Genome-wide mutagenesis of Zea mays L. using RescueMu transposons
- John Fernandes†1,
- Qunfeng Dong†2,
- Bret Schneider1,
- Darren J Morrow1,
- Guo-Ling Nan1,
- Volker Brendel2, 3 and
- Virginia Walbot1Email author
© Fernandes et al.; licensee BioMed Central Ltd. 2004
Received: 4 March 2004
Accepted: 5 August 2004
Published: 23 September 2004
Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.
MuDR/Mu transposable elements are widely used for mutagenesis and as tags for gene cloning in maize [1, 2]. The high efficiency of Mu insertional mutagenesis regulated by MuDR in highly active Mutator lines reflects four features of this transposon family. First, a plant typically has 10-50 copies of the mobile Mu elements , although some plants have over 100 copies. Second, they insert late in the maize life cycle, generating diverse mutant alleles transmitted in the gametes of an individual Mutator plant . Third, they exhibit a high preference for insertion into genes . And fourth, most maize genes are targets as judged by the facile recovery of Mu insertion alleles in targeted screens [1, 4–6]. In directed tagging experiments, the frequency of Mu-induced mutations for a chosen target gene is 10-3-10-5 . Interestingly, a bronze1 exon  and the 5' untranslated region of glossy8  contain hotspots for Mu insertion in specific regions, which may explain the higher frequency of mutable allele recovery for these genes.
Somatic mutability, visualized as revertant sectors on a mutant background, is indicative of transposon mobility. By monitoring maintenance of a mutable phenotype, it was established that the Mutator transposon system is subject to abrupt epigenetic silencing, which affects some individuals in most families [10, 11]. A molecular hallmark of silencing is that both the non-autonomous Mu elements and the regulatory MuDR element become hypermethylated [12, 13]. Without selection for somatic instability of a visible reporter allele and/or hypo-methylation, Mutator lines inevitably lose Mu element mobility.
The high efficiency of Mu mutagenesis has been exploited in several reverse genetics strategies. The first protocol described used PCR to screen plant DNA samples to find Mu insertions into specific genes using one primer reading out from the conserved Mu terminal inverted repeats (TIRs) and a gene-specific primer [14–17]. Alternatively, survey sequencing of maize genomic DNA flanking Mu insertions yields a list of tagged genes in each plant [18, 19]. A third method uses RescueMu, a Mu1 element containing a pBluescript plasmid, to conduct plasmid rescue by transformation of Escherichia coli with total maize DNA samples. To identify insertions in genes of interest, RescueMu plasmids can be screened or the contiguous host genomic DNA can be sequenced using primers permitting selective sequencing from the right or left TIRs of Mu1 .
Here we describe the initial results of a large scale RescueMu tagging effort conducted by the Maize Gene Discovery Project. The tagging strategy employed grids of up to 2,304 plants organized into 48 rows and 48 columns. Plasmid rescue was undertaken from individual pools of up to 48 plants per row or column. Genomic sequences next to RescueMu insertion sites were obtained for all the rows and for a subset of columns of six grids. Maize genomic sequences were subsequently assembled into 14,887 unique genomic loci using computational approaches. These loci were analyzed for gene content, the presence of repetitive DNA and correspondence to mapped maize genes and ESTs. Gene models were built by co-assembling the genomic sequence with ESTs and cDNAs by spliced alignment and by ab initio gene prediction. Identified gene models were tentatively classified using gene ontology terms of potential homologs .
Many features of Mu element behavior have been examined previously using hundreds of tagged alleles or by analyzing the population of Mu elements in particular plants and a few descendants. With single founder individuals for the analyzed tagging grids, we could examine the distribution of new insertion sites of RescueMu in large progeny sets. The contiguous genomic sequences were analyzed to determine if there were insertion hotspots, preferential insertion site motifs, routine generation of the expected 9-base-pair (bp) direct target sequence duplication (TSD) and evidence of pre-meiotic insertion events.
Like other Mu elements, RescueMu exhibits a strong bias for insertion into or near genes, as few insertions were recovered in retrotransposons or other repetitive DNA. In addition, for the set of RescueMu insertions into confirmed genes, a bias for insertions into exons (rather than introns) was observed, consistent with the well-established use of Mutator as a mutagen. The gene-enrichment exhibited by RescueMu was compared against two physical methods of gene enrichment, methyl filtration  and high C 0 t genome fractionation .
RescueMutransposition in active Mutator lines
Grid organization and analysis of mutant phenotypes segregating among selfed progeny of grid plants
Grid size‡ (row × col)
Independent mutations (% of families)¥
34 × 48
52 × 48
40 × 40
48 × 48
40 × 48
41 × 41
46 × 48
38 × 36
38 × 34
38 × 45
30 × 30
36 × 20
40 × 40
32 × 44
47 × 48
48 × 48
48 × 24
36 × 36
48 × 48
48 × 46
48 × 48
48 × 48
34 × 48
45 × 45
44 × 44
Materials were selected from the progeny of grid A plants for grids B through E using two criteria: there were visible seedling mutations in around 10% of progeny characteristic of a very active Mutator line  and the presence of trRescueMu. By DNA blot hybridization of individuals within grids B through E, the RescueMu transposition frequencies ranged from 0.1 to 0.26 (Table 1). By sequence analysis after plasmid rescue, trRescueMu were identified that had inserted into likely maize genes and generated the diagnostic 9-bp TSD characteristic of Mu transposition (data not shown). There were also events initially scored as transposition by blot hybridization that represented RescueMu rearrangements within the transgene array, and deleted forms of RescueMu were detected by blot hybridization and gel electrophoretic sizing of rescued plasmids (data not shown). Although RescueMu insertion frequency was low, overall Mu movement was very high in these grids; visible, independent seedling mutations were identified in 10.1-28.3% of the selfed progeny (Table 1), as high as the most active Mutator lines described to date .
Subsequently, surveys within each grid were used to identify plants with two or three newly trRescueMu and no evidence of Mutator silencing for construction of the next tagging populations. In this manner, the frequency of trRescueMu was increased in some grids to 1.0-1.4 per plant (Table 1) reflecting a frequency of 0.5-0.7 per parental element.
Library plate preparation and gene representation
As shown schematically in Figure 1, the trRescueMu insertion sites have been immortalized by preparing libraries from each of the row and column leaf pools from 16 grids, with three additional grid libraries under construction (Table 1). Briefly, total maize DNA was digested with BamHI and BglII, both of which recognize sites outside of RescueMu, and the fragment mixture was used to transform E. coli (see Materials and methods). The resulting library plates contain 56-96 individual row and column libraries representing the diversity of germinal trRescueMu and a sampling of somatic events present in the harvested leaf tissue (each well in a library plate is a pool of 20-48 plants from a row or column). The parental RescueMu insertion sites inherited from the grid founder(s) are present in every library.
Library plates contain a high diversity of genomic sequences. In a row of 48 plants, assuming random insertion, two segregating founder elements and a transposition frequency of 1.0, there will be 50 different plasmid types in the heritable class. Including heritable and somatic insertions, we estimate that each row or column library contains about 100-200 distinct plasmid types. Given these parameters, a library plate from a 48 row × 48 column grid with an average of 150 somatic plasmids per row or column library would contain 14,400 somatic insertion sites plus 2,304 germinal events and the two parental insertion sites. Because RescueMu shows a strong bias for insertion into genes , each library plate contains a substantial fraction of the predicted 50,000 genes of maize , provided the insertion sites are random. Ultimately, library plates for 19 grids derived from 33,000 plants and containing an estimated 30,108 heritable trRescueMu insertion sites (grid size × transposition frequency from Table 1) will be available online from the Maize Gene Discovery project through MaizeGDB .
Plasmid recovery analysis and identification of probable germinal insertions (PGIs)
Based on gel electrophoretic analysis of nearly 1,000 rescued plasmids, the genomic DNA flanking RescueMu averaged 3.5 kilobases (kb), with a range of 0.4-15 kb (data not shown). To accommodate the large size of some plasmids, a PCR template preparation protocol was devised to amplify genomic inserts of up to 16 kb for high-throughput sequencing ; primers were designed to amplify from within the right and left TIRs reading outward into the maize genomic DNA such that high quality sequence would be available to identify the TSDs flanking RescueMu insertion sites. Plasmids from all rows plus several columns of a grid were sequenced, with a routine yield of 80-92% success. A subset of plasmids could not be bidirectionally sequenced, because they lacked the TIRs at one or both ends. Deleted forms of trRescueMu were detected in several percent of the individuals surveyed by DNA blot hybridization (see Figure 2 for an example). If such derivatives retained the origin of replication and ampicillin-resistance marker, they could be cloned by plasmid rescue; if the TIRs were absent, they could not be sequenced.
Previous analysis of trRescueMu demonstrated that somatic insertion events, typically found in a tiny leaf sector, were sequenced just once from a leaf DNA sample while multiple instances of the germinal events could be recovered . Out of 28,988 non-parental plasmids sequenced, 41% (11,749) were recovered once (new trRescueMu somatic plus germinal insertion events) for each grid, and 59% (17,239) were recovered multiple times (probable new trRescueMu germinal insertion events). In addition, a total of 24,875 parental plasmids were transmitted from the founder plants. The percentage of parental plasmids within each grid varied from 17% for grid G to 61% for grid P. Some grids had more parentals than other grids and some parental plasmids were preferentially sequenced for unknown reasons. The parental insertion sites include the two or three known parental sites that each segregated into 50% of the progeny. Somatic sectors in the tassel or ear of the parental plant that generated plasmids found in multiple individuals within the grid are analyzed in a later section.
Probable germinal insertions (PGI) based on row and column matches
Transposition frequency (τ)*
Expected PGI (e)†
Row + column matches (m)
Percentage of expected‡
Transposition frequency (using row + column)§
Analysis of the row and column sequence data within grids demonstrates that the row sequencing was too shallow to recover some probable germinal insertions more than once and that a fraction of germinal insertions were not sequenced. For example, within grid G, 385 plasmids were identified twice in the available column data but were missing from the row sequences; this is over twice the number of plasmids identified by row and column matching. From the number of plasmids successfully sequenced per row within grid G, we estimated a 70-95% probability of sequencing the likely germinal insertion events at least once in the rows. For other grids, the sampling efficiency ranged from 30 to 95% per row. Grids in which some rows had sampling efficiency less than 60% are listed as partial in Table 1; sequencing was terminated in portions of these grids because of technical difficulties such as an excess representation of a parental insertion site, a large number of rearranged RescueMu elements that could not be sequenced with the standard protocol, or poor yield of RescueMu plasmids for unknown reasons.
Probable germinal insertions (PGI) based on multiply recovered plasmids
Multiple recovery (m)
Single recovery (s)
Expected frequency (τ)†
Expected PGI (e)‡
Percentage of expected§
Plasmids in multiple recoveries
Verification of germinal transmission
Individual grid plants with probable germinal insertions were identified on the basis of recovery of the same plasmid in both a row and a column. In addition, library plates containing all of the row and column libraries can be screened using PCR, with one primer designed to the Mu1 TIRs present in RescueMu and a second primer in the gene of interest, as illustrated in Figure 3. A probable germinal insertion plasmid should yield the same size product in at least one row and one column library of that grid plate; the row and column identifiers specify the address of the plant(s) containing this insertion. To test this method, 11 instances of duplicate plasmid recovery in grid G (N. Arnoult and G-L.N., unpublished data) and 14 such cases in grid H (K. Goellner and V.W., unpublished data) were verified to be represented in both a row and a column library by PCR screening of the corresponding library plate. Seedling progeny from the identified row and column plants were evaluated for the presence of the expected RescueMu insertion site. A germinal insertion was verified for 16/16 cases examined by DNA blot hybridization and/or PCR of individual progeny plants in the family (see Additional data file 2 for methods and for plants used to verify germinal transmission ).
Mutational spectrum of RescueMu
Of the 14,887 RescueMu insertion sites identified in six grids (multiple insertions into a gene from the same grid being counted only once because the majority are the same insertion event), 88% represent single instances of transposon insertion locations. There were 596 instances of a specific genomic sequence having two or more RescueMu insertion events. If the maize genome contains 50,000 distinct genes that are targets of Mu insertional mutagenesis, then far fewer cases of duplicate recovery would be expected by chance alone, given the number of events analyzed (p < 0.001); therefore, RescueMu exhibits some preference for particular genes.
Detailed analysis of insertion sites recovered multiple times
Number of same-base insertions that occurred in the indicated number of grids
Number of contigs with the indicated number of different insertion sites
Percentage of total
Sites per contig
In addition to the computational comparison in which an overlap of 50 bases (95% identity) was scored as insertion into the same gene, over 730 insertion sites were examined manually for 250 cases of genes with insertions from more than one grid. Of these insertion sites, 80% were at different locations within the same locus; we found 85 cases of insertions within a 1-10 bp region and 67 cases of insertions at the same base. Previously, Dietrich et al.  reported that 62 of 75 Mu insertions at glossy8 were in the 5' untranslated region, with 15 insertions at the same base; similarly, the beginning of exon 2 within bronze1 is the most frequent site of Mu insertion in that gene .
One RescueMu contig from the Genomic Survey Sequencing (GSS) section of GenBank, ZM_RM_GSStuc03-10-31.4765 , is a hotspot for RescueMu insertion, with six plasmids sequenced from row 42 of grid G and one each from grids H, I, and M. Insertion sites were identical across the grids. Sequences generated to both the left and right of the RescueMu element were aligned as demonstrated in Figure 3a. Many maize ESTs matching a maize acetohydroxyacid synthase were found near this insertion site; the closest (GenBank GI: 4966438) is less than 50 bp away. Because this RescueMu insertion site was recovered multiple times in grid G, a heritable insertion may exist. After PCR screening of grid G plasmid libraries, summarized in Figure 3a, the plant at row 42, column 22 was identified. To assess heritability of this RescueMu insertion site, total leaf DNA was extracted from selfed seed of this plant, namely G 42-22, obtained from the Maize Genetics Cooperation Stock Center. PCR screening of the DNA (Figure 3c) indicated that plant 5 is homozygous for the insertion and plant 7 is homozygous wild type. DNA blot hybridization with a 0.6-kb purified PCR probe amplified with primer pair 1 + 5 confirmed plant 5 to contain the homozygous insertion allele, plant 7 to be wild-type, and the rest to be heterozygous for the insertion (Figure 3e). Various mutant phenotypes were observed in plant 5 (Figure 3f), including retarded seedling growth, reduced plant height, discolored streaks on adult leaves and sterile tassel and ear. Because there are multiple Mu elements in this line, further characterization of selfed progeny of its heterozygous siblings will be performed to determine the true phenotype caused by this insertion.
Analysis of 9-bp TSD and insertion site preferences
Because a 9-bp TSD is characteristic of Mu insertion events, the 9 bp next to the left and right TIRs of an individual RescueMu plasmid were used to join the right and left flanking sequence provided they were complementary (Figures 1, 3); note that the sequences are complementary because they were generated from different strands. For non-parental plasmids, left and right sequence data were available for 13,966 plasmids, and the 9 bp was readily identified computationally for 47.2% (6,596) of these. The remaining non-parental plasmids did not have both right and left sequence data and/or the 9-bp motif could not be verified; 5.7% (1,816) contain only post-ligation sequences. Possible explanations for incomplete sequencing results include deletions next to Mu1 elements that remove a portion of the TIR as well as flanking host sequence [34, 35]; these events occur with about a 10-2 frequency at existing insertion sites and if they occurred during or subsequent to RescueMu insertion they would preclude identification of the 9-bp repeat. Alternatively, the lack of a 9-bp TSD could reflect sequencing error. Manual inspection of 300 of the unmatched cases indicated that for nearly 90% there was an 8/9-base repeat match with the mismatch being an undetermined base (an 'N') or a single missing or additional base. Given that all sequences were single pass but of high average quality (phred 35, equivalent to one base-calling error in 3,160 bases), we consider that 9-bp TSDs exist in virtually all trRescueMu insertion sites. A few cases showed anomalies in the TSDs, which probably reflect rearrangements near RescueMu.
Several groups have reported weak consensus insertion site preferences for Mu based on smaller data sets [9, 18, 20]. We have derived a site-specific frequency profile of the bases from 3,999 RescueMu insertion regions . The profile is in agreement with what has been reported earlier by Dietrich et al. , showing a strong bias for high G/C content in the 9-bp TSD within a flanking dyad-symmetrical consensus: CCT-(TSD)-AGG. The non-random insertion pattern strongly suggests that RescueMu targeting is at least partially dependent on sequence features. In addition, we have compared the profiles derived independently from insertion sites within confirmed exons, introns and uncharacterized regions, respectively, and found the same base preferences in all three sets (data not shown).
Of 14,887 genomic loci, 62% matched maize or other plant EST/cDNAs. As more genomic sequence becomes available that can be assembled with ESTs to annotate the non-coding portions of maize genes, it will be interesting to determine if the RescueMu insertion sites that do not match an EST or gene in another species represent introns or other non-coding genic regions. On the basis of the gene structure annotated by maize EST matching, we have located 968 TSD sites within genes. Of these, 849 are inside exons. To check if RescueMu has a preference for insertion into exons (that is, the above observed high frequencies of exon insertions is not the result of potential high exon proportion in the maize genes), a standard binomial test with normal approximation was performed. On the basis of the matching to ESTs, the lengths of all exons and introns observed from all RescueMu contigs were counted as 2,182,954 bp and 439,403 bp, respectively. Assuming that RescueMu does not have a preference to insert into exons (null hypothesis), the probability of observing an exon insertion event is proportional to the length of exons (single binomial trial probability 0.832). The probability of observing at least 849 exon insertion events was calculated (less than 0.001; reject the null hypothesis). This result suggests that RescueMu has some preference to target exon regions within genes.
As outlined in Materials and methods, the RescueMu GSS sequences were scanned and masked for repetitive elements as collected in The Institute for Genomic Research (TIGR) Cereal Repeat Database . The repeat content was compared with results for GSS sequences derived by methylation filtration (MF) and high C 0 t selection (HC) using the same repeat-masking criteria . The percentage of masked nucleotides was 16.5, 24.5 and 16.2% for RescueMu, MF and HC, respectively.
Matching of RescueMu genomic loci to other available databases to determine percentage of genic and repeat loci
Number of genomic loci
Total maize genomic loci discovered
Number of genic loci identified by:
Number of genic loci† (percentage of total)
Number of loci matching repeats:
45S ribosomal DNA (18S + 28S)
5S ribosomal DNA
Number of repeat loci‡ (percentage of total)
In addition, only 0.4% of the RescueMu insertions were found in either the approximately 10,000 copies of the 9.1 kb 28S + 18S rRNA genes  comprising 3.6% of the 2.5 gigabase (Gb) maize genome, or in the large number of tRNA and 5S rRNA genes in the maize genome (Table 5). These results demonstrate a strong bias against insertion into genes transcribed by RNA polymerases I and III.
Also shown in Table 5, about 62% of the RescueMu loci match strongly to maize or other plant ESTs or appear to encode proteins with high similarity to known proteins. In addition, about another 5% of the loci were predicted to be genic regions with high stringency by ab initio gene prediction programs. As a control, we matched ESTs to contigs assembled from unfiltered (random) maize GSS sequences . From about 33,000 of those unfiltered contigs, less than 20% of them show significant matching to ESTs. This shows that RescueMu contigs contain more than threefold enrichment of genic regions than random sequencing. This is consistent with our expectation that RescueMu preferentially inserts into genes. It is worth pointing out that plant EST collections contain ESTs from repetitive elements. Although we masked contigs using the annotated TIGR repeat database , it is possible that some contigs still contain unidentified repetitive elements, which might overestimate the number of genic regions by matching the same ESTs to different copies of repetitive elements. In particular, 18% of the EST matched regions show high similarity to transposon coding regions based on BLAST searches against the GenBank nucleotide and protein databases, suggesting that at most 14% of unfiltered contigs include protein-coding genes. The numbers of genic sequences from MF and HC was reported to be 27% and 22%, respectively . However, these numbers are not directly comparable to our RescueMu results, because these authors used much higher stringency for the EST spliced alignments with the BLAT program , requiring 95 and 80% identity, respectively, when matching to the TIGR maize gene index or other plant indices. We used the GeneSeqer program for spliced alignment of the RescueMu data, which tolerates less sequence matching without compromising gene structure prediction accuracy . The results using GeneSeqer for RescueMu, MF, and HC are very similar (data not shown).
Palmer et al.  evaluated the gene discovery rates of MF, EST sequencing and RescueMu by comparing the respective sequence sets to rice gene models. They concluded that unique gene discovery is most efficient with MF at a sequencing depth when EST sampling saturates. However, their reported low gene discovery rate for RescueMu does not reflect the RescueMu insertion bias, because their dataset included all sequences deposited in GenBank. That is, they did not remove the redundancy resulting from multiple sequencing of parental insertions.
Multiply recovered RescueMuinsertion sites in the progeny of a single founder plant
Single and multiple recovery of specific RescueMu insertion sites within the sequenced rows of grids G, H, I, K, M, and P
To calculate the extent and timing of pre-meiotic sectors, the sequenced plasmids from grids G, H, I, K, M and P were classified as occurring in a single row or in multiple rows. The development of the tassel and ear must be considered when evaluating these data. An insertion event that occurs during meiosis can be represented in two haploid cells. During microgametophyte (haploid plant) ontogeny, both of these cells survive, resulting in two pollen grains with the same event. In contrast, only one megagametophyte develops after megaspore meiosis; therefore, female meiotic and subsequent events in the haploid megagametophyte are always represented in just one progeny plant. Most grid plants resulted from male transmission of RescueMu and a minority (about 10%) from female transmission. Given that the founder plants produced copious pollen, there is a low probability that two grains carrying the same meiotic insertion will both result in seed; therefore, the same RescueMu insertion site found in two rows should usually be from a pre-meiotic transposition event. For all events found in three or more rows, the insertion event must be pre-meiotic.
The 103 insertions sites found in three or more rows of grid G must be pre-meiotic events (see Table 6). They represent 9% of the probable germinal insertion events (103/1,091) identified by the criterion of recovery of the same plasmid twice or more (see Table 3). The percentage was similar for all six grids: there were 321 events identified in three or more rows out of 3,138 probable germinal insertions. Surprisingly, 138 contigs were found in four or more rows in these six grids, including 34 events in 10 or more rows (Table 6). Therefore, occasionally there is a RescueMu insertion event very early in the somatic development of the inflorescence or in the apical meristems. The majority of trRescueMu insertion sites are found in only one row (92% of germinal plus somatic insertion sites, Table 6).
Insertions found in at least two rows or columns among plants with both row and column sequence data
Plants from sequenced rows + columns
Insertions found in 2+ rows or 2+ columns
RescueMu was introduced into maize by particle bombardment resulting in complex transgene loci containing multiple copies of the transposon and the Basta-resistance plasmid used for selection of transgenic lines . After crossing with an active Mutator line, RescueMu exhibited somatic excision from a 35S:Lc reporter allele resulting in a red-spotted aleurone but the heritable insertion frequency was very low. Progeny screening identified individuals containing two or three trRescueMu elements lacking the original transgene array by genetic segregation and unmethylated Mu1 and MuDR elements. Some of these individuals and subsequent derivatives with the same characteristics were used as founder plants to construct grids of plants organized into rows and columns for efficient generation and analysis of germinal mutations. Tagging maize sequences with RescueMu followed by plasmid rescue and sequencing of the flanking host DNA has identified 3,138 insertion locales from 17,239 plasmids (see Table 3). These plasmids represent 59.5% (17,239/28,988) of the total non-parental plasmids of the genomic loci found in each grid. Because sequencing depth was too shallow to identify all likely germinal insertions, the 40.5% of non-parental plasmids recovered just once (11,749 from Table 3) represent a mixture of somatic and germinal events. On the basis of the estimation of germinal insertion frequency from DNA blot hybridization, the six grids should contain more than 8,000 heritable trRescueMu insertion sites, but the sequencing depth was too shallow to identify all of these by multiple recovery of the same plasmid two or more times.
RescueMu is suited for both reverse and forward genetic strategies. Given the genomic sequence contiguous to any trRescueMu, a PCR screen can be designed to identify which plant contains the insertion of interest using 96-well plates containing the immortalized collection of row and column rescued plasmids. The row and column plant address can be used to order seed for further genetic and phenotypic analysis as illustrated by the RescueMu insertion into the acetolactate synthase gene (Figure 3). Alternatively, the phenotype database, which is organized by individual plant, can be searched to identify individuals segregating for mutations of interest. Active Mutator lines with multiple mobile Mu elements were used so most mutations will be caused by these Mu elements because they increase mutation frequency 50-100-fold above spontaneous levels . The high forward mutation frequency reflects the copy number of the elements and their preference for insertion into or near transcription units . From the DNA hybridization blots (data not shown) used to verify that grid founder plants had unmethylated Mu elements, the copy number of unmethylated Mu elements was estimated at 20-40 per founder; therefore, two mobile RescueMu elements would be expected to account for 5-10% of the newly generated mutations. Seed was ordered through the Maize Genetics Cooperation Stock Center  for further characterization.
RescueMu insertions were found in genes and ESTs mapped to all 10 maize chromosomes , and were found in all of the gene classifications for maize (Figure 4). These data confirm the empirical observations of maize geneticists that MuDR/Mu transposons are general and efficient mutagens for maize genes . Analysis of 14,887 loci defined by RescueMu insertions demonstrates that transposition is highly preferential for RNA polymerase II transcription units: about 62% of the sites match maize or plant ESTs. Because the EST collections are incomplete and lack intron and promoter sequences, it is likely that an even higher proportion of RescueMu insertion sites are in or near genes but cannot be currently assigned to a specific gene. Given the current efficiency, large tagging populations in excess of 200,000 plants would be required in order to recover RescueMu mutations in all maize genes (estimation is based on the calculation method in ). The numerous grids evaluated for phenotypic characteristics should approach saturation of visible mutations, although most of the mutations are caused by standard Mu elements.
Given that the maize genome comprises approximately 70% retrotransposons and other highly repetitive sequences, including around 10,000 copies of the rRNA genes , these components of the maize genome are significantly under-represented in RescueMu insertion sites. Only about 8% of the RescueMu insertion sites match repetitive elements and few insertions (0.4%) were recovered in genes transcribed by RNA polymerase I or III. These results suggest that a chromatin component associated with polymerase II transcription units or the absence of a structure in other classes of genes is important in targeting RescueMu and other Mu elements to maize genes. Similarly, recombination during meiosis and transcription per se is targeted to genes. It is likely that the parasitic Mu elements exploit an element of host gene packaging that evolved for other reasons to facilitate transposition into genes.
The biological specificity for maize genes exhibited by RescueMu is close to methyl filtration and high C 0 t fractionation. The probable germinal insertion class defines a collection of mutations of enormous potential for the phenotypic characterization of maize with specifically disrupted functions. However, the low cost of template production is a distinct advantage of both physical enrichment methods compared to the high cost of designing, sampling and self-pollinating tagging grids. Current levels of sample sequencing from the physical enrichment templates highlight the desired redundancy of the RescueMu method, which is important for distinguishing somatic from germinal insertions at individual loci. The physical enrichment methods are considerably below one times coverage of the transcriptome of around 250 Mb; hence the current efficiency of generating novel sequence (the likelihood that the next clone sequenced is new) is much higher with these methods than with RescueMu.
Using the RescueMu insertion site data, several parameters of Mu transposition behavior were investigated. We confirm that a 9-bp TSD is characteristic of virtually all Mu insertion sites. We confirm that a small percentage of trRescueMu suffer deletions, including loss of a TIR, as noted in previous studies of Mu1 . Through evaluation of several hundred Mu insertion sites [9, 18], consensus motifs have been proposed for insertion sites. The sequence profile derived from the much larger population of RescueMu insertion sites is consistent with the previously proposed motifs. A bias exists for G+C-rich sequence, reflecting the composition of maize exons. We confirm that there are hotspots for Mu insertion, identified by finding identical trRescueMu insertion sites in independent grids. A few loci were recovered in four or more of the six grids analyzed, and many more in two (1,295 genes) or three (233 genes) grids. There is no strong DNA consensus motif at these hotspots, and we consider it more likely that a specific DNA structure or a protein associated with genes establishes conditions for efficient Mu insertion at particular sites. It is important to note that active transcription is not a requirement for Mu element insertion; otherwise Mu would preferentially insert into genes active late in floral development and in gametophytes.
The trRescueMu insertion sites represent a mixture of non-heritable somatic insertions present in leaves, germinal insertions in single grid individuals, insertion events in pre-germinal sectors within flowers, and parental elements. Parental elements identified in a grid founder plant segregated 1:1 in the progeny as expected. In addition, some insertion events were found in three or more grid rows, and hence in three or more individuals, and must be pre-meiotic transposition events in the founder. This class represented 10.2% (321/3,138) of all the likely germinal insertions identified (calculated from Table 6). Given the clonal analysis model of the pattern of cell divisions establishing the ear and tassel of maize [48–50], the earliest events within the apical meristem could affect up to half of the ear or tassel, with subsequent events affecting progressively narrower portions of the inflorescence. The majority of the pre-meiotic events are consistent with RescueMu transposition in the floral cells a few cell divisions before the onset of meiosis, that is, in precursor cells that are still proliferating and could generate at least two and up to approximately 50 meiocytes. A smaller fraction of new insertions events occurred early enough to be represented in many progeny of a particular plant. These rare, early transposition events generate very large sectors within the developing inflorescence.
Mu transposon mutagenesis is highly efficient, primarily because the transposon targets genes and it is usually found in 10-50 copies per genome. How does the plant tolerate the large number of mutations generated by this agent? Within the diploid somatic tissues, most new mutations lack a phenotype; however, the haploid gametophytes are subject to stringent selection. Unlike animals, in which the phenotypes of the sperm and egg are set by previous gene activity in the parent, many characteristics of the haploid phase of the plant life cycle reflect haploid genetic activity, which requires overlapping but distinctive suites of genes in the mega- and microgametophytes . Consequently, the late timing of new Mu insertions generates gamete diversity, but the unfit genotypes are culled from the population before fertilization. Coe et al.  describe the general problem that lethals occur much more frequently in pollen than in the megagametophyte. Any method that relies on pollen transmission will therefore fail to recover certain types of mutations that would be recovered through female transmission. For this reason, a subset of maize genes required in both types of gametophyte is refractory to knockout mutagenesis.
A public resource of transposon-tagged maize alleles was constructed and evaluated. RescueMu is an efficient tag for mutagenizing and cloning maize genes, because 66% of insertion sites appear to be in genes. Sequencing from immortalized plasmid libraries organized into row and column plates reflecting the organization of fields of plants permit identification of probable germinal insertions; the library plates can be searched by PCR to verify germinal insertions and subsequently acquire seed of the corresponding plant. Alternatively, a searchable database of segregating plant phenotypes in seed, seedling, or adult tissues can be used to find plants carrying mutations of interest. Although RescueMu can target most, if not all, RNA polymerase II transcription units in the nuclear genome, the transposon does exhibit hotspots in particular genes. Neither the hotspots nor other insertion sites contain a motif(s) defining predictable insertion locations. RescueMu properties confirm attributes established with smaller populations of standard Mu elements.
Materials and methods
RescueMu contains all of Mu1 plus a 400-bp segment of Sinorhizobium meliloti and pBluescript (Stratagene), as described previously by Raizada et al. . The complete sequence of RescueMu was obtained in this study using PCR primers to amplify overlapping sections of the element  for bidirectional sequencing (GenBank accession AY301066). In the construct used to make transgenic plants, the RescueMu transposon was placed in the 5' untranslated region of a 35S:Lc expression plasmid where it blocked expression . Lc is a member of the R family of transcriptional regulators of the anthocyanin pathway . Transgenic maize lines in the A188 × B73 (r-r/r-g, A1, Bz1, Bz2) hybrid background were crossed to r-g testers and subsequently with r-g Mutator lines containing multiple copies of MuDR to visualize RescueMu somatic excision as red anthocyanin sectors in an otherwise white aleurone. The tagging populations used here were developed by screening for transposition of RescueMu from the original, complex transgene arrays to diverse genomic locations. Using DNA blot hybridization, these once-transposed RescueMu (trRescueMu) were closely monitored for subsequent transposition, and lines were monitored for Mu1 and/or MuDR methylation in the TIRs, a sign of incipient Mutator silencing. Details of line development and evaluation, including DNA blot hybridization methods, will be presented elsewhere. The anthocyanin tester lines (recessive for r-g, a1, bz1 or bz2) were in inbreds W23, K55, A188, or hybrid combinations of these lines. Some RescueMu lines used in tagging grids were crossed to inbreds A619 or B73, which are both r-g, A1, Bz1, Bz2. Grid backgrounds are presented in detail at .
Plasmid rescue and DNA sequencing
Detailed protocols are presented at , and a schematic is provided in Figure 1. Briefly, leaf tissue was collected from all plants in each row and from a different leaf in each column of a grid. A separate plasmid rescue library was constructed after BamHI plus BglII digestion of the genomic DNA preparations. These libraries were immortalized in library plates available from the project . Plated colonies were picked, grown overnight in liquid media, and sequencing templates prepared by a direct PCR method suitable for amplifying genomic inserts of up to 16 kb. Cycle sequencing was performed using Big Dye Terminator chemistry to read out from a position around 110 within the left or right terminal inverted repeat (TIR) of RescueMu; although the primers were selective for one TIR, there was some cross-priming. All grid rows plus several columns were sequenced. Three 96-well plates were normally sequenced for each row or column to obtain sequence information for a desired minimum of 200 plasmids; additional sequencing reactions were conducted if necessary. Matches of row and column sequences are designated as probable germinal insertions, because they represent an insertion site present in two leaves of that plant (designated by its row and column address); when only row sequences were available from a particular plasmid, probable germinal insertions were designated after recovery of the same sequence two or more times. Plasmid types recovered just once are a mixture of heritable and strictly somatic insertions. Parental RescueMu insertion sites present in a grid founder plant segregated in the grid progeny, and these insertion sites were expected to be found in all rows and columns. In some cases, particular parental plasmids were over-represented in the sequenced plasmid population. To reduce their contribution and increase recovery of new insertion sites, a rare-cutting restriction enzyme site was identified in the parental plasmid and the corresponding enzyme was included in the genomic DNA preparation to bias against recovery of that parental plasmid.
5'-CAT TTC GTC GAA TCC CCT TCC-3' (RescueMu)
Locations and directions with respect to the insertion site of RescueMu are shown in Figure 3a. PCR conditions were as follows: 5-20 ng of each plasmid library, 2.0-2.5 mM Mg2+, 0.4 mM dNTPs, 0.8-1.0 μM gene primer and 4-5 μM RescueMu L primer in a 50 μl reaction was first denatured for 2 min at 95°C followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C and 2 min at 72°C, and a final 2 min extension at 72°C. The same PCR conditions were used for screening using 5-100 ng samples of maize total genomic DNA.
DNA blot hybridization
Total genomic DNA was extracted from leaf tissues using a modified urea method . After overnight digestion, the restricted DNA was separated on a 0.8% agarose gel and transferred onto Hybond-N+ membrane (Amersham Biosciences) in 0.4 M NaOH. Blots were hybridized with non-radioactive probes labeled with AlkPhos DIRECT system (Amersham Biosciences) for chemiluminescence detection on X-ray film.
Initial clustering and assembly of genomic sequences
The sequences were screened to remove the TIR sequences using the program crossmatch  and then trimmed to achieve a minimum phred score >15 in sliding windows over 40 bases. Overall the quality scores averaged phred >35, or less than one error in 3,160 bases. The average length of the trimmed, high quality genomic sequence entering the assembly was 378 bases. The right-TIR primer yielded 22% more successful sequence than the left-TIR primer resulting in an excess of right side sequences. Trimmed sequences were then assembled into contigs using phrap  with the following parameters: -minmatch 35 -minscore 30 -node_seq 14 -node_space 9. The member sequences for each contig were extracted from the phrap output files and assigned to a row or column of a grid. Within each contig, only a single sequence from a plasmid was used to determine the row and column representation. For example, if both the left- and right-flanking sequence from a plasmid assembled into one contig, this was considered one recovery of the plasmid. If the left-flanking sequence from one plasmid and the right-flanking sequence from a separate plasmid assembled into the same contig, this was considered two independent recoveries of the same genomic locus. In the latter case, if the right- flanking sequence was from a different row, then the sequence was recovered in multiple rows as well. All sequences were deposited into the Genomic Survey Sequencing (GSS) section of GenBank .
Assembly of RescueMu-derived genomic sequence data
As shown in Figure 1, using the 9-bp TSD characteristically generated during Mu element insertion , the sequences to the right and left of a particular RescueMu element can be assembled into a continuous sequence. To do this, trimmed RescueMu GSS sequences were downloaded from GenBank , for comparison to raw GSS sequences containing the Mu1 TIR sequences. The TIRs were masked by the cross_match program  to determine the flanking 9-bp TSD sequences. The TSDs are the end-overlaps between GSS sequences generated from the left and right side of RescueMu insertion. Merging through TSDs using the reverse-complementary strand of the left and right sequences recovers the original genomic sequences flanking the RescueMu insertion. A special consideration in the assembly of the genomic sequences flanking the right- and left-TIRs of RescueMu is the presence of a GGATCC (BamHI), AGATCT (BglII), or a GGATCT (BglII/BamHI) or AGATCC (BamHI/BglII) motif. The two restriction digestion sites represent a true ligation site of sequence that was non-contiguous in the maize genome, but the post-restriction site sequences can unambiguously be assigned to the right or the left of RescueMu. On the other hand, the GGATCT or AGATCC motif could be contiguous genomic sequence or could have been generated during the ligation step of the plasmid rescue. Consequently, assignment of the position of the sequence beyond the GGATCT or AGATCC motif is ambiguous. If the RescueMu insertion site matched EST sequence across and beyond the GGATCT or AGATCC motif, the post-ligation sequence could be properly assigned (Figure 1). In the RescueMu plasmid sequences considered here, the average number of sequences reported to GenBank was 2.3 (131,364/57,022) per plasmid.
The 131,364 RescueMu GSS sequences deposited at GenBank were screened for vector sequences against the UniVec database at the National Center for Biotechnology Information (NCBI)  using the crossmatch program: -mismatch 12 -penalty -2 -minscore 20. The resulting 130,861 vector-trimmed sequences were then screened against the maize repeat database annotated by TIGR  using the Vmatch program  with the parameters -l 50 -h 3 -identity 95. The 127,708 repeat-free sequences were then used to identify parental insertions. Any given RescueMu-transformed plant contains the parental RescueMu elements that were recovered at a high frequency during sequencing (from every sequenced row or column). Because our goal is to analyze the gene discovery by newly inserted RescueMu (that is, we are interested in where those non-parentals inserted into the maize genome), we decided to filter out the parental sequences as much as possible. We used Vmatch to cluster near-identical left and right sequences for each grid. A parental cluster contains sequences from nearly all the row or column sequences. A total of 59,069 parental sequences were identified and were excluded from the subsequent assembly. All the non-parental sequences were first preassembled for each plasmid using the left and right 9-bp TSD overlap. The merged GSSs were first clustered by PaCE  (minimum exact match 36 bp, minimum score threshold 30%) and then consensus sequences (contigs) for each cluster were generated by CAP3  (overlap 40 bp; 90% identity cutoff). Because PaCE and CAP3 only pair sequence with the minimal overlap required to establish statistically significant identity, the number of contigs is probably an overestimate of the number of independent RescueMu insertion sites. For the particular case where TSDs were not recovered during sequencing, the left and right sequences could not be assembled together, even though they were from the same plasmid. Therefore, a Perl script was developed to conduct single-linkage clustering based on clone-pair constraints to assemble the GSS to the same 'genomic loci' if they were derived from the same plasmid clone.
Classification of insertion site context
To be successful as a gene-discovery tool, the transposon insertions must be predominantly into the genic regions of the maize genome. To quantify the potential enrichment of the RescueMu flanking sequences for genic regions, we matched all assembled contig sequences against various classes of known repetitive sequences, including retrotransposons, DNA transposons, centromeric and telomeric repeats, rRNA genes and plastid DNA. For this analysis, the non-parental sequences were used in their original form, with only vector sequences but not repeat sequences trimmed. The sequences previously discarded for analysis because they consist almost entirely of repetitive elements were assembled using the same procedure as described above for the repeat-trimmed sequences. Note, however, that this number of loci is unreliable and probably an underestimate of the true number of loci recovered because of the intrinsic difficulty with assembling repetitive DNA. To identify the repetitive elements in the contigs, Vmatch (-seedlength 14 -hxdrop3 -l 30 -identity 70) was used in combination with the TIGR cereal repeat database (version 2 consisting of maize, rice, barley, sorghum and wheat repeats). The contigs were also scanned from tRNA genes by tRANscan-SE program  with its default parameters.
Gene discovery in GSS contigs
Both similarity-based and ab initio approaches have been used to detect gene structures of the GSS contigs. For the similarity-based approach, GeneSeqer  programs were used to match plant EST contigs and cDNAs to GSS contigs. The plant EST contigs were regularly assembled by PlantGDB . For the ab initio prediction, GENSCAN  (with default parameter settings for maize) was used and only high exon score predications (≥0.90) were selected. The GSS contigs were compared against SPTR , a nonredundant protein data set collected by the European Bioinformatics Institute (EBI), using BLASTX  with an E-value ≤ e-20. The BLASTX top protein hits were used to assign putative functions to the unique regions and for classification into functional categories based on annotation in the Gene Ontology  database.
The genetically mapped maize ESTs were retrieved from MaizeGDB . These ESTs were spliced-aligned to GSS contigs using GeneSeqer as described above. The matched GSS contigs were then plotted on the maize IBM Neighbor genetic map .
Analysis of 9-bp TSD and insertion site preferences
For the analysis of RescueMu target sites, we retrieved the 9-bp TSD sequences from the confirmed insertion sites where both the left and right sequences match on the 9-bp TSD. We also retrieved the 20 bp up- and downstream sequences around the TSD. Then a 15-base long profile (9-base TSD and its three up- and downstream neighbors) was derived from the sequences and their reverse-complement orientation determined using the Expectation Maximization Algorithm .
Analysis of tentative unique contigs containing GSS sequences from multiple grids
The GSS seqeunces present in each tentative unique contig (TUCs) were extracted from  and assigned to a row or column within a grid. A sample of TUCs with GSS sequences from multiple grids was then selected for detailed analysis. For each GSS in the TUC (excluding post-ligation sequences), the exact location of the TSD was determined by visual examination of the sequence alignment file for the TUC and the untrimmed GSS sequence data. The number of GSS sequences for each grid at each transposition site was recorded.
Grid plants were self-pollinated unless male or female-sterile. The resulting F1 families were evaluated by inspection of ears and kernels, at weekly intervals for five weeks after germination in a sand bench in a greenhouse, and at weekly intervals throughout the life cycle in the field. Phenotypes observed were recorded and are assembled into a searchable database at . Unique phenotypes were documented with a digital image, and there are links to corresponding RescueMu flanking sequences where established. Instructions on how to obtain seed of grid plants is also provided.
Additional data files
We thank the Maize Gene Discovery team for the development of stocks and extensive DNA blot hybridization data that preceded construction of RescueMu tagging grids and for the phenotypic evaluation of seed and seedling mutations. Diane Chermak generated all of the RescueMu library plates and most of the sequencing templates; we thank China Lunde for grid H templates and Laura Roy for grid S templates. We thank Xiaowu Gai and Trent Seigfried, who contributed to the development of ZmDB. This research was supported by a plant genome research program contract from the National Science Foundation (98-72657), which initiated the Maize Gene Discovery project. An REU supplement supported undergraduate students Warren Chen and Justin Schaffer at Stanford and Fred Oakley and Laura Schmitt at ISU in 2001.
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