Methods 49 (2009) 248–254
Contents lists available at ScienceDirect
Methods
journal homepage: www.elsevier.com/locate/ymeth
Regional mutagenesis using Dissociation in maize
Kevin R. Ahern a,1, Prasit Deewatthanawong a,1, Justin Schares b, Michael Muszynski b, Rebecca Weeks b,
Erik Vollbrecht b, Jon Duvick b, Volker P. Brendel b,c, Thomas P. Brutnell a,*
a
Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, USA
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011-3260, USA
c
Department of Statistics, Iowa State University, Ames, IA 50011-3260, USA
b
a r t i c l e
i n f o
Article history:
Accepted 9 April 2009
Available online 24 April 2009
Keywords:
Dissociation
Activator
Transposon tagging
Regional mutagenesis
Maize
Functional genomics
a b s t r a c t
We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation
(Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the
maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are
described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion
alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target
gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a
PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail
an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a highthroughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA
from seedling tissues. We also describe several web-based tools for browsing, searching and accessing
the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
Class II transposable elements (transposons) are powerful
tools for dissecting gene structure and function in maize [1].
The autonomous Activator (Ac) and its non-autonomous deletion
derivative Dissociation (Ds) were the first transposons discovered
[2] and are endogenous to maize. They have been extensively
characterized using genetic, biochemical and molecular genetic
tools [3]. A detailed understanding of both the mechanism and
regulation of the Ac/Ds transposition process has enabled the
development of sophisticated gene tagging strategies in maize
that exploit these elements [4–8]. Importantly, both Ac and Ds
offer several advantages for fine-scale genetic mapping that
greatly extend their utility beyond that of a simple molecular
tag. For example, Ac has been used to define enhancer elements,
intron/exon boundaries and 50 - and 30 -regulatory elements in
genes [7,9–11]. Imprecise excision events of both Ac and Ds have
been exploited to generate stable alleles that encode proteins
with altered biochemical functions [12–16]. Additionally, the
* Corresponding author. Address: Boyce Thompson Institute, Cornell University, 1
Tower Rd., Ithaca, NY 14853, USA. Fax: +1 607 254 1325.
E-mail address: tpb8@cornell.edu (T.P. Brutnell).
1
These authors contributed equally to this article.
1046-2023/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2009.04.009
propensity of Ac and Ds for short-range transposition has facilitated the recovery of multiple insertions in a gene-of-interest
(goi) through regional or reconstitutional mutagenesis [11,17].
In an elegant study, Moreno and colleagues generated over 250
novel alleles of p using closely linked Ac elements as donor alleles [7].
Despite these many benefits of Ac/Ds for gene characterization, their use has been relatively limited in maize. As both Ac
and Ds tend to reinsert at sites closely linked to the donor element [11,18–20], an efficient mutagenesis can only be conducted
when the donor resides close to the target locus. Studies by Dooner and Greenblatt have shown that the majority of linked Ac
insertions are within 4–6 cM of the donor locus [18,19], and a
similar trend is observed with Ds (Vollbrecht and Brutnell,
unpublished). Until recently, relatively few Ac and Ds elements
were precisely positioned throughout the maize genome, limiting
the scope of Ac/Ds regional mutagenesis [5]. However, in recent
years the number of precisely mapped Ac elements has grown
tremendously [21,22], and efforts are underway to distribute
thousands of Ds elements throughout the maize genome at
approximately 8–12 cM intervals http://www.plantgdb.org/prj/
AcDsTagging/. Using these materials, it is now possible to harness the utility of Ds to create mutant alleles through both forward and reverse genetic approaches. Here we describe two
genetic strategies that use Ds in regional mutagenesis and
describe the molecular protocols that we have developed to
facilitate these screens.
K.R. Ahern et al. / Methods 49 (2009) 248–254
2. Description of method
We envision two general schemes that may be applied to exploit Ds in regional mutagenesis, forward genetic and reverse genetic. In a forward genetic screen, populations are screened for a
visible or biochemical phenotype (e.g., pink endosperm or accumulation of lycopene by HPLC [10,15]). Forward screens are likely to
be conducted when the mutant of interest has been identified
and additional alleles are needed to enable the cloning of the gene
or to confirm the identity of the gene through the recovery of independent mutations, or when the objective of the study is to conduct extensive mutagenesis of a target locus. In a reverse genetic
screen, the genomic sequence of a target locus is used in a PCRbased screen to identify Ds insertions that have been mobilized
into the target from a closely linked donor. Forward genetic
screens are, by far, the easiest to conduct, as putative mutant alleles can be identified without expensive and time-consuming
PCR-based screens. However, reverse genetic screens may prove
invaluable when genetic redundancy masks a mutant phenotype,
or if the homozygous recessive alleles are associated with lethality,
poor penetrance or low expressivity. In these instances, the ability
to molecularly track the insertion allele could prove essential in
phenotypic characterizations of the mutant allele [23].
2.1. Identifying the Ds donor sites
The first step in conducting a regional mutagenesis is the same
for both forward and reverse genetic schemes: the identification of
donor Ds insertions (dDs) that are in tight genetic linkage with the
goi (Fig. 1). We recommend that a targeted mutagenesis be conducted with at least two dDs insertion lines, as it has been shown
that not all Ds elements can be mobilized at the same frequency
[24] and there may be subtle insertion site biases that depend on
the genomic context of a Ds insertion. Thus, using two dDs loci
may increase the likelihood of success. Currently, approximately
Fig. 1. Summary of genetic screens for Ds regional mutagenesis. Both forward and
reverse genetic screens begin with the identification of Ds insertion(s) that map
close to the gene or region of interest (1). Insertion lines are ordered on the project
web site, and plants carrying the Ds insertions identified through a PCR screen (2).
Testcrosses to a mutant reference allele (goi-mut/+ or goi-mut/goi-mut) or the W22
allele (r-sc:m3/r-sc:m3) are performed using the dDs lines (dDs/+, Ac-im/+, r-sc:m3/rsc:m3) as male pollen donors (3). The dDs lines should also be self-pollinated to
recover lines homozygous for the dDs insertion. Progeny are screened to identify
mutant individuals (forward genetic) or the presence of a Ds insertion in the goi
(reverse genetic; 4). DNA is isolated from mutant individuals or from pools of
seedlings (5) and the goi characterized using Ds as a molecular tag (6).
249
83% of the maize genome is covered by Ds insertions in our collection that are less than 12 cM apart (Vollbrecht, unpublished).
Therefore, for the vast majority of genes in the maize genome, it
should be possible to identify at least two Ds insertions that are
within 6 cM of the target locus. We are currently operating under
the assumption that the closer the dDs is to the target locus, the
higher the frequency of insertion. Although this assumption has
not been rigorously tested for Ds transposition, this trend is evident from several regional mutagenesis experiments conducted
with Ac [7,9,10,15].
All mapped Ds insertions have been placed on BAC contigs and
can be searched or browsed on our project web site (http://
www.plantgdb.org/prj/AcDsTagging/). To date, over 1700 unique
sequence-indexed Ds insertion lines are available, with approximately 83% precisely positioned on the current build of the maize
B73 physical map. A comprehensive description of this collection
will be published elsewhere, but all lines are currently available
and can be ordered through our project web site. All Ds insertion
lines are maintained in a uniform W22 inbred line and contain a
low number of active Ds insertions, most often one or two elements, ensuring a uniform population with low mutational load.
This collection represents the most comprehensive collection of
Ac/Ds insertions in maize and will continue to grow as additional
Ds elements are anchored to the genome.
To initiate a reverse genetic screen using Ds as a molecular tag,
the target locus must first be aligned to the current maize genome
sequence. In these screens, the known target gene sequence can be
BLAST-searched against the sequences of BAC clones, which serve
as the templates for the maize genome sequencing project
(http://www.maizesequence.org). Ultimately, as the maize sequence becomes more refined, the search will be against assembled maize pseudo-chromosomes. BLAST resources are also
available at http://www.plantgdb.org/ZmGDB/cgi-bin/blastGDB.pl.
Once the accession number of the BAC contig aligning to the target
gene sequence is known, the Ds collection can be searched for a donor as detailed below.
In a forward genetic screen, the target gene sequence is unknown. To find a dDs insertion in the region of the target, it will
first be essential to correlate the genetic position of the target locus
with the physical map. This may be achieved by establishing linkage between the gene of interest and genetic markers that have
been mapped to the maize genome (e.g., SSR or overgo markers
displayed at the http://maizesequence or http://maizegdb web
sites).
Once a BAC accession or a FingerPrint contig (FPC ctg or ctg)
number is identified for the goi, the Ds collection can be searched
using one of these identifiers to determine the closest Ds to be used
as a donor. Navigate to http://www.plantgdb.org/ZmGDB/DisplayGeneAnn.php?ds=1 to view the current searchable browser ‘‘Maize
BAC Annotation”. Enter the BAC GI, BAC accession number, or FPC
ctg into the search box (e.g., AC211274; ctg269). If the box labeled
‘‘Show only fDs-BACs” is check marked, this will limit the search to
only BACs or FPC contigs that carry a Ds element. A new ‘‘Maize
BAC Annotation” window will open, displaying all the BAC contigs
that are associated with the search term (Fig. 2). Sequences flanking Ds elements that show homology to predicted rice gene models
are displayed as ‘‘fDs Matches”. Click on the button in the left-hand,
Genome Database ‘‘GDB” column that aligns with any Ds element
to view a graphical representation of the current BAC physical
map with the position of the Ds element highlighted as a light blue
track in the ‘‘BAC/Clone Context View”. Scrolling over or clicking on
the yellow, top track will provide the assembly status for the BAC
clone and its chromosomal position. Currently, most BAC clones
are in ‘‘working draft” sequencing stage and consist of multiple
unordered DNA sequence pieces. Thus, the absolute placement of
a gene within a BAC clone is usually uncertain. However, any Ds
250
K.R. Ahern et al. / Methods 49 (2009) 248–254
Fig. 2. Web-based tools for identifying dDs insertion lines. Several informatics resources are available to browse and query the dDs collection at the project web site (http://
www.plantgdb.org/prj/AcDsTagging/). Currently, the Ds collection can be searched most effectively by first identifying a BAC or FPC contig that is linked to the gene of
interest.
element that resides on the same BAC clone as the target locus is
likely to serve as an ideal donor site. There are numerous search
features and display tracks available on this page that have been
previously described [25]. If the target gene resides on the same
BAC clone, it may be possible to zoom out and scroll to gauge
the physical distance between the donor and Ds target locus. However, as mentioned above, the structure of the BAC clone may be a
composite of several DNA pieces, so an absolute distance determination may not be possible.
The next step should be to verify that the most likely BAC localization has been found, using BLASTn. Clicking on the Ds track
opens a ‘‘Zm-AcDs Record” page, on which the sequence flanking
the Ds insertion is shown. This sequence was recovered from one
end of the element and used to map the insertion to the B73 genome. This page includes a link, ‘‘BLAST@ZmGDB”, which opens a
BLAST page with the fDs sequence as query. Select ‘‘ZmBAC” as
the database and BLASTn as the analysis method. Note that this
dataset includes all maize BAC-sized sequences, not just those
from the B73 genome project sequence.
Once the Ds element has been identified, seed stocks can be ordered from the ‘‘Zm-AcDs Record” page. A link to order seed stocks
(‘‘Order Seed @ PlantGDB”) is also on this page. Selecting this link will
direct the user to a seed order page with a link to a pdf order form.
Seed stocks will be genotyped and sent to the user for a nominal
fee to offset the costs of propagation, genotyping, and shipping. There
is an additional surcharge for international shipments to recover the
costs of pathogen testing and phytosanitary certification. There are
no MTA forms necessary, and all lines are non-transgenic, greatly
facilitating interstate and international transfers of germplasm.
2.2. Confirming the presence and location of the dDs
The success of a Ds mutagenesis strategy rests on the proximity
of the dDs to the target locus and on the instability of the element
when a source of active transposase is present in the same genome.
Thus, confirming both the presence and genomic location of the
element in segregating populations is essential. Because seed
stocks are verified and propagated on demand, users will receive
up to 10 seeds that segregate the dDs insertion and if needed, up
to 50 seeds of the W22 tester line. Each line is also homozygous
for a Ds insertion at the r locus (r-sc:m3), and most stocks will carry
one copy of an immobilized source of Ac transposase (Ac-im) [26].
Ac-im contains a deletion of 10 bp at one end of the element. This
deletion prevents the excision of the element, but does not affect
the synthesis of the transposase protein. The presence and copy
number of Ac-im can be monitored by the variegation pattern in
the aleurone. If Ac-im is transmitted through the pollen to females
homozygous for the r-sc:m3 tester, the aleurone will be heavily
spotted due to the presence of a single active Ac. If the element
is transmitted through the female, the triploid endosperm will inherit two copies of the Ac element and will be coarsely spotted,
indicative of later and less frequent transposition events [26]. This
‘‘negative dosage effect” is a hallmark of Ac activity and is likely the
result of the formation of transposase aggregates that inhibit the
transposition process when the levels of transposase protein extend beyond a critical threshold [27–29]. Although the Ac-im element can easily be tracked using this kernel phenotype assay,
the dDs of interest will most likely not reside in a reporter gene.
Thus, the presence of the dDs must be tracked molecularly.
To confirm the presence of each dDs that is requested, we use a
PCR assay to amplify a Ds-flanking sequence junction using a Dsend primer and a primer specific to the genomic insertion site.
Primers are designed using the open-source Primer 3 software at
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. To enable rapid thermocycling, product sizes are selected that range
from 150–300 bp. We perform PCR confirmation on seedlings
grown from the same ear used to distribute seed, so it is highly
likely that the dDs insertion will be present in the populations that
K.R. Ahern et al. / Methods 49 (2009) 248–254
are distributed. However, the parents of all Ds donor lines are
hemizygous for the dDs allele, so the dDs will segregate: approximately 50% of the kernels will carry the donor insertion, while
the others will not. Therefore, to maximize the efficiency of mutagenesis, it is essential to perform a similar genotyping assay and
identify the field-grown plants carrying the dDs insertion. We have
consistently amplified junction fragments using a modified REDExtract-N-Amp Plant PCR KitÒ protocol (Sigma–Aldrich cat.# XNAPS;
larger kits available). Approximately 1.0–1.5 mg (4–5 mm) of
young leaf tip tissue is harvested from field-grown plants, and
DNA is extracted by using one-quarter of the extraction and neutralization reagents by volume as suggested in the recommended
protocol. A standard PCR reaction is assembled using the included
Taq polymerase ready mix (4 ll plant extract, 1 ll Primer 1 (Ds
specific) [10 lM], 1 ll Primer 2 (flanking-sequence specific)
[10 lM], 10 ll Extract-N-AmpÒ ready mix, 4 ll dH20) or alternatively with GoTaqÒ DNA Polymerase (Promega cat.# M3001: 4 ll
plant extract, 2.5 ll primer 1 (Ds specific) [10 lM], 2.5 ll Primer
2 (flanking-sequence specific) [10 lM], 2 ll DMSO, 5 ll dNTP’s
[2 mM], 10 ll GoTaqÒ Buffer, 3.75 ll dH2O, 0.25 ll GoTaqÒ [5u/
ll]). Thermocycler conditions are as follows: initial denaturation
94 °C 30 ; 35 cycles of 94 °C 3000 , 55 °C 10 , 72 °C 3000 ; final extension
72 °C 100 ; 4 °C soak. PCR products are visualized on a 1% agarose
gel. An amplicon of the expected size denotes a plant that contains
a dDs insertion at the predicted locus, and may be used in the
planned genetic screen. This rapid and cost-effective assay permits
the identification of all dDs-containing plants and should be performed early in the season when leaf tissue is still developing.
We recommend self-pollinating these plants in addition to outcrossing for mutagenesis, as selfing will generate seed that will
be homozygous for the dDs insertion. As discussed below, the heterozygosity of the dDs limits the mutagenesis to the 50% of the
chromosomes that carry the element. Thus, if a second round of
mutagenesis is necessary or desired, initiating the mutagenesis
with a line that is homozygous for the dDs insertion will double
the frequency of recovered insertions. It will also be possible to
identify seeds from self-pollinated ears that are homozygous for
Ac-im as finely spotted kernels. As up to 40% of the germinal excisions of Ds from the r locus occur during gametophytic development [26], the generation of a dDs/dDs Ac-im/Ac-im stock could
significantly increase the efficiency of mutagenesis.
2.3. Remobilizing Ds for genetic screens
Given that the seed for the dDs line will be limiting, we advise
that plants proven heterozygous for Ac-im and for the dDs are
self-pollinated for use in subsequent nurseries and used as the male
(pollen) donor onto females that are homozygous or heterozygous
for the ‘‘mutant-of-interest” reference allele (forward genetics –
see Section 2.3.1). If no reference allele pedigree is available for testcrossing, then pollen from the Ac/Ds-containing males should be
crossed onto female r-sc:m3 tester plants maintained in the W22
inbred (reverse genetics; Fig. 1). For either strategy, it is advantageous to stagger the plantings of the female tester, especially if
the females are in a mixed or non-W22 genetic background. Background effects, as well as temporal and spatial variation in soil
and weather conditions, can cause asynchronous nicking (the coemergence of the pollen and the ear silks). In optimum field conditions, each male plant will disperse pollen for approximately three
days; we are able to conservatively pollinate ten female plants on
each of these days. Thus, five dDs males can be used to pollinate
at least 150 ears to produce approximately 30,000 progeny.
2.3.1. Considerations specific to forward genetic screens
The odds of recovering a Ds insertion allele in a forward genetic
screen will double by using a homozygous reference stock. How-
251
ever, some alleles maintained as homozygotes will compromise
the viability of the female gametophyte or otherwise preclude
the production of viable progeny. Therefore, consider whether or
not the homozygous mutant allele affects the fecundity of the female plant. If this is indeed the case, a forward genetic screen
should NOT be used to recover Ds insertion alleles. A heterozygous
reference stock can be used as an alternative, or one that is segregating 1:1 for the mutation of interest. In this approach, it will be
necessary to testcross the mutant progeny as either males or females to retain the Ds insertion alleles.
2.3.2. Efficiency of Ds tagging
We are currently conducting several targeted mutagenesis
experiments to estimate the population size needed for efficient
tagging (Vollbrecht and Brutnell, unpublished). We have previously shown that the transposition frequency of Ds excision mediated by Ac-im is approximately 4% and is similar whether the Ds
elements are mobilized through the male or the female gametophytes [26]. In a recent pilot experiment, a dDs located approximately 30 kb from a target locus was used to recover five Ds
insertion alleles in a population of approximately 34,500 progeny,
equivalent to an insertion frequency of 1.4 104. It should be
noted that the target gene in this study, Bronze2 (Bz2) is a physically small target of approximately one kb [30]. If we extrapolate
these results to a gene of average size (e.g., 4 kb), we may expect
four times as many insertions. In addition, these dDs lines were
hemizygous for the Ds insertion (as discussed in Section 2.2). If
homozygous dDs/dDs lines were used as males, the frequency of
bz2 mutants recovered would likely double. Taking this study
and other Ac tagging studies into consideration [6], we estimate
that testcross populations of approximately 5000 individuals
should be generated (approx. 25 testcross ears) when using males
that are hemizygous for the dDs insertion and when a 4 kb target
locus is less than 4 cM from the dDs. At these population sizes and
target distances, at least one Ds insertion allele should be
recovered.
2.4. Forward genetic screens
Once the testcross ears have been harvested, the progeny can
be directly screened for Ds insertions in the goi. Putative insertion
alleles are readily observed when the mutant confers a kernel or
seedling phenotype. However, if seedling screens are to be performed, it is essential that they be conducted in such a way to enable the recovery of the mutants, because any Ds insertional
mutant will be represented as a single-kernel event. We typically
conduct our seedling screens in flats with a soil mixture to facilitate the transplanting of seedlings to pots in the greenhouse.
Once the mutant phenotype is apparent, the seedlings should be
immediately transplanted to larger pots and fertilized weekly to
ensure good seed set. If greenhouse conditions are unreliable, it
may be prudent to conduct the screens in a summer nursery if
sufficient field space is available for growing up to 5000 plants.
A population of this size would likely lead to the recovery of multiple Ds insertion events. It is important to note that both stable
and unstable insertion alleles could be recovered in these directed
tagging experiments. As the dDs lines are hemizygous for the Acim transposase source, not all recovered progeny will inherit Acim, resulting in non-variegated stable insertion alleles. In addition,
Ds excision is often imprecise [31], and insertions into exon sequences are unlikely to restore gene function following somatic
excision [7]. Thus, in these instances the Ds insertion allele will
result in a phenotypically-uniform mutant appearance. However,
if the Ac-im source is inherited with the Ds insertion allele and
the Ds inserts into non-coding sequences, somatic variegation
may be evident.
252
K.R. Ahern et al. / Methods 49 (2009) 248–254
2.5. Cosegregation analysis
It will be important to verify putative Ds insertion alleles in the
goi through cosegregation of the Ds insertion allele with the mutant phenotype. Thus, in addition to self-pollinating mutant individuals, mutants should also be outcrossed to wild-type lines.
Subsequent self-pollination of the F1 individuals will result in a
segregating population. Using PCR primers designed to the end of
Ds and the flanking sequences, it will be possible to quantify the
linkage between Ds and the goi. The identity of the goi can be easily
addressed if two or more Ds insertion alleles are obtained that map
to a single genic region and condition a similar mutant phenotype.
However, if a single Ds insertion allele is recovered, it may be necessary to characterize an independent mutant allele to confirm the
identity of the goi. One option is to sequence the reference allele,
once gene sequence is recovered using the Ds insertion as a molecular tag (see Section 2.6 below). Unfortunately, the pedigrees of
many reference alleles are unknown or were generated using
chemical mutagens that produce single nucleotide changes. Thus,
sequencing alleles from diverse accessions to identify causative
polymorphisms is often extremely problematic because of the
diversity inherent to the maize germplasm [32]. However, once a
Ds insertion allele has been obtained, it can be remobilized to create additional alleles. This technique was recently demonstrated
using Ac insertions at the ps1 locus to generate 83 excision alleles
resulting in 19 unique footprint alleles [15]. Thus, with a single
well-placed Ds insertion (e.g., exon insertion), multiple stable alleles can be generated confirming the identity of the mutant gene
starting from a single Ds insertion allele.
2.6. Molecular characterization of Ds insertion alleles identified in
forward genetic screens
If the goi has not yet been cloned, then the Ds insertion will
serve as a convenient tag for recovery of adjacent gene sequences.
When performing such a directed tagging experiment, two molecular steps are advised. First, DNA blot analysis is performed to
identify a novel Ds insertion band that is present in the mutant
individuals and is absent from the parental lines. Second, the
DNA flanking the new Ds insertion is isolated using an Inverse
PCR (IPCR) technique. Once the flanking sequence is recovered,
PCR primers should be designed (as described in Section 2.2) to
the flanking sequences and used with Ds-end primers to confirm
that the insertion is not a cryptic or parental insertion and that
any amplified fragment cosegregates with the mutant phenotype.
2.6.1. DNA blot analysis
The donor Ds used to create this collection of Ds insertion lines
is a 2 kb Ds6-like element inserted at the r1 locus on chromosome
10 of maize. [11]. The Ds6-like element is nearly identical in structure to Ac except for a 2.5 kb deletion in the center of the element,
which we exploit as a molecular fingerprint in our screens. DNA
blot analysis is used to identify novel Ds insertions in mutant individuals, by using a probe spanning the Ds6-like deletion breakpoint
[26]. Because the copy number of active Ds6-like insertions is low,
it is often possible to identify a single new insertion in F1 progeny.
Once the mutant individuals have recovered from transplanting (at
least 1 week) or are at the five-leaf stage in the field, genomic DNA
should be isolated from the youngest leaf tissues of mutants, wildtype siblings, and the parental W22 lines, and DNA blot analysis
should be performed to confirm cosegregation of the new Ds
restriction fragments with the mutants. We recommend digesting
genomic DNA using the methylation-sensitive restriction enzymes
PvuII, SacII and PstI to enrich for hypomethylated fragments, as Ds
is thought to preferentially reinsert into these regions of the genome. We have used each of these enzymes to detect multiple active
Ds6-like insertions throughout the maize genome. To facilitate PCR
amplification, the restriction enzyme that results in the smallest
Ds6-like containing restriction enzyme fragment length polymorphism should be used for inverse PCR (IPCR). DNA extraction and
blot analysis is performed essentially as described [22] with the
following modifications. DNA is fractionated using 1% agarose gels
prior to DNA gel blotting. Seven micrograms of salmon sperm DNA
is denatured by boiling for 5 min and snap cooling in ice before
adding to 7 ml digoxygenin (DIG) Easy-Hyb buffer (Roche Molecular Biochemicals) during pre-hybridization. A (DIG)-labeled Ds6like probe is synthesized and hybridized to Nylon membranes
containing the fractionated DNA and only one 20-min high stringency wash is performed. Bands are visualized using 10 ll of
CDP-Star reagent (Roche Molecular Biochemicals) before exposure
to film for 2 h.
2.6.2. Inverse PCR protocol
Once a Ds6-like containing restriction fragment has been identified, sequences flanking the Ds insertion can be amplified using
an IPCR protocol that has been developed to specifically amplify sequences that flank Ds6-like elements. Discrimination between DNA
flanking these elements and other Ac or Ds transposons that are
present elsewhere in the genome is made possible by incorporating a primer in the reaction (LC24) that spans the deletion breakpoint of only Ds6-like elements. Approximately 3 lg of genomic
DNA is digested with a threefold excess of restriction enzyme for
5 h and the reaction inactivated at 65 °C for 20 min. An intramolecular ligation is performed by diluting the restriction enzyme digest
reaction with 1.5 units of T4 ligase (Promega) in a volume of
120 ml (final DNA concentration approximately 25 ng/ll). The
reaction is incubated overnight at 4 °C and DNA precipitated with
95% EtOH. DNA is resuspended in 50 ll of dH20 prior to two rounds
of IPCR amplification. An IPCR protocol for SacII is described below.
When PvuII or PstI are used, alternative primer sets will be used
during the PCR amplification as listed in Table 1. In addition,
0.8 ll DMSO (Fisher Scientific) and 2 ll of 5 M betaine (Sigma
Aldrich) are added to PCR reactions when PvuII or PstI are
used.
2.6.2.1. First round IPCR for SacII fragments. Add the following reaction components on ice in a microfuge tube:
1 ll precipitated ligation reaction.
2 ll 10 High Fidelity buffer (Invitrogen).
0.8 ll [50 mM] MgSO4 (Invitrogen).
1.2 ll [5 mM] dNTP’s (Promega).
0.6 ll [10 lM] primer LC18.
0.6 ll [10 lM] primer LC24.
0.08 ll [5u/ll] Platinum Taq High Fidelity (Invitrogen).
13.72 ll dH2O.
Mix reaction components well by vortexing and perform PCR as
follows: Heat denature at 95 °C for 4 min followed by 35 cycles of
94 °C for 10 s, 59.5 °C for 1 min, 68 °C for 3 min (extension time
varies; add approximate 1 min per 1 Kb of target). Terminate PCR
with a 68 °C extension for 20 min and hold reactions at 4 °C.
2.6.2.2. Second round PCR for SacII fragments. Dilute 1st round products 500-fold and add 1 ll to microfuge tube on ice. Prepare reactions as above substituting primers LC18 and LC24 with LC45 and
JGp2. Perform PCR reactions as above and visualize products on a
1% agarose gel. If products of the predicted size (based on DNA blot
analysis) are obtained, DNA should be precipitated using 95% EtOH
and products sequenced directly. If multiple products are obtained,
products of the appropriate size can first be excised from a preparative gel prior to sequencing.
253
K.R. Ahern et al. / Methods 49 (2009) 248–254
Table 1
Primers for IPCR amplification of fDs.
Restriction enzyme
Round of PCR
Primer name
Sequence
SacII
First
SacII or PstI
Second
LC 18
LC 24
LC 45
JGP 2
50 -CCTTGTTTTGATTGGCTGCTA-30
50 -TTGTTGCAGCAGCAATAACAGCAT-30
50 -GTGCTGTACTGCTGTGACTTGTG-30
50 -CCGGTTCCCGTCCGATTTCG-30
PvuII or PstI
First
PvuII
Second
LC 18
LC 24
LC 18
JS-R01
50 -CCTTGTTTTGATTGGCTGCTA-30
50 -TTGTTGCAGCAGCAATAACAGCAT-30
50 -CCTTGTTTTGATTGGCTGCTA-30
50 -GTTCGAAATCGATCGGGATA-30
2.7. Identifying Ds insertion alleles generated in reverse genetic screens
2.7.1.2. Extracting DNA. Equipment:
Perhaps the greatest utility of the Ds insertion lines will be in
conducting reverse genetic screens to target predicted genes that
reside close to Ds insertions. In these screens, no a priori assumptions are made with regards to predicted phenotypes. Mutants
are recovered using PCR-based screens on pools of individual seedlings. Populations carrying rare transposed Ds elements are surveyed to identify single seedlings with an insertion in the goi. To
date, no reverse genetic screens using Ds have been reported in
maize, but several screens are currently underway in the authors’
laboratories. From these pilot studies, it appears that a successful
reverse genetic screen will require 5000 testcross progeny that
are surveyed using pooled DNA samples. To date, we have been
able to detect one Ds insertion allele in pools of 100 sibling samples
using two rounds of PCR with nested gene-specific and Ds-end
primers (K. Ahern, unpublished). As mentioned above, it is essential that plants carrying putative Ds insertion alleles are self-pollinated or outcrossed following seedling screens. Thus, care should
be taken to conduct the reverse genetic screens as quickly as possible so that, once identified, seedlings can be transplanted to larger pots while still young. To facilitate these screens we have
developed a low cost, high-throughput DNA extraction method
to rapidly screen through large populations of testcross progeny.
1. Water bath, set at 65 °C.
2. Fluid ManagementÒ paint shaker (Harbil; model No. 5800000,
PN# 4980011) 120 V, 60 Hz and 11.8 A.
3. Bench-top centrifuge with micro-plate swing buckets (Beckman-Coulter; model AllegraTM 6R).
4. Multi-channel pipettor (12 channels; 50–300 mL).
2.7.1. High-throughput DNA extraction method
This high-throughput DNA extraction protocol has consistently
yielded DNA of high quality suitable for DNA blot analysis and IPCR
cloning reactions. Using both DNA blot and PCR assays, we have
detected single Ds insertions alleles in pools of 10 individuals.
Thus, this method can serve as a foundation in preparing DNA
pools for reverse genetic screens.
2.7.1.1. Harvesting tissue. Harvest consumables:
96 well polycarbonate snap rack (Matrix Technologies, Cat. No.
4893).
1.4 ml polypropylene tubes (Matrix Technologies, Cat. No. 4140).
ScreenMates 96 well lid for tubes, (Matrix Technologies, Cat.
No. 4410).
3.2 mm (1/8”) diameter stainless steel ball bearings, Grade
2000. (Abbott Ball, AISI Type 430).
Dry ice.
Cut seedlings approximately 10 days after planting (DAP) just
above the coleoptile (Fig. 3). Unwrap the first leaf and sheath and
discard. Collect tissue from the exposed 2nd leaf sheaths (and enclosed inner leaves) by slicing 2–4 mm sections (up to 18–20 sections) from the base of the seedling with a scalpel. Leaf tissue from
up to ten seedlings can be pooled into one tube, but the total
weight of the tissue should not exceed 100 mg. Place tissue samples into 1.4 ml tubes chilled on dry ice in a 96-well rack. Add 2
ball bearings per tube when harvest is complete. Cover; store 96well plate at 80 °C until ready to continue DNA extraction.
Solutions and chemicals:
1.
2.
3.
4.
5.
6.
7.
8.
Urea extraction buffer (UEB1; Ref. [33]).
TE buffer (1X, pH 8.0).
Phenol:Chloroform:Isoamyl alcohol (25:24:1).
100% chloroform.
100% isopropanol (chilled at -20 °C).
95% ethanol (chilled at 20 °C).
70% ethanol (chilled at 20 °C).
Liquid nitrogen.
Protocol:
1. Incubate UEB1 buffer for 10 min at 65 °C.
2. Remove sample rack from storage at 80 °C and place in a
liquid N2 bath to keep the tissue frozen.
3. Place sample in paint shaker for 1.5 min of shaking. Remove
samples and place in liquid N2 bath to keep tissue frozen and
repeat process twice. Tissue should be ground to a fine green
powder.
4. Briefly centrifuge tubes to pellet the powder; tissue must not
thaw.
5. Add 400 ll of pre-warmed UEB1 to each sample, vortex and
centrifuge briefly.
6. Incubate the rack for 2 min. in a water bath at 65 °C. Be sure
that the lid is sufficiently loosened prior to warming or the
resulting pressure from heating will cause the lid to pop
off and cross-contamination of the samples will occur.
7. Tighten the lid, vortex samples, spin briefly, loosen lid and
incubate at 65 °C for 8 min.
8. Spin samples briefly and add 400 ll of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and shake vigorously.
9. Centrifuge at 3500 rpm for 15–20 min at 4 °C.
10. In a fume hood, use a multi-channel pipettor to transfer the
supernatant to a new rack of tubes. Add 400 ll of 100% chloroform to the aqueous solution and close the lid tightly.
Shake the rack by hand vigorously. Centrifuge at 3500 rpm
for 15–20 min at 4 °C.
11. Use a multi-channel pipettor to transfer the supernatant to a
new rack of tubes.
12. Add 0.7 volume cold (20 °C) 100% isopropanol to the tubes,
and gently mix by inversion several times.
13. Incubate the samples for 10 min at 20 °C and centrifuge at
3500 rpm for 20–30 min at 4 °C. Discard the supernatant,
taking care not to lose pellets.
254
K.R. Ahern et al. / Methods 49 (2009) 248–254
Fig. 3. Harvesting leaf tissue for high-throughput DNA extraction. Young leaf tissue is harvested at approximately 10 days after planting by cutting just below the tip of the
coleoptile (A). The coleoptile and the first leaf are removed and portions of the remaining young leaf tissues are collected by cutting approximately 2–4 mm segments (B). Up
to 20 pieces can be placed into a single tube corresponding to 10 seedlings (C). All tissues should be placed into tubes kept on dry ice (D).
14. Wash the pellets with 1.0 ml of cold (20 °C) 70% ethanol.
15. Centrifuge sample racks at 3500 rpm for 20–30 min at 4 °C.
Carefully discard the remaining supernatant and air-dry
(1–2 h) to remove all residual ethanol.
16. Suspend the pellets in 100 ll TE buffer pH 8.0 and make sure
the pellets are well dissolved in solution. Store samples at
20 °C.
2.8. Concluding remarks
As maize transformation remains expensive and technically
challenging, it is likely that transposon-based methods of gene
characterization will continue to play a major role in maize genetics in the years ahead. Ds insertional mutagenesis promises to be
an extremely powerful tool for functional genomics in maize. The
technique enables the recovery of gene sequence when Ds is used
as a molecular tag in forward genetic screens and facilitates the
recovery of multiple mutant alleles when a gene sequence is the
starting point in a reverse genetic screen. Though few examples
of Ds tagging have been described to date in maize, thousands of
sequence-indexed Ds insertions are now available for maize geneticists to exploit. The protocols described in this report will serve as
the foundation for several large-scale tagging experiments in the
years ahead.
References
[1] H. Candela, S. Hake, Nat. Rev. Genet. 9 (2008) 192–203.
[2] B. Mcclintock, Carnegie Instit Wash Year Book 46 (1947) 146–152.
[3] R. Kunze, C.F. Weil, in: N.L. Craig (Ed.), Mobile DNA, vol. 2, ASM Press,
Washington, DC, 2002, pp. 565–610.
[4] D.L. Auger, W. Sheridan, J. Hered. 90 (1999) 453–458.
[5] L.J. Conrad, K. Kikuchi, T.P. Brutnell, in: G. Kahl, K. Meksem (Eds.), The
Handbook of Plant Functional Genomics, Wiley-Blackwell, Weinheim, 2008,
pp. 267–290.
[6] S.L. Dellaporta, M.A. Moreno, in: M. Freeling, V. Walbot (Eds.), The Maize
Handbook, Springer-Verlag, New York, 1994, pp. 219–233.
[7] M.A. Moreno, J. Chen, I. Greenblatt, S.L. Dellaporta, Genetics 131 (1992) 939–
956.
[8] S.L. Dellaporta, I.M. Greenblatt, J.L. Kermicle, J.B. Hicks, S.R. Wessler, in: J.P.
Gustafson, R. Appels (Eds.), Chromosome Structure and Function:Impact of
New Concepts, Plenum Press, New York, 1988, pp. 263–282.
[9] P. Athma, E. Grotewold, T. Peterson, Genetics 131 (1992) 199–209.
[10] M. Singh, P.E. Lewis, K. Hardeman, L. Bai, J.K. Rose, M. Mazourek, P. Chomet,
T.P. Brutnell, Plant Cell 15 (2003) 874–884.
[11] M. Alleman, J.L. Kermicle, Genetics 135 (1993) 189–203.
[12] Y.H. Liu, M. Alleman, S.R. Wessler, Proc. Natl. Acad. Sci. USA 93 (1996) 7816–
7820.
[13] Y.H. Liu, L.J. Wang, J.L. Kermicle, S.R. Wessler, Genetics 150 (1998) 1639–1648.
[14] S.R. Wessler, G. Baran, M. Varagona, S.L. Dellaporta, EMBO J. 5 (1986) 2427–
2432.
[15] L. Bai, M. Singh, L. Pitt, M. Sweeney, T.P. Brutnell, Genetics 175 (2007) 981–
992.
[16] M.J. Giroux, J. Shaw, G. Barry, B.G. Cobb, T. Greene, T. Okita, L.C. Hannah, Proc.
Natl. Acad. Sci. USA 93 (1996) 5824–5829.
[17] E.R. Orton, R.A. Brink, Genetics 53 (1966) 7–16.
[18] H.K. Dooner, A. Belachew, Genetics 122 (1989) 447–457.
[19] I.M. Greenblatt, Genetics 108 (1984) 471–485.
[20] M.F. Dowe Jr., G.W. Roman, A.S. Klein, Mol. Gen. Genet. 221 (1990) 475–485.
[21] M. Cowperthwaite, W. Park, Z. Xu, X. Yan, S.C. Maurais, H.K. Dooner, Plant Cell
14 (2002) 713–726.
[22] J.M. Kolkman, L.J. Conrad, P.R. Farmer, K. Hardeman, K.R. Ahern, P.E. Lewis, R.J.
Sawers, S. Lebejko, P. Chomet, T.P. Brutnell, Genetics 169 (2005) 981–995.
[23] M.J. Sheehan, L.M. Kennedy, D.E. Costich, T.P. Brutnell, Plant J. 49 (2007) 338–
353.
[24] J.F. Eisses, D. Lafoe, L.A. Scott, C.F. Weil, Mol. Gen. Genet. 256 (1997) 158–168.
[25] J. Duvick, A. Fu, U. Muppirala, M. Sabharwal, M.D. Wilkerson, C.J. Lawrence, C.
Lushbough, V. Brendel, Nucleic Acids Res. 36 (2008) D959–D965.
[26] L.J. Conrad, T.P. Brutnell, Genetics 171 (2005) 1999–2012.
[27] M. Heinlein, Genetics 144 (1996) 1851–1869.
[28] M. Heinlein, T. Brattig, R. Kunze, Plant J. 5 (1994) 705–714.
[29] B. Mcclintock, Cold Spring Harb. Symp. Quant. Biol. 16 (1951) 13–47.
[30] K.A. Marrs, M.R. Alfenito, A.M. Lloyd, V. Walbot, Nature 375 (1995) 397–
400.
[31] L. Scott, D. Lafoe, C.F. Weil, Genetics 142 (1996) 237–246.
[32] M.I. Tenaillon, M.C. Sawkins, A.D. Long, R.L. Gaut, J.F. Doebley, B.S. Gaut, Proc.
Natl. Acad. Sci. USA 98 (2001) 9161–9166.
[33] J. Chen, S.L. Dellaporta, in: M. Freeling, V. Walbot (Eds.), The Maize Handbook,
Springer-Verlag, New York, 1994, pp. 526–527.