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The
EcoRI digests of the set of selected cosmid clones were
hybridized with the maize-specific repeated-sequence multi-
probe that was used to screen the cosmid library (Fig. 2c). The
multiprobe highlighted about 100 of the more than 200 EcoRI
fragments identified in this set of cosmid clones. On average
about three fragments per cosmid were homologous to various
repeated DNA sequences present in the multiprobe. At the
same time a portion of the EcoRI fragments did not hybridize
to the multiprobe nor to total oat DNA but did hybridize to
labeled total maize DNA. These fragments may be added to
the collection of maize-specific repeated DNA sequences to
make the multiprobe an even more efficient screening tool.
Maize-Specific Clones Originate from Maize Chromosome
9.
EcoRI subfragments that showed no readily detectable
hybridization to maize genomic DNA as the probe were
identified in 12 of the 29 cosmid clones. Presumably they are
low copy number or unique nucleotide sequences. Altogether
they comprise 11% of the EcoRI fragments or about 6.5% of
the total amount of maize DNA cloned in this set of cosmid
clones. Twenty-one of them were recloned in plasmids and, as
illustrated for five fragments in Fig. 3, used as probes on blot
panels of DNA samples from addition lines for maize chro-
mosomes 2, 3, 4, 7, and 9. Two parental maize and two parental
oat lines were also on this blot panel (Fig. 3). Nine of these 21
EcoRI fragments revealed one or several bands with DNA
specific for chromosome 9 of maize and did not hybridize to the
DNA of the other four maize chromosomes tested (Fig. 3 a–c).
Seven of these nine fragments revealed restriction fragment
length polymorphism (RFLP) in the two maize stocks, A188
and Seneca 60 (Fig. 3 a and b). Another 11 EcoRI fragments
produced multiple bands with DNA of all maize chromosomes
tested, which is characteristic for low-copy-number families of
dispersed repeated sequences (10–100 copies) (Fig. 3 d and e).
Eight of these sequences produced a chromosome-specific
pattern of hybridization and potentially could be used for
chromosome identification (Fig. 3 d and e).
Several EcoRI fragments belonging to medium and highly
repetitive classes of nucleotide sequences from different cos-
mid clones of maize chromosome 9 were used as probes. These
revealed a complex pattern of hybridization for all maize
chromosomes on the panel of chromosome-addition lines (Fig.
4b). Labeled highly repetitive DNA from cosmid 1 (Fig. 4c), as
well as the full set of 29 cosmids (data not shown), gave strong
hybridization to all maize chromosomes and did not reveal any
chromosome 9-specific repetitive nucleotide sequences. Only
the 185-bp knob-repeat revealed apparent chromosome spec-
ificity (Fig. 4d). Many copies of 185-bp repeats are located on
chromosome 9, and a small fraction is present on chromosome
4 in these materials. Several weak bands are seen with DNA of
maize chromosomes 2 and 3. This result is compatible with
reports that most 185-bp knob DNA sequences are organized
in the form of clusters on different chromosomes and relative
knob size can differ greatly among chromosomes in different
maize lines (31).
F
IG
. 1.
Blot hybridization of a labeled multiprobe, composed of 22
maize-specific repetitive DNA sequences, to genomic DNA digests of
2 maize and 2 oat varieties. Strong signal is seen over lanes with maize
DNA but not those with oat DNA. (a) EtdBr-stained 0.8% agarose gel
after separation of DNA samples cut with EcoRI. (b) Autoradiogram
after hybridization to the P-32 labeled multiprobe.
F
IG
. 2.
Blot panel of 29 maize-specific cosmid clones isolated from
a cosmid library of an oat–maize chromosome 9 addition line. (a)
Cosmids (1–29) are cut by EcoRI restriction enzyme and size-
fractionated in an 0.85% agarose gel stained with EtdBr (M
ϭ
molecular weight marker, 1-kb ladder). (b) Labeled, total genomic
maize DNA as a probe shows strong, medium, or weak hybridization
to almost 90% of all EcoRI subfragments present in the cosmid clones.
(c) Labeled multiprobe of highly repeated maize DNA sequences
shows hybridization to from one to five EcoRI fragments in each lane.
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Wolf Prize in Agriculture
Most Cloned Maize Nucleotide Sequences Are Maize-
Specific.
Seven out of 11 unique or low-copy-number se-
quences showed no cross-hybridization to oat genomic DNA
(Fig. 3 a–d). This result indicates that a significant portion
(
Ͼ50%) of low-copy-number maize sequences have diverged
enough to show no cross-hybridization to oat genomic DNA.
F
IG
. 4.
Hybridization of medium and highly repetitive cloned maize DNA sequences to a blot panel of oat–maize chromosome addition lines
carrying maize chromosomes 9, 7, 4, 3, and 2, respectively; m1 and m2 are maize stocks A188 and Seneca 60; o1 and o2 are oat stocks Sun II and
Starter-1. (a) EtdBr-stained 0.8% agarose gel; lane M is a 1-kb molecular weight marker ladder. (b) The 0.7-kb EcoRI fragment of cosmid 10 shows
multiple bands on different maize chromosomes as well as one common band for all chromosomes. (c) Cosmid 1, with a 40-kb insertion, shows
strong hybridization signal over maize (lines m1 and m2) and over chromosome addition lines 9, 7, 4, 3, and 2; no hybridization is seen over oat
DNA (lines o1 and o2). (d) With the 185-bp knob repeat, most of the hybridization signal is located on chromosome 9. No hybridization to oat
DNA is detected.
F
IG
. 3.
Hybridization of unique and low-copy-number DNA sequences isolated from maize-specific cosmids shown in Fig. 2 to a blot panel of
oat–maize chromosome addition lines carrying maize chromosomes 9, 7, 4, 3, and 2, respectively; m1 and m2 are maize stocks A188 and Seneca
60; o1 and o2 are oat stocks Sun II and Starter-1. (a) The 2.5-kb EcoRI fragment from cosmid 15 shows one band on the chromosome 9 addition
line. An additional polymorphic band is present in the parental stocks of maize. (b) The 1.8-kb EcoRI fragment from cosmid 28 detects two bands.
One band, polymorphic between m1 and m2, is present in the chromosome 9 addition line. An additional nonpolymorphic band is present on
chromosome 9 and in both parental stocks. (c) The 2.1-kb EcoRI fragment from cosmid 10 shows one band on chromosome 9. (d) The 1.4-kb EcoRI
fragment from cosmid 20 detects about 20 bands in parental maize stocks and several bands among the chromosome addition lines. The band pattern
is chromosome-specific. No cross-hybridization occurred to oat DNA in a–d. (e) The 2.9-kb EcoRI fragment from cosmid 6 detects several
polymorphic bands in parental maize stocks. One nonpolymorphic band is seen in maize, oat, and chromosomes 9, 7, 4, and 2 addition lines.
Additional bands are seen in other chromosome addition lines.
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