全 文 ：作物学报 ACTA AGRONOMICA SINICA 2011, 37(5): 772−777 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This work was supported by the grants of “863” High-tech Program (No. 2006AA10A106), the China National Fundamental Fund of Personnel
Training (No. J0730649) and partly supported by the open funds of the National Key Laboratory of Crop Genetic Improvement.
Corresponding author: ZHENG Yong-Lian, E-mail: zhyl@mail.hzau.edu.cn
Received(收稿日期): 2010-10-23; Accepted(接受日期): 2011-03-08.
DOI: 10.3724/SP.J.1006.2011.00772
Genome-wide Analysis of MuDR-related Transposable Elements Insertion
Population in Maize
FENG Jing1, FU Xue-Qian1, WANG Ting-Ting2, TAO Yong-Sheng2, GAO You-Jun1, and ZHENG Yong-Lian1,*
1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; 2 Hebei Research Station of
National Key Laboratory of Crop Genetic Improvement, Agricultural University of Hebei, Baoding 071000, China
Abstract: Insertional mutagenesis has now been widely used to knockout genes for functional genomics. The maize Mutator
transposons hold an advantage of high activity to construct large mutant libraries. In this study, a MuDR line was used to cross with
an elite Chinese maize inbred line Z31. A total of 1 000 M1 individuals were planted and self-pollinated to generate their M2 fami-
lies. Experiments were conducted to investigate the insertion specificity of MuDR related transposable elements. Six hundred and
ninety-five MuDR inserted flanking sequences were isolated with a modified MuTAIL-PCR method and analyzed with bioinfor-
matics. Three hundred and seventy-four non-redundant insertion sites were identified and 298 of them were mapped to a single locus
on the integrated maize map. The results revealed some prominent features of the MuDR-related insertions of maize: random dis-
tribution across the 10 chromosomes, preferential insertion into genic sequence and favoring some classes of functional genes.
Keywords: Zea mays; Mutator (Mu) transposons; MuDR elements; Flanking sequence; Insertion sites; MuTAIL-PCR
全基因组分析玉米MuDR转座因子插入突变体库
冯 静 1 傅学乾 1 王婷婷 2 陶勇生 2 高友军 1 郑用琏 1,*
1华中农业大学作物遗传改良国家重点实验室, 湖北武汉 430070; 2河北农业大学作物遗传改良国家重点实验室河北实验基地, 河北保
定 071000
摘 要: 在功能基因组研究中, 插入诱变被广泛用于基因敲除。玉米 Mutator转座子因其具有较高的转座活性常被用
于构建大型玉米插入突变体库。本研究利用具有活性 MuDR因子的玉米材料与优良玉米自交系 Z31杂交, 获得 1 000
个M1单株, 自交构建M2群体, 研究 MuDR因子在基因组中插入位点特性。利用优化的 MuTAIL-PCR方法分离出 695
条 MuDR 插入位点侧翼序列, 经初步生物信息学分析得到 374 条非冗余的插入位点, 其中的 298 条序列能够被定位
在玉米基因组物理图谱单个位点上。实验结果揭示了 MuDR因子插入的一些特性: 在 10条染色体上随机分布, 偏向
于插入到基因序列中, 并在某些功能基因中有明显插入偏好。
关键词: 玉米; Mutator转座子; MuDR因子; 侧翼序列; 插入位点; MuTAIL-PCR
Gene knockout has become a powerful and indispen-
sable tool in molecular genetics and functional genomics.
A comprehensive collection of gene knockouts allows us
to understand the relationship between the phenotypes
and mutations of genes[1].
Diverse approaches have been used to develop com-
prehensive gene knockout resources, which are necessary
for forward and reverse genetic analysis in plants.
Arabidopsis thaliana has provided a good model using
Flanking Sequence Tags (FSTs) approach via insertional
mutagenesis populations[2-3]. Several methods were pri-
marily applied to systematically amplify and sequence
the genomic DNA flanking the T-DNA tags from each
mutant. Then these FSTs were searched against the pub-
lic DDBI/EMBL/GenBank GSS database to obtain the
genome annotations. An improved FST approach was
adopted in rice[4-6] and the latest release of OryGenesDB
database contained 171 000 FSTs (http://orygenesdb.
cirad.fr/index.html), which greatly accelerated rice func-
tional genomic research[7].
Maize is comparable to rice as the model genetic sys-
tem for genome studies[8-9]. Since the Ac/Ds transposable
elements were discovered[10], the transposon mutagenesis
approaches have been widely utilized in disrupting genes,
isolating mutants and studying gene function in
maize[11-12].
第 5期 冯 静等: 全基因组分析玉米 MuDR转座因子插入突变体库 773


Transposon insertional mutagenesis can be classified
into low-copy and high-copy strategies[13]. The former
contains Ac and Spm, which produce low genome-wide
mutation rates and transpose preferentially to linked
sites[14-16]. These populations cannot remove the interfer-
ence of the background mutations due to the non-
autonomous Ds elements in the genome. The high-copy
system most commonly used in maize is Mutator[17].
Up to date, Mutator is the most active and mutagenic
plant transposon discovered in maize[18-20]. Its properties
of high copy numbers and high transposition frequency
make it suitable for forward and reverse genetic analy-
sis[21]. The Mutator transposon family is a two compo-
nent system[22]. All the maize Mutator elements contain
conserved −220 bp terminal inverted repeats (TIRs), but
each class of elements contains specific and unrelated
internal sequences[18,22]. Liu et al.[23] defined 21 novel Mu
TIRs using a DLA-454 strategy, different from the TIRs
reported previously. This system is regulated by autono-
mous MuDR elements, which control the transposition of
themselves and the other classes of the non-autonomous
Mu elements[24-25]. Besides, studies have shown that Mu
insertions are heavily biased for transcribed regions of
the genome[9,26-28].
The successful use of transposon tagging lies in the
identification and isolation of the genomic sequences
flanking the insertion sites. Many PCR-based methods
have been developed and some are optimized for Mu
elements according to the conserved sequences in the
inverted terminal repeats. Amplification of insertion
mutagenized sites (AIMS) was a ligation-mediated
method with the Mu primer biotinylated[29]. MuTAIL was
an adaptation of thermal asymmetrically interlaced
(TAIL) PCR[30] to amplify flanking fragments in a com-
plex pool of Mu-induced mutants[28]. Yi et al. combined
elements of both MuTAIL and AIMS into a procedure
called MuTA for co-segregation analysis[31]. Besides, an
adaptor-mediated PCR-based method, Digestion-ligation-
amplification (DLA), was developed to overcome diffi-
culties of amplifying unknown sequences flanking
known DNA sequences in large genomes[32]. Furthermore,
an improved draft nucleotide sequence of the 2.3-gi-
gabase maize genome has been released in 2009. All of
these would greatly facilitate the Mutator tagging
strategy in maize functional genomic research.
In our study, the M1 population was generated by
crossing the active Mutator transposon line as donor
parent with the recipient parent Z31. The number of copi-
es of the MuDR elements per mutagenic plant is one or
two in the population. We observed the phenotypes of the
M1 and the M2 generations and amplified the flanking
sequences of the MuDR with a modified MuTAIL-PCR
method. The TAIL products were cloned and sequenced
for further bioinformatics analysis. We attempt to con-
struct our own Mutator insertional mutant populations of
maize in China and create more novel mutants for the
maize functional genomic studies.
1 Materials and Methods
1.1 Plant materials
MuDR-active line was used as the pollen donor in a
cross with the maize inbred line Z31 (yellow kernel). The
progenies of the cross were self-pollinated to produce the
Mutator mutant stocks. The resulted kernels were planted
and young leaves were harvested for genomic DNA ex-
traction based on the protocol described by Settles [28].
1.2 Isolation of flanking sequences
Mu flanking sequences were amplified via a modified
MuTAIL-PCR[28]. Two nested specific primers were
modified according to the MuDR TIR sequence reported
as follows: TIR9-1: 5-ATAGAAGCCAACGCCATGGC
CTCCATTTCGTC-3; TIR9-2: 5-GGCCTCCATTTCGTC
GAATCCCTT-3.
The 12 arbitrary primers used in our experiment were
designed.
Either 5% DMSO or glycerin was added to the PCR
mixture. TAIL-PCR products were purified with QIA-
quick Gel Extraction Kit (QIAGEN, Germany) based on
the manufacturer’s instructions. The confirmed products
were ligated to the pGEM-T Vectors (Promega, USA).
Then the ligation products were transformed into the E.
coli DH5α by electronic transformer (Electroporator
2510, Eppendorf), and plated onto the LA medium con-
taining Ampicillin, X-gal and IPTG (40 mg μL−1).
Cloned inserts corresponding to the size of the fragment
from the PCR were full-pass sequenced by an
ABI3730XL sequencing facility (Applied Biosystems,
CA) and the sequences were analyzed bioinformatically.
1.3 Sequence analysis
A homology search was first performed against the
vector sequences of the plasmids using the NCBI Vec-
Screen tool (http://www.ncbi.nlm.nih.gov/VecScreen/
VecScreen.html). Then masked sequences were analyzed
for the presence of the conserved MuDR TIR sequence
using a BLAST program. Then a BLASTN was con-
ducted against the B73 RefGen_v1 database to get the
predicted location hits using the B73 Genomic Browse
Tools with the expectation score cut-off <10−12 (http://
www.maizegdb.org/).
2 Results
2.1 MuDR elements insertion sites
Mu-specific fragments were identified and cloned by
comparing its parental band patterns. Not all the 12 arbi-
trary primers were found to be suitable in our materials.
Only four of them, BAD5, CTG1, SAD11, and SW41
yielded large products from Mutator lines (Fig. 1).
Totally, 695 specific MuTAIL-PCR fragments were ob-
tained and sequenced from about 5 000 M2 individuals.
774 作 物 学 报 第 37卷


Fig. 1 MuTAIL products from the DNA of parents and mutants
using CTG1 arbitrary primer
M is DNA ladder marker DL2000. P1 and P2 are Z31 and Mutator ac-
tive parent. The other 12 lanes show the secondary products of the
TAIL-PCR. Arrows indicate the specific fragments.

Theoretically, a Mu flanking sequence should be ampli-
fied with 29 bp of Mu-TIR sequence downstream of the
specific primer of the TAIL-PCR. Presence of the
Mu-TIR sequence can identify the precise insertion site
of the elements. The results showed that, except for 150
of them without partial MuDR TIR sequence, each of the
remaining 545 sequences had a complete MuDR TIR
sequence and indexed a known individual. Vector and
quality-trimmed sequences were deposited to the NCBI
gss database (dbGSS_ID: 28569169 to 28569485).
2.2 Homology analysis of the flanking sequence
Analysis of 545 sequences by the ClustalX (V2.0.12)
software, 374 non-redundant insertion sites were ob-
tained. Of the 374 sequences, the overall average length
was 500 bp, including 25 sequences in the length ranged
from 25 bp to 100 bp, 153 sequences in the range of
100–499 bp, 183 sequences in the range of 500–999 bp
and 13 sequences longer than 1 000 bp. The homology
search of the trimmed sequences using the BLASTN tool
found that 298 sequences contained 29 bp TIR sequences
as expected and the others having significant similarity
(E-value<10−12) with the maize genomic sequence
mapped in the maize genome.
2.3 Distribution of MuDR insertions along
chromosomes
Based on BLASTN searches of the released BAC and
cDNA sequences, we used an in silico protocol to locate
MuTAIL sequences to make sequence information more
visual in functional genomics. Predicted locations of 298
MuTAIL are shown in Figure 2. Their distribution across
the 10 chromosomes of maize was nearly random.
2.4 Distribution of MuDR insertion sites rela-
tive to transcribed maize sequences
The genomic sequence could be roughly divided into
three classes: genic region, transposable element (TE)-
related and intergenic sequences. The observed numbers
in each of the three sequence classes are 497, 22, and 26,
respectively. Only 22 (~4%, 22/545) sequences contain
repeated sequences, having significant similarity with
TE-related sequences. In addition, the remaining 523
sequences could be uniquely mapped to the maize ge-
nome. Taken together, MuDR insertions observed
strongly disfavored the TE-related sequences and exhi-
bited a preference for transcribed regions.

Fig. 2 Distribution of MuDR insertion sites among chromosomes
The predicted locations of 298 MuTAIL sequences were determined by
BLASTN analysis as described in the materials and methods. The black
ovals show centromeres and the lines indicate the relative positions on
the chromosomes.

BLASTP results showed that 265 of the 374 non-
redundant insertion sites were found to be relevant to
predicted genes with function annotations. These inser-
tions were categorized into four disjointed regions: pro-
moters, 5-UTR, exons, introns, and 3-UTR. The ob-
served numbers of insertions that occurred in each of the
four regions are shown in Figure 3.

Fig. 3 Distribution of MuDR insertion sites within transcribed
maize sequences
Abscissa: positions of the insertion sites; longitudinal coordinates:
number of tested insertion sites.

2.5 Classification of MuDR target genes func-
tion using Gene Ontology (GO)
To gain a better perspective of the functional roles of
the insertion sites in maize, we looked for targets en-
richment in Gene Ontology (GO) molecular function and
biological process categories[33]. The targets were anno-
tated by using the GO annotations available from the B73
RefGen_v1. of the predicted targets, 71% had GO as-
signments whereas only 68% of the genes in the entire
refined set were associated with GO terms. BiNGO
(Biological Networks Gene Ontology)[34] was used to
study targets enrichment and to construct a hierarchical
ontology tree in Cytoscape[35], as shown in Figure 4. We
found that MuDR elements preferentially target genes
involved in a wide spectrum of regulatory functions and
第 5期 冯 静等: 全基因组分析玉米 MuDR转座因子插入突变体库 775



Fig. 4 Maize MuDR targets enrichment network based on GO molecular functions and biological processes
Significantly overrepresented GO terms were visualized in Cytoscape. The size of a node is proportional to the number of targets in the GO category.
The color represents enrichment significance: the deeper the color on a color scale, the higher the enrichment significance. White color nodes are not
enriched but show the hierarchical relationship among the enriched ontology branches.

selected biological processes including gene expression,
transcription, metabolism, catalysis, and transport.
3 Discussion
3.1 Efficiency of the modified MuTAIL-PCR
It has previously reported that MuTAIL-PCR is highly
effective to identify the causal Mu insertions rapidly[28].
We applied this method and made some improvement of
the specific primers in order to identify genes disrupted
by MuDR elements within a genome. We found that the
TAIL products were more specific with less but bright
bands in the agarose-gel (data not shown). In addition,
about 80% of the isolated sequences contained the au-
thentic 29 bp TIR sequence of MuDR elements. The data
demonstrated that the modified MuTAIL-PCR method is
effective to get the MuDR flanking sequences.
3.2 Transposition features of the MuDR ele-
ments
As more and more genomic insertion sites of Mu
transposons were amplified, the characterization of the
Mutator system was made clear. For example, the vp loci
of maize primarily acquires Mu1-related elements. Al-
leles of five loci (vp1, vp8, vp10/vp13, vp14, vp15) have
been confirmed[36-39]. However, previous experiments
showed that sh1 locus seemed to primarily acquire muta-
tions due to elements of the MuDR subfamily. Though
lacking of any evidence of hotspot genes in our experi-
ments, we found that some MuDR insertion sites were
located in the same predicted gene within dozens of
basepairs intervals. We conclude that MuDR elements
have a strong preference for insertion into low-copy se-
quences, especially into the transcribed sequences which
was consistent with the Mutator system.
3.3 Utilization of Mutator population
Thanks to the discovery of the maize Mutator system,
many large-scale genomic projects have been initiated. A
large number of Mu insertional populations were con-
structed to understand the phenotypic consequence of the
loss of any gene within the genome[27-28,40-41]. Z31, an
elite maize inbred line with high combining ability, was
used to construct our own Mu insertion mutant popula-
tion and get more novel mutants. Our strategy, based on
molecular analysis of mutant lines using MuTAIL se-
quencing, serves to anchor sequence-indexed insertional
mutations caused by Mu insertions. It dramatically sim-
plifies a reverse genetics project by allowing us to iden-
tify genes of interests and corresponding knockouts in
silico.
4 Conclusion
Six hundred and ninety-five MuDR elements flanking
sequences were isolated with a modified MuTAIL-PCR
method. Three hundred and seventy-four non-redundant
insertion sites were identified and 298 of them were
mapped to a single locus on the maize integrated map.
The results revealed some prominent features of the
MuDR-related insertions of maize. The construction of
the population and the analysis of MuDR insertion sites
were just part of our research. Two specific mutants will
be meticulously analyzed and further research is in pro-
gress.

Acknowledgements: We thank Prof. Lai J S of China
Agriculture University for supplying plant materials.
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