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Wolf Prize in Agriculture
(Zea mays L.) lines Seneca 60, A188, A619, B73, Mo17, the
(A188
ϫ W64A)F
1
hybrid, and a line carrying the allele bz1-
mum9. More than 200 putative F
1
-hybrid plants indicating
successful (oat
ϫ maize) hybridization were recovered and
analyzed for vigor, seed set, maize chromosome retention in
various tissues, and maize chromosome transmission to F
2
offspring (5, 10). F
2
plants were tested by PCR-based markers,
test crosses, genomic in situ hybridization (GISH), and chromo-
some counting for presence, stability, and transmission of maize
chromosomes added to the oat genome (15). F
2
plants were
propagated for production of F
3
and subsequent generations.
Backcross (BC
1
) plants monosomic for the maize chromosome
addition were produced by crossing disomic oat–maize chromo-
some addition plants back to their corresponding parental oat
lines. Batches of 150–300 BC
1
seeds were irradiated with
␥ rays
from a
137
Cs source at different intensities (20–50 krad) to
induce as many maize chromosome breaks as possible without
seriously damaging the vigor of the seeds or resulting seedlings.
BC
1
F
2
offspring with transmitted maize chromosome deficien-
cies or oat–maize chromosome translocations (RH plants) were
selected by a PCR assay with primers specific for Grande 1 (16)
and CentA (17) and used for physically mapping molecular
markers to the particular maize chromosome segments.
Genomic DNA Extraction.
For a limited number of PCRs (75 or
fewer) from single plants, DNA was extracted by the use of the
REDExtract-N-Amp Plant PCR kit (Sigma). For larger numbers
of PCRs (
Ͼ75) from single plants, DNA was extracted by using
either the DNeasy Plant Mini kit (Qiagen, Valencia, CA) or the
acetyltrimethylammonium bromide procedure (18). For labeling
and use as probe in GISH experiments, genomic maize DNA was
extracted from leaf cell nuclei and purified through a CsCl
gradient (19).
PCR.
PCRs were accomplished by the use of the REDExtract-
N-Amp Plant PCR kit, according to the vendor’s recommenda-
tions. F
1
plantlets and seedlings of the consecutive generations
were screened for the presence of maize sequences by using
maize-specific primers for the long terminal repeat of the highly
dispersed retrotransposon Grande 1 (16) and for the highly
centromere-specific retrotransposon-like repeat CentA (17).
Individual maize chromosomes or chromosome segments in F
1
plantlets, addition lines, and RH seedlings were identified by
using maize-specific primers for simple-sequence repeat (SSR)
markers (10) that were selected from the maize genetics and
genomics database (www.maizegdb.org). BC
1
F
2
plants (putative
RH plants) were tested for presence vs. absence of maize
chromosome segments by a PCR assay with 45 SSR markers
specific for maize chromosome 1 (p-umc1354 to p-umc2244
spanning 1,120 map units, according to the IBM2 map). Markers
were selected from the maize genetics and genomics database
mentioned above.
Cytology.
Root tips (1.5–2 cm) of oat, maize, and oat–maize
chromosome addition and RH lines were pretreated, fixed, and
stored as described in ref. 10. Root tips for chromosome counting
were prepared as described in ref. 20. Meristem cells were
squashed in 2% wt
͞vol Aceto-Orcein (Carolina Biological Sup-
ply). Root tips for GISH were prepared as described in ref. 10.
Further steps of RNase treatment, postfixation, and in situ
hybridization were as described earlier in ref. 21 except that total
genomic maize DNA was labeled by the use of the ULYSIS
Alexa Fluor 488 nucleic acid labeling kit (Molecular Probes) and
probed on slides without using unlabeled competitor DNA.
Hybridization was carried out in 40% formamide in 1.5
ϫ SSC
(225 mM NaCl
͞22.5 mM trisodium citrate, pH 7.0) at 37°C.
Posthybridization stringency washes were carried out in 40%
formamide in 1.5
ϫ SSC at 42°C. Chromosomes were counter-
stained with propidium iodide. Signals were visualized and
captured by using an Axioskop microscope equipped for epi-
fluorescence (Zeiss) and a Magnafire charge-coupled device
camera (Optronics International, Chelmsford, MA).
Results and Discussion
Maize Chromosome Elimination in (Oat
؋ Maize)F
1
Hybrids.
In oat
ϫ
maize crosses, maize chromosomes are occasionally retained (2).
This situation is distinct in that the maize genome is completely
eliminated in hybridizations between maize and wheat or barley.
There is only one report of a maize chromosome being retained
in wheat; however, the maize chromosome was not transmitted
to offspring (23). The timing of elimination differs as well. The
maize chromosome elimination process in oat sometimes ex-
tends over longer periods of time compared with that in F
1
hybrids generated from wheat
ϫ maize and barley ϫ maize
crosses (24). In 70% of (wheat
ϫ maize)F
1
embryos, one or more
maize chromosomes were eliminated at the first mitosis. By the
eight-cell stage, the embryos had lost all maize chromosomes
(24). Maize chromosome elimination from (oat
ϫ maize)F
1
embryos starts at an early stage in embryogenesis as well (4).
However, as an example of the extended time of maize chro-
mosome elimination from oat, in the (oat
ϫ maize)F
1
plant
F
1
-5133-1 with maize chromosomes 4, 7, and 10 all detected at
a young plant stage, a consecutive elimination of individual
maize chromosomes was detected by the PCR analysis of
genomic DNA extracted from tissues of flag leaves of different
tillers from the same plant shortly after meiosis. The first tiller
retained only maize chromosome 4, thus eliminated chromo-
somes 7 and 10. The second tiller eliminated chromosome 10,
thus retained chromosomes 4 and 7. The third and fourth tillers
eliminated chromosome 7, thus retained chromosome 4 and
chromosome 10 as a short-arm telosome (Fig. 1). We observed
further instances where maize chromosomes were lost in later
growth stages, particularly from plantlets with two or more
originally retained maize chromosomes in their complements
(results not shown).
(Oat
؋ Maize)F
1
Hybrids and F
2
Offspring in Different Genetic Back-
grounds.
Our initial work was conducted with the maize chro-
mosome donor Seneca 60 and the oat recipient Starter. In the
last 2 years we have tested several other combinations of maize
and oat lines. A total of 201 (oat
ϫ maize)F
1
plants have been
generated from various maize and oat backgrounds, of which 68
F
1
hybrids retained one or more maize chromosome(s) in their
complements. All 10 maize chromosomes could be recovered,
with each occurring at different frequencies as single additions
and in combination with other maize chromosomes. No obvious
preferential combination for two or more specific maize chro-
mosomes was detected in multiple additions in haploid oats. The
frequency of recovery of a particular maize chromosome, the
Fig. 1.
PCR products from DNA of the F
1
(Starter
ϫ B73) plant F
1
-5133-1 when
chromosome-specific SSR markers are used; electrophoresis in a 3.5% agarose
gel shows the different elimination of maize chromosomes in individual tillers.
Lanes 1, young plantlet; lanes 2, tiller F
1
-5133-1
͞a; lanes 3, tiller F
1
-5133-1
͞b;
lanes 4, tiller F
1
-5133-1
͞c; lanes 5, tiller F
1
-5133-1
͞d; and lane M, standard
100-bp ladder.
9922
͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403421101
Kynast et al.
38_2006-7 Phillips.p65
06-Mar-09, 7:49 PM
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