Group Title: BMC Genomics
Title: Sequence-indexed mutations in maize using the UniformMu transposon-tagging population
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Title: Sequence-indexed mutations in maize using the UniformMu transposon-tagging population
Physical Description: Book
Language: English
Creator: Settles, A. M.
Holding, David
Tan, Bao
Latshaw, Susan
Liu, Juan
Suzuki, Masaharu
Li, Li
O'Brien, Brent
Fajardo, Diego
Wroclawska, Ewa
Tseung, Chi-Wah
Lai, Jinsheng
Hunter, Charles
Avigne, Wayne
Baier, John
Messing, Joachim
Hannah, L. C.
Koch, Karen
Becraft, Philip
Larkins, Brian
McCarty, Donald
Publisher: BMC Genomics
Publication Date: 2007
 Notes
Abstract: BACKGROUND:Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.RESULTS:Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.CONCLUSION:We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.
General Note: Periodical Abbreviation:BMC Genomics
General Note: Start page 116
General Note: M3: 10.1186/1471-2164-8-116
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Bibliographic ID: UF00099993
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/
Resource Identifier: issn - 1471-2164
http://www.biomedcentral.com/1471-2164/8/116

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Research article C

Sequence-indexed mutations in maize using the UniformMu


31pe A-


transposon-tagging population
A Mark Settles*1, David R Holding2, Bao Cai Tan', Susan P Latshaw1,
Juan Liu3, Masaharu Suzuki', Li Lil, Brent A O'Brien', Diego S Fajardo',
Ewa Wroclawskal, Chi-Wah Tseung', Jinsheng Lai4, Charles T Hunter III1,
Wayne T Avignel, John Baier', Joachim Messing4, L Curtis Hannah1,
Karen E Koch', Philip W Becraft3, Brian A Larkins2 and Donald R McCarty'


Address: 'Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA, 2Department of Plant Sciences, University of
Arizona, Tucson, AZ 85721, USA, 3Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and
4Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA
Email: A Mark Settles* settles@ufl.edu; David R Holding dholding@ag.arizona.edu; Bao Cai Tan bctan@ufl.edu;
Susan P Latshaw latshaw@ufl.edu; Juan Liu juanliu1035@hotmail.com; Masaharu Suzuki masaharu@ufl.edu; Li Li lili1982@ufl.edu;
Brent A O'Brien bob2373@ufl.edu; Diego S Fajardo diegof@ufl.edu; Ewa Wroclawska ewroc@ufl.edu; Chi-Wah Tseung ctseung@ufl.edu;
Jinsheng Lai jlai@waksman.rutgers.edu; Charles T Hunter ibe@ufl.edu; Wayne T Avigne avigne@ufl.edu; John Baier baier@ufl.edu;
Joachim Messing messing@waksman.rutgers.edu; L Curtis Hannah lchannah@ufl.edu; Karen E Koch kekoch@ufl.edu;
Philip W Becraft becraft@iastate.edu; Brian A Larkins larkins@ag.arizona.edu; Donald R McCarty drm@ufl.edu
* Corresponding author



Published: 9 May 2007 Received: 29 August 2006
BMC Genomics 2007, 8:116 doi:10.1 186/1471-2164-8-116 Accepted: 9 May 2007
This article is available from: http://www.biomedcentral.com/1471-2164/8/1 16
2007 Settles et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Gene knockouts are a critical resource for functional genomics. In Arabidopsis,
comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking
insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the
genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate
unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also
generate somatic insertions that fail to segregate in subsequent generations.
Results: Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-
specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence
for somatic insertions within the I 1% of insertion sites that were not confirmed. Instead, this subset of
insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic
sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase
gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout
generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway
during grain-fill.
Conclusion: We show that FSTs from the UniformMu population identify stable, germinal insertion sites
in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse
genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant
insertion sites from UniformMu provide a genome-wide resource for reverse genetics.



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Background
An important aspect of functional genomics is under-
standing the phenotypic consequence of mutations in all
genes in a genome. A comprehensive collection of gene
knockouts allows a defined set of mutations to be system-
atically studied for more efficient association of genes to
functions (reviewed in [1]). Multiple approaches have
been used to develop comprehensive knockout resources.
Biological differences between organisms make specific
technologies such as homologous recombination, RNA
interference, or insertional mutagenesis more useful in
generating a resource for an individual species. In plants,
a comprehensive knockout collection was generated for
Arabidopsis thaliana via mutagenesis with insertion tags [2-
5]. Genomic DNA flanking each tag was systematically
amplified and sequenced from each mutant. These Flank-
ing Sequence Tags (FSTs) index each mutant to the
genome and are accessible through the SIGnAL T-DNA
Express database, which links the mutant stocks to
genome annotations [61. A similar FST approach was used
to develop a rice functional genomics resource based on
insertional mutagenesis populations [7-10]. The current
rice collections have more than 140,000 insertion lines
with associated FSTs that are integrated at the OryGen-
esDB database [11].

Maize is comparable to rice as a model grass species for
genome studies. Similar to rice, the maize genome is now
being sequenced with a minimal tiling path strategy [12].
There are also extensive gene-enriched sequences that are
estimated to include partial sequence from 95% of maize
genes with smaller introns [12,13]. In contrast to rice,
maize is a monoecious plant, and maize has a shorter life
cycle. These biological characteristics facilitate genetic
analysis. Also, maize inbreds are highly polymorphic.
Robust PCR markers, genetic maps, and recombinant
inbred lines have been developed that aid quantitative
trait studies and positional cloning (reviewed in [14]). A
comprehensive knockout collection of maize mutants
would complement the existing genome resources to
make functional genomics studies in maize simple and
rapid.

There are multiple insertion-tagged maize populations
that were generated with either Activator (Ac) or Mutator
(Mu) transposons (reviewed in [15]). There are >150,000
mutagenized lines among the combined Mu populations
[16-20]. These Mu lines are expected to have more than
1.5 million independent insertions, because Mu trans-
posons accumulate to high copy numbers within individ-
ual plants (reviewed in [21]). Moreover, Mu elements
show a bias for inserting into or near transcribed regions
of the genome and are associated with a high rate of for-
ward mutagenesis [17,19,22,23]. This high mutation fre-
quency makes Mu mutagenesis attractive for generating


knockout resources, but the high-copy nature of Mu ele-
ments presents a challenge in isolating individual inser-
tion sites for sequencing. Single plants have multiple
germinal insertions that represent both progenitor muta-
tions and mutations unique to the individual. In addition,
active transposon systems can generate somatic insertions
that fail to segregate in subsequent generations. Due to
these challenges, most Mu populations have been devel-
oped to conduct reverse genetics screens for only one gene
at a time [16,18,20].

FSTs have been generated from two Mu populations. Fern-
andes et al. [17] identified 14,887 non-redundant FSTs
using a transgenic Mul element that was engineered for
plasmid rescue of genomic flanking sequences. The plas-
mids were isolated from pools of actively transposing
plants and many of the FSTs are from somatic insertions.
A key challenge to sequencing FSTs from pools of actively
transposing plants is identifying germinal insertion sites
and associating the germinal insertions to individual seed
stocks. Fernandes et al. [171 sequenced from two dimen-
sional grids. Recovery of the same FST from both row and
column pools of plants was used to associate 528 of the
plasmid-rescue insertion sites to individual plants.

In contrast, FSTs from the UniformMu population were
generated with both a strategy to associate each FST to
individual lines and to select for germinal insertions
[19,23]. UniformMu is a Mu population that is intro-
gressed into the color-converted W22 inbred. Previously,
we showed that UniformMu is a robust forward genetics
resource for identifying visible mutant phenotypes [19].
UniformMu FSTs were isolated by amplifying native
transposon insertion sites with a modified TAIL-PCR pro-
tocol, MuTAIL [23]. MuTAIL products were cloned from
individual lines into small libraries. Sequencing ran-
domly selected clones, similar to an EST strategy, recovers
67%-75% of the insertion sites from each line. With this
sequencing strategy, each FST corresponds to an individ-
ual plant or maize ear.

The UniformMu FSTs also derive from plants in which the
Mu transposons were stabilized by selecting against
somatic transposition using the bronzel-mum9 (bzl-
mum9) mutation [24,25]. The selection against somatic
activity predicts that all of the FSTs should be from germi-
nal insertion sites. Database searches with 1,737 non-
redundant UniformMu FSTs indicated a bias for inser-
tions in or near transcribed regions of the genome[19].
Moreover, in silico mapping of the FSTs indicated that Uni-
formMu transposon insertions are distributed randomly
throughout the maize chromosomes suggesting the trans-
poson mutagenesis gave rise to mutations on a genome-
wide basis. These properties predict that UniformMu FSTs



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will be a resource of sequence-indexed mutations that is
simple to use for reverse genetics applications.

Here we test the utility of UniformMu FSTs for reverse
genetics. We assayed a sample of FSTs for germinal segre-
gation. We focused on FSTs that were predominantly
recovered from individual UniformMu lines. FSTs found
only in one plant have the highest possibility of represent-
ing somatic insertions and provide the strongest test for
whether somatic insertions exist within the UniformMu
FSTs. Our data indicate that 89% of the UniformMu FSTs
can be confirmed as germinal insertions by designing only
one or two locus-specific primers for PCR validation. The
most common problems for the 11% of insertions that
were not validated as germinal insertions were design
errors in the locus-specific primers due to low quality
sequence data or human error. One of the FSTs co-segre-
gates with a seed mutant that suggests an important role
for chloroplast-localized oxidative pentose phosphate
pathway enzymes in grain-fill, embryogenesis, and plant
development. These results demonstrate that the strategy
for sequencing UniformMu FSTs creates effective reverse
genetics resources for characterizing gene knockouts in
maize.

Results
A sample of 106 sequence-indexed insertion sites from the
UniformMu population were tested for germinal inherit-
ance. The UniformMu FSTs represent novel insertion sites
from the individual lines as well as progenitor sequences
that are found throughout the population. The FSTs ini-
tially were clustered by both blastcluster analysis [19] and
CAP3 assembly [26]to identify non-redundant sequences.
A total of 1,434 FSTs unique to individual UniformMu
seed mutants and 283 FSTs found in two to three mutants
were identified with these clustering methods. We
selected 79 FSTs found in individual mutants, 19 FSTs
found in two mutants, and 8 FSTs found in multiple Uni-
formMu lines for PCR validation. The 98 low redundancy
insertions found in 1-2 mutants were recovered from 50
of the lines analyzed by MuTAIL sequencing. Thus, the
106 insertions sites tested were a significant sample of
both UniformMu lines and MuTAIL FSTs.

The FSTs selected for PCR validation were compared to
the maize assembled genomic islands (MAGI) database as
well as the TIGR Zea mays Gene Index (ZMGI) to deter-
mine the position and identity of the insertion sites
[27,28]. Both the MAGI and ZMGI databases are enriched
for maize gene sequences. The insertions were categorized
as being within exons, introns, promoters, or unknown
sequences (Fig 1A). Sixty-three percent of the insertion
sites are found within 850 bp of transcribed sequences as
defined by either a match of greater than 95% sequence
identity between the FST and a ZMGI sequence or a match


to a MAGI sequence that shows sequence identity to a
ZMGI sequence (see Table 1 and Additional file 1). The
remaining 39 FSTs either matched 1-2 MAGI sequences
that contained no EST support for a transcribed sequence
or did not show a significant match to MAGI or ZMGI
sequences.

The FSTs were also analyzed for the presence of the con-
served Mu Terminal Inverted Repeat (MuTIR). MuTAIL-
PCR products are amplified with 29 bp of MuTIR
sequence downstream of the transposon specific primer
[23]. Presence of the MuTIR sequence identifies the pre-
cise insertion site for a FST (see Table 1 and Additional file
1). MuTIR sequences were present in 91 FSTs and absent
in the remaining 15 insertions sites selected for valida-
tion. MuTIR sequences are expected to be absent from a
proportion of FSTs, because the MuTAIL products were
cloned in random orientations and are frequently larger
than single-pass sequence reads. Locus-specific PCR prim-
ers were designed based on the location of the MuTIR and
MAGI sequence matches. We used three strategies to
design the primers (Fig 1B). First, left and/or right primers
were designed when the precise insertion site was placed
within a MAGI sequence. The left primer was based on the
MAGI sequence, and the right primer was designed using
an alignment of the MAGI and FST. Second, a single right
primer was designed when the MAGI showed partial over-
lap with the FST. These primers were designed using either
an alignment between the FST and MAGI or were
designed with the FST alone. Third, a presumptive right
primer was designed for the 15 FSTs that did not have
MuTIR sequences based on the hypothesis that these
MuTAIL products were not completely sequenced.

The locus-specific primers were used in combination with
MuTIR primers to amplify the insertion sites from individ-
ual mutant lines. The primers were tested with the initial
DNA template used to generate the FST, as well as with
siblings or progeny of the individuals used for templates.
Figure 2 shows examples of PCR validation assays for 10
FSTs. In these examples, the plant used for MuTAIL
sequencing came from a backcross of an individual Uni-
formMu line to the W22 inbred. Germinal insertions are
expected to be found in all siblings or to segregate in a 1:1
ratio, depending on whether the progenitor plant was
homozygous or heterozygous for individual insertion
sites. Overall, we confirmed 94 insertion sites from the
sample of 106 FSTs, using an average of 1.6 locus-specific
primers per FST (Additional file 2). In many cases, two
primers for an individual FST were tested simultaneously
and both of these primers amplified the Mu insertion site
(see Additional file 2). For 84 of the insertion sites, a sin-
gle primer was sufficient to confirm and track the inser-
tion. Importantly, all 94 Mu insertions confirmed in these
assays showed germinal segregation. Overall, our data


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Table I: Annotations of the insertion sites confirmed in Figures 2 and 31

Blastcluster MAG14.0 hit Insertion site ZMGI hit Insertion type


1097 no hit
731 TC300585
748 TC301994
TC300035
394 no hit
4515 TC290606
3281 TC314694
8436 TC304662
731 no hit
550 TC302939


Annotation/Expression Pattern


No EST support for gene
exon plastid 6-phosphogluconate dehydrogenase
exon Mixed tissues
exon Probable prefoldin subunit 2
No EST support for gene
exon Putative ATP dependent copper transporter
intron Putative calmodulin-binding protein
exon Putative cytosolic chaperonin delta-subunit
No EST support for gene
exon SAM & endosperm expression


A complete table of confirmed insertions is given in Additional file I Annotations of Confirmed Germinal Insertion Sites. The Blastcluster
identifiers are from the UniformMu website [49], and correspond to one or more FSTs in Genbank. The Insertion site base number is relative to
the matching MAG14.0 sequence. The Insertion type was classified as exon or intron based on an alignment of the relevant FST, MAGI and ZMGI
sequences.


indicate that the vast majority of these sequence-indexed
mutants are simple to recover from the UniformMu pop-
ulation.

There were two noticeable trends in the FSTs that failed to
amplify in the PCR assays (Table 2). First, the locus-spe-
cific primers for five insertion sites (bc41, bc182, bc1042,
bc1509, and 03S0467-14) were not designed to anneal at
the insertion site. Three of these FSTs included repetitive
or chimeric sequences that identified MAGI sequences
with only a partial sequence match to the FST. In these
cases, the specific primers were designed from MAGI
sequences that were not part of the FST. For bc182 and
bc1042, the locus-specific primers were designed to
amplify from the non-MuTIR end of the insertion site
sequences. The second trend was observed for a group of
four other FSTs (bc1073, bc1382, bc1435, and bc1537).
The locus-specific primers for these insertions showed
base pair differences between the most recent MAGI or
FST consensus sequence suggesting that the primers were
originally designed from lower quality sequence reads or
assemblies. Mu-specific products were not observed from
a final group of three insertions sites (bc93, bc1571, and
bc1526) for unknown reasons. Overall, 75% of the inser-
tion sites that failed to be confirmed had errors in the
locus-specific primer design, suggesting that sequence
errors or human errors were the primary causes for diffi-
culties in confirming an insertion site.

The PCR validation assays also identified an insertion site,
bc199, that co-segregates with a rough endosperm (rgh) seed
mutant phenotype. The bcl99 insertion disrupts the open
reading frame of a predicted 6-phosphogluconate dehy-
drogenase (6PGDH) gene at the Pgd3 locus, and we
named this insertion allele pgd3-umul. There are three
known loci in maize for 6PGDH enzymes: Pgdl, Pgd2,
and Pgd3 [29]. Pgd3 maps to the short arm of chromo-


some 4 in bin 4.03 [30]. We designed both left and right
locus-specific primers to develop a co-dominant PCR
assay for this insertion (Figure 3A). Co-segregation
between the pgd3-umul insertion site and the rgh*-OOS-
005-14 mutant was observed in a test of 29 progeny from
backcrosses to W22 (Figure 3B) as well as 28 homozygous
rgh/rgh mutant individuals from a segregating self-polli-
nation (Figure 3C). We expanded the linkage analysis to
test a total of 260 meiotic products and observed no
recombination between pgd3-umul and the rgh mutant
(data not shown). These data indicate that the map dis-
tance between pgd3-umul and the rgh phenotype is less
than 0.4 cM. This tight linkage raises the hypothesis that
the rgh mutant phenotype may represent the pgd3-umul
knockout phenotype, and we characterized the rgh*-OOS-
005-14 mutant further.

The linked rgh mutant shows highly reduced grain-fill,
along with a characteristic etching or pitting at the
endosperm surface (Figure 4A-B). Longitudinal hand sec-
tions of mature mutant kernels showed both reduced
endosperm development as well as embryo defects, with
most mutants failing to develop embryonic roots or leaves
(Figure 4C). Rare embryos with a visually apparent shoot-
root axis were observed in homozygous mutants. A small
fraction of these seeds were able to germinate when cul-
tured prior to seed desiccation. These mutant escapes
developed into morphologically normal seedlings and
plants (Figure 4D). However, the plants showed a pale
green leaf phenotype that was less severe near leaf veins.
The homozygous rgh individuals were also confirmed as
homozygous for the pgd3-umul allele using the locus-spe-
cific PCR assay (data not shown). Self or sibling pollina-
tions of these mutant plants showed all mutant seed,
which confirmed that the plants were homozygous rgh
individuals and not the result of hetero-fertilization (Fig-
ure 4E).


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bc0047
bc0199
bc0234
bc0472
bc0862
bc0881
bcl 133
bc1266
bc1474
bc1542


MAG14_20201
MAG14_45584
MAG14_4699
No hit
MAG14_29768
MAG14_30440
MAG14_155851
MAG14_43305
MAG14_118260
MAG14_139298


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Left/
Right


Right
Only


No
MuTIR


Promoter Exon Intron Unknown
Position of Insertion Site


TIR


Left : : Right

TIR


Right



Right


Figure I
Informatic analysis of UniformMu FSTs validated by
PCR. (A) Distribution of Mu insertion sites relative to tran-
scribed maize sequences. (B) Schematics of the locus-specific
primer design as constrained by available sequences. Left and
right specific primers were designed when genomic sequence
was available on either side of the insertion site. Right spe-
cific primers were designed when the insertion site defined
novel maize genomic sequence. A presumptive right primer
was designed for FSTs without a MuTIR sequence.



6PGDH enzyme activity was measured from 25 days after
pollination (DAP) endosperm extracts of W22, rgh*-OOS-
005-14 homozygous mutants as well as single and double
pgdl; pgd2 mutants (Figure 4F). The W22 sample showed
two major isozyme activities. The faster migrating activity
corresponds to three bands: PGD1 homodimers, PGD1/
PGD2 heterodimers, and PGD2 homodimers [31]. A sub-
set of these bands are absent in the pgdl and pgd2 single
mutants, and all three bands are lost in pgdl; pgd2 double
mutant samples. The rgh mutant sample lacks the slower
migrating activity. Serial dilutions of the W22 extract sug-
gest that the rgh mutant endosperm has <5% normal
activity for this 6PGDH isozyme (data not shown). We
conclude the slower migrating 6PGDH isozyme is likely
to be PGD3 as no reduction of this isozyme activity was


observed in the other pgd mutants. These data suggest that
the FST for pgd3-umul identified an enzymatic knockout.

6PGDH enzymes are found in both the cytosol and chlo-
roplast in plants (reviewed in [32]). To predict the likely
subcellular localization of the PGD3 protein, a complete
Pgd3 locus was assembled from EST and MAGI sequences.
The predicted peptide for the Pgd3 locus contains an N-
terminal extension that is absent from the predicted
PGD1 and PGD2 proteins in a multiple sequence align-
ment (data not shown). PGD3 clusters with a known
chloroplast-localized 6PGDH protein from spinach in a
ClustalW sequence similarity tree ([33,34]; Figure 5). Fur-
thermore, the PGD3 protein is predicted to be targeted to
the chloroplast by the protein localization programs: Tar-
getP, PSORT, and Predotar [35-37]. These sequence anal-
yses suggest that the pgd3-umul allele disrupts a
chloroplast-localized 6PGDH in maize. This prediction is
consistent with previous observations that the remaining
6PGDH activity in pgdl; pgd2 double mutants is entirely
plastid-localized [29].

Discussion
The current maize knockout resources have enough inser-
tions to disrupt every gene within the genome. FSTs will
make these maize mutants far more available to the
research community, and sequencing MuTAIL products is
a high throughput approach to generate maize FSTs. How-
ever, FSTs alone do not make a reverse genetics resource
easy to use. The tags need to have low entry barriers for
tracking an insertion site of interest as well as for obtain-
ing the line or strain that corresponds to the insertion site.
FSTs from the UniformMu population are predicted to
have low entry barriers due to the genetic markers
included in the population and the high-throughput
sequencing strategy used to generate the FSTs [19].

The bzl -mum9 mutant was used to select against somatic
activity, and here we have shown that the vast majority of
the resulting FSTs segregate as germinal insertions. We did
not observe any evidence of somatic insertions. Somatic
insertions would be expected to amplify from the DNA
template used to generate the FSTs, but not to amplify
from any other related individuals. In the cases where we
could not confirm an insertion, the insertion site did not
amplify from the template DNA used to generate the FSTs.

A secondary analysis of the specific primers indicated that
three factors led to a small fraction of insertion sites that
are more difficult to track. First, differences between avail-
able maize genomic sequences and the FSTs caused the
precise insertion site to be mis-identified for 6.7% of the
FSTs. These mis-identifications led to errors in locus-spe-
cific primer design. The sequence differences are likely to
result from sequence errors, incomplete maize genomic


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02S-2018 siblings


02S-2018 siblings


o 04 02S-2018 siblings
= l T I


SM r


02S-2018 siblings


bc199






bc472






bcl266






bc862


0


02S-2034 siblings


4IT


o 003S-3050 siblings
--q>1 T


03S-3060 siblings


02S-2028 siblings


S03S-3061 siblings


bc881


Figure 2
Examples of PCR validation assays for UniformMu FSTs. Each panel shows PCR products that were amplified using a
MuTIR primer and a FST locus-specific primer. Each primer was tested on the initial template DNA used to develop the Uni-
formMu FSTs (lane T) as well as DNA extracted from siblings (denoted by brackets). No DNA template controls were
included in all assays (lane H20). W22 DNA was used to confirm that the insertion site did not amplify in the recurrent inbred
parent of the UniformMu population.


sequence, and sequence divergence between the W22
inbred background used to develop UniformMu and the
B73 inbred chosen for genomic sequencing. Comparative
genomic sequence studies of maize inbreds suggests that
there are significant differences in gene content and repet-
itive elements between inbreds [38-40]. A second factor
that contributed to insertion sites that were not confirmed
as germinal was human error in identifying the TIR-
genomic DNA junction. These errors led to primer design
errors for 1.9% of the FSTs. Finally, PCR or sample track-
ing errors occur at a low frequency in any high-throughput
sequencing project. We speculate that a small amount of


cross-contamination between sequencing libraries can
explain the remaining 2.8% of insertion sites that were
not confirmed. Importantly, 89% of the FSTs were readily
confirmed as germinal insertions by locus-specific PCR.
Furthermore, the MuTAIL FSTs were generated from indi-
vidual plants or lines that were successfully bulked, and
each sequence read name corresponds directly to an indi-
vidual. These characteristics make it simple to identify the
seed stocks for each FST. Combined, the characteristics of
the MuTAIL FSTs and the UniformMu population make a
robust resource for reverse genetics.


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O 4


bc234






bc47






bc1474






bc1542


bc1133


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0 N
_- C


o04
04








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Table 2: Annotations of insertion sites that were not confirmed with PCR assays


Blast Cluster/Read
Name

03S-0467-13A2-G01 I
bc0041
bc0093
bc0182
bc1042
bc1073
bc1382

bc1435
bc1509

bc1526

bcl537

bc1571


MAG14.0 hit Insertion site ZMGI hit Insertion type


repeat hit only
MAG14 104
MAG14_65387
MAG14_ 121677
MAG14_ 128194
MAG14_94877
repeat hit only

MAG14_ 132661
repeat hit only

MAGI4_13062

MAG14_5849

MAGI4 154704


Annotation/Expression
Pattern


DR906457 exon egg cell expressed
252 no hit No EST support for gene
128 DN229926 exon leaf & SAM expressed
no MuTIR no hit No EST support for gene
1502 no hit No EST support for gene
3164 TC303220 exon TM20/Dek34 gene
no MuTIR no hit Novel maize genomic
sequence
no MuTIR DT943333 unknown thioesterase superfamily
no hit Novel maize genomic
sequence
no MuTIR TC292893 unknown 26S protease regulatory
subunit 7
481 TC291433 intron cold stressed seedling
expression
734 TC282381 -40bp short-chain dehydrogenase/
reductase


Likely cause for failure to
amplify

primer design error
primer design error
unknown
primer design error
primer design error
low quality sequence
low quality sequence

low quality sequence
primer design error

unknown

low quality sequence

unknown


The pgd3-umul insertion site that co-segregates with a seed
mutant phenotype illustrates the utility of the Uni-
formMu FST resource for generating hypotheses about
gene function. The pgd3-umul insertion is in a predicted
chloroplast-localized 6PGDH enzyme in the oxidative
section of the pentose phosphate pathway (OPPP). The
linked rgh*-OOS-005-14 seed mutant lacks a 6PGDH iso-
zyme activity suggesting pgd3-umul causes an enzymatic
knockout. These linkage and biochemical data suggest the
hypothesis that Pgd3 has an essential role in seed develop-
ment. Consistent with this hypothesis, the OPPP has been
shown to be involved in hexose cycling and starch synthe-
sis in developing seeds [41]. Also, previous studies with
maize pgdl and pgd2 mutants argued that the plastidic
enzymes for the oxidative section of the OPPP can com-
pensate for loss of 6PGDH in the cytosol [29,31]. Moreo-
ver, a study in maize root tips found that the oxidative
section of the OPPP to be most active in plastids [42].
Combined these data are consistent with plastidic OPPP
enzymes being required for seed development. However,
linkage data alone cannot prove that the rgh phenotype is
a result of the pgd3-umul mutation. Additional experi-
ments are necessary to test this hypothesis such as identi-
fying multiple mutant alleles of Pgd3. Alternatively, a Pgd3
transgene could be developed to test for complementa-
tion of the 6PGDH enzyme defect and the rgh*-OOS-005-
14 phenotype. The maize genetics community has multi-
ple resources to assist in identifying alleles or in generat-
ing transgenics for individual genes of interest (reviewed
in [151).

The pgd3-umul example also illustrates the complemen-
tary nature of having reverse genetics resources in multiple


plant species. The Arabidopsis genome contains two copies
of genes predicted to encode plastid-localized 6PGDH
enzymes and one gene predicted to encode a cytosolic
6PGDH (Figure 5; [32]). Each of these genes have multi-
ple sequence-indexed mutations that disrupt exon
sequences and are likely to cause null alleles [43]. Gene
redundancy in Arabidopsis may be one reason why mutant
phenotypes associated with plastid-localized 6PGDH
have not been reported previously. If plastidic 6PGDH
enzymes are essential in plants, a double mutant for
knockouts of Arabidopsis orthologs, Atlg64190 and
At5g41670, would be expected to show a visible pheno-
type.

The rice genome has a single locus that is predicted to
encode plastid-localized 6PGDH, Osl g29400, similar to
maize. However, the current rice knockout resources do
not have FSTs that disrupt this locus [44]. The rice
ortholog for cytosolic 6PGDH, Os06g02144, has three
insertions, but these insertions map to promoter and 3'
UTR regions and may not be null alleles. Thus, the maize
mutants in 6PGDH enzymes suggest an agricultural rele-
vance for the plastid-localized OPPP that would not be as
simple to identify using the current Arabidopsis or rice
resources.

Conclusion
The pgd3-umul allele is just one example of the current
collection of 1,882 non-redundant insertion sites
sequenced in UniformMu. We conclude from our analysis
that the vast majority of the UniformMu insertion sites are
germinal. Importantly, multiple studies have shown that
Mu insertions are heavily biased for transcribed regions of



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A
TIR
pgd3-umul 200 bp
98%
TC300585
I I99%

MAGI4 45584
Left Right


B rghl+ +/+
N BC2 BC3 BC2 BC3


Left/Right




TIR8/Right






C



Left/Right





TIR8/Right


1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031


0 +/+


rgh/rgh


1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031

Figure 3
Co-segregation of pgd3-umul with rgh*-OOS-O05-14. (A) Schematic alignment of the bc 199 UniformMu FST site (pgd3-
umu ), the TC300585 Zea mays Gene Index EST contig, and the maize assembled genomic island, MAGI4_45584. The sche-
matic shows the location of the MuTIR sequence (black arrow, not drawn to scale) and percent nucleotide identity is noted.
The left and right primer sites are marked by gray arrows; primer sizes are not drawn to scale. (B) Co-segregation analysis
with progeny from backcross generations 2 and 3 (BC2 and BC3) in a W22 introgression. Left/Right products amplify normal
alleles. TIR8/Right products amplify the insertion site. (C) Co-segregation analysis with self progeny. PCR was completed with
DNA extracted from homozygous normal (lanes 2-3) or rgh mutants kernels (lanes 4-31).






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owes







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normal mutant
E"


pgdl;
W22 rgh pgdl pgd2 pgd2
4PGD3
PGD1
low k# PGD2


Figure 4
Phenotype of the rgh*-OOS-005-14. (A) Self-pollinated
ear segregating for rgh kernels (arrows). (B) Mature normal
and rgh sibling kernels. The top row shows the abgerminal
side of the kernels, and the bottom row shows the germinal
side of the kernels. Scale bars are 5 mm. (C) Longitudinal
hand sections of normal and rgh kernels. Aleurone, starchy
endosperm, and embryo tissues are denoted by a, s, and e,
respectively. Arrows indicate embryonic shoot and root tis-
sues in the normal kernel. Scale bars are I mm. (D) Normal
and rgh mutant plants 50 days after initiating tissue culture.
(E) Homozygous rgh mutant ear at 35 DAP. (F) Native PAGE
assay for 6PGDH activity from 25 DAP endosperm extracts.
Black arrow indicates the inferred PGD3 isozyme and the
open arrow indicates the PGDI and PGD2 homo- and het-
erodimer isozymes.


- _'r .



OV V


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the genome [17,19,22,23]. In addition, the UniformMu
population is in an inbred background that simplifies
phenotypic analysis [19]. Combined, these features pro-
vide a robust, genome-wide resource for reverse genetics
in maize. Further sequencing of UniformMu FSTs would
enhance this resource by expanding it towards a compre-
hensive knockout collection.

Methods
Sequence Analysis
The UniformMu FSTs were clustered onto maize
sequences using the methods described in McCarty et al.
[19]. In addition, the FSTs were assembled using CAP3
[26], and the FST contigs corresponding to the blastclus-
ters chosen for PCR analysis were retrieved from the
assembly for more detailed annotation. The FST contigs
were queried against the MAGI4.0 release of the maize
genome survey sequence assembly as well as release 16.0
of the Zea mays gene index (ZMGI) using BLASTN searches
[27,28,45]. Top matches were visually inspected to deter-
mine if the sequence alignment was consistent with a pre-
cise match to the maize genome or tentative consensus
EST contig. An insertion site was considered precisely
identified when the top match had >95% identity
between the FST and the MAGI or ZMGI. For matches to
MAGI sequences, alignments were expected to extend
through the full length of the MAGI or FST sequence with
no insertion or deletion polymorphisms >20 bp. When a
FST showed significant identity with more than one
MAGI, the MAGI sequence that defined the precise inser-
tion site was reported. The MAGI sequence matching each
FST was also queried against the ZMGI to identify MAGIs
with evidence for a transcribed region.

Each FST was scored for the presence of the MuTIR by vis-
ual inspection of the terminal end sequence of the FST
contigs. Insertion site positions were classified as follows.
Promoter insertions had TIR-genomic DNA junctions that
were <850 bp 5' of the ZMGI tentative consensus EST con-
tig. Exon insertions had a TIR junction within a ZMGI
sequence. Intron insertions had a TIR junction in genomic
DNA that could be positively identified as intron
sequence based on an alignment of the FST, MAGI, and
ZMGI sequences. Unknown insertions did not have a
ZMGI match with either the FST or corresponding MAGI
sequence. FSTs without a clear TIR sequence were also
classified as unknown.

The Pgd3 locus was assembled by aligning the bc199 FST
contig with MAGI4_45584, MAGI4_45583, and the ZMGI
TC300585 contig. The largest open reading frame (ORF)
was selected and used to generate the protein similarity
tree with ClustalW and TreeDraw, which were run on the
SDSC Biology Workbench [34,46,47].


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Figure 5
Sequence similarity tree of 6PGDH enzymes.
Sequence similarity tree of 6PGDH enzymes from maize
(Zm), rice (Os), Arabidopsis (At), spinach, Chlamydomonas
reinhardtii, and Synechocystis sp. PCC 6803. Rice and Arabidop-
sis proteins are identified by their locus numbers. The pro-
tein sequences used to generate the ClustalW alignment and
tree were from the ORF of the Pgd3 locus and Genbank
accessions: AAC27702, AAC27703, AAK49897, AAK51690,
AAL76323, AA042814, ABA93694, NP 198982,
NP 442035, NP 850502, NP 910282.


PCR Assays
The PCR assays were completed with multiple methods,
and no significant difference was observed between
approaches. Detailed methods for specific insertion sites
can be obtained from the primary contacts given in Addi-
tional file 2 for each insertion site and primer. In general,
DNA was extracted as described in Settles, et al. [23]. The
locus-specific primers were designed with Primer3 for 20-
25 base oligomers with a predicted Tm between 60-65 C
[48]. The specific primers were paired with a MuTIR spe-
cific primer. MuTIR primers used in this study included
TIR4, TIR6, and TIR8 from Settles, et al. [23] as well as the
primers 5'-CGCCTCYATITCGTCGAATCC-3' and 5'-
GCCTCCATITCGTCGAATC-3'. PCR was typically com-
pleted with Taq DNA polymerase and buffers from either
Invitrogen or Promega with either 5% DMSO or 1 M
Betaine added to the final reaction. Thermocycling condi-
tions were generally 94C for 1 minute, 60C for 1
minute, 72 C for 1 minute for 40 cycles. Annealing tem-
peratures were adjusted when the predicted Tm of a spe-
cific primer was lower than 60 C. Most specific primers
gave products in the first set of PCR conditions tested, and
a careful study of optimal general methods for amplifying
specific insertions sites was not completed.

rgh*-OOS-005-I4 Mutant Phenotypic Analysis
The longitudinal hand sections of mutant and normal rgh
kernels were completed with mature kernels from a self-
pollinated heterozygous individual. The kernels were
imbibed in water overnight and sectioned with a fresh
razor blade and unstained sections were imaged. To


recover homozygous mutant plants, rgh mutant and nor-
mal seed were removed from mature, segregating self-pol-
linations (30-35 DAP) prior to drying the ears. The seed
were surface-sterilized in 70% ethanol for 2 min, trans-
ferred to 20% bleach, 0.1% Tween 20 for 15 min, and
washed in sterile water four times. The pericarp was
removed from the kernels under sterile conditions and
transferred to Murashige and Skoog salt and vitamin mix-
ture at pH 5.7 with 0.4% Phytagel and 1% sucrose. The
seeds were cultured at 32/25C (day/night) with a 16-h
photoperiod. Germinated seedlings were transferred to
soil after 2-4 weeks of culture, and grown in greenhouse
conditions with a similar temperature and light regime.

6PGDH enzyme activity was measured by native-PAGE
assays essentially as described by Bailey-Serres et al [31].
Briefly, 0.1 g of endosperm tissue was ground in extrac-
tion buffer (20 mM Tris, pH 8.0, 4 mM DTT, 5 mM MgCl2,
15% (v/v) glycerol, 0.001% Bromophenol blue) at a ratio
of 0.2 mL extraction buffer to 0.1 g tissue fresh weight. The
protein extracts were cleared prior to gel loading by cen-
trifugation at 10,000x g for 10 min at 4C. The extracts
were loaded based on equal fresh weight of starting sam-
ples and the proteins were separated at 4 C with native-
PAGE (7% acrylamide, 0.375 M Tris, pH 8.8, 12% glyc-
erol(v/v)). After electrophoresis, the gel was stained for
6PGDH (0.1 M Tris-HC1, pH 7.5, 0.1 mg/mL NADP, 0.1
mg/mL nitro blue tetrazolium, 0.01 mg/mL phenazine
methosulfate, 0.5 mg/mL 6-phosphogluconate) at room
temperature for 30 min. The gel was de-stained with water
prior to drying and digital imaging.

Authors' contributions
AMS completed germinal PCR segregation analysis
including locus-specific primer design for 30 of the inser-
tion sites tested, completed phenotypic analysis of pgd3-
umul, integrated the data from the other authors includ-
ing informatics analysis to annotate the insertion sites,
supervised the experiments completed by LL, DSF, EW,
and CWT, and wrote the manuscript. DRH, BCT, SPL, JLiu,
MS, LL, BAO, DSF, and EW completed germinal PCR seg-
regation analysis including locus specific primer design
for 76 of the insertion sites tested. CWT and LL completed
the tissue culture experiments, enzyme activity assays, and
pgd3-umul co-segregation analysis. JLai participated in the
informatic analysis of the FSTs. CTH, WTA, and JB gener-
ated the segregating UniformMu populations. JM super-
vised the experiments completed by JLai and edited the
manuscript. LCH supervised the experiments completed
by JB and edited the manuscript. KEK supervised the
experiments completed by BAO, CTH, and WTA as well as
edited the manuscript. PWB supervised the experiments
completed by JLiu and edited the manuscript. BAL super-
vised the experiments completed by DRH and edited the
manuscript. DRM supervised the experiments completed


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by BCT, SPL, and MS, participated in the informatics anal-
ysis of FSTs, and edited the manuscript. All authors read
and approved the final manuscript.


Additional material


Additional File 1
Annotations of Confirmed Germinal Insertion Sites. Excel table of all
the insertions confirmed in this study including the insertions in Table 1.
The Blastcluster identifiers are from the UniformMu website[49], and
correspond to one or more FSTs in Genbank. The Insertion site base
number is relative to the matching MAGI4.0 sequence. The Insertion type
was classified as exon, intron, or unknown based on an i ....... .. f the
relevant FST, MAGI and ZMGI sequences. The exact number of bases 5'
(negative bases) or 3' (positive bases) are given for insertions that were
found to be 5' or 3' of a ZMGI cDNA assembly.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-8-116-S1.xls]

Additional File 2
Locus-specific primers used in this study. Excel table of the locus-specific
primers and germinal segregation results from this study. The Blastcluster
identifiers are from the UniformMu website [49], and correspond to the
insertion sties in Table 1, Table 2, and Additional file 1. Specific contact
information for each primer/insertion site is given.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-8-116-S2.xls]


Acknowledgements
The authors thank Julia Bailey-Serres for providing homozygous mutant
seeds for the pgd and pgd2 single mutants as well as the pgd I; pgd2 double
mutant. The authors also thank the anonymous reviewers for critical read-
ing of the manuscript. This research was supported with funding from the
NSF Plant Genome Research Program (grant #0076700), the Vasil-Mon-
santo Endowment, and the University of Florida Genetics Institute Seed
Grant Program.

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