DNA RESEARCH 1, 139-148 (1994)

Determination of RAPD Markers in Rice and their Conversion intoSequence Tagged Sites (STSs) and STS-Specific Primers

Lisa MONNA, Akio MIYAO, Takakazu INOUE, Shuichi FUKUOKA, Muneo YAMAZAKI, Hui Sun ZHONG,

Takuji SASAKI,* and Yuzo MINOBE

Rice Genome Research Program, National Institute of Agrobiological Resources, 1-2 Kannondai 2-chome,Tsukuba, Ibaraki 305, Japan / Institute of Society for Techno-Innovation of Agriculture, Forestry and Fisheries,

446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305, Japan

(Received 13 May 1994)

Abstract

We produced 102 randomly amplified polymorphic DNA (RAPD) markers mapped on all 12 chromo-somes of rice using DNAs of cultivars Nipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers 10 nucleotides long were used bothsingly and in random pairs and about 1,400 primer-pairs were tested. Using both agarose gel and poly-acrylamide gel electrophoresis enabled us to detect polymorphisms appearing in the range from <100 bpto 2 kb. The loci of the RAPD markers were determined onto the framework of our RFLP linkage mapand some of these markers were mapped to regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs were determined by cloning, identifying andsequencing of the mapped polymorphic fragments.

Key words: molecular markers; polymerase chain reaction (PCR); random amplified polymorphicDNA (RAPD); sequence tagged site (STS); Oryza sativa

1. Introduction

Genetic markers with sequence information, such asSTSs, are very useful for constructing a physical mapand for map-based cloning of genes of interest. Poly-merase chain reaction (PCR) primers can be designedbased on the sequence to amplify the mapped region.Using these primers, one can easily pick up yeast ar-tificial chromosome (YAC) or cosmid clones containingthat region. A large number of mapped STSs makesit possible to construct a genetic physical map cover-ing all regions of the genome. In the Rice Genome Re-search Program (RGP), a genetic linkage map of rice hasbeen constructed by using the restriction fragment lengthpolymorphism (RFLP) method (Kurata et al., in prepa-ration), and sequencing of the mapped RFLP markershas been performed to generate STSs.1 Sequenced cDNAclones have also been used as RFLP probes to generatelarge numbers of mapped STSs. However, the RFLPmethod is time- and labor-intensive and difficult to lo-cate clones containing repetetive sequences on specificsites. Therefore, we use unique genomic or cDNA clonesfor mapping and there are still some regions with veryfew or no markers.

Communicated by Satoshi Tabata* To whom correspondence should be addressed. Tel. +81-298-

38-2176, Fax. +81-298-38-2302

The random amplified polymorphic DNA (RAPD)method is based on the polymerase chain reaction (PCR)using short (usually 10 nucleotide) primers of arbitrarysequences. Polymorphism of amplified fragments arecaused by: (1) base substitutions or deletions in the prim-ing sites, (2) insertions that render priming sites too dis-tant to support amplification, or (3) insertions or dele-tions that change the size of the amplified fragment.2

RAPD markers have been demonstrated as useful ge-netic markers3 for a variety of eukaryotic organisms, in-cluding humans, fungi and plants.

This method has been utilized for setting of geneticmarkers or segregation analysis in yellow birch,4 tomato,5

conifers,6 wheat,7-8 lettuce,9'10 and rice.11 This methodhas also been utilized for genetic fingerprinting.8'12'13

Recently, development of sequence-characterized ampli-fied regions (SCAR) from RAPD markers has beendemonstrated.10 In this method, cloned and sequencedpolymorphic fragments are used to design PCR primersto amplify the polymorphic regions. The inconvenienceof using RAPD markers for screening of YAC or cos-mid clones can be solved by establishing SCAR or STS-specific primers. A sufficient number of STSs enables usto generate YAC or cosmid contigs or to combine geneticlinkage maps with physical maps.

In this study, RAPD analysis comparing japonica andindica rice and of 186 F2 individuals from a single cross

140 Determination of RAPD Markers in Rice and their Conversion [Vol. 1.

of these parents was carried out using 60 random primers10 nucleotide long. We tested these primers pairwise, forapproximately 1,400 patterns, and generated 102 RAPDmarkers on all of the 12 chromosomes of rice. The loci onthe map were determined by placing them onto a frame-work map of RFLP markers. Twenty of these mappedfragments were cloned and sequenced to determine STSsand STS-specific primers.

2. Materials and Methods

2.1. Plant materials and total DNA isolationJaponica rice, cultivar Nipponbare, indica rice, culti-

var Kasalath and the 186 F2 segregants derived from across of these parents were kindly provided by Dr. M.Yano (Hokuriku National Agricultural Experimental Sta-tion). DNA was extracted from green leaves by CTABmethod.14

2.2. PrimersAll oligonucleotide primers were synthesized using a

DNA synthesizer (either model 394 or 380B, AppliedBiosystems, Mountain View, CA, USA). The sequencesof random primers used in this study are listed in Table1. Sixty arbitrarily designed 10 nucleotide primers weresubjected to RAPD analysis. Then these primers werepaired randomly for approximately 1,400 combinations(out of 1,800 possible combinations) and were also usedfor detection of RAPD markers. The advantage of ran-dom pairing of primers lies in increasing the variabilityof polymorphic fragments and also in reducing the costof designing primers.5'8

2.3. Conditions for polymerase chain reactionPCRs were carried out in a 20 /A reaction solution

containing 20 ng of genomic DNA template, 1 /zM ofeach primer (2 /xM in cases of single-primer PCR), 200HM each of dATP, dCTP, dGTP, and dTTP, 50 mM KC1,10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl2, 0.1% TritonX-100 and 1 unit of Taq DNA polymerase (Promega orWako, Pure Chemical Industries, Osaka, Japan).

Amplification was carried out in a PTC-100 Pro-grammable Thermal Controller (MJ Research, Inc.).Ninety-six-well MicroTestHI Flexible Assay Plates (Fal-con) were used to hold the reaction mixtures.

Sixty decamer primers with arbitrary nucleotide se-quences with G+C content of 40-70% were synthesizedand used for RAPD analysis. First, single-primer PCRswere performed using each of these primers, and thencombinations of two primers were tested. Amplificationwith two primers often showed different electrophoreticpatterns from those with a single-primer and the chanceof obtaining polymorphisms per primer increases greatly

Table 1. The sequences of 10-nucleotide random primers used inthis study. RA1 to RA8 were designed based on the publishedsequences of rice genes, and others were designed randomly.RA1 to RA30 contain 4-6 G+Cs and RA31 to RA60 contain 7G+Cs.

RA01RA02RA03RA04RA05RA06RA07RA08RA09RA10RA11RA12RA13RAMRA15RA16RA17RA18RA19RA20RA21RA22RA23RA24RA25RA26RA27RA29RA28RA30

GTCTGACGGTCAGCTCAAGTCGATCGAGGAGCAGAGCATCAAGCAGCAAGTCTTCGAGGAAGCACTTCGGCACCGTTCTGACTCCGCAGTGTCCTCTGAACGGTTTAACGTGAGTACGTCTATTGTCAGCCGCGATTTGAGACCATAGTCCCGACAGCTTATTTTACGCGATGGCCTTTATAGACAGTCGAATCGATACGAGGCCGTATCATGAGTCCACCTAGCTGACGTCAAACTCGGTACATGACAGCTGCAAAGATTAGCCGTCAATGCGGTCAACATTTGATCGCACGCTGATCA

RA31RA32RA33RA34RA35RA36RA37RA38RA39RA40RA41RA42RA43RA44RA45RA46RA47RA48RA49RA50RA51RA52RA53RA54RA55RA56RA57RA58RA59RA60

AACCGACGGGTGCCCTGCCTTGCGGACGTCCTTGCCTCCCAAGCTCCCCGGGGGGTCGTTTGTGGCCGGTCGGCAGTACCTGCTACGCCCGGCGGACTGTGAGTGCGCAGCCGGACTGAGCGCGGACGATTGGTCGCACGTACCACCCCGCCAGACCCTGCGGGAACCGAGAAGGCGCGTGTCACTCCCCGCTGTGCAGCTGTCCGGGTGGGCACCACCATCCGTCGTGGCGTAGCGCGAGGGCTCTAGCGGTGGGCAGATGTCCACCGGGCCCCATCACCGGGCAACGTCATCGGCCCT

and reduces the cost of synthesizing or purchasing a largenumber of primers.

The PCR conditions for RAPD analysis were as fol-lows: for pre-denaturation 1 min at 94°C followed by45 cycles of polymerization reaction each consisting of adenaturation step for 1 min at 94° C, an annealing stepfor 2 min at 35° C and an extension step for 3 min at72° C. The last cycle was followed by pause of 7 min at72° C to ensure that primer extension reactions proceededto completion. The PCRs using 20-mer primers specificto cloned polymorphic fragments were performed in thesame way but the annealing temperature was changed to60°C and the cycle number was decreased to 30.

2.4- ElectrophoresisFragments generated by amplification were elec-

trophoresed on 2% agarose gels and/or 4.5% polyacry-lamide gels (19:1 acrylamide/bis) with 0.5xTBE buffer.Each gel was stained with ethidium bromide, visualized

No. 3] L. Monna et al.

RA31 mix RA32 RA35 mix RA36 RA45 mix RA46

M N K N K N K M N K N K N K M N K N K N K

141

Figure 1. Efficiency of primer combination for detecting polymorphism. The DNAs of rice cultivars Nipponbare and Kasalath wereamplified with random primers indicated above and separated on 2% agarose gels. Primers were used singly and then mixed ('mix').See Materials and methods for the details of reaction conditions and Table 1 for the sequences of random primers. M: molecularweight marker 0X/Hae I. N; Nipponbare DNA was used as template. K; Kasalath DNA was used as template.

by illumination with UV light and photographed.

2.5. Linkage analysisThe map positions of polymorphic markers were deter-

mined using MAPMAKER15 by placing them onto a mapof approximately 1,000 RFLP markers previously estab-lished by the Rice Genome Research Program. Our previ-ous map containing 605 markers was already published.16

The latest map will be reported separately (Kurata et al.,in preparation).

2.6. Cloning and sequencing of polymorphic fragmentsPolymorphic fragments were purified after separation

by electrophoresis. Purified fragments were kinated byT4 DNA kinase, blunted by T4 DNA polymerase andligated to Smal site of pBluescriptll SK+(Stratagene).We transformed E. coli NM522 with the resultant con-structs. Single-stranded DNA extracted from 12 clonescontaining inserts of the appropriate length were usedfor the sequencing reaction using universal Dye-primer(Applied Biosystems) and Bca BEST DNA polymerasesequencing kit (Takara, Tokyo, Japan) according to themanufacturer's instructions. Sequencing was performedwith an automated fluorescent DNA sequencer (Model373A, Applied Biosystems).

Based on the determined sequences, pairs of 20-merprimers specific to polymorphic fragments were selectedusing OLIGO version 4.0 (National Biosciences, Inc).

3. Results

3.1. RAPD analysis by primer pairingWe synthesized 60 primers for RAPD analysis, 30 of

which having a 40-60% G+C content and the other 30

with ca. 70% G+C. Random pairing of these primersfor RAPD analysis is effective for detecting polymor-phic fragments which are not detected when using eachprimer singly (Fig. 1). Although purchase or synthesis ofprimers for RAPD analysis is expensive, random pairingenables us to screen 0.5 x N2 primer pairs with N primersinexpensively. Besides, sequencing of cloned fragmentsrevealed that polymorphic fragments often have differ-ent random primers on both ends when mixed primerswere used in the reaction. Thus, primer mixing makes itpossible to detect much more polymorphisms than usingprimers singly.

High (70%) G+C content of primers gave better resultsthan the primers of 40-60% G+C content (33 versus 18markers, respectively). Pairing of the high and low G+Ccontent primers produced 51 markers. The length of themapped fragments varied in the range 120-1700 bp, withan average of about 500 bp.

3.2. Generation of RAPD markersOne hundred and two RAPD markers were generated

in all areas of the 12 chromosomes of rice (Fig. 2). Al-though we determined the RAPD markers randomly,some regions were densely mapped, such as the distalend of chromosome 4, the middle of chromosome 5 andthe middle of chromosome 6. The RAPD markers ob-served on chromosomes 1 and 8 were widely scattered.The number of mapped markers per chromosome variedfrom 3 (chromosome 3) to 16 (chromosome 6).

Several markers determined in this study were local-ized in the regions where no previous RFLP markers hadbeen established. For example, a linkage group with sev-eral RFLP markers, which was previously unlinked to anyof the 12 chromosomes, was connected to the upstreamof chromosome 9 by generating RAPD marker P33. P13

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No. 3] L. Monna et al. 143

a) M 1 2 3 4 5 6 7 8

2.01.3

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b) M 1 2 3 4 5 6 7 8

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Figure 3. Two percent agarose gel (a) and 4.5% polyacrylamide gel (b) electrophoresis of RAPD products amplified from rice DNAsof F2 individuals (cross of cultivars Nipponbare and Kasalath). RA33 and RA43 were used as random primers. Sequences of RA33and RA43 are shown in Table 1.

and P24 made the length of chromosome 7 almost twiceas large as considered formerly. The telomeres of chro-mosomes 1, 6 and 8 were extended by RAPD markers.This shows that our method is useful to fill the gaps orto extend the RFLP linkage map of rice chromosomes.

The sensitivity to detect polymorphism between Nip-ponbare and Kasalath depends largely on the resolutionin electrophoresis. With 2% agarose gel electrophore-sis 0.6-2 kb fragments could be distinguished. However,polymorphisms often appear in the shorter fragments af-ter PCR and they could not be unambiguously detected^With 4.5% polyacrylamide gel electrophoresis, fragmentsof <100 bp-lkb are clearly separated (Fig. 3). Joint useof both gels for electrophoresis enabled us to detect atleast one polymorphism in almost every analysis. Thusa single F2 analysis could generate 1-8 RAPD markers.

Although RAPD markers are considered to be domi-

nant, one co-dominant marker was found (P74) as shownin Fig. 4. In this case, the third band larger than either ofparental bands appeared heterozygous. The third bandcould be generated by misannealing between denaturedssDNAs of both parental fragments.

3.3. Determination of STSs and STS-specific primers

Twenty STSs were determined and STS-specificprimers were designed (Table 2). Polymorphic bandswere cloned and sequenced from both ends, and the re-sulting sequence data was used for primer design. Us-ing OLIGO 4 software, an optimal primer-pair can beselected which has an appropriate Tm value and G+Ccontent, and which should not form a primer dimer orhairpin loops. Among the combinations of several primerpairs, specific pairs which produced only one fragment ofPCR product were selected as STS-specific primers.

144 Determination of RAPD Markers in Rice and their Conversion [Vol. 1.

M N K 1 2 3 4 5 6

Figure 4. Electrophoresis profile of co-dominant RAPD marker P74. RAPD products were separated on 4.5% polyacrylamide gels in0.5xTBE buffer (see Materials and methods). RA22 and RA32 were used as random primers and DNAs of Nipponbare, Kasalathand 6 of F2 individuals were used as templates. Note that heterozygous F2 individuals No. 2,4,5 and 6 have an additional fragmentlarger than either of parental fragments. See Table 1 for the sequences of RA22 and RA32.

Table 2. The RAPD markers mapped into the RFLP map which were converted into sequence tagged sites (STSs). The shown sequencesof selected 20-mer primer pairs can specifically amplify these mapped polymorphic fragments.

P name primers fragmentlength

positionchr/cM

specific primer 1 specific primer 2

P6P8P9P15P27P28P29P32P33P34P35P36P37P42P53P54P56P57P61P65

RA3+RA31RA3+RA31RA4+RA15RA7+RA12RA31+RA41RA42+RA46RA43+RA47RA43+RA49RA45RA45RA45RA45RA50+RA51RA23+RA27RA53+RA58RA53+RA58RA58+RA60RA46RA42+RA48RA33+RA43

280bp140800160800800

1100650

1100700310280600

11001500700270

1100350300

2/107.95/56.25/54.18/2.48/35.34/103.26/74.911/36.89/28.34/124.97/57.512/73.55/55.51/52.18/124.011/59.96/69.73/120.31/0.03/76.4

LP0109LP0115LP0081LP0119LP0103LP0093LP0111LP0089LP0135LP0083LP0085LP0087LP0101LP0315LP0319LP0324LP0145LP0137LP0139LP0321

GCAATCCAGTTTTTCCATTT LP0110CGAGGAGATAGTGCGTATTA LP0116GCAGAGCATCAGCCACTAAA LP0082CTTATCGTGTAAATCTGCTC LP0120CAGTTGTCTGTCTTTTTACA LP0104GAGAGGAGGCACTGATTTAG LP0094AGACCTTCAGACCGCTATGT LP0112CATCTTTTGAACCCATCTCG LP0090TAGTGCTAACAAATCAAAGC LP0136ATGCCATCATCTTGGTTTAC LP0084TCCTCTCCATCGTTCCTGAG LP0086AGAAAAACGGCTGAAAATGC LP0088GGTAGATAGTTCCTCTCATT LP0102AGGGATACATTGTGTCTTGC LP0316CATCACCTGCCACCAACTCC LP0320ACACCCACAGTCTCCATCAG LP0325TTCTGGTCAAACTGTGAAAG LP0146AAGAGAAAGTGAAGTGTAAC LP0138GGCAAGAGAACATCGGTAGC LP0140CCAATACAGCAGATAGACAT LP0322

AACCTTCCCTCTTCATCAAGCTATTAGCGGAAAGAGCAAGTGCAAGTGTGTCTTACCTAGTATGTCACCTATCATTGGAACCTCTAATCTGCGGAGTCATTGATAGCAGCAATGGTTTTTCGGACGATGGTGGTTCTGATGTCTCCATCTCTTTTACCTCTCACAAGAGGTTTTCATCCAAATCATCTTCCACATTCCTCTTGACCGACTGATGAGGACAACGCTGACTAATCCCTCCTATTTACTGCGTGGCTTGCTGACACGGTATGGAAAGAGGTGAATCGCCCTAAACAAAACCTGCAAGGCATTATTATTACCATGGTGATACCTTGGAGAAATCCTGATGGTAGCAAGAGTAACGTGTCAGATACAACCTTTTTCAGGAAGAACCAGTAAAATC

The fragments amplified with selected 20-mer primers(STS-specific primers) were much shorter than the orig-inal polymorphic bands because of the position of STS-specific primers settled inner side of the cloned RAPDfragments. These primers could be used for screening aYAC library.

Although we did not design STS-specific primers togive polymorphisms between Nipponbare and Kasalath,

4 of 20 pairs of STS-specific primers (P9, P36, P33, andP61, see Table 2) produced dominant polymorphisms,with a fragment in Nipponbare and with no band inKasalath, which agreed with the pattern of RAPD anal-ysis. In these cases, additional F2 analyses were per-formed using newly selected 20-mer primers. The segre-gation pattern of the new polymorphic bands amplifiedwith the STS-specific primers were identical to that of

No. 3] L. Monna et al. 145

a)M N K 1 2 3 4 5 6 M 7 8 9 10 11 12 13 14

b)

Figure 5. An example of F2 analyses both with original random primers (a) and with STS-specific primers determined in this study(b). a) RAPD analysis was performed using DNAs of Nipponbare, Kasalath and 13 F2 individuals as templates and RA4 and RA15as random primers. Polymorphic fragment showed by an arrow was mapped as RAPD marker P9 (Table 2). b) PCR analysis wasperformed using sequence tagged site (STS)-specific primers LP0081 and LP0082. Polymorphic fragment mapped as P9 was clonedand sequenced and the primers were designed based on the determined sequence. Table 2 shows the sequences of LP0081 and LP0082.

the original RAPD (Fig. 5). Other (16 out of 20) STS-specific primers amplified the same sized fragments fromboth parents, Nipponbare and Kasalath. To identify thecloned fragments, hybridizations of the cloned fragmentsto Southern blots of RAPD of F2 individuals was car-ried out using the cloned fragment as a probe. Then weconfirmed that the hybridization pattern was identical tothe segregation of RAPD (Fig. 6). As described by Paranet al.,10 this hybridization check is critical when gener-ating STSs from RAPD markers and when determiningSTS-specific primers.

Thus, we checked all 16 cloned fragments by hybridiza-tion. Together with the 4 pairs which showed polymor-phisms, 20 pairs of STS-specific primers were determined(Table 2).

4. Discussion

The advantages of RAPD mapping are: (1) cost effec-tiveness, (2) the fact no sequence information of templateDNA or synthesizing of specific primers is required, (3)relatively low amount or purity of template DNA can beused, (4) rapidity and technical ease, and (5) ability togenerate markers in the regions containing repetitive se-quences. On the other hand, the disadvantages are: (1)relatively low accuracy of linkage analysis because of itsdominant nature, (2) high sensitivity to PCR conditions,and (3) difficulty of direct use for screening YAC or cos-mid libraries. The third disadvantage, however, can beovercome by determining specific primers based on thesequence of the polymorphic fragments, i.e., determiningSTSs.

In this study, we produced 102 RAPD markers onall 12 chromosomes of rice using DNAs of Nipponbare,Kasalath and the F2 population generated by a singlecross of these parents. We synthesized only 60 primers

146 Determination of RAPD Markers in Rice and their Conversion

a ) M N K ^ F2 individuals *•

[Vol. 1,

b)

Figure 6. Identification of the cloned fragments by Southern hybridization, a) Electrophoresis of RAPD products amplified from F2DNAs using RA50 and RA51 as random primers (See Table 1 for sequences of RA50 and RA51). The fragment indicated by thearrowhead was mapped as a RAPD marker P37 (see Table 2). b) Hybridization of cloned fragment to the Southern blot of the gelshown in a). Note that the patterns of hybridization signals were consistent with the segregation of the mapped fragment.

for RAPD analysis, and over 1,400 screenings were pos-sible by using these primers pairwise.

We cloned mapped fragments for determination ofSTSs, but the possibility of miscloning an adjacent frag-ment could not be discounted. For identification ofcloned fragments, we performed Southern hybridizationof RAPD products of F2 individuals using each clonedfragment as a probe. Then we sequenced the identifiedfragments and designed 20-mer primer pairs specific tothe determined sequences (STS-specific primers). Six-teen of 20 pairs of STS-specific primers designed in thisstudy amplified fragments of the same size from all geno-types. These primers have the advantage that they canbe utilized for screening a library derived from eitherparental DNA. The remaining 4 pairs amplify fragmentsonly from a single allele. In these cases, these primerscan be used directly for verification of mapped loci (Fig.5) and identification of fragments may not be necessary.Although the library screening will be useful only for theparent DNA possessing the amplifiable allele, redesigningthe primers may solve this problem.

STSs mapped by RFLP or RAPD analysis, will bevery useful because YAC or cosmid libraries containingthe region of interest can be screened rapidly using syn-

thesized primer pairs which are capable of amplifying thespecific region. If sufficient numbers of mapped STSs areavailable, a complete physical map comprising the en-tire genome can be constructed. In the Rice GenomeResearch Program (RGP), sequenced cDNA clones havebeen used as probes for RFLP mapping, and mappedRFLP markers have already been converted into STSs.1

Screening and connecting of YAC or cosmid clones forconstruction of the physical map using mapped STSs hasalso been started.

So far we have not found obvious repetitive sequencesin the cloned fragments of RAPD markers. However,in preliminary experiments, hybridization of cloned frag-ments to the blots of restriction endonuclease digestedNipponbare DNA often showed smeared signals. Thissuggests that these fragments contain repeated or high-copy sequences (data not shown).

Although the RAPD markers determined in this studywere scattered on all chromosomes, there are still someregions possessing no markers for 10-20 cM (Fig. 2). Ournext challenge is to generate linkage markers in theseregions to fill the gaps or to tag genes of interest, by suchmeans as bulked segregant analysis, as well as to providesufficient numbers of linkage markers and mapped STSs.

No. 3] L. Monna et al. 147

Table 3. The loci of RAPD markers mapped in this study. Centimorgan data indicates the distances from the first marker of eachlinkage group shown in parentheses. The last marker of each linkage group is shown in brackets and the attached centimorgan dataindicates the total length of each chromosome of rice determined by RGP.

chr.lP61P62P26P22P42P77P23P102P67P70[R687

chr.2(C1470P49P117P86P6P17[C1357

chr.3(L375P65P60P57[R1468

O.OcM4.0

22.241.952.183.593.1

102.2122.0124.5193.6]

0.0)45.5

101.2107.0107.9112.7155.7]

0.0)76.476.6

120.3168.8]

chr.4(C445P28P74P46P16P66P30P85P34[R2373

chr.5(TEL1P25P21P47P10PIP9P37P8P71P105P83P68[C1230

0.0)103.2104.9108.8115.4118.2118.4123.0124.9128.8]

0.0)27.938.853.554.154.154.155.556.258.773.373.792.3

123.5]

chr.6(W160P5P3P56P7P81P69P121P29P31P78P59P72P41P12P98[P73

chr.7(R1744P24P13P132P58P35P120P82[C794B

0.0)60.366.069.769.769.769.769.774.974.977.484.693.295.896.9

129.8134.9]

0.0)26.646.554.156.357.559.087.3

128.7]

chr.8(C83P15P2P27P18P63P99P39P103P53[P122

chr.9(C4P107P104P33P52P88P93P100P134[L984

0.0)2.4

35.335.353.664.667.898.2

124.0124.0125.1]

0.0)10.325.928.366.071.971.971.974.9

102.5]

chr.lO(L769P133P14P89P44P91[C223

chr.l l(TEL3P119P76P l lP90P79P94P101P32P64P54P97P131P45[TEL2B

0.0)22.224.326.927.327.385.5]

0.0)2.6

15.629.534.535.035.035.036.837.359.967.968.7

121.1129.2]

chr.12(C901P38P80P96P4P36[TEL2A

0.0)58.761.163.067.673.5

110.5]

Conversion of the rest of the R A P D markers determinedin this study into STSs is also in progress.

A c k n o w l e d g m e n t s : We thank Dr. Ilkka Havukalaand Dr. Nori Kura ta of the Rice Genome Research Pro-gram for their critical reading and help revising thismanuscript.

References

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