Cloning and characterization of repetitive DNA sequences from genomes of

Oryza minuta and Oryza australiensis

H. ASW~D~NNOOR~
Department of Agronomy, University of Missouri-Colum bia, MO 65211, U.S.A.
R. J. NELSON
Division of Plant Pathology, International Rice Research Institute, Manila, Philippines
J. F. DALLASAND C. L. MCINTYRE
Department of Agronomy, University of Missouri-Colum bia, MO 65211, U.S.A.

Division of Plant Pathology, International Rice Research Institute, Manila, Philippines

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AND

U.S. Department of Agriculture, Agricultural Research Service, and Department of Agronomy,

University of Missouri-Columbia, MO 65211, U.S.A.
Corresponding Editor: G. Fedak
Received January 24, 1991
Accepted May 29, 1991

ASWIDINNOOR, H., NELSON,R. J., DALLAS,J. F., MCINTYRE, C. L., LEUNG,H., and GUSTAFSON, J. P. 1991. Cloning
and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis. Genome,
34: 790-798.
The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentia-
tion was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOml,
For personal use only.

pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone,
pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different
rice genomes showed strong hybridization of all five clones to 0. minuta genomic DNA and no cross hybridization
to genomic DNA from Oryza sativa (AA genome). The pOml and pOmA536 sequences showed cross hybridization
only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences
showed cross hybridization to 0. australiensis genomic DNA in addition to showing hybridization to the 0. minuta
genomic DNA.
Key words: rice, genome-speci fic repetitive sequences, Oryza.
ASWIDINNOOR, A., NELSON,R. J., DALLAS,J. F., MCINTYRE, C. L., LEUNG,H., et GUSTAFSON, J.P. 1991. Cloning
and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis. Genome,
34 : 790-798.
La valeur des sequences repetitives d'ADN specifiques aux genomes et employees comme marqueurs dans les etudes
de differenciation genomique a ete investiguee. Cinq sequences repetitives d'ADN provenant d'especes de riz indigenes
ont ete clonees. Quatre clones ont ete isoles de 1'Oryza minuta accession 101141 (genomes BBCC), savoir les pOml,
pOm4, pOmA536 et pOmPB10, et un clone, le pOa237, a ete isole de 1'Oryza australiensis accession 100882 (genome EE).
Les transferts Southern a differents genomes de riz ont montre une forte hybridation des cinq clones avec I'ADN geno-
mique de 1'0. minuta, mais aucune hybridation croisee avec I'ADN genomique de 1'Oryza sativa (genome AA). Les
sequences pOml et pOmA536 ne se sont hybridees de facon croisee qu'avec toutes les especes de riz indigenes qui con-
tenaient le genome C. Toutefois, les sequences pOm4, pOmPBlO et pOa237 se sont hybridees de facon croisee avec
I'ADN genomique de 1'0. australiensis, en plus de montrer de l'hybridation avec 1'ADN genomique de 1'0. minuta.
Mots cles : riz, sequences repetitives specifiques aux genomes, Oryza.
[Traduit par la redaction]
Introduction of genes for improvement (Sitch 1990). Wide crossing
More than 20 wild species of rice (Oryza) have been iden- between species aims at transferring traits from distant
tified. The genomes of several of them have been character- relatives into cultivated varieties and is becoming an impor-
ized and assigned to groups, whereas others have not been tant part of rice breeding programs (Sitch et al. 1988; Jena
characterized. The wild species of rice represent a rich source and Khush 1989).
It has been suggested that the various species of rice iden-
tified contain six genomes: A , B, C, D, E, and F (Morishima
h his paper reports the results of research only. Mention of a 1984). This genomic classification has been based largely on
proprietary product does not constitute an endorsement or a recom-
mendation for its use by the USDA or the University of Missouri. the analysis of chromosome pairing behavior in interspecific
2 ~ h ipaper
s is a contribution of the U.S. Department of Agri- hybrids. Recently, molecular markers have been utilized to
culture, Agricultural Research Service, and the Missouri Agricul- study genome differentiation in various plant species. Both
tural Experiment Station, Journal series No. 11 438. single-copy (Song et al. 1988; Bonierbale et al. 1988) and
3 ~ r e s e naddress:
t Department of Agronomy, Faculty of Agri- repetitive D N A sequences (Evans et al. 1983; McIntyre et al.
culture, Bogor Agricultural University, Bogor 16143, Indonesia. 1988) have been utilized.
Printed in Canada / Imprime au Canada
 ASWIDINNOOR E T AL.
TABLE1. Rice species, chromosome number, and genome group
IRGC*
accession
Species No.
0 . sativa
0. nivara
0. rufipogon
0. perennis
0. punctata
0. punctata
0. officinalis
0. eichingeri
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0. rninuta
0. rnalarnphuzaensis
0. latifolia
0. alta
0. grandiglurnis
0 . australiensis
0. brachyantha
*International Rice Germplasm Center.
+From Sitch (1990).
IFrom Nayar (1973).
It has been shown that a substantial fraction of the
genomes of higher eukaryotes are composed of moderately
and highly repetitive DNA sequences that are unique to cer-
For personal use only.
tain genomes. Many such genome-specific sequences have
been isolated from higher plants (Rayburn and Gill 1986;
Sonina et al. 1989; Junghans and Metzlaff 1988; Metzlaff
et al. 1986; Saul and Potrykus 1984; Schweizer et al. 1988;
Appels and Moran 1984; McIntyre et al. 1988). There is
evidence that such genome-specific sequences also occur in
rice (Zhao et al. 1989). The value of such DNA sequences
as markers in analyzing alien-genome introgression into
wheat (Triticum) has been reported by Appels and Moran
(1984).
Oryza minuta, a tetraploid wild species of rice, is known
to have resistance to brown planthopper, whitebacked plant-
hopper, green leafhopper, blast, and bacterial blight (Sitch
1990). These and other pests are important problems in
world rice production. This species has been crossed with
cultivated rice (Oryza sativa) to transfer the above resistance
traits. Backcross progeny with resistance to blast and
bacterial blight have been recently obtained (Amante-
Bordeos et al. 1991).
This paper reports on the cloning and characterization of
five DNA sequences that are abundant in the 0. minuta
genome and absent from the 0. sativa genome. The cloning
of another sequence, pOm6, had previously been reported
(Aswidinnoor et al. 1991). Oryza minuta genome-specific
repetitive DNA families are currently being tested for their
potential as markers in analyzing genome introgression in
the backcross derivatives.
Materials and methods
Plant materials
Rice species used in the present study are listed in Table 1. Seeds
were obtained from the International Rice Research Institute
(IRRI). Plants were grown in a growth chamber (12 h light : 12 h
darkness, 29:21 "C (light : dark), 70-75% relative humidity) or in
a greenhouse in Columbia, MO.
Chromosome Genome
no. constitution
Genornic DNA isolation and preparation
Leaf material was harvested, lyophilized for 3-4 days in a
Labconco freeze-drier, and powdered using a Tectator Cyclotec
sample mill (Fisher Scientific). Nuclear genomic DNA was extracted
according to the method of Saghai-Maroof et al. (1984). Chloro-
plast and mitochondria1 DNA of 0. sativa cv. IR36 were obtained
from Dr. K. Tsunewaki of Kyoto University, Japan, and from
Dr. V. Walbot of Stanford University, respectively. Restriction-
enzyme digests and gel electrophoresis were done using standard
methods (Sambrook et al. 1989).
Cloning of repetitive DNA
Clone pOa237 from Oryza australiensis was obtained as follows.
Genomic DNA of 0. australiensis accession 100882 and 0. sativa
cv. IR36 were digested with TaqI enzyme and electrophoresed on
an agarose gel. After staining with ethidium bromide, a distinct
band of approximately 1.3 kb was prominent in the 0. australiensis
lane and absent from the 0. sativa lane. Eluted DNA from this
band was then ligated into the AccI cloning site of pUC19.
Cloning and selection of genome-specific repetitive sequences
from 0. rninuta were done using three different approaches. First,
total genomic DNAs from 0. rninuta accession 101141 and
0. sativa cv. IR36 were digested with the restriction enzyme
BarnHI, run together on an agarose gel, and then stained with
ethidium bromide. Two prominent, distinct bands of approximately
0.6 and 1.7 kb were present in the 0. rninuta lane and absent in
the 0 . sativa lane. DNA from these bands was cut from the gel
and electroeluted. To quickly assess their genome specificity, these
crude band preparations were then hybridized onto dot blots of
genomic DNA from 0. rninuta and 0 . sativa, which were prepared
according to the method of Raeder and Broda (1984). The crude
preparations showed either specificity or differential hybridization
signal to 0. rninuta as compared with 0. sativa. They were then
used as probes to screen a pUC19 library containing size-selected
( < 1 kb) TaqI fragments of genomic DNA from 0. rninuta ligated
into the AccI cloning site. Size selection was done by glycerol-
gradient centrifugation, and the library screening was accomplished
using the method of Grunstein and Hogness (1975). Clone pOml
was obtained using the 0.6-kb fragment as probe. Clone pOm4
was obtained by selecting the same library, using the 1.7-kb frag-
ment as probe.
The second approach involved the construction of a random
genomic library of Eco RI fragments of 0. rninuta accession 101141
 GENOME, VOL. 34, 1991

TABLE2. Sources of library and insert sizes of the five repetitive DNA clones

Source of Insert Cloning

Clone* library size (bp) site Vector

pOa237 Tag I prominent band 1300 Acc I pUC19

pOml Tag I glycerol gradient 239 Acc I pUC19
fractionation
pOm4 Tag1 glycerol gradient 428 Acc I pUC19
fractionation
pOmA536 Eco RI random genomic 400 Eco RI pUC 13
pOmPBlO Sau3AI PERT 305 BamHI pUC13
*Clone pOa237 was isolated from 0. australiensis accession 100882, and the others were isolated from
0. minuta accession 101 141.
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CGAAACCACA CAAGTGTTTT TTGGGCAAAA GGGAATATGT TTGGCCCCAT TGGTCGCGAA

AATTAATTTT TACACGAGTC GGTGGGCCCT CCGGAAATTT GTGTGCTGCG AAAACGAAAC

CGCTCAATAG TGCTTTTGTG TCAGATGGGA ATGTGTTTGG CACCATTGGT TGTGAAAATG

AATTTTTAAA CGAGTCGGTG TGGCCGAAGA AAGTTCGTGT GCTACGAAAC GAATCCGCT

CGATATGTAT GAATCTTTTC TAATTGATCC CTGATCATAT ATATCA'I'TTA TTGGAACTAA

TATGATTCTA AGATCGGCAT GTCTATTGAT TGATGATCAT GTCTCATAGA TCATAGGTAT

For personal use only.

GGAGATACCA AATCAATAAA CATGGACATA TGTGTTAGAG AACACATTAT TGGATAGACC

CACCATGAGA CACTACAGGA ATTAATGTGC CATTAGTTGG TCTCAGGTAG TGTTGGTACA

AAGTCCTTAG ACCTGAGATC ACCATGGATT CCAACATGTG TAGTAGCCTA CTTTGGGACT

ACCAAACGCT ATTCCGTAAC TGGGTAGTTA TAAAGGTAGT TTTCGGGTTT GCTATGAAAC

ATGGGGTGGG GATGTGAGTG ATCAAGATGG AA'I'TTGCCCC TC(X"I'TGGAG AGATATCTCT

AGGCCCCT

GATCCAGAAT CGCCTGGAAA GATATGCAAG CTACAGAAAT CCATTTATGG ATTGAAGCAA

GCATCTCGGA GTTGGAATAT TCGTTTTGAT GAAGTAATCA AAGGGTTTGG TTTCATCAAA

AATGAAGAAG AGGCCTGTGT TTACAAAAAG GTCAGTGGGA GCGCAATTGT ATTTGTAATC

TTATATGTGG ATGACATATT GTTGATTGGA AATGATATCC CTATGCTAGA ATCCGTCAAG

TCTTCATTGA AAAATAGTTT TTCCATGAAA GACTTAGGGG AGGCAGCATA CATATTGGGC

ATTCG
FIG. 1. Nucleotide sequence of three repetitive DNA clones isolated from 0. minuta accession 101141: (A) 239-bp pOml; (B) 428-bp
pOm4; (C) 305-bp pOmPB10.

in the vector pUC13. Screening was done using labeled genomic
DNAs from 0. minuta and 0. sativa, and clone pOmA536 was
obtained.
Third, an additional library was constructed using the phenol
emulsion reassociation technique (Kohne et al. 1977; Kunkel et al.
1985), and was called a PERT library. In this library, selectively
enhanced species-specific Sau 3AI fragments of 0. minuta accession
101141 were cloned into the Bam HI site of pUC13. Plasmid mini-
preparations were screened by cross hybridization with genomic
DNAs of 0. minuta and 0. sativa, and clone pOmPBlO was
obtained.
DNA sequencing
The nucleotide sequence of the clones pOml, pOm4, and
pOmPB10 was determined using the chain-termination method of
Sanger et al. (1977). The T7 sequencing kit (Pharmacia LKB) was
used with a double-stranded recombinant plasmid DNA template
(Mierendorf and Pfeffer 1987).
Southern blot hybridization
Restriction enzyme digested chloroplast, mitochondrial, and
genomic DNA samples were electrophoresed on agarose gel. South-
ern blotting onto a nylon membrane was done using standard
 ASWIDINNOOR ET AL.
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For personal use only.

FIGS. 2A and 2B. Southern blot hybridization of clones pOml and pOmA536 to various genomes of Oryza. Genomic DNAs (7.5 pg)
of respective rice species were digested with Eco RI restriction enzyme, electrophoresed, and blotted onto nylon membrane as described
in the Materials and methods. Isolated ;l.;c;t of cloned DNA was labeled with [ 3 2 ~ ]and d hybridized
~ ~ ~ to the filters. (A) Probed
with pOml; (B) probed with pOmA536. Filters were washed at 2 x SSC, 0.1 % N-lauroylsarcosine for pOml and at 0.1 x SSC, 0.1 %
N-lauroylsarcosine for pOmA536. All washing was carried out at 65OC. IR36 = 0. sativa cv. IR36.
 IR36 n
P
w
0. punctata 103888

0. punctata 103906

0. officinalis 100896

0. eichingeri 10 1422

0. eichingeri 101425
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0. malamphuzensis
h 100957
IR36 0 0. punctata 101409
0. punctata 103888 0. latifolia 100963

0. punctata 103906 0. latifolia 100966

0. grandiglumis 101405
0. officinalis 100896
0. alta 101395
0. eichingeri 10 1422
0. australiensis 100882
0. eichingeri 101425
B
0. minuta 101089 ? ' II)
0. brachyantha 10 123 1

lambda DNA / Hindlll

For personal use only.

0. minuta 101125

0. minuta 101141

0. malamphuzensis
100957
a 0. punctata 103888

0. punctata 101409 0. punctata 103906

0. latifolia 100963 0. officinalis 100896

0. latifolia 100966 0. eichingeri 101422

0. grandiglumis 101405 0. eichingeri 10 1425

0. alta 101395 0. minuta 101089

0. australiensis 100882 0. minuta 101125

0. brachyantha 101231 0. minuta 101141

lambda DNA/ Hindlll 0. malamphuzensis

100957
0. punctata 101409

0. latifolia 100963

0. latifolia 100966

0. grandiglumis 101405

0. alta 101395

0. australiensis 100882

0. brachyantha 101231

lambda DNA / Hindlll

 ASWIDINNOOR ET AL.
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For personal use only.
FIG. 4. Hybridization of pOml to 0. minuta accession 101141
genomic DNA digested with (lane 1) Alu I, (lane 2) HaeIII, (lane 3)
HinfI, (lane 4) RsaI, (lane 5) Sau3A1, (lane 6) TaqI. Marker was
PAT, digested with Hin fI. Filter was washed at 0.1 x SSC, 0.1%
N-lauroylsarcosine, at 65°C.
methods (Sambrook et al. 1989) except that 25 mM NaPO,
pH 6.5 was used as the Southern transfer buffer. Cloned inserts
were cut and separated from the recombinant plasmids and then
labeled with [ 3 2 ~ ] dusing ~ ~ ~ the, random-primer method
(Feinberg and Vogelstein 1983). The hybridization buffer contained
5 x SSC (1 x SSC: 0.15 M NaCl plus 0.015 M sodium citrate),
5 x Denhardt's solution, 50 mM Tris pH 8.0, 10 mM EDTA
pH 8.0, 0.2% N-lauroylsarcosine, and denatured salmon sperm
DNA. Hybridization was carried out at 65°C.
Results
Cloning of repetitive DNA sequences
Five repetitive DNA sequences that hybridized to
0. minuta DNA but not to 0. sativa DNA were cloned.
A summary of their sources of library and their insert sizes
is given in Table 2.
The nucleotide sequence of pOm1, pOm4, and pOmPB10
is presented in Fig. 1. The other two clones, pOa237 and
pOmA536, were not sequenced. The insert size of pOa237
was too large, and there was another 3.3-kb EcoRI fragment
in the recombinant plasmid of pOrnA536. The G + C content
of pOm1 was 45%, that of pOm4 was 40%, and pOmPBlO
was 39%. According to the expected melting temperature
formula, T , = 81.5 + 16.6(log M) + 0.41(% G + C )
(Hayes et al. 1970; Bender et al. 1978), the expected melting
temperature of the pOm 1, pOm4, and pOmPBlO sequences
at 0.015 M Na' were 70.0, 67.7, and 67.2"C, respectively.
795
FIG. 5. Hybridization of pOmA536 to 0. minuta accession
101141 genomic DNA digested with (lane 1) BamHI, (lane 2)
EcoRV, (lane 3) HindIII, (lane 4) KpnI, (lane 5) PstI, (lane 6) SalI,
(lane 7) Sau3A1, (lane 8) SstI, (lane 9) TaqI. Marker was X DNA,
digested with HindIII. Wash condition was the same as for Fig. 4
Taxonomic examination by Southern blot hybridization
Southern blot hybridization analysis was used to examine
the degree of specificity or extent of cross hybridization of
the five sequences to DNA of various rice species. Figures 2
and 3 show the extent of hybridization of the five cloned
DNA sequences to DNA samples representing various
genomes of Oryza. All five DNA clones showed strong
hybridization to genomic DNA of 0. minuta (BBCC
genomes), but none hybridized to genomic DNA of 0. sativa
cv. IR36 (AA genome). Absence of hybridization of these
clones was also observed when they were hybridized with
genomic DNAs from several other A-genome carriers,
0. sativa cv. IR54 and cv. IR64, as well as the wild species
0. nivara accession 103839,O. rufipogon accession 100907,
and 0.perennis accession 104453 (data not shown). None
of the five DNA clones show hybridization to the genomic
DNA of Oryza brachyantha (FF genome).
In addition to hybridizing to DNA of 0. minuta (BBCC
genomes), clones pOml and pOmA536 showed cross hybrid-
ization to other rice species containing the C genome. There
was an almost equal degree of hybridization of Oryza
officinalis (CC genome) and 0. minuta (BBCC genome)
with these two sequences, while hybridization varied con-
siderably with the other C genome containing species.
Hybridization signal of pOml occurred to larger fragments
of Oryza eichingeri (CC genome), Oryza malamphuzaensis
(BBCC genomes), tetraploid Oryza punctata (BBCC
genomes), and accessions containing CCDD genomes
(Fig. 2A). Probe pOmA536 showed similar hybridization
patterns with 0. officinalis (CC genome), 0. eichingeri
(CC genome), 0. minuta (BBCC genome), and 0. malam-
phuzaensis (BBCC genomes) (Fig. 2B). A different hybrid-
 796 GENOME,
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FIG. 6. Hybridization of pOm4 to 0. minuta accession 101141
genomic DNA digested with (lane 1) AluI, (lane 2) HaeIII, (lane 3)
Hin f I , (lane 4) RsaI, (lane 5) EcoRV, (lane 6) Sau3A1, (lane 7)
TaqI. Marker was PAT, digested with Hin fI. Wash condition was
For personal use only.
the same as for Fig. 4.
ization intensity to this probe occurred among rice species
having C and D genomes. Oryza latifolia showed a con-
siderable amount of hybridization, whereas it was almost
absent in 0. alta and 0. grandiglumis.
The other three clones, pOm4, pOmPB10, and pOa237
(Figs. 3A-3C), with the 0.1 x SSC wash showed strong
cross-hybridization signal with 0. minuta (B and C genomes)
and 0. australiensis (E genome). Only very light hybridiza-
tion appeared on accessions having C and D genomes, and
there was no hybridization with genomic DNA from
0. malamphuzaensis and tetraploid 0. punctata with these
three repetitive DNA clones.
Southern blot analysis of cross hybridization among the
five DNA clones showed that they did not cross hybridize
with each other, indicating that each is unique and represents
a different family of repeats. However, clone pOm4 showed
some cross hybridization with pOa4, a repetitive sequence
isolated from 0. australiensis, reported by Zhao et al.
(1989). From the DNA sequence analysis, there was 50.1 %
random cross homology between these two clones. Further-
more, none of the five sequences hybridized t o chloroplast
and mitochondria1 DNA from 0. sativa cv. IR36.
Organization of the cloned DNA sequences in the 0. minuta
genomes
Genomic DNA from 0. minuta accession 101141 was
digested with several different restriction enzymes. A ladder
pattern was observed from hybridization of pOml to
BamHI- (data not shown), Sau3AI-, and TaqI-digested
DNA (Fig. 4.), which indicated a tandemly repeated organi-
zation of this repetitive sequence in the genome of 0. minuta.
For pOmA536, EcoRI digestion gave the most nearly even
distribution of signal in a large number of bands (see
VOL. 34, 1991
FIG. 7. Hybridization of pOmPBlO to 0. minuta accession
101141 genomic DNA digested with (lane 1) BamHI, (lane 2)
EcoRV, (lane 3) HaeII I , (lane 4) Hin fI, (lane 5) RsaI, (lane 6)
Sau3A1, (lane 7) SstI, (lane 8) TaqI. Marker was PAT, digested
with HinfI. Wash condition was the same as for Fig. 4.
Fig. 2B). Several bands were resolved from digestion with
other restriction enzymes (Fig. 5).
Strong bands were also revealed for pOm4, pOmPB10,
and pOa237 when they were hybridized t o 0. minuta
genomic DNA digested with several restriction enzymes
(Figs. 6 , 7 , and 8). A single band was observed from hybrid-
ization to Tag1 digestions for pOm4, SstI, and RsaI for
pOmPB10, while Tag1 also resolved most of the signal into
one band for the probe pOa237.
Discussion
The cloning and characterization of five families of
repetitive DNA from the genomes of 0. minuta and
0. australiensis have been reported. Two of the DNA
families, pOml and pOmA536, are probably from the
C-genome complement, since they cross hybridize only with
the species containing this genome. The other three families,
pOm4, pOmPB10, and pOa237, show cross hybridization
to both 0. minuta and 0. australiensis genomes. From cyto-
logical studies, it has been suggested that 0. minuta is an
allopolyploid with a genome designation BBCC. Oryza
officinalis (C genome) is generally agreed to constitute one
of the two genomes in 0. minuta. Even though different
results and opinions exist concerning the other genome com-
plement, it was thought to be from the diploid form of
0. punctata (B genome) (Nayar 1973; Ogawa and Katayama
1974). However, Vaughan (1989) noted that it is difficult
to understand the role of 0. punctata in the evolution of
0. minuta, based on the current distribution of 0. punctata
in Africa.
The origin and dispersion of repetitive DNA sequences
in the genomes of wild rice species may be useful in monitor-
ing genome introgression. From the analysis using probe-
enzyme combinations, different patterns of hybridization
 ASWIDINNOOR ET
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FIG. 8. Hybridization of pOa237 to 0. minuta accession
101141 genomic DNA digested with (lane 1) BamHI, (lane 2)
EcoRV, (lane 3) HaeIII, (lane 4) Hin fI, (lane 5) RsaI, (lane 6)
For personal use only.
Sau3A1, (lane 7) SstI, (lane 8) TaqI. Marker was PAT, digested
with HinfI. Wash condition was the same as for Fig. 4.
among the repetitive D N A families were found. T h e families
showed a number of distinct bands a s well as smear patterns.
T h e highlighting of a number of distinct bands suggested
that sequences complementary t o t h e probe(s) a r e present
in several discrete configurations, presumably next t o other
repetitive sequences (Evans et al. 1983). Hybridization also
occurred t o a smear of fragments over a wide range of sizes.
This pattern was most probably d u e t o the hybridization of
repeats that are dispersed, o r adjacent t o single o r low-copy
sequences (Evans et al. 1983; Saul a n d Potrykus 1984; Rivin
e t al. 1986). Furthermore, the distribution analysis of
repetitive families over different chromosomes could be
accomplished by hybridization t o D N A samples from mono-
somic alien addition lines (MAAL), each representing a
single chromosome of a genome complement o r by utilizing
in situ hybridization of t h e respective probes t o cytological
preparations. A complete-set M A A L of 0. officinalis
( C C genome) in cultivated rice ( 0 . sativa) background has
been produced (Jena a n d Khush 1989), a n d the production
of 0. australiensis ( E E genome) M A A L is in progress (Sitch
et al. 1990). Dispersed repetitive D N A sequences f r o m the
wild species of the genus Oryza will be of interest in monitor-
ing alien genome introgression into cultivated rice.
Acknowledgments
H. Aswidinnoor was supported by the Indonesian Second
University Development Project. T h e authors thank
Dr. V. Walbot of Stanford University and Dr. K. Tsunewaki
of Kyoto University, J a p a n , f o r mitochondria1 a n d chloro-
plast D N A of 0. sativa cv. IR36, a n d Dr. R. W u of Cornell
University f o r the clones pOa4, pOo2, a n d p O b l . This
AL. 797
research was supported by grant RF860593 ( t o J.P.
Gustafson) f r o m t h e Rockefeller Foundation.
AMANTE-BORDEOS, A.D., NELSON,R.J., OLIVA,N.P., DALMACIA,
R.D., LEUNG,H., and SITCH,L.A. 1991. Transfer of blast
bacterial blight resistance from Oryza minuta (acc. 101141) to
Oryza sativa. Proceedings of the 2nd International Rice Genetics
Symposium, May 1990. International Rice Research Institute,
P.O. Box 933, Manila. Philippines. In press.
APPELS,R., and MORAN,L.B. 1984. Molecular analysis of alien
chromatin introduced into wheat. In Gene manipulation in plant
improvement. Edited by J.P. Gustafson. Plenum Press,
New York. pp. 529-557.
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