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Wolf Prize in Agriculture
inserts from only maize chromosome 9 was isolated and
characterized. Based on the large difference in nucleotide
sequence composition between oat and maize and the means
to efficiently isolate and identify maize DNA fragments from
a chromosome addition line, we propose oat as an effective
host for the cloning of maize DNA segments to construct
physical maps for maize chromosomes.
MATERIALS AND METHODS
Maize and Oat Strains.
Oat–maize disomic addition lines
for maize chromosomes 2, 3, 4, 7, and 9 were derived from
plants recovered following sexual crosses of oat by maize (8, 9).
In these crosses about one-third of the recovered plants
contained a haploid set of 21 oat chromosomes plus one or
more maize chromosomes as a result of incomplete maize-
chromosome elimination during early embryo development.
Partial self-fertility due to the production of unreduced ga-
metes by these plants yielded disomic maize-chromosome-
addition oat plants (2n
ϭ 42 ϩ 2). The presence of maize
chromosomes was verified cytologically for each plant in the
current study. The chromosome addition lines, the maize
parent lines Seneca 60 and A188, and the oat parent lines
Starter-1 and Sun II were used for DNA extraction.
Isolation and Analysis of DNA.
Leaves of 2- to 4-week-old
seedlings grown in a growth chamber were used for nuclei
isolation in pH 9.5 buffer according to the protocol of Liu and
Whitter (32). High molecular weight DNA was purified by
phenol extraction from nuclei after lysis of the nuclei suspen-
sion in an equal volume of the same buffer supplemented with
2% sarkosyl. After two phenol extractions, DNA was precip-
itated with two volumes of ethanol in the presence of 0.3 M
NaOAc, dissolved in TE buffer (10 mM Tris
⅐Cl͞1 mM EDTA,
pH 8.0), treated with RNase (50 mg
͞ml), and extracted with
phenol-chloroform.
Sau3A genomic DNA fragments were cloned into the
BamHI site of dephosphorylated plasmid vector pBlueScript
(pBS) II KS (Stratagene). EcoRI subfragments from cosmid
clones were cloned into the EcoRI site of the dephosphory-
lated pBS KS vector according to standard procedures (33).
Insertions were amplified with the help of forward and reverse
primers (Stratagene) and purified by agarose gel electrophore-
sis.
Gel-blot analysis of plant and cosmid DNA was carried out
as described by Sambrook
et al. (33) with several modifications
(34). DNA fragments and total plant DNA were labeled by
random primer extension (35).
Seventeen clones carrying maize repetitive nucleotide se-
quences (30) were kindly provided by J. Bennetzen (Purdue
University, West Lafayette, IN). A clone containing the maize
185-bp knob repeat (36) was provided by W. Peacock (Com-
monwealth Scientific and Industrial Research Organization,
Canberra, Australia).
Cosmid Library Construction and Screening.
Cosmid li-
brary construction and screening were done using the cosmid
vector SuperCos 1 (Stratagene) and packaging extract Giga-
Pack II (Stratagene) with protocols provided by the manufac-
turer. Total nuclear DNA was partially digested with Sau3A,
dephosphorylated, and ligated to the cosmid vector. Ligation
products were packaged and the library propagated in Esch-
erichia coli XL1-Blue MR. Cosmid libraries were constructed
for genomic DNAs of the parental maize (Seneca 60) and oat
(Starter-1) lines, as well as for the oat–maize chromosome 9
addition line.
The size of the insertion in a cosmid clone was determined
as the sum of EcoRI subfragments after fractionation in gels
with different concentrations of agarose from 0.6% up to 1.5%
to achieve satisfactory resolution of long and short DNA
subfragments.
RESULTS
Cloning of Maize-Specific Repetitive Sequences.
When la-
beled maize total genomic DNA was used as a probe in blot
hybridization, little cross-hybridization to oat genomic DNA
occurred under standard conditions (65
ЊC in 6ϫ SSC) in
comparison with strong hybridization to maize genomic DNA
(data not shown). This hybridization pattern indicated that a
significant portion of the repeated nucleotide sequences of
maize and oat are not shared. To isolate maize repeated DNA
sequences specific to maize relative to oat (hereafter referred
to simply as ‘‘maize-specific’’ sequences) a maize plasmid
genomic library was constructed with fragments from a com-
plete Sau3A digest and screened with labeled total maize
DNA. Clones were isolated that gave a high signal with total
labeled maize DNA as the probe. Labeled oat genomic DNA
hybridized only to a small extent to the same set of clones on
a replica filter. Purified plasmids with Sau3A insertions,
together with a group of maize repetitive DNA sequences in
plasmids (see Materials and Methods), were cut with appro-
priate restriction enzymes to release insertions. They were
screened by blot hybridization with labeled maize and oat
DNA. Clones showing a high signal similar to the signal of an
18S–26S rDNA sequence were considered as highly repetitive.
From this screening, 15 plasmids from the Sau3A maize
genomic library, 6 Zpr clones (from Bennetzen), and the one
185-bp knob repeat clone were selected as highly repetitive
maize-specific nucleotide sequences. Insertions from these 22
selected plasmids were combined to form a composite multi-
probe. This multiprobe revealed very strong hybridization to
maize genomic DNA on a Southern blot and no detectable
hybridization to oat genomic DNA (Fig. 1). The same multi-
probe was used to screen cosmid partial libraries of genomic
DNA of maize and oat (about 10,000 clones each). Almost all
colonies (90–95%) in the maize cosmid library revealed strong
or moderate signals. At the same time, only a few colonies, all
with relatively weak signals, were detected in the oat cosmid
library. Taken together, these results indicate that this com-
posite multiprobe is highly specific for maize DNA and is
suitable for detecting maize DNA fragments in a cosmid
library made from an oat–maize chromosome-addition line.
Isolation of Maize-Specific Cosmids from an Oat–Maize
Addition-Line Cosmid Library.
The multiprobe was used to
screen a cosmid library made from an oat–maize chromosome
addition line carrying maize chromosome 9. The screening and
rescreening of about 5,000 clones led to the isolation of 29
hybridization-positive individual colonies. The resulting clones
constituted a maize chromosome 9 library. The cloned maize
DNA fragments in the selected 29 clones had a median size of
about 39 kb. Together they comprised more than 1 Mb of
maize genomic DNA. EcoRI restriction enzyme digestion
enabled cutting out the cloned DNA fragments from the vector
and identification of the number and size of EcoRI subfrag-
ments in the original insertions (Fig. 2a). Fractionation of the
samples on gels of various agarose concentrations from 0.6 to
1.5% enabled detection of doublets and additional small
subfragments below 0.5 kb not detected in the 0.8% agarose gel
blots shown.
Blot hybridization of labeled oat genomic DNA to purified
DNA recovered from each cosmid clone cut by EcoRI did not
reveal strong cross-hybridization to any of the EcoRI subfrag-
ments (data not shown). Only after substantial overexposure
were weak signals revealed and then only for a portion of the
DNA fragments. At the same time, most (
Ͼ90%) of the EcoRI
fragments revealed strong, medium, or weak hybridization to
labeled maize genomic DNA (Fig. 2b). The difference in
hybridization to oat vs. maize genomic DNA indicates that the
selected cloned DNA fragments likely originated from the
maize chromosome, with no evidence that any were chimeric
in origin.
Agricultural Sciences: Ananiev et al.
Proc. Natl. Acad. Sci. USA 94 (1997)
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