re-
gions are not amplifiable by Sau3A adaptor-mediated PCR
due to the Sau3A’s sensitiveness to cytosine methylation
(Chen and Armstrong, 1995; Liu et al., 1997).
3.3 The quality of DNA libraries
The quality of DNA libraries depends on the enrich-
ment of unique/low copy sequence and repetitive
sequences. In the four libraries of chromosome 6B, 42%-
47% recombinant clones were unique/low copy sequences,
whereas 43%-48% were high-copy repetitive sequence.
On average, in a chromosome, over 80% DNA should be
repetitive sequence. The discrepancy probably was due to
the fact that Sau3A used for chromosome DNA restriction
is cytosine methylation sensitive. Some repetitive se-
quences with cytosine methylation spread on the chromo-
some especially in the centromere region might be selec-
tively amplified. Similar results were obtained by Chen and
Armstrong (1995), Liu et al. (1997), Stein et al. (1998), and
Zhou et al. (1999; 2001). So far, there has been no report on
microsatellite markers being developed from PCR products
of microdissected chromosomes and subsequent libraries.
In this study, several known microsatellite markers located
on chromosome 6B were proven to be present in the PCR
products of microdissected segments. Therefore, new re-
gion-specific microsatellite markers could be developed
from the libraries of chromosomal segments.
The number of recombinant clones and the size of
inserts are other factors influencing the quality of chromo-
somal DNA libraries. Only Stein et al. (1998) reported the
construction of two chromosomal region-specific DNA li-
braries of maize chromosome 6, in which the number of
recombinant clones were 4×103 and 4×106, respectively,
and the size of insert averaged 430 bp (ranging from 200 to
1 000 bp). In our study, the number of recombinant clones
for all four segments ranged from 2.1× 105 to 2.9×105,
and the size of insert ranged from 300 to 1 800 bp. The latter
are much larger than those described by Stein et al. (1998),
Jung et al. (1992), Schondelmaier et al. (1993), Chen and
Armstrong (1995), but similar to those described by Zhou
et al. (1999; 2001). The size of inserts was influenced by
many factors, such as acetic acid used for root treatment
and chromosome preparation. To avoid acid depurination,
we fixed root tips in 3 ethanol:1 acetic acid for just 5 min and
transferred them into 70% ethanol for at least one day.
In the four libraries of chromosome 6B, new microsatellite
markers and other DNA sequences specific to chromosomal
segments will be obtained and used for high-density link-
age mapping of chromosome 6B and the isolation of agro-
nomically useful gene. Further work on selection of poly-
morphic clones as molecular markers is now being
undertaken.
Acknowledgements: We thank Dr. Richard Wang (USDA-
ARS, Utah State University) for critically reading the manu-
script and Prof. Michael Denis Gale (John Innes Centre) for
kindly providing primers of wheat microsatellite markers.
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(Managing editor: ZHAO Li-Hui)