Microdissection of the Ah01 chromosome in upland cotton and microcloning of resistance gene anologs from the single chromosome
Cao et al. Hereditas
Microdissection of the A 01 chromosome h in upland cotton and microcloning of resistance gene anologs from the single chromosome
Xinchuan Cao 0
Yuling Liu 0
Fang Liu 0
Zhongli Zhou 0
Xiaoyan Cai 0
Xingxing Wang 0
Zhenmei Zhang 0
Yuhong Wang 0
Renhai Peng 0
Kunbo Wang 0
0 State Key Laboratory of Cotton Biology/Institute of Cotton Research of Chinese Academy of Agricultural Sciences , Anyang, Henan 455000 , China
Background: Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. Results: Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. Conclusions: Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.
Upland cotton; Chromosome microdissection; Microcloning; RGA
Chromosome microdissection is one of the most
important techniques in molecular cytogenetic research. Specific
chromosome or chromosomal sections are isolated using
a glass needle or laser under a microscope, and then are
enzymatically digested and amplified to construct DNA
library of a single chromosome or chromosomal section.
Research focusing on a single chromosome or a
chromosomal subsection can greatly reduces subsequent work,
such as identifying, screening and minimizing the whole
genome screening. This technique has been widely used
in Drosophila, humans and many other animals since its
establishment [1–8] Subsequently, the technique has been
widely adapted to apply in herbaceous plants including
barley, wheat, rice, and tomato [9–18] and woody plants
such as pomelo and poplar [19, 20].
Plants have developed defensive mechanisms to protect
themselves from pathogen infection through a number of
evolutionary processes. The gene-for-gene hypothesis
proposed by Flor is based on the interactions between
pathogenic fungi and host plants and constitutes the theoretical
basis of cloning avirulence genes from pathogens and
resistance genes (R genes) from plants . So far, many R
genes have been cloned from different host plants using
positional cloning and transposon tagging methods.
However, considering the large number of physiological races
of pathogens, transposon tagging and positional cloning
methods are clearly inefficient. Thus new strategies and
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methods should be adopted to accelerate the cloning of
disease R genes. Due to the conserved domains of R genes,
homologous sequence amplification or the homologous
sequence-based candidate gene approach would be a good
choice; actually, these techniques have been quickly
adopted by the scientific community. A great progress has
been made in recent years for obtaining disease RGAs
from many plant species [22–28]. Additionally, some of
these RGAs were used as probes for linkage analysis and
positioning [22–24, 27].
As the primary natural fiber crop, cotton (Gossypium
hirsutum) plays an important role in the world’s
economy. However, cotton cells contain large amounts of
secondary metabolites, and their chromosomes are small
in size and nearly identical to each other. These prevent
in somehow to well prepare the chromosomes from the
cells and clearly distinguish them from their karyotypes,
and thus cytogenetic research of cotton is still lagging
behind other plant species, such as rice and wheat. As a
typical tetraploid plant species [29–35], there are two
sub-genome (A1A1D1D1, 2n = 4× = 52) and high
number of nucleotide sequence repeats in cotton genome.
There are greater uncertainties in interpreting whole
genome while assembling or annotating [33, 34].
Microdissection of a single chromosome or its subsections using
direct micromanipulation techniques and gene
microcloning through molecular biology should be one easy way to
slove this problem. However, currently, there is only one
report about single chromosome microdissection that was
from somatic cells , there is no any report on
chromosome microdissection from pollen mother cells (PMC)
and on gene microcloning from single chromosome.
There are many important genes are closely related to
disease resistance, fiber development, fiber quality and
yield in the Ah01 chromosome of TM-1 upland cotton
[37–39]. In this study, the Ah01chromosome was
microdissected from the Ah01 monosome materials derived
from TM-1 (a genetically standard line of upland cotton)
using the laser method. A DNA pool was constructed
from the single chromosome by amplifying DNA using
linker adaptor polymerase chain reaction (LA-PCR).
RGAs from this chromosome were then cloned.
A accession of Ah01 monosome, developed from the
genetically standard line of upland cotton at Texas A&M
University (U.S.) , was used as the primary plant
materials. The accession was grown in the Greenhouses at the
Institute of Cotton Research, Chinese Academy of
Agricultural Sciences, (ICR-CAAS) (Anyang, Henan,
China) and is also maintained in the National Wild
Cotton Nursery located at Sanya City, Hainan Island,
SSR markers and primers
The chromosome-Ah01-specific BAC clone 52D06 was
provided by Professor Tianzhen Zhang of Nanjing
Agricultural University. The primers of corresponding
simple sequence repeat (SSR) marker BNL3580 (F
primer: CTTGTTTACATTCCCTTCTTTATACC; R
primer: CAAAGGCGAACTCTTCCAAA), degenerate
specific primers P1 (5′-GATCCTGAGCTCGAATTCG
ACCC-3′) and P2 (5′-GGGTCGAATTCGAGCTCAG-3′)
were synthesized by Shanghai Sangon Biotech Inc. .
Preparation of mitotic metaphase chromosomes
Mitotic metaphase chromosomes were prepared
according to a previous report  with a few modifications.
The slides prepared were kept at −20 °C for a long-term
storage or at 4 °C for a short period storage. Slides were
baked at 60 °C overnight immediately before use.
Preparation of film-slides
Sampling, fixing, and enzymatic hydrolysis of flower
buds were performed according to the protocol of Peng
et al. . Enzymatically digested anthers were smeared
on film-slides as previously described .
Microdissection of single chromosome and LA-PCR
Single chromosome was microdissected using the
CellCutPlus Laser micromanipulation system (MMI
Company, Swiss) and LA-PCR amplification was conducted
as previously described . Positive (~10 pg of genomic
DNA added to the initial template) and negative controls
(no genomic DNA added to the initial template) were
also set up.
Two rounds of LA-PCR products were separated through
electrophoresis with 1% agarose at 100 V for 30 min.
LAPCR products were observed and photographed under
UV light after 40 min staining with ethidium bromide.
Southern hybridization was conducted with PCR
products, partially digested genomic DNA, positive control
(PCR product from genomic DNA as template) and
negative control (no template PCR reaction) .
The amplified pool of Ah01 chromosomes and second
LA-PCR products were amplified using the
chromosome- Ah01- specific SSR primer respectively. The
amplified products were checked by polyacrylamide gel
Fluorescence in situ hybridization
Dual-color FISH (fluorescence in situ hybridization) and
the detection of metaphase chromosome specimens
were performed according to a previous . The
second round of LA-PCR products labeled with DIG
(Digoxigenin-11-dUTP, Roche) and specific Biotin
(Biotin-16-dUTP) labeled bacterial artificial chromosome
(BAC) clones (52D06) were used as probes, which were
detected by Anti-Digoxigenin-Rhodamine (red) and
FITC-Anti-Biotin (green) (Roche Diagnostics, USA),
respectively. Cot-1 DNA was used to pre-hybridize for
blocking the repetitive sequences. Chromosomes were
counterstained by 4′, 6-diamidino-2-phenylindole (DAPI)
in VECTASHIELD anti-fade solution (Vector
Laboratories, Burlingame, CA). The hybridization signals were
observed using a fluorescence microscope with a
changecoupled device (CCD) camera (Zeiss Axiokop2 plus). The
images were adjusted using Adobe Photoshop CS3
Cloning and analysis of RGAs
RGA sequences were obtained by PCR with the Ah01
chromosome second round LA-PCR product as
template and P1 and P2 as degenerate specific primers.
Positive control (about 10 pg of genomic DNA was added to
the initial substrate) and negative control (no template)
reactions were also performed. The PCR products were
examined by agarose gel electrophoresis and Southern
hybridization. The target bands of the PCR products
were recovered. Positive clones were obtained, and
sequenced, and the sequences were used as a probe BLAST
search of homologues in NCBI Genbank database..
Screened homologous RGA clones were sequenced by
Shanghai Sangon Biotech Inc. Introns were annotated
with ORFinder. The sequences were queried against
the tetraploid Gossypium hirsutum genome sequcence
[33, 34]. The final obtained sequences were submitted
to Genbank. BlastN search was performed in GeneBank
using these sequences, and a sequence cluster was created
Chromosomes preparation and microdissection
PMCs moderately digested in an enzymatic mixture
were stained with carbolfuchsin. The PMCs at
metaphase I were used for chromosome preparation (Fig. 1a).
The target chromosome Ah01was initially found under
low magnification, and then captured under high
magnification for collection in a tube containing10 μL
proteinase K (50 ng·μL−1) solution. The protocols for cutting
and collecting chromosomes are shown in the Figure 1.
For comparison, other chromosomes in metaphase I
were simultaneously collected in different tubes for
SSRamplified proof after second LA-PCR amplification.
LA-PCR amplification of chromosomal DNA
Two rounds of LA-PCR were conducted to amplify the
Ah01 chromosomal DNA. Electrophoresis results (Fig. 2)
revealed that a weak DNA smear with sizes ranging
from 200 to 1000 bp after the initial LA-PCR (Fig. 2,
lane 3), and a strong DNA smearing pattern with sizes
ranging from 300 to 2500 bp were generated after the
second LA-PCR (Fig. 2, lane 5, 6), because of more
products. For the negative controls, there were no bands
(Fig. 2, lane 1, 2). The positive control produced a weak
initial band (Figure 2, lane 4) and an obvious smearing
pattern after the second LA-PCR (lane 7 in Figure 2).
These results indicated that the Ah01 chromosome was
Southern blot analysis
Enzyme-labeled upland cotton genome was used as a
probe, and the second LA-PCR products were verified
by Southern hybridization with negative and positive
Fig. 1 Microdissection and collection of single mono-chromosomes by CellCut Plus laser manipulation. a Film-slide preparations of meiotic metaphase
I chromosomes with one monomer chromosome (Ah01). b Film-slide preparations of meiotic metaphase I chromosomes with one microdissected
chromosome. c The microdissected chromosome on the cap of a collection tube. Arrow indicates the Ah01 chromosome. Bar: 5 μm
Fig. 2 Agarose gel electrophoresis of LA-PCR products. 1, 2: Negative
controls 3 Product from the first round LA-PCR. 4, 7: Positive controls.
5, 6: Products from the second round LA-PCR. M: DNA marker
controls (Fig. 3). Results of Southern blot showed that
the second products of LA-PCR (Fig. 3, lane 4–6) and
positive control (Fig. 3, lane 2, 3) had obvious bands,
indicating that the amplification products from G.
hirsutum genome were ranging from 300 to 2000 bp; that
was consistent with the results from agarose gel
electrophoresis. There were no bands in the negative control
PCR (Figure 3, lane 1).
Verification of SSR amplification
Specific SSR primer from Ah01 chromosome was selected
to amplify the second LA-PCR products of chromosome
Ah01 and some other chromosomes. Results were checked
by PAGE, and it was observed that the Ah01chromosome
could amplify a specific band (240 bp), as shown in Fig. 4
(Fig. 4, lane 10). The similar band was obtained using the
genome DNA as positive control (Lane 12), no band in
negative control (Fig. 4, lane 11) and partly others
chromosomes (Fig. 4, lane 1–9).
Fluorescence in situ hybridization
Dual-color FISH was performed using DIG-labeled
products of LA-PCR II and specific Biotin-labeled Ah01
chromosome BAC clone (52D06) to probe the
metaphase chromosome slide. As shown in Fig. 5, the target
chromosomes were accurately identified by means of the
specific BAC clone as well as products of LA-PCR II.
Meanwhile, partial other chromosomes had weak signal
(red light), indicating homologous sequence on these
Isolation of RGAs
RGAs were isolated using PCR with P1 and P2 as
primers, and TM-1 upland cotton genomic DNA and
Fig. 3 Southern blotting of products from the second round LA-PCR. 1:
The negative control. 2, 3: Positive controls. 4, 5, 6: The second round
LA-PCR products. 7: EcoRI digested genomic DNA. M: DNA marker
Fig. 4 PAGE of SSR primer amplification product from single
chromosome pool. 1–9: SSR primer amplification products from partial
other chromosomes pool with Ah01 chromosome specific primer. 10:
SSR primer amplification products from single chromosome pool with
chromosome Ah01 special primer (arrow indicated). 11: The negative
control. 12: The positive control. M: DNA marker
Fig. 5 FISH signals of products from the second round LA-PCR. a Chromosomes stained with DAPI. b Signals fromproducts of LA-PCR II (red).
c Signals fromchromosome Ah01 specific BAC (green, arrow indicated). d Signals from dual-FISH
the second round LA-PCR Ah01 chromosome pool as
templates, respectively. Products were detected by gel
electrophoresis and Southern hybridization. Using
genomic DNA as positive control, a slightly wider DNA
smear with sizes ranging from 400 to 1000 bp and a major
band of 550 ~ 700 bp was generated (Fig. 6a, Lane3). A
narrow DNA smear with sizes ranging from 550 to 800 bp
and a main band of 650 bp was generated using the
second LA-PCR products as template (Fig. 6a, Lane 2).
Southern blot results demonstrated that the products
come from the genome of upland cotton (Fig. 6b).
Cloning and validation of RGAs
Main bands of the PCR products were recovered and
cloned. Two hundred positive clones (PS001 ~ PS200)
were obtained and sequenced, followed by BLAST
analysis. Three sequences [(Additional file 1) named PS016
(Genbank ID: KU051681), PS054 (Genbank ID: KU0
51682) and PS157 (Genbank ID: KU051680)] contained
a conserved domain common to the NBS-LRR R genes
in plant. Clustering results showed that they were
homologous to these published RGAs (Fig. 7). Alignment
was made with others RGAs from NCBI (Additional file
2), the results also definitely confirmed that the three
RGAs were the NBS-LRR class of RGAs in cotton.
Identification and microdissection of a single
Accurate identification of the target chromosome is a key
step in chromosome microdissection and cloning.
Identification of the target chromosome has mainly relied on the
morphological features such as monosome, trisome,
nullisome and shape-specific chromosomes [9–11, 43–47].
Fig. 6 Agarose gel electrophoresis (a) and Southern blotting (b) of P1/P2 primer PCR products. A-1, B-1: Negative controls. A-2, B-2: Single Ah01
chromosome as DNA template. A-3, B-3: Positive controls using 10 pg G. hirsutum genomic DNA as template. B-4: EcoRI digested genomic DNA
of G. hirsutum. M: DNA marker
100 AY747332.1Arachis hypogaea
75 90 AY331206.1Gossypiumbarbadense
100 AF402768.1Theobroma cacao
100 AY746420.1Poncirus trifoliata
100 95 AY130803.1CitrusgrandisxPoncirustrifoliata
70 100 FM992103.1Gossypium arboreum
100 55 90 FAJY736391810953..11GGoossssyyppiiuummbhairrsbuatduemnse
100 AF420476.1Brassica nigra
40 AK230460.1Arabidopsis thaliana
Fig. 7 Cluster analysis of single chromosome RGA nucleotide sequenceswith those from other species
Chromosome banding technique has also been reported
as a method to identify chromosomes , but this
method has not widely used in plants. In this study,
monosome chromosome in meiotic metaphase I were easy
to identify and isolate from other chromosomes.
There are three approaches reported for chromosome
isolation. One is flow cytometry, which has facilitated
the dissection of large genome into smaller and defined
segments for the purpose of gene discovery and genome
sequencing in plants . Nevertheless, this method not
only requires expensive instrumentation, but also fails to
distinguish chromosomes with similar morphological
characteristics from one to another, which limit its
application in plants to some extent. The second approach is
the glass needle method, which involves in dissection of
the target chromosome under an optical microscope by
a glass needle. The approach is easily operate and
independent from high-end instrumentation, which has
resulted in effective and widespread application in plants
[16, 20]. However, the approach requires the operator to
be trained well enough, or there should be much
deviation operated by different persons or even in different
personal statuses by an operator. The third method is
laser cutting [44, 49], in which chromosome specimens
are dispersed onto a special carrier covered with a
membrane for dissection and collection. In most cases,
dissection is much easier than collection. In this study, the
CellCutPlus Laser microdissection system was applied to
isolate the target chromosome. Initially, cotton
chromosomes were spread on the microscope slide coated with
film, and then a single chromosome was dissected and
automatically collected in a microcentrifuge tube with a
Confirmation of the chromosomal DNA
LA-PCR is a powerful tool for the amplification of long
DNA segments, and it has been widely used in molecular
biology [18, 50, 51]. In this research, Ah01 chromosomal
DNA was acquired by LA-PCR after microdissection of
the target chromosome. Prior to subsequent steps, the
PCR products were examined by agarose electrophoresis,
Southern blot analysis, SSR primer confirmation and
confirmed by FISH. Combining several confirmation methods
could achieve multiple analyses, and ensure that
amplification products were from the target chromosome.
Significance of generating RGAs from specific single
chromosome of cotton
R genes have been isolated from the whole genome or
its cDNA in woody plants [7, 19, 20, 39]. In this study,
RGAs were isolated from a single chromosome dissected
from upland cotton, clarifying the source and location.
Efficiency of downstream work was greatly improved
due to the isolation of a single chromosome from the
entire genome. It has been reported that the R genes
family frequently clusters on a certain chromosomal
segment . Acquired RGAs could be transformed to
molecular marks, and serve to construct a genetic map
due to the clear linkage relationship from one
chromosome. In addition, it will contribute to the development
of map-based cloning, thus, generating RGAs from a
specific chromosome has many advantages.
Although cotton is one major crop in the world like rice,
wheat and maize, its cytogenetic studies falls much behind
others due to the smaller and identical chromosomes in
morphology as well as large amounts of secondary
metabolites within its cells. All these factors make the cotton
chromosome preparation difficult. Here, we successfully
developed a technique to separate a single chromosome
from upland cotton PMC (monosome cells) with the laser
cutting. Using this technology, we also microcloned three
RGAs from the DNA pool constructed with the single
chromosomes (Ah01). The three RGAs belong to the
nucleotide binding site-leucine rich repeat (NBS-LRR)
gene family. The techniques will promote the cloning of
cotton R genes and marker assistant improvement of
cotton genetics and breeding.
Additional file 2: Alignment of the three RGAs. (PDF 143 kb)
We deeply thank Prof. Tianzhen Zhang (Nanjing Agricultural University,
China) for kindly providing the chromosome-specific BAC clone.
The research was supported by grants from the National Natural Science
Foundation of China (No. 31471548), State Key Laboratory of Cotton Biology
Open Fund (No. CB2014A07), the Program for Science & Technology
Innovation Talents in Universities of Henan Province (13HASTIT026).
RP and KW designed the study; XC, YL, ZL, FL, YW, ZZ, XC, XW, CW, YW and
ZL performed the experiments; YL wrote the manuscript, RP and KW
proofread the manuscript. All authors read and approved of the manuscript.
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