Botanical Studies (2007) 48: 263-272.

*

Corresponding author: E-mail: bomchung@gate.sinica.edu.

tw; Tel: 886-2-27892701; Fax: 886-2-27827954.

INTRODUCTION

Cultivated rice is one of the most important staple food

crops in the world. In addition to two cultivated species,

O. sativa and O. glaberrima, the genus Oryza is comprised

of more than twenty wild species (Aggarwal, 1997; Ge

et al., 1999; Vaughan et al., 2003).

Oryza species are

classified genetically into ten genome types, i.e. the AA,

BB, CC, BBCC, CCDD, EE, FF, GG, JJHH and JJKK

according to the chromosomal pairing behavior at meiosis

of interspecies hybrids, genomic DNA hybridization, and

DNA sequence analysis of nuclear and chloroplast genes

(Moringa, 1964; Oka, 1988; Vaughan, 1994; Khush, 1997;

Ge et al., 1999), thus the phylogenetic relationships of the

genus Oryza was described (Ge et al., 1999).

Wild rice species are important genetic resources and

have been broadly introduced into rice breeding programs

for a long time (Chang et al., 1975; Sitch et al., 1989; Brar

and Khush, 1997; Khush, 1997; Nakagahra et al., 1997;

Xiao et al., 1998). Various molecular markers have been

proven efficient for discriminating specific genomes in

hybrids and monitoring genome introgression in some of

the breeding programs mentioned above.

Eukaryotic genomes contain abundant repetitive DNA

sequences. Most repetitive sequences spread throughout

genomes; however, a few repetitive sequences cluster

at unique chromosomal positions, which are useful

landmarks for chromosome identification. Because

most repetitive DNA sequences do not encode proteins,

mutations on such sequences usually will not make

significant changes in the phenotype. Therefore, repetitive

DNA sequences could rapidly accumulate a great diversity

in comparing with the unique coding DNA sequences

during evolution.

However, they are also constantly

homogenized via a molecular process called "concerted

evolution", so that few variants can be fixed (for review

see, Elder and Turner, 1995). Such a rapid divergence and

homogenization process results in some of the repetitive

sequences to become highly specific to a species (Dover

1982; Grellet et al., 1986; Ganal et al., 1988; De Kochko

et al., 1991), or even specific to a chromosome (Willard

and Waye, 1987; Wang et al., 1995). Some repetitive

sequences play important roles in chromosomal functions,

such as centromeric repeats (Ananiev et al., 1998; Dong

et al., 1998; Page et al., 2001) or telomeric repeats

mOleCUlaR BIOlOgy

a repetitive sequence specific to Oryza species with BB

genome and abundant in Oryza punctata Kotschy ex

Steud

Yueh-Yun CHENG, Shao-An FANG, Yao-Cheng LIN, and Mei-Chu CHUNG*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan, Republic of China

(Received August 23, 3006; Accepted March 28, 2007)

ABSTRACT.

Molecular markers are capable of discriminating specific constituents of genome and

monitoring genomic introgression of interspecific hybrids. In this study, we isolate and characterize a BB

genome specific RAPD, Opun210, from Oryza punctata Kotschy ex Steud (W1593), an African native wild

rice. We demonstrate the Opun210 as a highly species-specific marker. The

Opun210 sequence is 789 base

pairs in length and estimated at 5.3 ¡Ñ 10

4

copies in O. puntata (W1593) haploid genome, which contains the

most repeats of Opun210 among Oryza species. The results of DNA sequence alignments among Opun210

and a few hits in the GenBank found that a relatively high similarity was in position ~500 nucleotides regions

at the 5¡¦ ends, but a low similarity was in the rest of the nucleotides at the 3¡¦ ends. SCAR-PCR profiles

indicates that this fragment was specific to BB genome. Furthermore, the Opun210 sequence at position

430~480 nucleotides putatively encodes a peptide with 88% identity to a Ty3-gypsy retrotransposon protein or

a peptide with 94% identity to a hypothetical protein. The results of Southern hybridization and fluorescent

in situ hybridization (FISH) indicated that the repetitive Opun210 sequences dispersed throughout the entire

genome of O. punctata. The origin and divergence of the Opun210 sequence in genus Oryza is discussed

based on the investigations in this study.

Keywords: Fluorescent in situ hybridization (FISH); Oryza punctata; Repetitive sequence; Species-specific

RAPD.

264

Botanical Studies, Vol. 48, 2007

(Richards and Ausubel, 1988). Therefore, the organization

of repetitive DNA sequences and the correlation among

different repetitive sequences and unique sequences in the

genome are very important aspects of eukaryotic genome

characterization.

More than 20 repetitive DNA sequences have been

isolated and characterized from various Oryza species.

Some of the repetitive sequences were highly genome-

specific (for review see: Yan et al., 2002), and some of

them were physically localized to individual chromosomes

by using (fluorescent) in situ hybridization (Wu et al.,

1991; Wang et al., 1995; Ohmido and Fukui, 1997; Uozu

et al., 1997). However, none sequence with specificity to

BB genome has been previously described. In this study,

we firstly report a BB genome specific RAPD (random

amplified polymorphic DNA), which was amplified from

O. punctata.

The RAPD method allows investigating genomic

variation without prior knowledge of DNA sequences

(Williams et al., 1990). Most RAPD bands are known to

be generated from repetitive DNA sequences (Williams

et al., 1990; Devos and Gale, 1992; Echt et al., 1992);

however, a few RAPDs were derived from low-copy

sequences in the Hordeum species (Marillia and Scoles,

1996) and in the Triticeae species (Svitashev et al.,

1998). Several interspersed, species-specific repetitive

elements isolated from RAPD products were useful for

phylogenetic relationship studies and for interspecific

hybrids identification; some of them even can be used as

FISH markers for chromosome identification (Skinner

1992; Ko et al., 2002).

Here we report the isolation and characterization of

the first BB genome specific sequence, Opun210, which

was a RAPD generated from O. punctata (W1593).

Oryza

punctata, a wild species of rice native in Africa,

together with O. grandiglumis, O. latifolia, O. minuta,

and O. officinalis belong to Oryza ser. Latifoliae. Two

morphological types exist in O. punctata; the diploid

species (2n=24) with BB genome, while the allotetraploid

species (2n=4x=48) with BBCC genome. Our results

reveal that Opun210 sequence disperses throughout

O. puntata (W1593, BB) genome and presents more

frequently in this genome than in other Oryza species.

Opun210 can be use as a specific marker for characterizing

BB genome.

maTeRIalS aND meTHODS

Plant material

The accessions used in this study are shown in Figures

1 and 4, and Table 2. The seeds of wild species of rice

were kindly provided by the late Professor H. I. Oka,

National Institute of Genetics, Japan (Wild Rice Database,

http://www.pgcdna.co.jp/cgi-bin/wrdb/content.cgi). Plants

have been propagated for many years at the experimental

field of the Institute of Plant and Microbial Biology,

Academia Sinica, Nankang, Taipei, Taiwan.

Isolation and cloning of genome-specific DNa

Total genomic DNA extracted from young leaves of

plants following the protocol described by McCouch et al.

(1988). Protocol for RAPD analysis follows the descrip-

tion in our previous report (Wu et al., 2002). The primers

were obtained from the University of British Columbia,

Vancouver, BC, Canada (UBC Kit 1-4). Each 25 £gl reac-

tion mixture contained 25 ng of template DNA, 1¡Ñ PCR

buffer (10 mM Tris-HCl, pH8.8, 1.5 mM MgCl

2

50 mM

KCl, and 0.1% Triton X-100), 200 £gM dNTPs, 0.2 £gM of

a given pair of primers, and one unit of HotstarTaq DNA

polymerase (Qiagen). The amplification was carried out

in a thermal cycler (GeneAmp PCR System 9600, Perkin-

Elmer, Norwalk, USA),

programmed for an initial denatur-

ing for 15 min at 95¢XC, followed by 45 cycles of 5 sec at

94¢XC, 20 sec at 36¢XC, 90 sec at 72¢XC, and a final extension

for 10 min at 72¢XC. Reaction products were separated

by electrophoresis through 1% Nusieve GTG plus 1%

SeaKem LE agarose gels (Cambrex Bio Sciences Rock-

land, Inc. Rockland, USA) in 0.5X Tris-borate-EDTA buf-

fer (1X TBE: 89 mM Tris-borate, 89 mM boric acid, and 2

mM EDTA), and visualized by ethidium bromide staining

under UV illumination. The sizes of bands were estimated

by referring to 100-bp ladders (New England Biolabs) in

each gel.

Putatively genome-specific RAPD bands were excised

from the gel after electrophoresis; DNA fragments were

eluted and purified by a QIAquick Gel Extraction kit (Qia-

gene), then cloned into pCRTMII vector using a TA clon-

ing kit (Invitrogen, Carlsbad, California, USA) following

suppliers¡¦ instructions. The positive colonies (white) were

verified by PCR to identify cloned fragments. Inserts were

sequenced by using an Autoread sequencing kit and an au-

tosequencer (Amersham Pharmacia Biotech).

Southern Hybridization

Genomic DNA samples were completely digested

with various restriction enzymes (New England Biolabs),

then separated by electrophoresis at 40 kV (1 kV/cm) for

overnight on a 1% agarose gel in 0.5X TBE buffer, and

then blotted to Hybond-N

+

nylon membranes (Amersham

Pharmacia Biotech) by Southern transfer. Probe

preparation, membrane hybridization, and signal detection

were performed following the instructions of ECL

TM

direct

nucleic acid labeling and detection system (Amersham

Pharmacia Biotech).

Copy number estimation

To estimate the copy numbers of cloned DNA

sequences within each genomes, total genomic DNA of

each accession and a series of diluted recombinant DNA

were applied to a Hybond-N

+

nylon membranes through

a slot-blot template (Bio-Dot Slot Format, Bio-Rad

Laboratories, Philadelphia, PA) and performed Southern

hybridization as described above. The intensities of

hybridization signals on X-ray film were quantified with

a densitometer (Molecular Dynamics) and ImageQuant.

CHENG et al. ¡X Genomic specific sequence of

Oryza punctata

265

software (Amersham Pharmacia Biotech). The copy

number of the repetitive units estimation depended on the

relative intensity compared between the signal derived

from the genomic DNA and that from the series dilution

of plasmid DNA as previously described by Rivin et al.

(1986).

Chromosome preparation

Healthy root tips were harvested from germinating rice

seeds, pretreated in 2 mM 8-hydroxyquinoline at 20¢XC for

2 h to accumulate prometaphase cells, rinsed with distilled

water, then fixed in fresh prepared Farmer¡¦s Fluid (95%

ethanol : glacial acetic acid = 3:1) at room temperature

for overnight. Chromosome preparations were carried

out following the protocol described in Wu et al. (1991).

Root tips were macerated with 6% cellulose (Onoauka

R-10, Yakukt Honsha, Japan) and 6% pectinase (Sigma

Chemical Co., St. Louis, Mo.) in 75 mM KCl, (pH=4.0)

at 37¢XC for 70 min. After rinsing with water, tissues were

squashed onto slides in the same fixative. Slides were air-

dried and stored at -80¢XC until used for FISH. Slides were

dehydrated through an ethanol series (70%, 95%, and

100%, five minutes each) prior to be used in FISH.

Fluorescence in situ hybridization

The FISH procedure was performed following

the protocol described previously (Kao et al., 2006).

Digoxigenin-11-dUTP-labeled probes were detected using

a rhodamine-conjugated anti digoxigenin antibody (Roche

Diagnostics GmbH, Penzberg Germany). Chromosomes

were counterstained with 4¡¦, 6-diamidino-2-phenylindole

(DAPI). All images were captured by using a CCD

camera (Cool SNAPfx, Photometrics, Tucson, AZ), which

was driven by Image-Pro Plus software (version 4.5.1,

Media Cybernetics,

Yorktown, VA, USA), attached to a

Zeiss axioplan epifluorscent microscope (Axioplan, Carl

Zeiss AG, Germany). Final image adjustments were done

with Adobe Photoshop 6.0 (Adobe Systems Incorporated,

San Jose, CA, USA).

ReSUlTS

Identification and isolation of genome specific

DNa sequence in rice

A total of 131 decameric random primers were screened

for RAPD with highly polymorphic and well-resolved

genome-specific bands. Abundant polymorphism was

detected with all primers used in this study. Forty-three

among these primers could generate 243 products, which

showed distinctive and satisfactory amplification profiles.

The average number of discrete bands generated per prim-

er was 5.65, ranging from a single band (from W1577 by

UBC 189) to 16 bands (from W1564 by UBC 101). The

amplified products were approximately 300 to 2800 base

pairs (bp) in size. Reproducible amplification profiles

were further evaluated for the specificity of RAPD. Poly -

morphism is defined as the presence/absence of a particu -

Figure 1. RAPD profiles showing inter-specific/genomic, intra-

genomic/specific polymorphism detected among 14 Oryza spe-

cies with primer UBC 210. DNA fragments ca. 800 bp amplified

from O. punctata (W1593; arrow) were eluted and cloned for

further characterization.

Figure 2. SCAR-PCR profiles showing fragments ca. 800 bp

specifically amplified from accessions with BB genome. SCAR

primers were designed according to the sequences of the cloned

Opun210 sequence and UBC 210 primer. Products amplified

from cultivars Nipponbare and IR36 were far less than those

from accessions with BB genomes.

266

Botanical Studies, Vol. 48, 2007

lar band generated from different accessions by the same

primer.

In this study, polymorphism presents among genome

types and species, even among the different accession

numbers in same species. As shown in Figure 1, these

RAPD profiles amplified from 14 accessions with primer

UBC 210 (5¡¦-GCACCGAGAG) presented several distin-

guishable patterns of intragenomic/intergenomic varia-

tions. RAPD profiles (Figure 1) were distinguishable

between O. glaberrima (AA, lane 1) and O. sativa (AA;

lanes 2-4), between O. punctata and O. minuta (BBCC;

lanes 7 and 8), and between O. grandiglumis and O. lati-

folia (CCDD; lanes 11 and 12). Polymorphism even pre-

sented among different cultivars of O. sativa (lanes 2-4).

RAPD profiles of indica type cultivars IR36 (lane 3) were

different from those from japonica type cultivars TNG

67 (lane 2) and Nipponbare (lane 4). Although the same

primer was used, the RAPD profiles (Figure 1) generated

from diploid O. punctata (BB genome), both W1977 (lane

5) and W1593 (lane 6), and tetraploid O. punctata (BBCC;

lane 7) were similar but not identical. Polymorphic pro-

files were also generated from two O. officinalis acces-

sions, W1275 (lane 9) and W0567 (lane 10) in this case.

The remaining accessions, O. australiensis (EE; lane 13)

and O. brachyantha (FF; lane 14) showed unique amplifi-

cation profiles with primer UBC 210.

One distinct band ca. 800 bp commonly presented in

those profiles (Figure 1) of Oryza species with BB (lanes

5 and 6) and BBCC genomes (lanes 8 and 9) and two cul-

tivars of O. sativa (AA genome), indica cv. IR 36 (lane

3) and japonica cv. Nipponbare (lane 4). The DNA frag-

ments amplified from O. punctata (W1593; lane 6) were

chosen to be eluded from agrose gel and cloned for further

characterization.

One clone, designated as Opun210 was further charac-

terized in this study.

Opun denotes its origin species O.

punctata and 210 refers to the number of primer UBC 210

in UBC kit. The nucleotide sequence of Opun210, 789

base pairs in length, appears in the GenBank nucleotide se-

quence databases under the accession number DQ104697.

This sequence has been compared with 3,243,863

sequences in a databank including GenBank+EMBL+

DDBJ+PDB sequences (but no EST, STS, GSS, environ-

mental samples or phase 0, 1 or 2 HTGS sequences) by the

similarity searching method with the Basic Local Align-

ment Search Tool (BLAST) program from the Web at

http://www.ncbi.nlm.nih.gov/BLAST/. The results of this

similarity search with EXPECT threshold value better than

10 obtained 77 high-scoring segment pairs (HSP) without

gapping. The expectation values (E-values) reported by

similarity programs indicate nearly exact matches between

query sequences with each of the database sequences. The

results of alignments between the Opun210 sequence and

the top 50 matches showed about 90% similarity in ap-

prox. 500 nucleotides regions at the 5¡¦ ends, but of relative

low similarity to the rest at 3¡¦ ends.

Furthermore, the results of the similarity search for

coding proteins by BLASTX showed that the Opun210

sequence at position 430~480 nucleotides putatively

encoded a peptide with 88% identity to a Ty3-gypsy sub-

class retrotransposon protein annotated by O. sativa,

japonica cultivar-group (gi|62734207|gb|AAX96316.1|)

or a peptide with 94% identity to a hypothetical protein

LOC_Os11g22630.

We designed a pair of Sequence Characterized Am-

plified Region (SCAR) primers based on the sequence

data obtained above. Each SCAR primer contained the

original ten-bases of the UBC 210 primer at the 5¡¦ end

and the subsequent 14 internal bases from the end. The

synthesized 24-mers were: SCAR 210a (5¡¦-GCACC-

GAGAGAAGGAAGGAAGGGG) and SCAR 210b

(GCACCGAGAGTATATTGAACTGGT-3¡¦). SCAR-

PCR amplification of the rice genomic DNA of each spe-

cies was performed in a standard PCR. The SCAR-PCR

profiles are shown in Figure 2. A distinct band ca. 800

bp was amplified from several species with either BB or

BBCC genomes, including O. punctata (W1577, W1593,

and W1564; Figure 2, lanes 5-7) and O. minuta (W0045;

Figure 2, lane 8), respectively. The bands amplified from

cultivars IR36 and Nipponbare (O. sativa; Figure 2, lanes

3-4) were relative faint. No products were amplified from

the remaining accessions by this SCAR 210a/b primer pair

(Figure 2). These results confirmed that the Opun210 se-

quence was specific to the BB genome.

The abundance of Opun210 repeats in the rice

genome

The nucleotide sequence of Opun210 was also BLAST

searched against new genomic survey sequence (GSS)

databases with BLAST identity length > 120. The results,

as shown in Table 1, revealed that this fragment appeared

repeatedly, although at low hit percentages, in those

rice species with AA genome. The copy numbers of the

Opun210 sequence in Oryza genomes were estimated

by comparing the relative intensities of the signals of

quantitative slot-blot hybridization between standard

(Figure 3, left) and each genome (Figure 3, right). A

dilution series of cloned Opun210 DNA was referred as a

copy number standard in these estimations (Figure 3, left).

The haploid genome size of each species referred to the

data published in Plant DNA C-values Database (Bennett

and Leitch 2001, release 3.1, http://www.rbgkew.org.uk/

cval/homepage.html).

Table 1. The frequencies of Opun210 sequence found in GSS

database of rice genomes with BLAST identity length > 120.

O. sativa (japonica) O. nivara O. rufipongon

Library name OSJNBa OSJNBb OR_Bba OR_Cba

Reads NO. 73362 54097 106130 71006

Hits number

in database 43 17

88

74

Hits %

0.0586 0.0314 0.0829 0.1042

CHENG et al. ¡X Genomic specific sequence of

Oryza punctata

267

Oryza

punctata (W1593), from which Opun210 was

cloned, was found to have the most repeats of Opun210

among all accessions (Table 2). There were estimated

5.3 ¡Ñ 10

4

repeats of Opun210 in the O. punctata (W1593)

haploid genome, approximately 7.6% of its haploid

DNA contents. For the convenience of comparison, the

copy number of Opun210 sequences in each genome

is presented as a relative percentage of that found in

O. punctata (W1593), which is referred as 100%. The

relative copy numbers (%) are ranged from 4% in IR36

(AA) to 19% in O. minuta (W0045, BBCC) among the

Oryza species. In comparing with O. puctata (W1593),

less Opun210 repeats were found in another O. puctata

accession (W1577) and in tetraploid O. punctata (W1564,

BBCC), 14% and 11% in respective genome. In O .

officinalis (W1275, CC) and Nipponbara, the relative

copy numbers were 12% and 13%, respectively. In O.

glaberrima (AA) and O. gradiglumis (W1194, CCDD),

the relative copy numbers are below 10%. No Opun210

homologous sequences were detected in the O. latifolia

(W0542, CCDD) genome or in the O. australiensis

(W0008, EE) genome.

Distribution of clone Opun210 sequence in O.

punctata genome

The results of Southern hybridization showed multiple

distinguishable bands with a smear background in all

lanes of different restriction enzyme digestions (Figure

4). Those bands were not in ladder pattern, thus Opun210

sequences were not considered as the typical pattern of

tandem repeats. These results also indicated that the

Opun210 sequences moderately repeated and dispersed

throughout the entire O. punctata genome.

In situ hybridization results also demonstrated that

Opun210 sequences dispersed over all chromosomes of

the O. puntata (W1593). Although several chromosomes

obviously have more Opun210 sequences than the rest,

the localizations of Opun210 sequences were evenly

distributed on individual chromosome. No banding

patterns showing conspicuous signals as repeats clustering

sites were observed on individual chromosome (Figure

5). FISH results indicated that Opun210 sequence was

not a chromosome specific repetitive sequence and was

unsuitable for chromosome identification.

Figure 3. Quantitative slot-blot hybridization for estimation of

the copy numbers of Opun210 sequences in rice genomes. A

diluted series of plasmid Opun210 DNA (left) and 2 £gg genomic

DNA of each accession (right) were blotted on nylon membrane

and hybridized to Opun210.

Table 2. The copy numbers of Opun210 sequence found in rice genomes.

Species

Genome designation Genome size

(pg/1C)

a

Copy number/haploid

(relative copy number, %)

O. sativa, Japonica, cv. TNG 67

AA

0.43

b

3.2 ¡Ñ 10

3

(6)

O. sativa, Indica, cv. IR36

AA

0.48

1.9 ¡Ñ 10

3

(4)

O. sativa, Japonica, cv. Nipponbare

AA

0.43

6.2 ¡Ñ 10

3

(12)

O. glaberrima (W0025)

AA

0.43

7.5 ¡Ñ 10

3

(6)

O. punctata (W1577)

BB

0.55

7.4 ¡Ñ 10

3

(14)

O. punctata (W1593)

BB

0.55

5.3 ¡Ñ 10

4

(100)

O. punctata (W1564)

BBCC

1.13

c

6.0 ¡Ñ 10

3

(11)

O. minuta (W0045)

BBCC

1.18

1.0 ¡Ñ 10

4

(19)

O. officinalis (W1275)

CC

0.73

7.4 ¡Ñ 10

3

(13)

O. gradiglumis (W1194)

CCDD

1.00

3.4 ¡Ñ 10

3

(6)

O. latifolia (W0542)

CCDD

1.15

Not detectable

O. australiensis (W0008)

EE

0.98

Not detectable

a

Genome sizes as listed in Plant DNA C-values Database (http://www.rbgkew.org.uk/cval/homepage.html).

b

Estimated according to the genome size of O. sativa, Japonica, cv. Nipponbare.

c

Genome sizes as previously reported by Salles e al. (2001).

268

Botanical Studies, Vol. 48, 2007

DISCUSSION

In our studies, except for the Opun210, most of the

putative species-specific RAPDs finally showed no obvi-

ous genomic specificity by Southern hybridization (data

not shown). Our results suggested that RAPDs, although

polymorphic in size, often presented high sequence simi-

larity, as it has been reported previously (Williams et al.,

1993). RAPDs with similar sequences were thought to be

amplified from segments flanked by same priming sites,

but separated in different distances. Such RAPDs were

considered as the consequent events of insertion/dele-

tion within these regions during evolution. However, co-

migration RAPDs in the electrophoresis gel may contain

unrelated DNA sequences (Thorman and Osborn, 1992).

Nevertheless, RAPDs are efficient and inexpensive mo-

lecular markers, and have been proven successfully in

various taxonomic and phylogenetic studies (Kazan et al.,

1993; Wilkie et al., 1993). In this study, we have proven

the RAPD method as an efficient approach for isolation a

genome/species-specific repetitive sequence. Our results

indicated that Opun210, which was amplified from O.

punctata (W1593) with UBC 210 primer, was a whole ge-

nome dispersed repetitive sequence and was specific to the

BB genome.

In this study, we want to evaluate the efficiency of

RAPD method in differentiation genome/species-specific

RAPDs and investigation the organization of rice ge-

nomes. Although there were very few primers could differ-

entiate among three accessions of O. punctata, including

W1577, W1593, and W1564, UBC 210 primer could gen-

erate reproducible and distinguishable RAPD profiles from

each accession (Figure 1, lanes 5-7). This primer also

could discriminate two O. officinalis accessions W1275

and W0567 (Figure 1, lanes 9-10). These results suggest

that those segments flanked by UBC 210 primer are vari-

ous in copy number and size among genomes, therefore,

Opun210 can represent as a genome specific marker. Our

Figure 4. Electrophoretic fractionation (A) and Southern hybridization analysis (B) of the organization of Opun210 sequence in O.

puntata (W1593) genome.

Figure 5. The distribution of the Opun210 sequences on O. pun-

tata (W1593) mitotic chromosomes. The Opun210 sequence was

mapped to mitotic nuclei by FISH with a digoxigenin labeled

probe and immunologically detected by rhodamine-conjugated

anti digoxigenin antibody (red). Chromosomes were counter-

stained with DAPI (blue). Scale bar = 10 £gm

CHENG et al. ¡X Genomic specific sequence of

Oryza punctata

269

results suggest that RAPDs can provide not only detailed

analysis for verification at species level, but also an effi-

cient approach to isolate genome/species-specific markers.

Although the Opun210 sequences showed no specific dis-

tribution on individual chromosome, it still could be used

as an O. punctata specific RAPD marker. Such markers

will be useful for monitoring genome introgression in in-

terspecies hybridization breeding programs involving this

accession.

As shown in Figure 1 and Table 2, Opun210 is com-

monly found in species with BB genome, especially in O.

punctata (W1593, BB). In O. officinalis (W1275, CC),

although the band corresponding to Opun210 was absent

from lanes 9 and 10 in Figure 1, amount of Opun210

sequences could be detected by quantitative slot-blot

hybridization (Table 2). This implies that those distinct

bands mentioned above were amplified from fragments

with similar sequences but in polymorphic sizes. Oryza

officinalis (W1275, CC) and O. minuta (W0045, BBCC)

both geographically distribute in Asia. This may suggest

a close relationship between them based on the amount

of Opun210 repeats found in their genomes. According

to the phylogenetic relationships of the genus Oryza, spe-

cies with AA, BB, and CC genome types were grouped in

the same clade (Ge et al., 1999). The accessions with AA

genome, except a japonica type cultivar Nipponbare, have

less Opun210 repeats than those accessions with BB or

CC genomes. Nipponbare has as many Opun210 repeats

as O. punctata (W1577, BB; W1564, BBCC) and O. of-

ficinalis (W1275, CC). It indicated that AA genome was

eventually less close to BB genome than CC genome did,

while Nipponbara may have progenitors with BB or CC

genome. Opun210 repeats were less, or even absent in

species geographically distributed in central/south America

or Australia, such as O. gradiglumis (W1194, CCDD), O.

latifolia (W0542, CCDD), and O. australiensis (W0008,

EE) (Table 2). These species were grouped in different

clands from that contained AA, BB, and CC genomes (Ge

et al., 1999). Therefore, those bands, either conspicuous

or faint, present in profiles of CCDD (Figure 1, lanes 11

and 12), EE (Figure 1, lane 13), and FF (Figure 1, lane 14)

genomes were amplified from unrelated sequences with

the same priming sites.

About half of the rice genome is composed of repeti-

tive sequences (Kurata et al., 1994). The polymorphic

distributions of retrotansposons have been found different

among rice varieties. Retrotransposons are considered to

play important roles in rice genome diversity (Wang et al.,

1997; 1999). Based on the full genome draft sequences,

in silico survey of different kinds of repetitive sequences

revealed that there are approximately 38 Mb of long re-

petitive sequences and 150 Mb of short repetitive DNA in

the rice genome (Goff et al., 2002). A large fraction of the

moderately repeated sequences comprises transposon- and

other mobile DNA-related sequences (Mao et al., 2000).

Sequence analysis revealed that Opun210 might originate

from one of the retrotransposons, which commonly existed

in ancestral Oryza species. Mutations occurring at the 3'

end flanking sequences and consequentially rapid ampli-

fication with other relative elements might drive them to

become abundant and specific to O. punctata (W1593)

during evolution. Such putative possibilities can be re-

flected in the results of Southern hybridization. Southern

hybridization analysis showed that repetitive Opun210 se-

quences presented as several distinct bands or as smeared

patterns in restricted digestions (Figure 4). The distinct

bands, larger than Opun210 in size, may suggest that

those fragments contained sequences complementary to

the probe were present in several discrete configurations,

presumably next to other repetitive sequences. Smeared

hybridization signals were most probably due to the probe

sequence being hybridized to dispersed repeats or being

adjacent to single or low-copy sequences as previously re-

ported (Evans et al., 1983; Saul and Potrykus, 1984; Rivin

et al., 1986).

A comparative genomics program entitled the ¡¥Oryza

Map Alignment Project¡¦ (OMAP) has been embarked

(Wing et al., 2005). The OMAP aims to construct and

align BAC/STC -based physical maps of ten wild rice

species and one cultivated rice to O. sativa ssp. japonica

c.v. Nipponbare genome sequence finished by IRGSP.

The results of our study suggest that genome/species

specific repetitive DNA sequences are various in types and

amounts among the Oryza species and may play important

roles in the organization and speciation of rice genomes.

Therefore, such kinds of repetitive sequences may

consequentially determine the efficiency of alignments.

Acknowledgements. This work was funded by the

Institute of Plant and Microbial Biology, Academia Sinica,

and the National Science Council, Taiwan, ROC (NSC91-

2313-B-001-024) to Mei-Chu Chung.

lITeRaTURe CITeD

Aggarwal, R.K., D.S. Brar, and G.S. Khush. 1997. Two new

genomes in the Oryza complex identified on the basis of

molecular divergence analysis using total genomic DNA

hybridization. Mol. Gen. Genet. 254: 1-12.

Ananiev, E.V., R.L. Philips, and H.W. Ring. 1998. Chromo-

some-specific molecular organization of maize (Zea mays

L.) centromeric regions.

Proc. Natl. Acad. S ci. USA 95:

13073-13078.

Brar, D.S. and G.S. Khush. 1997. Alien introgression in rice.

Plant Mol. Biol. 35: 35-47.

Chang, T.T., S.H. Qu, M.D. Pathak, K.C. Ling, and H.E. Kauff-

man. 1975. The search for disease and insect resistance in

rice germplasm. In O.H. Frankel and J.G. Hawkes (eds.),

Crop Genetic Resources for Today and Tomorrow. Cam-

bridge University Press, Cambridge, England, pp. 183-200.

De Kochko, A, M.C. Kiefer, F. Cordesse, A.S. Reddy, and M.

Delseny. 1991. Distribution and organization of a tandemly

repeated 352-bp sequence in the Oryza family. Theor. Appl.

Genet. 82: 57-64.

270

Botanical Studies, Vol. 48, 2007

Devos, K.M. and M.D. Gale. 1992. The use of random amplified

polymorphic DNA markers in wheat. Theor. Appl. Genet.

84: 567-572.

Dong, F., J. Miller, S.A. Jackson, G.L. Wang, P.C. Ronald, and

J . Jiang. 1998. Rice (Or yza sativa) centromeric regions

consist of complex DNA. Proc. Natl. Amer. Sci. USA 95:

8135-8140.

Dover G.A. 1982. Molecular drive: a cohesive mode of species

evolution. Nature 299: 111-117.

Echt, C.S., L.A. Erdahl, and T.J. McCoy. 1992. Genetic

s egregation of random amplified polymorphic DNA in

diploid cultivated alfalfa. Genome 35: 84-87.

Elder, J.F. Jr. and B.J. Turner. 1995. Concerted evolution of

repetitive

DNA sequences in eukaryotes. Q. Rev. Biol. 70:

297-320.

Evans, I.J., A.M. James, and S.R. Barnes. 1983. Organization

and evolution of repeated DNA sequences in closely related

plant genomes. J. Mol. Biol. 170: 803-826

Ganal, M.W., N.L.V. Lapitan, and S.D. Tanksley. 1988. A mo-

lecular and cytogenetic survey of m ajor repeated DNA

sequences in tomato (Lycopersicon esculentum). Mol. Gen.

Genet. 213: 262-268.

Ge, S.T. Sang, B.R. Lu, and D.Y. Hong. 1999. Phylogeny of rice

genomes with emphasis on origins of allotetraploid species.

Proc. Natl. Amer. Sci. USA 96: 14400-14405.

Goff, S.A., D. Ricke, T.-H., Lan, G. Presting, R. Wang, M.

Dunn, J. Glazebrook, A. Sessions, P. Oeller, H. Varma et al.

2002. A draft sequence of the rice genome (Oryza sativa L.

ssp. japonica). Science 296: 92-100.

Grellet, F., D. Delcasso, F. Panabieres, and M. Delseny. 1986.

Organization and evolution of a higher plant alphoid-like

satellite DNA sequence. J. Mol. Biol. 187: 495-507

Kao, F.I., Y.Y. Cheng, Chow TY, H.H. Chen, S.M. Liu, C.H.

Cheng, and M.C. Chung. 2006. An integrated Map of Oryza

sativa L. Chromosome 5. Theor. Appl. Genet. 112: 891-902.

Kazan, K., J.M. Manners , and D.F. Cameron. 1993. Genetic

relationships and variation in the Stylosanthes guianensis

species complex assessed by amplified polymorphic DNA.

Genome 36: 43-49.

Khush, G.S. 1997. Origin, dispersal, cultivation and variation of

rice. Plant Mol. Biol. 35: 25-34.

Ko, J .M., G.S . Do, D.Y. S uh, B.B. Seo, D.C. Shin, and H.P.

Moon. 2002. Identification and chromosomal organization

of two rye genome-s pecific RAP D products use ful a s

introgression markers in wheat. Genome 45: 157-164.

Kurata N, Y. Nagamura, and K. Yamamoto. 1994. A 300 kilobase

i nterval genet ic m ap of rice i ncluding 880 e xpres sed

sequences. Nature Genetics 8: 365-372.

Mao, L., T.C. Wood, Y. Yu, M.A. Budiman, J. Tomkins et al.,

2000. Rice trans pos able el ements : a s urvey of 73,000

sequence-tagged-connectors. Genome Res. 10: 982-990.

Marillia, E.F. and G. Scoles. 1996. The use of RAPD markers in

Hordeum phylogeny. Genome 39: 646-654.

McCouch, S., G. Kochert, Z. Yu, Z. Wang, G.S. Khush, W.

Coffman, and S. Tanksley. 1988. Molecular mapping of rice

chromosomes. Theor. Appl. Genet. 76: 815-829

Moringa, T. 1964. Cytogene tica l inves tiga tions on Or yz a

species. In IRRI (ed.), Rice Genetics and Cytogenetics ,

Elsevier, Amsterdam, pp. 91-110.

Nakagahra, M., K. Okuno, and D. Vaughan. 1997. Rice

genetic res ources : history, conservation, investigative

characterization and use in Japan. Plant Mol. Biol. 35:

69-77.

Ohmido, N. and K. Fukui. 1997. Visual verification of close

disposition between a rice A genome-specific DNA

sequence (Trs A) and the telomere sequence. Plant Mol.

Biol. 35: 963-968.

Oka, H.I. 1988. Origin of Cultivated Rice. Elsevier, Amsterdam,

255 pp.

Page, B.T., M.K, Wanous, and J.A. Birchler. 2001.

Characterization of a maize chromosom e 4 centromeric

sequence: Evidence for an evolutionary relationship with

the B chromosome centromere. Genetics 159: 291-302.

Richards, E.J. and F.M. Ausubel. 1988. Isolation of a higher

Eukaryotic telomere from Arabidopsis thaliana. Cell 53:

127-136.

Rivin, C.J ., C.A. Cullis , and V. Walbot. 1986. Evalua ting

quantitative variation in the genome of Zea mays. Genetics

113: 1009-1019.

Saul, M.W. and I. Potrykus. 1984. Species-specific repetitive

DNA used to identify interspecific somatic hybrids. Plant

Cell Rep. 3: 65-67.

Sitch, L.A., R.D. Dalmacio, and G.O. Romero.1989.

Crossability of wild Oryza species and their potential use

for improvement of cultivated rice. Rice Genet. Newslett. 6:

58-60.

Skinner, D.Z. 1992. A PCR-bssed method of identifying species-

specific repeated DNAs. Biotechniques 13: 210-214.

Svitashev, S ., T. Bryngelss on, X. Li, and R.R. Wang. 1998.

Genome-specific repetitive DNA and RAPD markers for

genome identification in Elymus and Hordelymus. Genome

41: 120-128.

Thorm ann, C.E. and T.C. Osborn. 1992. Use of RAPD and

RFLP markers for germplasm evaluation. In Applications

of RAPD Technology to Plant Breeding: Proceedings of the

Joint Plant Breeding Symposia Series, Minneapolis, Minn.,

In D. Hoisington and A. McNab (eds.), Crop Science Soci-

ety of America. American Society for Horticultural Science.

American Genetic Society, Minneapolis, Minn., pp. 9-11.

Uozu, S., H. Ikehashi, N. Ohmido, H. Ohtsubo, E. Ohtsubo, and

K. Fukui. 1997. Repetitive sequences: cause for variation

in genome size and chromosome morphology in the genus

Oryza. Plant Mol. Biol. 35: 791-799.

Vaughan, D.A. 1994. The wild relatives of rice: a genetic re-

sources guide book. International Rice Research Institute,

Los Banos, The Philippines.

Vaughan, D.A., H. Morishimay, and K. Kadowaki. 2003. Diver-

sity in the Oryza genus. Curr. Opin. Plant Biol. 6: 139-146.

CHENG et al. ¡X Genomic specific sequence of

Oryza punctata

271

Wang, S., N. Liu, K. Peng, and Q. Zhang. 1999. The distribu-

tion and copy number of copia-like retrotransposons in rice

(Oryza sativa L.) and their implications in the organization

and evolution of the rice genome. Proc. Natl. Acad. S ci.

USA 96: 6824-6828.

Wang, Z.X., N. Kurata, S. Saji, Y. Katayose, and Y. Minobe.

1995. A chromosome 5-specific repetitive DNA sequence in

rice (Oryza sativa L.). Theor. Appl. Genet. 90: 907-913.

Wang, S, Q. Zhang, P.J. Maughan, and M.A. Saghai Maroof.

1997. Copia-like retrotransposons in rice: sequence hetero-

geneity, species distribution and chromosomal locations.

Plant Mol. Biol. 33: 1051-1058.

Wilkie, S.E., P.G. Isaac, and R.J. Slater. 1993. Random amplified

polymorphic DNA (RAPD) markers for genetic analysis in

Allium. Theor. Appl. Genet. 86: 497-504.

Willard, H.F. and J.S. Waye. 1987. Hierarchical order in chro-

mosome-specific human alpha satellite DNA. Trends Genet.

3: 192-198.

Williams, J.G.K., M.K. Hanafey, J.A. Rafalski, and S.V. Tingey.

1993. Genetic analysis using random amplified polymor-

phic DNA markers. Methods Enzymol. 218: 704-740.

Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and V.

Tingey. 1990. DNA polymorphisms amplified by arbitrary

primers are useful as genetic markers. Nucleic Acids Res.

18: 6531-6535.

Wing, R.D., J. S.S. Ammiraju, M. Luo, H. Kim, Y. Yu, D. Ku-

drna, J. L. Goicoechea, W. Wang, W. Nelson, K. Rao, D.

Brar, D. J. Mackill, B. Han, C. Soderlund, L. Stein, P. San-

Miguel, and S. Jackson. 2005. The Oryza Map Alignment

Project: the golden path to unlocking the genetic potential

of wild rice species. Plant Mol. Biol. 59: 53-62.

Wu, H.K., M.C. Chung, T.Y. Wu, C.N. Ning, and R. Wu. 1991.

Localization of specific repetitive DNA sequences in indi-

vidual rice chromosomes. Chromosoma 100: 330-338.

Wu, J.Y., H.K. Wu, and M.C. Chung. 2002. Co-dominant RAPD

markers closely linked with two morphological genes in

rice (Oryza sativa L.). Bot. Bull. Acad. Sin. 43: 171-180.

Xiao, J., J. Li, S. Grandillo, S.N. Ahn, L. Yuan, S.D. Tanksley,

and S.R. McCouch. 1998. Identification of trait-improving

quantitative trait loci alleles from wild rice relative, Oryza

rufipogon. Genetics 150: 899-909.

Yan, H.H., G.Q. Liu, Z.K. Cheng, X.B. Li, G.Z. Liu, S.K. Min,

and L.H. Zhu. 2002. A genome-specific repetitive DNA se-

quence from Oryza eichigeri: Characterization, localization,

and introgress ion to O. sativa. Theor. Appl. Genet. 104:

177-183.

272

Botanical Studies, Vol. 48, 2007