Download A Deletion Affecting Several Gene Candidates is

Journal of Heredity 2004:95(5):436–444
DOI: 10.1093/jhered/esh057
ª 2004 The American Genetic Association
A Deletion Affecting Several Gene
Candidates is Present in the
Evergrowing Peach Mutant
D. G. BIELENBERG, Y. WANG, S. FAN, G. L. REIGHARD, R. SCORZA,
AND
A. G. ABBOTT
Address correspondence to D. G. Bielenberg, at the above address, or e-mail: [email protected].
Abstract
Evergrowing (EVG ) peach is one of only two described mutants affecting winter dormancy in woody perennial species. EVG
peach does not set terminal buds, cease new leaf growth, nor enter into a dormant resting phase in response to winter
conditions. The EVG mutation segregates in F2 progeny as a single recessive nuclear gene. A local molecular genetic linkage
map around EVG was previously developed using amplified fragment length polymorphism (AFLP) and simple sequence
repeat (SSR) markers, and a bacterial artificial chromosome (BAC) contig that contains the EVG mutation was assembled.
A MADS box coding open reading frame (ORF) was found in a BAC of this contig and used as a probe. The probe detected
a polymorphism between the wild-type and mutant genomes, and the polymorphism is indicative of a deletion in EVG
peach. The EVG gene region contained six potential MADS-box transcription factor sequences, and the deletion in EVG
affected at least four of these. The deletion was bracketed using RFLP analysis, which showed that it is contained within
a segment of the genome no greater than 180 kb.
Perennial woody plants are the dominant species in many
ecosystems of the world and have significant ecological and
economic importance. A major life-history trait of perennial
species is the ability to become physiologically dormant in
order to avoid unfavorable climatic conditions, such as the
low temperatures and limited water availability associated
with winter. Mutants that fail to cease growth and enter dormancy under dormancy-inducing conditions have been described in only two woody perennial species, Corylus avellana L.
(Thompson et al. 1985) and Prunus persica (L.) Batsch
(Rodriguez et al. 1994). The best described mutant of these
two species is the Evergrowing (previously known as Evergreen,
USDA PI442380) peach mutant, a nondormant genotype
identified from southern Mexico, where killing frosts do not
occur (Rodriguez et al. 1994; Werner and Okie 1998).
In Mexico, terminal growth on Evergrowing trees is
continuous under favorable environmental conditions, and
leaves are retained until they are lost due to drought and/or
disease (Diaz 1974). At more northern latitudes, the
Evergrowing peach does not appear to respond to winter
dormancy cues, exhibiting persistent shoot growth and
a lack of leaf abscission when experiencing short days and
low temperatures in the fall until these tissues are killed by
freezing temperatures (Rodriguez et al. 1994). In addition,
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the frost hardiness of Evergrowing trees is half that of wildtype dormant trees (Rodriguez et al. 1994). For example,
Evergrowing trees show some cold acclimation and accumulation of bark storage proteins and dehydrins; this occurs
later in the fall and to a lesser degree than in wild-type trees
(Arora and Wisniewski 1996; Arora et al. 1992, 1996). A
number of crosses of Evergrowing (nondormant) trees with
different wild-type dormant trees were made by Rodriguez
et al. (1994), and all nine of the resulting F2 progenies fit
a 3:1 (dormant:nondormant) ratio, suggesting that the
Evergrowing phenotype is controlled by a single recessive
nuclear gene.
A genetic map of the Evergrowing (EVG ) locus was
previously generated from a segregating F2 population
derived from a cross between wild-type dormant (‘‘Empress’’
dwarf) and mutant, nondormant (Evergrowing, PI442380)
parents and reported by Wang et al. (2002b). Subsequently
a physical map of the EVG region comprised of bacterial
artificial chromosomes (BACs) was initiated from the closest
sequence tagged site (STS) marker found in that study (Wang
et al. 2002a). Analysis of simple sequence repeat (SSR)
markers developed from this contig confirmed the positions
of the genetic makers relative to one another (Wang et al.
2002a). BAC PpN018F12 (Prunus persica ‘‘Nemared’’ 018F12)
Downloaded from http://jhered.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
From the Department of Horticulture, Clemson University, Clemson, SC 29634 (Bielenberg, Wang, and Reighard);
Department of Genetics, Biochemistry, and Life Science Studies, Clemson University, Clemson, SC 29634
(Fan and Abbott); and USDA-ARS Appalachian Fruit Research Station, Kearneysville, WV 25430 (Scorza).
Bielenberg et al. A Deletion in Evergrowing Peach
Methods
Genomic DNA Isolation
Peach leaves for DNA extraction were obtained from
individuals of an F2 mapping population and from moderate
chilling cultivars (Musser Fruit Research Center, Clemson
University, Clemson, SC). All other peach tissue was shipped
overnight on ice from the USDA-ARS Southeastern Fruit
and Nut Research Laboratory (Byron, GA). Leaves were
weighed as 1.0 g fresh weight samples, wrapped in aluminum
foil, frozen in liquid N2, and stored at 808C.
Total DNA was isolated from the frozen leaves using
a CTAB (hexadecyltrimethylammonium bromide) extraction
buffer protocol modified from Doyle and Doyle (1990). For
each sample 1.0 g of frozen leaf tissue was ground in a mortar
and pestle under liquid N2. This ground tissue was transferred to 15 ml CTAB buffer [2% (w/v) CTAB, 1.4 M NaCl,
20 mM EDTA, 100 mM Tris-HCL pH 8.0] in a 50 ml plastic
culture tube at 608C and incubated for 30 min at 608C. After
incubation, one volume of chloroform:isoamyl alcohol (24:1)
was added and the contents gently mixed. The sample was
then clarified by centrifugation at 1600g for 10 min at 48C.
The upper aqueous phase was transferred to a clean 50 ml
culture tube and another volume of chloroform:isoamyl
alcohol (24:1) was added and gently mixed. The sample was
again clarified by centrifugation at 1600g for 10 min at 48C,
and the resulting upper, aqueous phase transferred to a clean
50 ml tube. The DNA was precipitated from this solution by
the addition of one-tenth volume 3.3 M sodium acetate (pH
5.2) and one volume of cold isopropanol. Recovered nucleic
acids were transferred to a 2.0 ml microcentrifuge tube,
washed in 70% (v/v) ethanol, and resuspended in 1.0 ml TE.
RNase A (Sigma-Aldrich Corp., St. Louis, MO) was added to
a concentration of 10 lg/ml and the sample was incubated at
378C for 30 min. RNase A-treated samples were ethanol
precipitated, resuspended in 500 ll of 0.13 TE, and
quantified by spectrophotometry.
BAC DNA Isolation
All BAC clones used in this study were obtained from
a genomic library of the peach rootstock Nemared (wildtype dormant), digested with HindIII and cloned into the
pBeloBAC11 vector (Georgi et al. 2002). BAC DNA was
isolated from Escherichia coli cultured overnight at 378C in
10 ml LB media using the alkaline lysis protocol for
large insert clones described by Sambrook and Russell
(2002). DNA quality and quantity was assessed by spectrophotometry.
BAC End Sequencing
Cesium chloride-purified BAC DNA was used as a template
for end sequencing of PpN018G07 and PpN089G02.
Sequencing reactions were performed using Big Dye v2.0
terminator chemistry (Applied Biosystems, Inc., Foster City,
CA) according to the manufacturer’s instructions for large
insert DNA. Sequencing reactions were analyzed on an ABI
Prism 377 sequencer. BAC end sequences were submitted to
GenBank and assigned the following accession numbers:
PpN018G07SP6 (CL311195), PpN018G07T7 (CL311196), PpN089G02SP6 (CL311194), and PpN089G02T7
(CL311193).
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was found to contain the EAT/MCAC marker (Wang et al.
2002a), which was mapped at a distance of 1 cM from the
EVG gene (Wang et al. 2002b). BAC PpN109L12 was shown
to overlap with the T7 end of BAC PpN018F12. An SSR
marker (pchgms29) was developed from BAC PpN109L12
and mapped 2.9 cM from the EAT/MCAC marker, in the
opposite direction from the map location of the EVG gene
(Wang 2002). The position of pchgms29 confirms the contig
walk initiated from the SP6 end of BAC PpN018F12 is in the
direction of the EVG gene (Wang 2002).
End sequencing of PpN018F12 revealed a potential open
reading frame (ORF) with homology to a MADS box-type
transcription factor and was designated bes_18F12B01 (BAC
end sequence of BAC PpN018F12 clone B01) (Georgi et al.
2003). MADS box transcription factors are found in a wide
variety of organisms and regulate important developmental
processes (Garcia-Maroto et al. 2003). In plants, MADS box
genes have been shown to regulate reproductive traits such
as the timing of meristem transition from vegetative to
reproductive growth in response to environmental conditions, floral structure, fruit ripening, and abscission of
mature fruit (Fang and Fernandez 2002; Lohmann and
Weigel 2002; Mao et al. 2000; Mouradov et al. 2002;
Vrebalov et al. 2002). In addition, a number of plant MADS
box genes have been found that are expressed in vegetative
tissues (Alvarez-Buylla et al. 2000; Garcia-Maroto et al. 2000;
Johansen et al. 2002; Kim et al. 2002; van der Linden et al.
2002). However, with the exception of ANR1 in Arabidopsis,
which is in the signaling pathway for increased root
elongation in response to nitrate (Zhang and Forde 2000),
and POTM1, which is involved in suppression of axillary bud
growth in potato (Rosin et al. 2003), most of these genes do
not yet have a defined function. Nonetheless, the role that
MADS box genes play in major developmental transitions in
plants made the MADS sequence found in PpN018F12
a candidate for the Evergrowing mutation.
Here we report that a hybridization probe developed
from the MADS box coding sequence found in
bes_18F12B01 is polymorphic between genomic DNA from
wild-type dormant and mutant nondormant EVG peach
trees. Southern analyses revealed a cluster of six genes
homologous to the sequenced ORF and four of these genes
were missing in the mutant, indicating that a possible
deletion of these genes was present in the Evergrowing peach
mutant. In addition, we demonstrated that these six MADS
box genes were located within the approximately 180 kb
region spanned by three overlapping BAC clones and that
the hypothesized deletion was bracketed by the ends of this
BAC contig.
Journal of Heredity 2004:95(5)
Probe Amplification and Labeling
Figure 1. Sequence of peach BAC partial sequence
bes_18F12B01. Sequence in uppercase is the 196 bp MADS
box ORF. Underlined and bolded sequences denote the
primers used to generate the 121 bp DNA fragment used in
subsequent Southern hybridizations.
Southern Analysis
Genomic or BAC DNA was digested at 378C overnight with
10 units of enzyme (HindIII, EcoRI, or BamHI; Promega
Corp.) and size fractionated in a 0.8% (w/v) SeaKem LE
agarose gel (BioWhittaker Molecular Applications, Rockland,
ME) using 13 TAE (40 mM Tris-acetate, 1 mM EDTA, pH
8.0) as a running buffer. After separation was completed, gels
were depurinated by incubation in 0.125 N HCl followed by
a 30 min incubation in denaturing buffer (1.5 M NaCl, 0.5 M
NaOH), and finally a 30 min incubation in neutralization
buffer (1.5 M NaCl, 0.5 M Tris pH 7.5). DNA was
transferred to Hybond-XL membrane (Amersham Pharmacia Biotech, Little Chalfort, Buckinghamshire, England) by
capillary blot using 103 SSC (pH 7.0) as a transfer buffer.
Following transfer, DNA was fixed to the membrane by
baking at 808C for 2 h.
Membranes were prehybridized at 658C with a 0.5 M
sodium phosphate, 7% sodium dodecylsulfate (SDS)
hybridization buffer for a minimum of 30 min. Radioactively
labeled and denatured DNA probe was added directly to the
prehybridization solution, and the membranes were hybridized with the probe overnight at 658C. All hybridized
membranes were washed at 658C twice with 23 SSC (pH
7.0), 1% (w/v) SDS for 30 min, and twice with 13 SSC,
0.1% (w/v) SDS for 30 min. The Southern blot containing
DNA from nonpeach species were prehybridized and
washed at 508C. Membranes were then exposed to autoradiographic film (Kodak X-Omat Blue XB-1, Perkin-Elmer
Life Sciences, Inc., Boston, MA) for 3–10 days as determined
by signal strength.
Results
Primers were designed for the conserved regions of the
MADS box sequence found in bes_18F12B01 (BAC end
sequence of BAC PpN018F12 clone B01) (Figure 1). A 119
bp fragment of the MADS box sequence was amplified by
PCR from PpN018F12 for use as a DNA probe (Figure 1).
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The amplified probe was hybridized to restriction enzymedigested DNA from wild-type dormant and nondormant
mutant trees from the F2 population used to map the locus.
In the HindIII digested wild-type DNA, the probe
detected what appeared to be five strongly hybridizing bands,
one each at 3.3 kb, 2.7 kb, 2.4 kb, 2.1 kb, and 1.5 kb (Figure
2). However, the smallest hybridizing band was of greater
intensity and wider spread than the other four bands,
indicating that it was either the sequence of highest similarity
to the hybridizing probe or potentially two or more
overlapping bands (Figure 2). In the HindIII digest of the
mutant DNA, the two bands present corresponded in size to
two of the bands in the wild-type DNA—2.7 kb and 1.5 kb
(Figure 2). The 1.5 kb band in the HindIII-digested mutant
DNA was of lower intensity and spread than the band at the
same position in the wild-type DNA. This interpretation is
consistent with the presence of two overlapping bands at
1.5 kb in the wild-type DNA and the presence of only one,
slight larger band in the mutant DNA (Figure 2).
The EcoRI-digested wild-type DNA showed six strongly
hybridizing bands, one each at approximately 12.0 kb, 7.2 kb,
4.9 kb, 3.5 kb, 3.3 kb, and 2.2 kb (Figure 2). In the EcoRIdigested EVG mutant DNA, one band corresponded in size
to a band existing in the wild-type digestion, that is, 4.9 kb,
but the larger band did not correspond to one in the wild
type (Figure 2).
Three overlapping BAC clones from a previously created
EVG locus physical contig (Wang et al. 2002a) were digested
with HindIII and separated on a gel with HindIII-digested
DNA from wild-type dormant and nondormant mutant
trees, and hybridized with the amplified MADS box probe
(Figure 3). HindIII was used for analysis of the BAC clones
because the BAC clones were prepared from partial HindIIIdigested fragments of the peach genome and therefore
should produce a banding pattern identical to the genomic
DNA. The three BAC clones together possessed bands
corresponding to all six of the HindIII-digested MADS box
hybridizing bands in the wild-type DNA (Figure 3). These
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Forward and reverse PCR primers for each of the
amplifications were designed from BAC end sequences
using Primer3_www.cgi v 0.2 (Rozen and Skaletsky 2000)
and obtained from Integrated DNA Technologies (Coralville, IA). The BAC from which primer pairs were designed
was used as a template to amplify probes. Amplification
products were size fractionated on an agarose gel
(PpN089G02SP6, 260 bp; PpN018G07SP6, 235 bp;
bes_18F12B01, 119 bp). The amplification products were
excised from the gel and purified using the QIAquick Gel
Extraction kit (Qiagen, Inc., Valencia, CA).
DNA probes were labeled with a32P-dCTP (PerkinElmer Life Sciences, Inc., Boston, MA) following denaturing
and incubation with random DNA hexamers and the
Klenow fragment of DNA polymerase (Promega Corp.,
Madison, WI) at 378C for 3 h (Sambrook and Russell 2002).
Bielenberg et al. A Deletion in Evergrowing Peach
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Figure 2. Southern hybridization of digested genomic
DNA from wild-type dormant (DE) and Evergrowing
mutant (EVG ) F2 siblings. Genomic DNA samples (10 lg)
were digested with HindIII, EcoRI, or BamHI. Membranes
were probed with DNA amplified from the bes_18F12B01
MADS box ORF (121 bp). Lines represent positions
of bands from the HindIII-digested k DNA
size marker.
Figure 3. Southern hybridization of HindIII-digested
genomic DNA from wild-type dormant (DE) and
nondormant mutant (EVG) peaches and of three
BACs from the EVG contig (PpN089G02, PpN018F12,
and PpN018G07). Membranes were probed with DNA
amplified from the bes_18F12B01 MADS box
ORF, as in Figure 2. Lines represent positions of
bands from the HindIII-digested k DNA size
marker.
439
Journal of Heredity 2004:95(5)
bands were labeled, from largest to smallest, as A, 3.3 kb; B,
2.7 kb; C, 2.4 kb; D, 2.1 kb; E, 1.5 kb; and F, 1.5 kb. The four
missing bands in the nondormant mutant DNA (A, C, D,
and F) corresponded to fragments observed in PpN018F12
or PpN089G02, or both. The hybridizing fragment (E)
observed in PpN018G07 was not altered between the wildtype and mutant genomic DNAs (Figure 3).
EcoRI digestion of the BACs and subsequent hybridization with the MADS box probe resulted in nonidentical band
patterns relative to the genomic DNA (data not shown).
Therefore the analysis of this digest could not be used to
confirm the molecular structure of the EVG locus in the
wild-type and mutant genomes.
The SP6 and T7 ends of BACs PpN089G02 and
PpN018G07 were sequenced, and these sequences were used
to identify amplification primers for the production of BAC
end specific probes for the EVG contig. T7 ends of
PpN018G07 and PpN089G02 were previously determined
to overlap with PpN018F12 by chromosomal walking during
the generation of the BAC contig (Figure 4) (Wang et al.
2002a). Therefore the extreme ends of the BAC contig are
represented by the SP6 ends of PpN018G07 and
PpN089G02. The probes were separately hybridized onto
a Southern membrane containing HindIII-digested wild-type
genomic DNA, mutant genomic DNA, and the three BACs in
the EVG contig (Figure 5). The PpN089G02-SP6 end probe
hybridized to a fragment of similar size (1.9 kb) in the wildtype genomic DNA, mutant genomic DNA, and
PpN089G02, but did not hybridize to a fragment in either
of the other two BACs (Figure 5A). The PpN018G07-SP6
end probe hybridized to a fragment of similar size (0.7 kb) in
the wild-type genomic DNA, mutant genomic DNA, and
PpN018G07, but did not hybridize to a fragment in either of
the other two BACs (Figure 5B). Thus the BAC contig, which
was developed from wild-type dormant Nemared genomic
DNA, spans the deletion in the mutant EVG genome.
In order to determine if multiple copies of this gene
sequence were characteristic of the specific peach cultivars
used in this study or a more general characteristic of peach,
440
Figure 5. Southern hybridization of HindIII-digested
genomic DNA from wild-type dormant (DE) and nondormant
mutant (EVG) peach and of three BACs from the EVG gene
contig. (A) Membranes hybridized with a DNA probe amplified
from the SP6 end of BAC PpN089G02. (B) Membranes
hybridized with a DNA probe amplified from the SP6 end of
BAC PpN018G07. Lines represent positions of bands from the
HindIII-digested k DNA size marker.
EcoRI-digested genomic DNA from several peach cultivars,
along with a limited selected of individuals from the
Evergrowing F2 mapping population, were hybridized with
the bes_018F12 MADS box probe (Figure 6). EcoRI was
selected for use in the genomic analyses due to the clearer
separation of hybridizing bands observed with this enzyme
(see Figure 2). All wild-type dormant peach DNA analyzed
had from five to seven fragments that hybridized with the
MADS box probe (Figure 6). The individuals from the F2
population used to map the Evergrowing gene all showed the
same pattern of hybridization, six fragments in the wild-type
dormant trees and two fragments in the mutant nondormant
trees (Figure 6). Additional cultivars (i.e., Carogem, Biscoe,
Maycrest, Goldcrest, Contender, and Peen-To) were also
analyzed and the hybridization patterns were similar to the
patterns of the other wild-type dormant trees (data not
shown).
EcoRI-digested and HindIII-digested genomic DNA
from four additional species within the Rosaceae that enter
a winter dormant state were hybridized with the bes_018F12
MADS box probe to determine if these species also
contained multiple copies of a similar sequence. Woody
perennial species related to peach (plum [Prunus domestica L.],
apricot [Prunus armeniaca L.], cherry [Prunus avium L.], and
apple [Malus domestica Borkh.]) contained a number of DNA
fragments hybridizing to the MADS box probe from
bes_018F12B01 (Figure 7).
Discussion
The MADS box coding sequence from bes_018F12B01
hybridized to six fragments in EcoRI- and HindIII-digested
wild-type DNA and showed polymorphism between the
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Figure 4. Structure of the BAC contig covering the EVG
gene region. Lines designating the individual BACs are scaled to
show their relative sizes with respect to one another. The
degree of overlap is not known. Letters corresponding to bands
in the HindIII digestion of wild-type genomic DNA are show
in the positions along the contig deduced from Southern
hybridization (see Figure 3). Letters positioned above overlaps
are found on both BACs. The parentheses enclosing A and C
signify that their positions could be reversed.
Bielenberg et al. A Deletion in Evergrowing Peach
wild-type and mutant DNAs (Figure 2). The presence of six
hybridizing bands in the EcoRI digestion and the absence of
an EcoRI restriction site in the DNA probe provide
confirmation that there are six bands in the HindIII-digested
wild-type DNA as well, with the smallest band actually being
a doublet. RFLP analysis showed the complete absence of
four of the bands in the mutant. Sequence polymorphisms
that result merely in the gain or loss of restriction enzyme
recognition sites would be expected to produce bands of
different sizes, as the restricted DNA fragment to which the
probe hybridizes would be smaller or larger in size. However,
in the case presented here, the complete loss of hybridization
to the genomic DNA suggested that the genomic DNA to
which the probe has homology was not present in the genome
of the EVG mutant (Figure 2). The four MADS box genes,
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Figure 6. Southern hybridization of EcoRI-digested genomic
DNA from peach cultivars of varying chilling requirements
with selected individuals from the EVG F2 mapping
population. Cultivar name or sample identification is at the top
of each lane. F2 individuals are designated by a number and
noted as either wild-type or mutant phenotype. Membranes
were probed with DNA amplified from the bes_18F12B01
MADS box ORF (121 bp). Lines represent positions of bands
from the HindIII-digested k DNA size marker (from top: 9.416
kb, 6.557 kb, 4.361 kb, 2.322 kb, and 2.027 kb).
similar to the MADS box probe from bes_018F12B01, are
therefore the first candidate genes for the Evergrowing
mutation. However, sequencing of the EVG gene region
will certainly uncover more candidates for this mutation.
The nature of the mutation that has resulted in the loss
of the four MADS box hybridizing fragments cannot be
definitively determined from the experiments we present
here. A number of structural rearrangements could be
responsible for the absence of hybridization bands in the
mutant (Figure 2). Since we have not performed comprehensive hybridizations with probes from throughout the
BACs in our contig, we cannot definitively state the nature of
the deletion(s). These sequences may be a series of tandem
duplicated loci, of which certain pairs may be independently
lost in the mutant trees. Such a series of closely linked small
deletions would still potentially result in the simple 3:1
Mendelian inheritance of the Evergrowing trait (Rodriguez
et al. 1994) seen in the F2 mapping population. However,
a simpler explanation would be presence of a single large
deletion in the mutant that results in the four missing bands
(Figure 3). We have based our analyses of the bracketing of
the mutation on this assumption, but are cognizant that
subsequent sequencing of the genomic region of the mutant
will provide ultimate proof of the structural arrangement of
the region.
The sequence of the MADS box probe from
bes_018F12B01 was very similar to that of other MADS
box transcription factors (Table 1). The most similar gene
sequences were all MADS genes of the MIKC structural class
( Johansen et al. 2002), however, since the probe sequence is
comprised of only the highly conserved MADS box coding
region, these results may change upon sequencing of the
complete genes from the EVG region. Two of the top five
paralogous genes have been well studied: Arabidopsis
Agamous-Like 24 (AGL24) and Tomato Jointless (LeJointless).
AGL24 is involved in the vernalization pathways of
Arabidopsis, which regulate the timing of the terminal
meristem transition from vegetative to reproductive growth
in response to cold treatments (Yu et al. 2002). LeJointless
regulates the formation of abscission zones in the pedicels of
tomato fruit (Mao et al. 2000). Both of these traits deal with
reproductive events in annual species, while the Evergrowing
mutant is disrupted in its ability to undergo winter dormancy,
a vegetative trait in a perennial, indicating that there may not
be useful analogies to be drawn between the three pathways,
despite apparent similarities between the protein sequences.
The six related MADS box coding sequences detected in
the RFLP analysis appear to be grouped in a relatively small
region of the genome. We confirmed this grouping by
hybridizing the bes_018F12B01 MADS box probe to three
HindIII-digested BACs (PpN089G02, PpN018F12, and
PpN018G07), which were previously determined to form
a contig through the EVG region, digested wild-type, and
EVG mutant genomic DNA (Figure 3). Despite the better
separation of the hybridizing MADS box probe achieved
with the use of EcoRI as a restriction enzyme (Figure 2),
HindIII was used as an enzyme in the BAC analysis.
The Nemared BAC library was prepared using partial
Journal of Heredity 2004:95(5)
HindIII-digested fragments (Georgi et al. 2002), therefore
digested fragments present in the genomic DNA should all
be the same size in the BAC clones. Across the BAC contig,
all six of the hybridizing fragments that were seen in the
genomic wild-type DNA were accounted for in the BAC
clones (Figure 3). Therefore the MADS box hybridizing
sequences detected in the genomic Southern analysis appear
to be grouped in one discrete region spanned by the BAC
contig. However, it is not known whether there are full gene
structures associated with the MADS box sequences or
whether these sequences are simply gene fragments. Full
sequencing of the BACs in the contig will be required to
determine the true arrangement and structure of the genomic
region.
Arabidopsis contains more than 100 predicted MADS box
transcription factors (Garcia-Maroto et al. 2003). Despite
this high number, there is only one cluster of four MADS
box genes, the MADS affecting flowering (MAF ) gene family
(Ratcliffe et al. 2003). In tomato, the Rin (ripening inhibitor)
locus was discovered to contain a pair of closely spaced
MADS box genes that had become fused due to a deletion of
2.6 kb of intervening sequence (Vrebalov et al. 2002). The six
putative MADS box genes reported here would be the first
group of clustered MADS box genes identified in a tree
species.
The proposed arrangement of the BAC contig created
for this region positions PpN089G02 and PpN018G07
overlapping with PpN018F12, but no overlap with each
other (Figure 5). Therefore these BACs extend away from
PpN018F12 in opposite directions (Wang et al. 2002a). The
MADS box probe hybridization supported this arrangement.
PpN018F12 shared two hybridization bands (D, 2.1 kb; F,
1.5 kb) with PpN089G02 that were not found in
PpN018G07 (Figures 3 and 4), and PpN018F12 shared
one hybridization band (E, 1.5 kb) with PpN018G07 that
was not found in PpN089G02 (Figures 3 and 4). In addition,
there were three bands (A, 3.3 kb; B, 2.7 kb; C, 2.1 kb) in
PpN018F12 that were not represented in either PpN089G02
Table 1. The top five most similar protein sequences to the translated MADS box ORF from bes_18F12B01 using tBLASTp
from the National Center for Biotechnology Information (NCBI) database
e-value
4.0
7.0
9.0
9.0
2.0
3
3
3
3
3
442
10
10
10
10
10
20
20
20
20
19
Gene
Species
GenBank accession
MADS box transcription factor
MADS box transcription factor
MADS box protein AGL24
MADS box transcription factor JOINTLESS
Putative MADS domain transcription factor
MpMADS1
Canavalia lineata
Ipomoea batatas
Arabidopsis thaliana
Lycopersicon esculentum
Magnolia praecocissima
AF144623.1
AAK27150.1
CAB79364.1
AAG09811.1
BAB70736.1
Downloaded from http://jhered.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Figure 7. Southern hybridization of HindIII- and EcoRI-digested genomic DNA from selected species probed with DNA
amplified from the bes_18F12B01 MADS box ORF (121 bp). The probe was hybridized at 508C. Lines represent positions of
bands from the HindIII-digested k DNA size marker (from top: 9.416 kb, 6.557 kb, 4.361 kb, 2.322 kb, and 2.027 kb).
Bielenberg et al. A Deletion in Evergrowing Peach
the Clemson University Experiment Station. This material is based on work
supported by the CSREES/USDA under project no. SC-2003055.
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or PpN018G07, confirming that there was a middle region of
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The limit of the deletion present in the EVG trees was
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mutation between the probes used and the mutation.
Therefore the mutation (i.e., deletion) appeared to be
contained wholly within the region spanned by the three
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The size of the region that may contain the EVG
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and annotation of the three BACs in the contig will allow
identification of the structural nature of the deletion and
undoubtedly increase the number of candidate genes.
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Received September 15, 2003
Accepted April 1, 2004
Corresponding Editor: Reid Palmer
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