CA2269215A1 - Genetic markers for rpsl-k gene and method of use - Google Patents

Abstract

The present invention regards genetic markers for the Rps1 gene of the soybean plant.
The invention further includes a method for identifying and developing genetic markers for the Rps1 gene. Additionally, methods for using the genetic markers in selective breeding and development of soybean plants and cloning of the Rps1 gene using map-based techniques are described.

Description

GENETIC MARI~RS FOR TH)EHt R,psl-k GENE AND THE METHODS OF USE
CROSS- ~ ~ n NCE TO RELATED A>PPL)GGATION
This is a 111 (a) Patent Appn of copending U. S. Provisional Application SJN
601083,439 filed on April 29, 1998.
TECHrIICAL FFIELLD OF THE INVENTION
The imrention described herein relates to the field of geaetics a~ the tion of genetic marlaers for the selective breeding of soybean plants.

BACKGROUND OF THE INVENTION
Soybean is a major cash crop and investment commodityr throughout the World.
For example; soybeans serve as a universal ant for both animal feed and human food production, and soybean oil is one ofthe most widely used edible oils in the World.
Moreover, the monetary value of soybean harvested in 1994 was to be at $30,390,000,000 (U.S. currency). Thus, factors that reduce soybean product~n, such as disease, ins~s, weeds and weather, can influonce the vv~el~e of entire nations.
Diseases have been a major problem of soybean production in the United States.
Phytop~o~a sojae (P. ,sojoe), which causes root and stem rot of soybean, is one ofthe most destructive diseases against soybean. (Kaufl~m) M. J.,1~t Gerdemann, J. W., (PhytopJethora megaspernta Dtechs. f. sp. glyicinea T . Kuan 8t D. C. Erwin). P. sojae is not continent specifics and therefore occurs in most of the soybean-growing areas of the World, which in addition to the United States) inchuies Canada, Austzalis, Hungary, Japan) and New Zealand (Athow, K.L., FroigrslDiseasesln Soybean: Imprav~ne~, Productiwt) and Uses., J.R.
Wilcox) ed. American Society of Agronomy, Madison, WL, pp 689-727 ( 1987). It has been discovered that minor of soybean confer diirn levels of ieaistaax to P.
so,~ae.
(Wallaer, AK., Schrnitthenner, AF., JYeritabik'ty of Toleraaoe to Phyto~thWrr rot in Soybean. Crop Sci 24: 490-491 ( 1984)). Such resistance, which conti~es toward defending the soybean against the pathogen P. sojae, is often referred to as field resistance, toleiarrre) or rate-seducing resistance. (Olah) AF., Scbmitthermer) AF.) and Wallcsr, A.K., Glyceolli~e Aac~amrlatian in Soybeans Lines Tolerma' to PhyRophtha~ nregr~ernra f. sp.
gfa.
Phytopathology 75: 542-546 ( 1985); Schtnittl~ner, AF., Cornpof Soybean Diseases.
aid ad. J.B. Sinclair and :P.A Baclm~n, American Phytopathological Society, St. Paul, M.N., pp 35-38 (1980)). The soybean gene most directly imrolved in providing resistance against the P. sojae pathogen is the appropriately labeled Rps (resistance to P.
s~ojae) gene.
Mole speciixalty a series of single-don»t Rps genes provide the race-specific resistance among soybean cultivars. As to date, at least Fourteen (14) Rps genes which originate fi-om seven di~nent loci, have been reporDed to provide resistance against thirty-seven (3~ recorded P. ,sojae races. (Schmittheaner, AF., Conrper~dira~r 'Soybean Diseases.
3rd ed. JB. Sinalair and P.A Beckman) American Phytopathological Society, St.
Paul, MN..) pp 35-38 (1989 Wand E.W.B., The l~eradion of Soya Bemns With Phytophthora megrrsperma f. sp. gfiycinea. PathoBenicity, pp 311-27 ( 1990); In Biological Control of Soil-Borne Plant Pathogeens. D. Hornby, ed. CAB Int., Walling~onl, UK; Anderson, T.R, and Buzael), RL, hiheritance arnd.L~~lrag~e of the Rps! Gene far Resistance to Phytophthora Rot ofSoy~rean. Plant Drs. '76:958-959; PoLdn) KM.) Lo~enaen, L.L., Olson) T.C.) and Shoemaker, RC., An UnusraT Po~Tym~ic Locxs Useful far Taggingllpsl Resistance Alleles in Soybean. Theor. Appl. Genet. 89:226-32 (1994)). Ofthese ~nurteen (14) R,~s siac reside at the llpsl locus (Polzin~ KM., Lorenz) L.L.) Olson, T.C., and Shoemaker, RC., Theor. Appl. Genet. 89:226-32 (1994)).
Recently, the ~~otics ofP. sofas avirul~e have been studied. (Wbiason) S.C.) Dnen~ A, Macdean, D.J) and hvvio, J.AG., Evidence far Outcrassing ire Phytophthora sofas aadl.gofa 1)NA Marker to TivoAvirrrlence Genes. Cue. Genet. 27:77-82 (1994)). Furthermore) p~enetic stud>es of the lips genes as they exist vv~in soybeans and the corresponding avini>e~e ofP. sojcre sug8est a classical gene-for-gene infraction.
(Flor, H.H., The Complementary Genic Systems in Flaac arrd Flax Rust. Adv.
Genet. 8:29-54 (1956)).
In soybean) Rpe 8e~-controlled mcan be exp~nessed as a variety of defense responses. A pm~emed method for detecting the Rps gene coMnolled resjatanoe is to measure the acaunulation of phytoalexin glyceollin. Phytoalexin glyceollin is a by-product resulting from an i~Ct>on of a soybean pleat containing the Rps 8ene by a P. sojae race c~tnying a corresponding avnulence gene. (Keen) N.T., ani Yoshikawa) M, ?Sre Expression ~' Resistmsae fn Soya Bearer to Phytoph~ora megasperma f. sp glyc~ecr. pp 329-44 ( 1990) as found in Biological Cornrol of Soil-Borne Plant Pathogens. D. Hornby, RJ Cook, Y. Hears, W.H. Ko, AD. Rovira, H. Schippersy and P.R Scott, eds. CAB International, Wallingfiord, UK). Thus, a soyb~n cuttivar coataimng the Rps gene is desired, however, determining whether the soybean cultivar contains the Rps gene through bioche~ic~l analysis and classical genetics is time consuming and expensive.
A metllud for expeditiously and inexpensively deng whether a soybean cultivar contains the Rps gene is needed. The invention descn'bed herein, however, answers such need by identifying and applying genetic markers ~or the Rpsl gene. In general, the use of genetic markers can assist in the indinect selection of agcvnomically favorable geness among segregating irldividuala in plant br~oeding populations. Marker-assisted selection is partiat>arly useful when the desired trait is largely ai~Ctod by the environment, which often cannot be adequately controlled to optim~e the expmssion of the trait. For e~le, the greater the elect of an environment on a trait and the less that environment can be controlled, the less will be that traits heritability and conoomi~rt predictability of phenotypes.
Genetic marloera~
additionally, prove the researcher with the genotypes ofdii~ent plant specs, which, in ei~Ct, limits the need for progeay testing. Moreover, the heritability of molecular marloa~s can be 100~/0. Very tightly linloed molecular marinas, for example, can serve as very useful criteria in praiicting the phenotypes or genotypes of attr~utss with low lsu~abibity. If a polymorphic 8enetic marber occurs within a fiew centimorgans of a gene acting a desired trnit, combination events between the marker and gene would occur nu~ely, providing the high he~itabi)ity and low error rate for reliable indimct selections of a favorable 8ene ZO and the trait it 8overns.
Marker assisted selection ~or Pkytopr~ona root and stem rot (PRST) n~sistance can only be accom~ished after one or more markers are found that are geaeticalty linlaed to one or mom PRST resistance lea. The Applicarrt of the invention descn'bed herein has isolated and genetic markers for PRST resistance and developed a method for using such msrlaers in breeding soybean plants that ate PRST resistant. Additionally, the Applicant has applied a method of using the previously unknown geaetic marlrers fir map-based cbning of the Rpal gene.

Other novel chaiscteristics of the im~ention which includes not only the physical sauctun3 of the markers but the method of their usa, together with fiuther objects and advar~tag~es ofthe ikon will be better understood fibm the following descriptions, examples and figures. Each e~le and figum, however, are mecety for the purpose of better dlusfraxing the markers and their use and not intended, in any fashion, to be a definition ofthe limits of the imrention.

The inveritifln provided herein includes a phua>ity of rnicleic adds molecules, each having a DNA sequence ofvarying lengths) for assisting in the selection and development of soybean plants canying the Rpsl gene. Each of these nucleic acid molecules function as a genetic marlaer for the Rpsl gene, in particular, the Rpsl-k allele.
The invention provided herein further includes a method for isolating each of the nucleic acid mold that fl~nction as gbnetic marlaers for the Rpsl gene.
The imrention pa~ovided herein fbrther includes for a method for cloning the Rpsl gene by isolating large DNA fragments from a soybean bacDerial arafiGal chromosome along with at least two (2) nucleic acid molecules to form a DNA bridge. The bridge is formed betvween the two (2) nucleic acid molecules and in between the molecules is the Rpsl locus.
Furthenmom, the i~rnion provided hendn inchides a method for identifying genetk marloers for the Rpsl gene which are located within SEQ m N0:38.
The invention provided heroin further inchules a phua>rty of nucleic acid molecules that assist in the development of genetic markers specific to the Rps locus.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a genetic mapping of modular s linlaed to Rpsl-k, indicating the map position of OPRK15 in relation to the Rpsl-k allele and RFLP rrmioers.
FIG. 2 depicts a mapping ofmolecular rnarlaers linked to Rpsl-k using near-isogenic lines.
FIG. 3 depicts the organ~tion ofTgmr, presenting the base pair sequences ofcertain fegdires: left (SEQ m NO:1) and right (SEQ ID N0:4) flanking tar8et sites and 3' end (SEQ 1o N0:2) and 5' end (SEQ D7 N0:3) for internal domain based on PCR-amplified product from line PI 103.091 (~sl-d); TS--tar8et site (SEQ ID NO:S); imper~Ct ter~l imbed mpeats underlined and comprising five (5) bp; the 5' end of the internal domain showing a twelve (12) by sequence ides~r to the 3' end sequencx ofthe beaJn initiator tRNA"' (SFQ ID N0:6~ a polypurine tract at the 3' end of interrml domain in bold letters.
FIG. 4 depicts the overall organization ofTgr~ showing LTRs~ 4 ORFs and internal domains. There are ten ( 10) mutations that resulted in stop colons (~) between the gag and RNaseH and one fi~ameshift mutation between ORF2 and ORF3. Ash the point mutation I~ding to &st stop colon between ORF2 and ORF3 resulted in a fismeshitt, then snc (6) mutations for stop colons ocxtn~d betvv~aen gag a~ RNaseH. RB= RNA
binding motif PT=protease; IIVT~nbegrase; RT=reverse tnmscriptase; and RH RNaseH. Tlre positions ofthe OPRK1S marker and different ial dotrmins ofthe Tgmr are shown.
FIG. 5 depicts a comparison of amino acid sequences across conserved domains of Tgrmr, SIRE-l, Tal 3, Tntl, and cqvia. Amino acid residues conserved among retrotransposons and nKxovirvses arse indicated by asterisks (*). Idemical amino acid residues are shown in bold. RNA binding Tgmr, (SEQ ID N0:7); (S,EQ 1D N0:8); ,~
.11~ (Sl Q ID N0:9); T~ -~ (SEQ ID NO:10); Ta L~, (SEQ I17 NO:11~ (SEQ ID
N0:12).
I~egrase: Tgmr, (SEQ m N0:13); Tnt ~ (SEQ I<? N0:14); Ta ],~, (SEQ lt? NO:15);
die, (SEQ ID N0:16).

Protease: Tgmr, (SEQ ID N0:17); ,~" (SEQ ID N0:18); Tnt LQ9, (SEQ ID N0:19);
Ta .1.:~ (SEQ ID N0:20); ~, (SEQ ID N0:21}.
Reverse tianscriptase: 'Tgmr, (SEQ ID N0:22); Tnt 1:Q4, (SEQ ID N0:23~ TA ~, 3 (SEQ
ID N0:24); ~ (SEQ iD N0:25).
FIG. 6 depicts the target sequences of Tgmr (SEQ ID N0:26) and sequencxs of equivalent regions from lines canyiag other Rp~sl alley. Tgmt (SEQ >d N0:26)) Williams 82; Rpsl-a (SEQ ID N0:27~ Mulo~en; Rp~sl-b (SEQ ID NO: 27), PI 84637; Rp~l-c (SEQ ID
N0:27~ Lee 68; Rp~sl-d (SEQ ID N0:28)) PI 103.091. The downward poirning open triangle (~ indicates the position of Tgmr.
FIG. 7 depicts a random ampli8od polymorphic DNA (RAPD) map ofthe ltpsl legion.
FIG. 8 depicts strategies used in isolating ar~lified fragment length polymorphism (AFLP} markers linked to ~rsl-k The strategies are presented as a schen~mtic representation of genotypes in F3 bullaed segnegant poolsy and recombinant pools used fur AFLP analysis.
Chromosome fragments of cv. Wiltiarn$ 82 were derived from cv. Kingwa and are denoted as th~k lines; chromosome fragments of cv. Willianis are shown as thin Lines.
Pool A is homozygous for l~sl-k and RAPD271(+) alleles; pool B is homozygous fdr rpsl-k and RAPD271(-); Pool C consists of recombinant homozy8ous resistant plants heternzy~us fvr RAPD271; and Pool D consists of recombinant homozygous siiscepri'ble pants heterozygous 2U for RAPD271.
FIG. 9 depicts a genetic map ofthe Rpsl r$gion ilhistrsting two (Z) random amplified polyrnorphic DNA (RAPD) mariloers (RAPD271 and Tgmr), seventeen ( 17) amplified fiagment length polymorpl>isms (AFLP) marlaers) and Fourteen (14) restriction fiagment length polymorphisms (RFiP) marloers originating from duplicated sequenczss.
Genetic ZS distances in centiimigaria are presented to the left of the map. AFLP
markers AA3 and CC3 are repetitive sequences that ale not polymorphic between resistant and susceptible parents.
Therefore, the map positions of these two (2) marlaers were determined only by the AFLP

mapping of sixteen (16) recombii~~a (rpsl-k rpsl-1~ RAPD271 [+/ ]~ whereas TC
1 and CGl were mapped by RFLP analysis from a large segregating p~pulabon.

11137ro3903 DETAILED DESCRZPT>iDN
Markers to a locus for the Rpsl gene, which is associated with Phytophthora root and stem mt (PRST) resistance were genetically identified and mapped. The markers include OPRK15, Tgmr, TC 1, CGl, RAPD 217 and RAPD 271, which pmvide new a~ vahiable tools to soybean breeders For selecting and developing future soybean cultivars having PRST
resistarxx. The described herein additionally provide the ~nundation for the initiation of a chromosorre landing or walking experimesrt, thereby enabling the isolation and ident>scation of the agranomically important soybeaa resistant 8ene, R,p~!-k.
The cbning of this Rps gene can contribute toward the elucidation ofthe recognition proces$
and the signal transduction pathway involved in the expression of racx-specific resistance in the soybean-P.
soiae irrter~rction. Furthermore, the cloned Rpsl-k gene can also be introducxd via direct tnmlsformation into soybean culuvars carrying alternative Rpvsl functional alleles to incorporate maistance to addit~r~gl P. sojae races T)~ OPRK15 and Tgr~r Markers Etiolated soybean cultivars or Fa seedlings oft6e cross Elgin (rpsl-k) x E420 (Rpsl-k) were preferably gniwn as deb by Ward, et. al. (Ward E.W.B., La~trov'rts G., Umvin C.H., Buzaell R.L, Hypo~catyl Reaatienss acrd GtyCeouin in Soybeans Inoaclated With Zoo~spnres of Phytaphthara nregaspernra oar. sojae. Phytopathology 69:951-55 (1979)). The etiolated hypocotyls were then inoculated with zoospores ofP. so~Qe racx 1 and preferably evahrated for symptom davelopmont two (2) days after ino<u>ation. The prefer inoculation and symptom evaluation were also performed according to the techniques set-forth in Ward, et. al., Phytopatholo~r 69:951-55 (1979). Susceptible plants to P. sojae were separated firm ZS all others. In totai, fifty .four (54) susceptible plants were selected for initial mapping of molecular markers and random amplified polymorphic DNA (RAPD).

11137!03903 In a separate e~xperinne~ Fx seedlings of the cross E300 (rpsl-k) x OX717 (Rpsl-k) wane preferably grown In light and unifoliate leaves were pne~erably irnculated for symptom development as descn'bed in Bhattacbaryya, M.K., Ward, E.W.B., F~ressioa ofGene-Sp~ec fic arid Age Related Resistance crr~d the Aacianulution of Glyceollin in Sayaerat Leaves Infected >Yith Phytopi~ra megrrsperma f. sp. glycinea. Physiol. Mol. Plant.
Path 29:105-13 (1986). Plants susceptible to P. sojae were SeparaDed from rrinet~fiour (94) others.
Leaves from 163 of the susceptr~le plarrts wane harv~ed for DNA preparation.
RAPD analysis was perform~i following the p~natocol of Williama et a1 (Wil>~
J.G.K., Kubelih AR; Livah K.J.) Rafalski, J.A, Tingey, S. V., DNA Polyneorp~rs Amplified byArbitraryPrimers are Tlseful as Genetic JIIarkers,. Nucl. Acid Ices. 18:6531-35 (1990)). The decamer aligns used in the RAPD procedure were obtained from Operoa Technobgles, Inc. in A>aareda, CA The OPRKI S msrkar is named after the oligo Kl 5, which has the sequence CTCCTGCCAA (SEQ ID N0:29). The OPRKI S-specific band (amplif ed from Williams 82, which lack the Rps 1-k gene, but not from Winiamss which irrchrdes the Rpsl-k gene) DNA template was 8e1-purified and used as a probe in Southern bbt analysis.
DNA was preferably isolated from etiolated cotyledons or leaves following the protocol of Whine and Kapper. (Vyllite, J.L., Kaper, J.M., A Simple Method far Detection of Viral Satellite RNAs in ~~rrall P&ag Tissue Srmrple~ J. Virol. Meth. 2'i:83-94 (1989)). An 8.0 E,ig portion ofDNA from individual samples was digested with individual restriction endoaucleasee~ run on a 0.8% agarose gel and blotted onto a nylon membrane using a preferned solution of 0.4 M NaOH and 1.5 M NaCI. The hybridization aril washing ofblots were preferably carried out following surd protocols, which can be found at Mamatis, T., Fritsch) EF., Sambmok, J., Moleculor Clarung: A LabaratoryMamral) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 3 83-89 ( 1982).

Molecular masker OPRK15 was initially mapped using fifty-four (54) arsarptible Fz seedlings of the cross Elgin (rpsl-k) x E420 (Rpsl-k). An additional 163 susceptible Fz seedlings ofthe cross E300 (rp~sl-k) x OX717 (Rp~!-k) wane used in mapping OPRK15. The restriction fiagment length polyimrphism (RFLP) markers pA 71 and pA 280, which am known to map in the Rpsl region (biers, B.W., Manson, L., Insande, J, and S>wemaker, R.C., MuppingPhyt~phth~a Res~cmce Loci in Soyibearr with Restridian Fra~nent I,e~th Polymarpldsms, werE kindly provided by Dr R. C. Shoernaloer) USDA ARS, Amen Iowa.
The map s and standard errors were calculated by the Map Manager program (emar'1:
mapmg~r(~mcbio.med.bu~lo.edu).
Higl~rmolea~lar-weight DNA was isolated from harvested leaves of two-week old plants and ground in an ice cold mortar and pestle with a pr~rred ion buKer comprising (0.1 M Tris-HCl (pH 9.4~ 0.1 M EDTA, 0.8 M KCI, 0.5 M sucrose, 1.0 mM
pheaylmethylsulforryl fluoride (PMSF), 0.5% Triton X 100, 0.1% 2rpboethanol, 40.0 mM speraridine and 10.0 mM spcrn>ine. The result was an fact, which was then filtered through a 100 ~n ~rlon membrane. Cells in the filtrate were cxahifuged down in a Sorvall RT6000 B centrifuge (DuPorlt, Boston MA) at an rpm that would form a call pellet, pm~erably 2000 rpm. The cell pellet was then rasuspended in an extinction bu~'er without PMSF. Centrifugation and resuspension of the cell pellets is buKer without PMSF were repeated two (2) additional times to wash the cell pellet. The cell pellet, after the final wash was next resuspended in an equal vohrme of extraction burr without PMSF to form a cell suspension. Tlra suspension was then preferably mixed with an equal volume of 2.0% agarose (low-~ng-point agarose, Sea Plaque GTG, FMC Bioproducts, Rockland ME) in TE50 (10.0 mM Tris-HCl pH9.4, 50.0 mM EDTA) at 45°C to form a . The mi~une was placed in a pref~d mold (BioRad Laboratories, Cambrid8e, MA) and arose plugs (760~c.1) wane obtained. The plugs were first preferably treated with proteirmse K ( 1 mg/ml) in 0.5 M

1113~~03903 EDTA (pIi 9.4) and 1.0~/o sarkosyl for 48 hours at 55°C with one chan8e of enzyme solution.
The plugs wam than preferably frosted with PMSF and then washed thoroughly with TE
bug'er before use. The high-molecu)ar-weight (HMW) DNA was psrdally di8ested withMboI
and sine-fisctior~ated in a sucrose ~radient. Other restr~ion endonuclsuch as B~ etc., can be used to digest the HIVlW-DNA
Sized-fiacrion~l DNA fragments ofappro~mately 50-75 kb were next pmfezably lito a Super Cos 1 vector (Stratagene Inc.) LaJol~, CA) and packa~.
Approximately 240, 000 color forming units (cfu) nod fi~nm the ligation of 1.0 ~cg lambda arms to the soybean DNA inserts. Inserts oftwenty (20) randomly cbnes that each of the clones carried soybean DNA with an average insert size of 38 kb. The tibtaty was plated preferably on 120 Petri plates (145.0 mm diatnetex) std grown overnight. The cobnies from individual plates were then pnefietably stored in glycerol ( 18.0'/0) at -70°C and used to propane DNA DNA fi~am each cosmid pool was preferably digested with H~ndlli, although t~estriction endonucleases that produce polymorphisms for OPRK 15 between Williams and Willatns 82 will work (e.g. EcaRi). Southern blot fivers were ptepar~ed and the blots were then hybridized with OP:EtKI S to ide:t~r cosmid pools carrying this msrloer.
Selected cosrtrid pools were scxeened by r"olony hybridization, and individual cosmids c~rryiag this were purified.
DNA was preferably sequenced by the dideoxy sequencing method . A Taq DyeDeoxy Terminator Circle Sequencing Kit (Applied Biosystems Inc., Foster City, CA) was pmfetably used according to the ma~s.ir~riuction. The polymetase chain ran (PCR) products wet~e separated electrophoretically, and resulting data was subsequently pnx~ssed by an AHB73A automated DNA soquencer (Applied Biosystetns). DNA and deduced amino acid sequences were anatyzed using GCG programs.

The DNA samples from soybean lines Mulcden (~sl-a), PI 8463? (Rpal-b), I,ee 68 (Rpsl-c) anti PI 103.091 (Rp~sl-d) were PCR-ausing two preferred oli8os based oa nucleotide sequences ilanldng the insertion sibs (5'-end oligo GTAATCTCTTTATAGTATGCATG (SEQ ID N0:30) and (3'-end olio CATTGTACCA
ATAATGATTG (SEQ m N0:31)) at a annea>mg temperature of 50°C for one (1) tninuOe and an extension at ?2°C for five (5) mirnttes. The PCR
products of less then 1 1~
were then purified from an agarose gel. A second PCR step was per>iormed using a second pair ofp~eferned oli8os (5 =end oligo GATTTGA-CATCATGTGAATTT GGAG (SEQ 1'D
N0:32 and (3'~nd oligo CCACTTCAGC TAGTGCAACT TGTATG (SEQ 1D N0:33)) at a preferred annealing temperature of 60°C for one (1 ) m>nute and an extension at 72°C for one (1) ~rnne: Products from the second PCR reaction of apptvximately 215 by were cloned into a T vector.
The'1" vecxor used to clone the PCR f ages was prepared pre6erably by digesting a modif ed BluescxiptII KS(+) plasmid withXcmI to 8enelate the 3'-end T
overhangs. The plasmid was modifed by inserting one of two pcefetted adaptors into the Nat1/BcaxHI and HXIroI sites of the plasmid, v~ely. The first pneferned adaptor, adaptor 1, inchtdes DNA sequences as presented in SEQ 1'D N0:34 and SEQ 1'D N0:35. The second prefexted adaptor, adaptor 2, inchtdeg a DNA sequence as presented in SEQ ID
N0:36 and SEQ ID N0:37. The preferred adaptots wee also designed to have 2 XcmI sites in opposite orns to obtain T-overhangs at the 3'-end al~erXcmI di~ion) In addition to XanI
sites, either an Mhd or SftI site excel to the XcmI sites was preferably included . The ~-~actosidase reading fisme of the 'T' vector was maintained to allow blueJwhite selection of tecombirmnt clones. We have termed the new plasmid vector, pRG51.

Near-isogenic lines (NILs) of Williatns and. WilBams 82 were used in ident~ag the RAPD marker OPRK15) which approxiamtely inchuies bases 3223-4080 of the Tgmr sequence (SEQ ID N0:38). The OPRK15 nsrloer was preferably obtained after scn~ning 175 decameroligos in P(:R n;actions accozding to the protocol in (Williams, J.G.K., Kubelik, AR., Lival~ R.J, Rafalsk~ J.A, Tingey, S.V., DNA PolymarphismsAmplifred byArbitrary Primers Are Useful as G:endic Mcaker~ Nucl. Acid Res. 18:b531-35 (1990)).
Oligos aad primers were obtained from Operon Technologies) Alameda, CA Southern bbt analysis of the OPRKI 5 marker showed several common bands in both the Wz'8iams DNA and Williams 82 DNA) and two additional bands only in Williams 82 DNA digested with H~dIIt.
The dominant OPRK15 marker, and two (2) previously mapped RFLP marlaers pA 71 a~i pA
280, (Died B.W., Mansur, L., Imsande, J., Shoema)aer, R.C., Mapping PhNo~thora Resistmzae Loei in Sayibean With R~strirxia~t Fragment Length PolymoMmvers:
Crop Sci. 32:377-83 (1992) ~w~ere mapped using fifty-four (54) susoephble Fa plants obtained from a cross between Elgin (rpsl-k) and E420 (Rpsl-k). OPRK 15 co-segregated witty the lipsi-k gene. An additional 163 susceptible F~ plants obtained from a different cross betweea E300 (rps!-k) and OX717 (Rp~rl-k) were evaluated ~or possible zeoombination betvveezz OPRK15 and the Rpsl loci. Only one z~ocotion event was zeconied between these 2 loci.
FIG. 1 depicts a genetic map ofmolecular marlaers linlaed to the Rpsl-k 8ene indicating the map position of OPRK15 in relation to the Rpsl-k allele. FIG. 1 also z'llustrates the map position of PA 'dl and PA 280 in zelstion to the Rps1-k allele. The map of pA 71 and pA 280 was based on the analysis of Sfly-~our (54) susceptible Fs plants of a cross between Elgin and E420.. For mapping pA280, genomic DNA was digested with Hind while far pA 71, 8enomic DNA was digested with TagI. The map distances were calculated by using the MaP M~B~' PmB~

OPRK15, pA 71, and pA 280 were also mapped using a series ofNILs ofdi~ent genetic backgrounds. These ptefemed NILs diiFer at the Rpsl-a, Rps!-c, or l~sl-k alleles. A
dominant resistance-specific OPRK15 band was observed in all lines carrying Rps!-k but not the.Rpsl-a or Rp~s!-c allele. FIG. 2 depicts a mapping of molecular roarlaecs linked to the Real-k 8ene using the NIL.S. DNA from fourteen ( 14) peas of NILs dii~ng at Rpsl-a, eight (8) pairs at Rps!-c, and eleven (11) pairs at Rp~sl-k were drably di8esbed with D9ral;
Taql; and D~ mspe~ively. Southern blots pn~ared fivm the DraI-di8ested DNA
were h~ybridi~d to RFLP msrke~rs pA 280 and OPRK15 individually, whereas blots carrying the TaqI digested DNA were l~rbridi~ed to gA 71. Polymorphic band patterns between recurrent and donor parents were ecornd as allelic cormast, while similar band patterns between mcurnent and donor lines were scomd as non-allelic contrast. Pairs of lines showing allelic conhast am in the histogram of FIG. 2. For example, only two (2) out of fourteen (14) pairs of NILs dii~'ering at Rp~sl-a showed allelic oornrast or polymorphism for pA ? 1.
Additionally, dissimilar band patterns for a RFLP marker between the donor parent and the NIL carrying the introgressed region indicated a crossing over everrt betvuecn the ~Sl and RFLP loci. For ele) a single pair ofNIL out ofelevea (11) pairs di~edng at Rpisl-k showed recombination between Rp~sl-k and pA 71. The upper part of the histognun in FIG.
2, dhist~es no recombination ofNIL pairs while the lower part r7hrsttates pairs ofNIL,s that do not show allelic contrast among themselves but show allelic contrasts with their corresponding donor parems ~or the sper~c a~rker. FIG. 2 further ilhrstzates the absence of the OPRKI 5-specific band in lines cx<rrying either ~l-a or Rp~sl-c. Atmlysis of Harosoy isolines carrying five (5) dint Rpsl alleles also revealed that dorrrinart OPRK15-specific bands were present only in lines carrying the Itpsl-k allele. Sequence analysis high qty of OPRK15 with the reverse ttanscriptase of Tntl.
To characterise the DNA sequence ofthe OPRK15 marloer, a co~nid lrbrary about eight (8} soybean genome e~quivalerrts of DNA was pmferably constructed in the Super 11137!03903 Cos 1 vector (Stratagene) and stored in 120 pools each carrying approximately 2000 cfu.
DNA was isolated iivm each ofthese pools and digested with HindIii. Southern blot analysis) as known in the art, was perBormed on the HindJ~ digested DNA using the OPRKI 5 marl~r. The results ids nine (9) independent cosrnid pools that carried the specific sequences. One pool was screenod and cosmid 31) wh~h contains the sequencea~ was . Additionally, Southern blot analysis of DNA from the cosa>id 31, which was digested by four (4) restriction erdonucleases, cor~rmed that this clone cont$ined the OPRKI 5 sequence. From this clone, OPRK15 spxif>c HF.~RV, and DmI
fiats were sub-cbned into the p~lasmrd vector Bhiescript II (SK(-)) (StrataBene) fior further characteri~t~n of this .
ID>BrNT)T'ICATION OF THE RETROTRA1~1SPOSON Tsar MARKER
DNA was preferably sequenced by the dideoxy sequencing method. A Taq DyeDeoxy Termirmtor Cycle Sequencing Kit - (Applied Biosystems) was used according to marw~cdu~er's instructions. Sequencing wan performed along most of the approximately 6 kb DraI iiagneat) which was cloned into BhieScript II vector. The resuhs ide~ified a 4965 by copies-lilae retrotzanspoeon element. The sequence ofthis 4965 by copia~h'lae retrotransposon elen»t approximately comprises nucleotides 306-5270 (SEQ TD N0:38) ofthe Tgmr sequence. This element, additionally has two (2) almost identical long teal repeats (LTR) with the (5'-e~ LTR having 259 by at nucleotides 300 to 564 of SIrQ >D N0:38;
and the 3'-end LTR having 248 by at nucleotides 5023 to 5270 of SEQ ID N0:38.) On a final note, both LTRs carry an imperfect tenrrinal repeat sequence (5 =TCTTA.......TAATA
3'~
which is illustrated in FIG. 3.
The Tgrvriequence also shares a twelve ( 12) nucleotide (TGGTATCAGAGC) (SEQ
ID N0:39) iderrtit3r with bean tRNA;~ at the 5' end. (Canaday, J, Gur3lemaut, P., WeB, J.H., The Nrrcleat~de Sequences of the hritiatar Ti~arrsfer RNAs from Bemr Cytopla~r card Chlaropbsts. NucL Acid Res. 8:999-1008 ( 1980)). This identity is further illustrated at rnicleotides 565-576 of SEQ ID N0:3 8. Additionally, the Tgmr carr~s a purine-rich sequence (GA~GGGGGC'~Cr; nucleotides 5013 to 5021 of SEQ 1o N0:38) adjacent to the 3'-end LTR, which is common in other plant netroelemerrts (Grandtrastien, M.A, Retroelements fn Higher Plants. Trends Genet. 8:103-08 ( 1992)).
Upon imertion of the cvpia-like mdvtnu~c~son element, the five (5) by (TAGGG) target site wan duplicated. The element apparently carries four (4) non-overlapping open reading (ORFS), presumably insulting fiom at least six (6) point mutations, which produced six (6) stop cadons. Such results appear acxeptable ifwe assume that the point mutation leading to the diet stop codon) between ORF2 and O1ZF3, induced the fi~ameshift.
FIG. 4 inusaates such a &ameshift. As a result ofthis frameshift, the element did not carry the functional prot~se, reverse transcriptase and RNaseH genes Found is other retrotransposons (Flavell, AJ., Retroelenrents; Re»erse Th~xiptase ar~d.Evwlrrtton. Comp.
Biochean. Physiol. 1108:3-15 (1995); Grandbastieo, M.A, Retmelem~ts in Higher Plus.
Trends Genet. 8:103-08 (1992)). The elerneut, how~aver, did carry all the es~ial irreernal domains that show sib id~tity to those of other -like retmdements SIR~l, Tntl, Tal and copra. (Bi) Y.A) Lath H.M., Sequence Analysis of a cDNA
Containing the Eag and prat Regions of the Soybeme Retrovirus-Like F.hnennt, SIR~I. Plant Mol. Hiol.
30:1315-19 (1996); Grandbastien, M.A, Spielma~, A, Cabochey M., Tht~ A Mobile Retroviral~ii<e ale Element of Tobca;co Isobted by Pdmrt Cell Genetics Nature 337:376-80 (1989); Mount, S.M., Rubin, GM., Canplete Nucleotide Seq~ce of the Dra~ila T frrnt~a~able Element Copra: Ho~noloRy Between Copra and .Retroviral Proteins.
Mol. Cell Biol. 5:1630-38 (1985); Voytas, D.F., Ausubel, F.M., A Copra Z.uEe T~vp~able Element Family in Arabidopsis thaliarx~ Nature 336:242-44 (1988)). Table 1) along with FIG. 4 and FIG. 5, further illustrate the similar general structure betvinen the copra-like ietrottan~son element and other copra-lilae retrnelements. Thus, based on the similarity of general stnu~ture and identity of the amino acid sequences of the Tgrrs' with that of other copies-like retroelernerits and the presence ofa non-functional protease, a reverse and a RNaseI~ we have concluded that Tgmr is a non-autonomous copies-like retrotlansposon.

Table 1. Comparison other of Tgmr with copies-like elemerrts Homology Amino Tgmr (%)' acid identity with domain Tntl-9~f Tal3 cawia GAG 26.3 29.2 23.7 ILVT 37.3 36.0 37.3 RT 38.3 34.0 36.1 RH 39.3 31.7 41.6 P'er~t amim said identity as measured by BESTFTT of the GCG pacl~g~.
G~T~ 8a8 Pi'o~~ ~T. ~8~~ RT, wares bansaiprtase; RH, RNaseH.
It is generally understood that retrotn3nsposona transpose through RNA
intermediates and, with the assistance of the enzyme ~werse transcriptase) produce a DNA
copy in a new chromosomal location. Although the original element remains independent (thereby increasing its copy number), the inten~al domains of the elemerrt can be deleted due to reansn.
For example, Vegh, et al. have reported that the retnntzanaposon-lilac sequence Tmsl of Mediaago sativre rearranges. (Vegh, Z., Ymcae, E., Kadirov, R, T6th) G., Kiss, G.B.) Tfie Nucleotide Seqm~ of a Norlerle.,~ecf,~c Gent Nms-2S ofMec~ago satires: Its Primary Evnhrtia~ Yia Fxare~Sht~ling and Relrae~po~sae~l~ledia~ted DNA
Recsrcmgerrtents Plant Mnl. Biol. 15:295-306 ( 1990)). Thus, to rule out the possrbility that the observed lack of a Tgmr element bnloed to Ppsl alleles (other than Rpsl-k) could be a result of similar reanangetnents in the 8anldng regions of the other four Rpsl alleles equivalent flanking target regions of the Tgmr sequence from lines carrying dii~erent Rp~sl al~les were PCR

1113?/03903 amplified in a sub-PCR reaction. The ie~dting PCR-product was then s~uenced either directly or after cloning into a plaamd vector. Analysis of the equivalent Banking target sequerrxs from diet lines corrfirrned that Tg~ was inserted only in the flanking region of the R~1-k allele. These findings an further illustrated in FIG. 6. Southern blot analysis, perfpnned as described above, demonstrated that only DNA of soybean cultivacs comprising the Rpsl-k allele hybridized to the Tgmr specific bands.
T'HE TCi AND CGi MARKERS
to soybean cv. wiuiams and ics N>1, will;ams 82) c~. Elgin and its Nlz. Eazo, c~. E3oo and its N1I. 0x7171, and the ltpsl-k source cv. Kingwa were used. The NIis Wil)iams 82, E420, and 0X7171 contain the ltPsi-k allele and are resistant to at least 21 races) inchuiing rice 1 ofP. sojae. (Scbmitthenaer, AF.; Hobe, M, and Bhat, R.G., Phytaphthara sojae Races in Ohio Over a lO-Yearlntervrrl. Plant Drs. 78:2fi9-7b (1996)). Willisms 82 is a backcross-derived variety devebped at the Univezsity of Il>;noisy Urbane.
Willisms was crossed with Kingwa as the source ofRp~sl-k Followed by six backcrosses. The ii>ials were selected ~or P. sofas race I resistancx. From this baclacross and selection, four (4) BC6F~
lines homozygous for ~l-k wens pmfesably bulked for Williams 82.
Elgin 87 is a backcnoss-derived variety developed at Iowa State University, Ames. It is derived by crossing Elgin with Williams 82. After four baclacrosses and selection for resistance, tvve~rty-one (21) BC4Fs lines homozygous fvr R,psl-k were pre~rably bullood for Elgin 87.
0X717 was obtained at the Harrow Research Center, Ontario, Canada, by backciossing Elgin 87 to Elgin and selecting BCS F~ lines homozygous Sot Itpsl-k E300 and E420 wee obtained at the Harrow Research Center by mitagenizing Elgin and Elgin 87, respeL~tively. A pnefer~d compound for mutagenizing Elgin and Elgin 87 is ethylmetbane sul~onate. After this tteatrnent, super-modulating lines were selected. E300 is homozygous for rpsl and E420 is homozygous for Rpsl-k.

For linka8e analysis, three independent populations were analysed. The populations analysed consisted ofthe FZ arxl F3 pro8e~r of the Elgin x E420 cross, the Fa and F3 progeny of the E300 x OX717 c .roes, and the F3 and F4 progeny of the Williarns x Williams 82 cross.
The F4 fannies were oM~ained from selected F3 s that wane heterozygous for both l~sl and the RAPD 271 loci. Other populations can be used as long as they segregate for Rpsl-k Although several methods of growing seeds ~cist, etiolated seven (7) day old seedlings or two (2) week-old green seedlings wem grown under conditions pmviously descn'bed by Ward, e:
a1 and Bhattacharyya, et a1 (Ward, E.W.B., Lazarovitss G., Unvvin, C.H., and Buz~l, RL, Hypocotyl Reactions and Glyr,~eollin in Soybeans Inoculated With Zoo-Spies o,~
Phyta~hara megnspeTma vim: sojae: Phytopathobgy 69:951-55 ( 1979);
Bhattaclmryya) MK, and Ward, E.W.B., Expression of Gene-Spec mad Age~telated Resis~ce and tI~
A~iaa of Gly~cedl~ in Sopbec~ ~eaHes l~'eded with Phytophthora raegrrsperma f.
sp.
gty~a. Physiol. ~rol. Pant Pathol. 29: los-13 (19s6)).
Sagmgating materials were pre~arably tested for their responses to P. sojae race 1 by inoculating unwounded, etiolated or detached leaves with P. so, jae. (Wand) E.WB., et. al., Phytopatbology 69:951-55 (1979) (Bhatrac6aryya, M.K., and Ward, E.WB., Physiol.
Mol. Plant Pathol. 29:105-13 ( 1986)). The P. sofae race 1 was prefaabty grown in the dark at 20 °C and ~ospores were obtained from six (6) day old culture. Seven (7) day old etiolated seedlings were inoculated and preferably maintained at 100% relative lnu~dity in the dark at 25 °C. Re$istarn and susceptible msponses were scored twenty (20) to four (24) hours after inoculRtion. After disease evaluation) tissues fi~ee of fungal imrasion were harvested fivm each individual seedling and inunediately frown for DNA
isolation. The tissue was preferably fio~en in liquid nitrogen. Twenty (20) etiolated seedling of each F3 family were evaluated for disease developm~t and DNA was prepared from cotyledons ofthese sue. Unifoliate leaves of two (2) week old F= seedlings were detached and placed in Petri plates, inoculated with zoospores, and scored for disease development between three (3) and five (5) days following inoculation. The leaf GIs from the susceptible plants were collected for DNA preparation.

Soybean genomic DNA was pnepaced by the method of White ~ Kaper. (A Simple Method for Detection of Viral Satellite RNAs in Small Plant Tissue Samples. J.
Virol.
Methods 23:83-94 (1989)). Primers of 10-mer oligpmrcleotides were purchased from the Oligonucleotide Synthesis Laboratory, University of British Cohunbia, Vancouver, Canada, and Operon Technologies Alameda, CA Although several possible PCR procedures are available, the PCR procedure reported by Williama et al. (1990) was pre~n~ed but for a ~ew minor modifications. Amplification mactions were in 25.0 p 1 volumes cod 10.0 mM
Tris-HC1 (pH 8.3~ 50.0 mM KC1, 2.25 mMMgClb 150.0 EtM each dNTP (Pharmacia, Piscataway; NJ), 0.2 pM primer, 25.0 erg of 8enomic DNA, and 0.5 unit of Taq DNA
polymerase (Perlan-EImer/Cetus, Norvva)k CT}. Amplification was performed in an Ericomp DNA Thermal Cycler (Eric~mp) San Diego, CA) prod for one cycle of two (2) minutes at 96°C, and 45 cycles ofo~ (1) minute at 94°C, one (1}
mite at 36°C, two (2) minrtes at 72°C, and seven (7) minutes at 72°C. Reaction products were then separated by electrophoreses in 1.5% agarose gels containing ettridimn bromide.
The amplified fragment length Polymorphism (AFLP) tachuique was pe~nrmed acxording to the protocol reported by Zabeau & Vos, except for sfight mo~i&~tions.
(Selective Re~iction Fragment Amplification: A General Method for DNA
FinBerptinbng.
2D European Paterrt Application No. 92402629.7; Publ. no. EP 0534858 A1 ( 1993)). A 0.5 Irg sample of soybean genomic DNA was di8ested withMseI and Hfrr~ restriction enzymes for one (1) hour. AMseI adaptor and the biotiny>ated HindIQ adaptor, along with ATP and T4 DNA ligase, were then added to tire restriction digests and incubated Sor three (3} hours.
(Zabeau 8t Vos, 1993). 'The sequence ofthe MseI adaptor is r7htstisted in SEQ
ID N0:40 and SEQ m N0:41. Additionally, the DNA sequence of the biotinylated HindIQ-adaptor is r'lltin SEQ ID NO: 42 and SEQ m N0:43. After ligation of the M.seI adaptor and the HirrdJ~ adaptor, the biotinylated fragmerns were prEferably separated from non-bioti~rlated fiagments by binding to streptavidin bids (Dynal) Oslo, Norway). The Hmd)B
primers and MseI primers used for AFLP amplifications consisted of a core sequence and selective rnrcleotide (SN) as illustrated below and at SEQ 1D N0:44 and SEQ 1D N0:45, n~spectivel~r.
cone SN
H'~ primer 5-AGACTGCGTACCAGCTT NNN..3 (SFQ ID N0:44) llweI prioser 5-GACGATGAGTCCTGAGTAA NNN,3 (SFQ ID N0:45~
A preferred cascade amplification protocol was per6nrn~ed according to Zabeau 8t Vos. European Pgtent Application No. 92402629.7 ( 1993). The protocol used primers contairring one (+1) a~ three (+3) selective nucleotides. In the first ampl~~tiOn, a total set of 16 (4 x 4) a~li~ations, each with two primers and ore selective nucleotide, was performed using a reaction mncture of25.0 pl oftempiate DNA, 0.5 U Taq polymerise (Boeb<in8er Mannheim, Indiarmpolis,1N), 10.0 mM Ttis-HC1 (pH 8.3), 1.5 mM
MgCl~ 50.0 mM ICC1, and 0.2 mM ofeach dNTP. Twenty (20) amplification cycles were performed under the conditions that fellow: a thirty (30) second DNA denatuiatioa step at 94°C, a thirty (30) second an~aling step of609C, aid a one (1) minute ion step at 72°C. After completion ofthe twenty (ZO) amplification cycles, 10.0 ul ofthe macton was dihrted with 190.0 pl ofTris-EDTA, wh~h was then used for a second PCR
In the second amplification, H~-primers with thn~ selective nucleotides (+3) were end-labeled with [Y-~PJ ATP and T4 polymicleOtide ltinase. Amplifications wec~e ~
in a total vohime of 10.0 ul with 2.25 pmol MseI-primer (+3)~ 0.38 pmol labeled Hindlff primer (+3)~ and 1.0 a 1 of template DNA These reacrbns wane performed for thirty-six (36) cycles as hollows: a thirty (30) second DNA denahuation step at 94°C, a thirty (30) second am~ag step (see below and a one (1) minute extension step at 72°C. The anneabng te~atu~ for the first cycle was 65°C. This atmoaling temperntute was subsequently induced at a rate of -0.7°C per cycle for the next twelve ( 12) cycles.
Thereafter, the PCR was cod at an annealing temperature of 5690 for the remaining tvvenhr three (23) cycles. All AFLP ro were performed in a PTC 1-100 thermal cycler (MJ Research Inc, Watertown, MA). The conditions for the second amplification am also not inflexible and can be modified according to the user's need.

Folbwing the ampl~Cation products were mixed with an equal volume of formacnide dye (98% formamide, 10 mM EDTA pH 8.0, aad bromophenol blue and xylene cyanol as tracking dyes;i. The resulting lures were heated fdr three (3) minutes at 90°C, and quenched on ice. A 2.0 It I sample of the tmixture was then loaded on a 4.5%
polyacrylamide sequencing gel. After electmphomsis~ the gel was vacuum dried and exposed to Kodak BIOMAX MR X ray film.
Polymorphic RAPD or AFLP bands wem isolated from agarose or sequencing 8elas re-amplified, and tadiolabeled with [~ ~P]dATP. Radiolabeling was pc~efe~ably performed according to the random-hexamer iadiolabeling method as presented by Feinberg et a1 (Feinberg) A.P., Yogelstein, S., A Technique For Radio-LabelingDNA Frager»ents to High Sp~eci, f~'c Adivtty. Anal. Biochem 137:266-67 ( 1984)). The genom~ DNA was s~ibsequently digested with one (1) often (10) restriction enzymes (B~ Bcl~ Bglll; Dral;
F.caRl;
EcoRV, HinrllQ Pstl, Rs~al, or Taq1). The iesulring fiagmelrts were then electrophoietically separated on 0.8°% agarosa gels, and capillary blotted onto a Zeta Probe blotting membzane (Bio-Rail) using a permed tracer solution of0.4 M NaOI~ 1.5 M NaCI. The filters wem next vacuum dried and piehybridi~ed in a preferred solution comprising (pH
7.2) O.SM Naa HPO,, 1.0 mM EDTA) and 7.0'/o sodium dodecyl sulfate (SDS~ at 65°C for two (2) to thc~ee (3) hours. The hybridi~tion was perfonmed in a ~es~h solution of 0.5 M Na=
HPO,, 1.0 mM
EDTA, and 7.0°% SDS with the addition of a ~P-labeled probe at 65°C for over sixteen (16) hours. Filters wem then pnefelably washed with Zx SSC (lx SSC is 0.15 M NaCI
ph~s 0.015 M sodium cite), and 0.1% SDS twice at room be~ature. Fibers were then p~ably washed with Zx SSC and.1.0~/o SDS for thirty (30) minutes at 65°C
~nlbwed by a brief rinse in 2x SSC at ivom tea~erature.. After the rinse, the fitters were exposed to Kodak BIOMAX
MR X ray film For a period of one ( 1) to four (4) days.

Segregation of the ltpsl-1~ RAPD, and ItFLP markers in the F~ F~, F4 populations was scored. Scoring was perJi'rmed, using either etiolated or leafinoculatinns procedures, to distinguish resistance fmm susceptib>e genotypes. RAPD marlaers were scored for different progenies for the presersre or absence of a RAPD-specific DNA fiagment. RFL.P
markers were either do~mnt (presence of only one fragments) or co-dominant, which is characttri~d by the presence oftwo (2) polymorphic bands oornesponding to a resistant and suscxptlble parent. 314 suscept~le genotypes were identified from 1,289 segregating genotypes preferably from either F.z or F3 progeny of the Willian~s x Williann 82 cross, the Elgin x E420 CRON, and the E300 x OX717 cross.
RFI,P analysis of this subset population with RAPD271 revealed nineteen (19) n~ombinants betvveenl~psl and R.APD271, ofwhich sixteen (16) were used in the AFLP
mapping of the resistance-dominant or co-dominant AFLP marlaers. Susceptible-dominant or co-domin~t AFLP marlaers, or AFLP marloxs that co-segregated with l~l-k in AFLP
nipping were converDed to RFL,Ps. The prefermd conversion technique required the isolation of the AFLP f agmeirts through use of PCR The isolated fragments were they pmferably cloned in a 'T' vector. (Bhattaclxuyya, M) K., Gonzales, R A., Kraft, M., and Bull, R L
Copies-Like Retroiran~~t T'gnrr ClaselyL~d to the Rp~sl-,~~Illele That Confers Ray Speck Resistance of Sad to Phytqphthora sofas. Plant Mol. Biol. 34:255-64 (199?)).
The resulting clonod fiagmerrts were used as probes for both Southern blot analysis and for mapping the AFLP rnsrlaers. A prefer toque ~or using the fisgrne~ts as probes ~or mapping AFt,P markers allows for use of either the whole segregating material or part theieo~ which include$ the Fa F~, and F4 popu)ations. Duplicated sequences ids by AFi.P or adjacent regions ofRAPD or AFLP marlaers were pn~rably mapped by gel bbt analysis of recombinant plar>ts. Although several methods are available, genetic maps were then constructed by the aid ofthe Map Marmger ~og~am (Manty, K., and Cudmore, R 1995.
Map Manager Version 2..6.5. Roswell Park Cancer Institutes Department of Cellular and Molecular Biobgy.

High molecular weight DNA of soybean NILs Williams and Williams 82 was isolated following the preferred protocol of Liu 8t Whittler except for a few suitab~
modifications.
Rapid Prepr~ation ofMegabase Plant DNA F3~an NrecTei in Agarase Plrgs andMicraaeals.
Nucleic Acid Res. 22:2168-69 (1994). The final nuclei suspension was then preferably mixed with an equal vohune of 2.0'/0 low melting point Sea Pique GTG agarose (FMC
Biopmducts), pmpamd in 50.0 mM EDTA and 10 MM Tris-HCI (pH 9.4), and poured into a mold to form plug. The ag~rose plugs were treated with 0.5 M EDTA (pH 9.3~
1.0%
Sarlaosyl and 1.0 mg of pnoteinase K per rot. The plugs wane then preferably washed with i .0 mM PMSF, 10.0 mM Tris-HCI (pH 7.5) and 50.0 mM NaC 1 and subsequently dialysed in 10:0 mM Tris-HCI (pH 7.5) and 50.0 mM NaC 1.
For clenhomogeneous electric field (CHEF) gel electrophoresis, approximately 5.0 pg of high rrolecular weight DNA was dige$ted separately with the following rare cutting enzymes; B,su361) BssHT, Bsbrl, Nc~t) Nrul, Mlul, SAIL, ~m~aI) S~ Xma111;
a~i~aI
(Strata8ene, La Jolla) CA). For compleoe restriction d 4 ~ plu8s containing mol~lar weight DNA ~wene preferably incubated overnght in a 200.0 p1 res~on cocktail (ZO unite of enzyme + 1x restriction enzyme-specific buB'er) at 4° C
and later incubated at the appropriate ire for six (6) to eight (8) hours.
Restriction digested high molecular weight DNA, along with the Megabase I and II
DNA standards (Gibco BRL~ Grand Island, NY), were preferably separated on 1.0~/o pulsed field cerdged agarvse (Bio-Rad) in O.Sx Tiis-borate-EDTA, using a Bio-Red CHEF
rr~apper.
CHEF electrophoresis was performed for ?,G.56 hours using an auto algorithm program having a pre~nnd switch time of 2.98 seconds (initial) and 3 5.3 8 seconds (~nal~ an angb of 1204 and a gradient of 6.0 Vlcm. The gel) af3er ele~rophor~esis, was then sta>ned with ZS ethidium bromide folbwd by exposure to UV fight. After a desired period of time, the Bel was photographed. The DNA method in the gel was then preferably trarrs6en~d to a Zeta probe blotting membrane (Bio-Rad) and hybr~ized with radiolabeled AFLP probes.

The marlaer sequences isolated above wem cloned from the PCR products of an AFLP
gel. The specific band was amplified from soybean cultivar Williams or from both Williams and Williams 82. The specific bead, however, could be amplified from other soybean cultivars. In a sub-PCR. using a band from the AFi.P gel as a template, the marlaer fragment was isolated as described above and pcefr~ably cloned into the vector pRG5l.
(Kastiga, T., Salimatl~ S.S., Shi J., Crij~ M., Buz~l, R., and Hhattaclmryya, M.K.) Higle Resohrtion Genetic and Physical Mapping ~MolecudorMar~frerr~ Lied to the Phytop~thora Resistm~ce Gene lips 1-Ir in Sopbean, MPMI Vol. 10, No. 9, 1997, pp. 1035-1044. Pub. no.

1020-01R). The marlaer specific cbne was then pm~e~ably soquen~d by the dyedeoxy sequence method aid its identity confmned by using tire clone as a DNA probe fur RFLP
~PP~&
I90LATION AND MAPPING OF TCl AND CGl The screening of 400 decamer primers against the cv. Williams (rpsl-k) and its NIL
W82 (Itp~sl-k) r~ultad in the amplification of approximately 2,000 discrete DNA
fragments ranging from 200 to 3,000 bp. Ofthe primers screened, six 8eneiated fish that wane preeer~t in resistant cv. Williams 82 but ~t in suscc~ble cv.
Williams. With respect to Elgin (rspl-k) and its NIL.E420 (Rpsl-k~ 350 primers'wene screened. 250 of these primers wane the same as those used ~or W>7liams and Willisms 82. The majority ofthe a~iiied fiagments were identical between these two pairs ofNILs. All snc primers except the primer UBC123 that produced polymorphic fagments is resistant cv. Wi>>iams 82 also generated the same fragments in resistatrt E420 but not in suscep~le cv. Elgin. Two primers (UBC330 and UBC348), which did not reveal polymorplrism$ between Williams and Willisms 82, generated fiagmeats that were pnesemt in resistant cv. E420 but not in suscepb'ble cv.
Elgin. The polymorphic fragment produced with primer UHC348 did not segregate in a Mer~delian fashion in a segregating population obtained from the cross Elgin x E420. The polymorphic fragment generated with primer UBC330 showed a 3:1 segregation ratio but was not linked to the Rpsl locus.
Linkage analysis, of the five RAPD markers that produce E420-specific bands along with two RFLP markers (biers B.W., Mansur L., Imsande J., Shoemaker RC., Mapping Phytopl~ra Resis~caice Loci in Soybean with Re~tridion Fragme~ Length Polyrrtarphisr»
Markers. Crop Sci 3 2:377-83 ( 1992)) relative to the ~sl locus, was conducted on fifty-four (54) susceptible FZ plants for the crass Elgin x E420. All RAPD markers) along with two RFLP amrlaers, mere positioned on one side ofRpsl as dlustzated in FIG. 7 and Table 2.
RAPD271 (GCCATCAAGA; SEQ )D N0:46) and RAPD217 (ACAGGTAGAC; SEQ m~
N0:47) wane then comrerted to co-domin~t and dog RFLP markets, respectively, and mapped by Southern blot analysis as described inKasr~ et al., (1997). Map positions were determined by the Map Manager Program. (Meaty, K.F., Gudtmre, R, Jr.) Kohler, G., Map Manager Version 2.6.5. Roswall Park Cancer Institute, Dept. ofCell. & Mol.
Bio., (on-line,), (1995). The rest ofthe resistance-domirmrrt RAPD markers were pnefi~e~ably mapped byr polymetase chain reaction (PCR) Followed by Southern hybridisation ofPCR
products using the RAPD fragments as probes. The PCR techniques used are discussed above while the Southern hybridi~tion tecluriques used are known in the art. RFLP marlaers pA71 and pA-280 am those previously mapped in the Rpsl-k region by Dierss et al., Crop Sci 32:377-83 (1992)).
Akhough markets RAPD271 and RAPD217 co-segregated with RFLP markers pA-71 and pA Z80, respectively, they shoved di~'ettat hybtidi~tion patberas on DNA
gel blots when the RAPD fi~awere used as probes. Such a findit>$ indicxrtes that the and RAPD217 ma,rlaers arse diB'erent fiom these two previously reported RFLP
markers (Dier$
et al. 1992). Table 2 sutnmari~es these difi$.

Table 2. Rps!-linked marlaers isolated by random amplified polymorphic DNA
(RAPD) analysis.
RAPD RFLP Map distance Marker phenotype' phenotype;' (cM)°
RAPD217° RDd RD 2.8 RAPD271 RD CD 1.9 RAPD274 RD Mono 8.5 RAPD206 RD ND 2.8 RAPD304 RD ND 2.8 RD, resistance-domi~t; CD, co-dominant; Mono, monomurphic; ND, not RAPD marloers were converDod to restriction fragment length polymorphism (RFLP) marlaers by using RAPD frays as probes in Southern blot analysis.
' Map distancxs in centimorgans (cM) were determined by RFL,P (RAPD217 and RAPD271) and RAPD (RAPD274, RAPD206 and RAPD304) analysis of 54 Fa susceptible segregants of the ewes Elgin (rp~l-k.rpsl-k) x E420 (Rpsl-k R,~sl-k).
° All five primers that produce the RD phenotype in E4Z0 were mapped but those in Elgin were not mapped.
A high density genetic map ofthe region carrying Rpwl-k is required ~or map-based cloning ofthe Bene. AFLp analysis has recently been applied to soybean withEcoRI and MseI restrict~n enzymee~ with a total of six selective nucleotides (Lire, J
J., Kuo, J., Ma, J., Saunders, J.A, Beard, H.S., MacDor~ld, M.H., K~enworthy, W., Ude, G.N.) Matthewa) B.F., Identi,~aat~'o~e ofMdecu~r Makers in Soybean Carnp~aring RFZP, RAPD ar>dAFLP
DNA
Mapwing ?'eaaineiqroes. Plant Mol. Biol. Rsp. 14:156-69 (1996)). We selected the restriction enryme Hid instead ofEcarRl, because HfndIB provided a more complete di8estion of the soybean 8enomic DNA
To 8eneiate tightly linlae<1 Rpsl-k markets eiFciently two steps of AFLP
analysis vv~ere performed. In the first step, in addition to the cvs. Wdliams and Williams 82, two bulked segregant pools (Michehnore, R.W., Punu>, L., Kessel; R.V., Identificxrtiore ofMca~s Linked to Disease Resistance Genes by Bulked Segregrmt Analysis: A Rapid Method to DietecxMarkers is SpeciJlc GenarnicRegions by UsingSegreg~~ngPopulatia~rs, Proc. Nat'1 Acad. Sci. USA 88 9828-832 ( 1991 derived from F~ progeny of the cross Williams x Williams $2 were used in an AFLP analysis. One bulk was composed oftvventy (20) F3 >iunilies homozygous for the dominant allele Rpsl-k (pool A~ and the other was of twenty (20) F3 families homozygous ~or the recessive allele rpsl-k (pool B). Pool A
and Pool B acre depicted in FIG. 8. None ofthe F3 ofthese two (2) bulks were heterozy8ous or co-dominant for the RFLP marker RAPD271. The four DNA templates-William~s, Williams 82, ant pools A a~ B--were scxeened with 1,240 out ofthe possible 4,096 ninidomly selected Hi~I x MseI primer combinations. On average, eighty (80) DNA
fragments were ar~lified per pnrner; themfone, approximately 100,000 loci were evaluated for polymorphism) Thirty~four (34) primer oombinatio~ revealed polymorphisms between the psre~~ts, and between the two pools. These primer combinations were then reassessed in the second AFLP
step to confirm reproducibility and also to help eliminate loosely linked markers.
In the second AFLP scn~ing step, three (3) additional DNA templates-pools C
and D and donor parent Kingwa~ene used. Pool C and Pool D ane depicted in FIG. 8.
Pool C
was composed ofthc~ee F3 fami>~s homozygous for the i~sl-k allele and heberozy8ous ~or RAPD271, while pool D was composed of five (5) F3 fads homozygous ~or the reel-k allele but heterozygous for RAPD271.
FIG: 8, furthermore, illustrates the strategies employed in the second AFLP
scn~g step. The seven (7) DNA templates, consisting of two (2) NILs, four (4) bulk pools, and the donor parent, were assayed with thirty-four (34) inforn~ative primer combinations obtained from the first screening step. Recombinant plants have crossover points in the interval n the I~sl and RAPD2T 1 loci. Kasuaga) T, et. al. ( 1997). Markets present in pools A

and C but absent from pool H and D, or presern in pools B and D but absent fl~om pools A and C, represent marlaers most to the Rpsl closely linked locus.

Twenty seven (2'1} out of thirty four (34) primer combinations used in the AFi.P

analysis mproduced AF'LP
marloers. The AFLP markers were classified into three d classes: resistance-dominarrt;
susceptibility-dominant;
and co-domirmnt. Table 3 fiutber depicts the findings of the ns reproducing AFL,P
twenty seven (27) primer marlaers.
combinatio An interesting observation primer combinations worth noting is that seven (7) of the producing polymorpmsms in the first scr~een;ng did not show reproducible insults in the second sctee~ng.

Table 3. Primer combinations used in the isolation of AFLP rnarlaers AFLP RFLP Primer Madoer Phenotype' Phenotype'~ combination AAl RD' dd ACAIAGA

AA3 RD mono ATTIAAG

AA4 RD mono ATT/ACT

AAS RD CD ATT/ATC

AC1 RD dd AAC/CTG

AC2 RD dd AGAICAA

AT 1 RD dd AGAfTAG

AT2 RD dd AGA/TAG

AT3 RD dd AGA~TGG

CAl RD mono CAT/ACT

CC3 RD mono CCAICAT

CGl CDR CD CGAIGTT

CG2 CDs dd CCT/GTC

CT2 RD RD, SD tit CGA/TCA
CD

CT3 SD mono CAGITGT

GA3 CD'' dd GCAIAGG

TAl RD dd TAC/AGT

TC3 SD mono TGTICGA

TGl RD nld TGA/GTT

TG2 RD n/d TGT/GTT

TT3 RD dd TGAlTTC

TT4 SD nild fTACITGT

RD, resista»~ominant; CD, co-domiasat; SD, susceptibility domirmr~t; mono, monomorphic; rJd, not deteimirred.
AFLP marlaers wane com~erted to RFt~ marlaers by using PCR amplified AFLP DNA
fiagments as probes in Southern blot analysis.
' Map distances betvveen CGl, TC 1 and Rpsl were deed by RFLP analysis.
Other map distaaces were determined by AFT.P analysis.
° Resistar~o- and susceptible-specific bands are observed for CGI, CG2 ami GA3 in AFLP gels. Sequence analysis confined that CGl is a co-dominarrt marker.
Sequence information fur CG2 arid GA3 was not available. There~om, they azE
considered to be putative co-dominant niarlmrs.
' ACA/AGA represents NNN (selective nucleotide, SN, see AFLP analyses of gslley S/page 5 of the paper by Tahao et al.). ACA is 3 selective nucleotides of the H
primer, while AGA is 3 selective nucleotides ofthe MseI pritner. In simple terinas if we use the following two primers we will a~mplif~r the AA marker: Hin~dllI
primer 5-AGACTGCGTACCAGCTT ACA 3 (SEQ iD N0:48); MseI primer 5-GACGATG-AGTCCTGAGTAA AGA 3 (S~EQ I,D N0:49).
Silnilatiy) if we use the following two primers we will amplify the T4 marker:
Htnc~
primer 5-AGACTGCGTACCAGCTT TAC-3 (SEQ ID NO:50); MseI primer 5-GAC-GATGAGTCCTGAGTAA TGT-3 (SEQ D7 NO:51).
Additionally, nineteen (19) ofthe thirty-four (34) primer combinations amplified products flvm s or pools carrying the Rpsl-k allele. These markers are farmed resistance-dominant marloers. However) only five (5) ofthe primer combinations amplified fisgmec~ts from susceptible psrerrt or pools B and D carrying the reel-k allele. This class of AFLP markets is termed suscept~ility-dominant. Three (3) primer combinations 8anetated co-dominant marlaers.
In this second step of AFIp analysisy inclusion of pools C and D aided the id~rtification of markers that are linked bosely to the Rpel locus. (See ICasuga, T., et al.
(1997)).
A high density genetic map ofthe AFLP markers was constricted through AF1LP
ads by mapping the resistance-dominant and oo-dominant marlaers of the susceptible ZO segregants that are recombinants ~or RAPD. Sixteen (1fi) r~eco~manx individuals with genotypes rpsl-klrpsl-k and RAPD271(+)IRApDZ'71(-) were e<tbjx~d to AFLP
analysis, as descn'bed herein, to place resistance-dominant and co-dominant market loci in the RAPD271-Reel-k interval. FIG. 9 illustrates the 3.0 cM distance between the RAPD271-Rpsl-k interval. Among the sixteen ( 16) rocombinar~ts, no r~ocombination events wars detected between the Rp~sl-k allele and rrasrlaers CGI, AA3, and CC3.
Several AFLP mariners, inchiding resistance-dominant, co-dominant, and ibility dominant markers, were also mapped by ItFLP analysis of the segregating population Fragznents ofDNA, vornesponding to the polymorphic fragments identified by AFLP anat5rsis, were purified from the polyacrylarnide gel aryl then re-amplified by polymeta~e chain reaction (PCR) using the same primer combinations tln~t g~ethe original polymorphic fragments. Comrentional Southern blot arsilysis was performed using the NII,s Williams DNA and the Williaras 82 DNA The DNA was separately digested using the following restriction endonocleases: BmnH>; Bolt) Bglll; Dra>; F,corRS, F~corRV, H
Pst>; Rsa>; and TaqI. The radiolabeled AFI,P probes revealed that eight (8) out of fourteen ( 14) AFLP rnarke<s produced polymorphiscns between NILs with at list one restriction enzyme. The mmsining six (6) AFLP probes prndt~ced rnonomorphic patterns with all ten (10) tested restriction enzymes. Additionally, four n.sistance-dominant or susceptibrlrty domiz~t AFLP behaved as co-dominant marlaers in the RFLP armlysis. Such Srdings are ids in Tabbe 4.
Total populations of Fa F~) and F4 pro8eny, or only recombinant plar~s in the Rpsl-k and RAPD271 ir~rval, were then digested with suitable enzymes. The segregation of alleles at ararloer loci was tested using RFLP analysis previously diswssed. (See Kasuga, T., et. al., (199?)).
Table 4. l~sl-linimd s isolated by amplified fiagrrrent length polymorphism (AFLP) analysis AFIrP RFLP Map distatsae Msr)rer phenotypes phenotype's (cM)' AAl RD'" ND 3.0 AA3 RD Morn 0 AA4 RD Mono 1.59 AAS RD CD 0.80 AC 1 RD ND 1.27 AC2 RD ND 2.23 AC3 RD CD 0.80 CAl RD Mono 0.$0 CC 1 RD RD 0.48 CC3 RD Mono 0 CG1 CD CD 0.06 CG2 CDd ND 2.70 CT2 RD RD and SD 0.80 CT3 SD Mono ND

GA3 CDd ND 2.07 GT1 RD SD 0.32 TAl RD ND 0.64 TC1 SD CD 0.07 TC3 SD Mono ND

TGl RD ND ND

RD, ~sistance-dominant; CD, co-doa~; SD, susceptibility-do~rt; Mono, monomorphic; ND, rat determ>aed.
AFLP marloets were oomerted to RFLP mars by using PCR amplified AFLP DNA
fisgments as probes in Southern blot analysis.
° Map distances in ~morgans (cM) between CGI, TC 1 and Real were daterndned by RFLP analysis. tether map distances were determined by AFLP analysis.
Resistance- and susceptible-specific bands are observed fir CGI, CG2 and GA3 in AFLP 8els. Sequence analysis confirmed that CGl is a co-dominant marlmr.
Sequence in~onrnation Ear CG2 and GA3 is not available. ThereEare, they are considered to be putative co-donrinaflt marlaers.

Additional RFI:P mapping was performed for the suscepa'bilityr-dog marlaer TC
1.
The sus~ptibility~-dominant marker TC 1 was comrertsd to a co-dominant RFL,P
rnarlaer and mapped with segregating materials equivalent to 1,386 chromosoms. A single recombinant between the Rp~sl and TC 1 loci was identified when the BgliZ restriction enzyme was used in RFI:,P mapping) indicating a map position of 0.07 cM fnom the I~sl locus. FIG.

9 further illustrates the distance iiam the TCl marlaer to the Rpsl locus. The co-dom~ar~ marker CGl was then comrerted to a co-dominant RFLP marker. An RFLP an~alys;s (as pn;viously descn'bed) of segcegatiqg materials r~epne1,770 chromosomes rev~ied a single recombinant between the CGl and Rpsl-k allele. This nesutted in a map position of 0.06 cM from the Rps 1 locus. As illustrated in FIG. 9, the CGl marker is on the opposite side of the Rpsl locus when considered against the other known Rps 1-hnlaed marloers. The beret of having marl~rs on both sides of the Rpsl-k allele allows Sor a more precise mapping which can facilitate in the cbning of Rps 1-k) Com~eatiorml Sauthern bbt analysis reviled several Rpsl-)mloed probes derived from AF1.P n~atioea or from clmractetization of ad~rcent regions of AFI,~' or RAPD
marlaers as having hypervat;able polymorpfric bands betweea NILs Williams and Williams 82.
RFLP' ar~alysjs, as descrbed hen3in~ indicated that sac of the irybridizsag DNA
fisgme~T2-C) CT2-D,CT2-E,CT2-1V.~CT2-N, and CT~O-are linloed to the Rpsl locus. The map distances from tbrEe marlaers (CT2-C, -D, and -0) were determined by appl»ng the map program (Manly, K.F., et al., ( 1995)) and are shown in FIG. 9.
ZS A soybean bacterial artificial chromosome (BAC) h'brary was scwith tl~
marloer TC1. Salimath, S.S., Bhattacharyya, MK., Generatiate of a Soyhemr BAC
Lr7rrary) and Iden~tf,~ation ofDNA Sequenc~R TFgI~lyLfnked to the Rpsl-,it Dfseose Rash Gene) Theor. Appl. Genet. (in print), ( 1999). One BAC clone (TC 1-BAC) carrying TC
1 sequences was identified. Comrentional Southern blot analysis revealed that an end ofthe 11137!03903 to seven (7) additional restriction fragments specific to Williams 82. Six (6) of these seven (7) fragments, TC 1-A, TC 1-B, TC 1-C, TC 1-D, TC 1 E, and TC 1-G were mapped to one locus at 0. 8 cM distance from l~sl. The other f agsnent, TC 1-F, was mapped at a locus 0.64 cM from the Itpsl locus.
The thins probe that showed hypervariable polymorphisms) as a resuh of comnal Southern blot analysis was derived from the flanking sequences of the RAPD
marker OPRK15. Sequence analysis ofthis marloer established that the dominant RF1LP
n~arloer is part of a copra-Else rotrotransposon Tgsnr integrated in the linked chromosomal region of the Real-k allele. The dominant RF1.P marloer, however, is not integrated in the linked chromosomal mgion of other anal Rpsl allele. (Bhattachsryya, M. K, Gon~ales, R
A, Kraft) M, and Buzaell, R L, Plant Mol. Biol. 34:255-64 (1997)).
Comnentional Southern blot analysis of the Tgmr Banking soque~ncea star (4) Williarns 82- specdfic and two Williams-speafic fiats. Ofthe four (4) Williams specific fiag<ne~, these (3) co-segre~ted with the Tgmr locus. The fourth Fragment) Tgmr F, was napped at a 2.0 cM distance from the Rpsl locus:
The AFI,P markers TC 1 (SFrQ 1D N0:52) and CGl (SEQ ID NO:53) were snapped on either side of the Rplel locus at 0.07 and 0.06 cM distances) respeL~ively.
The AFLP
markers AA3 and CC3 co-segregated with the IiRsl in AFLP analysis, as indicated by FIG. 9.
These AA3 and CC3 markers hybridised to mater monomoiphic DNA fragments in core! Southern blot analysis. Therefore, they could not be mapped more prxisely by ltFLP analysis.
The TCl and CGl sequences were cloned from the PCR products of an AFLP gel.
Bands sp~fic to each sequence were ampli$sd from the soybean ca~ltivar l~lian~s but not from Williams 82. In a sub-PCR using the original Williama~spec~c baud from the AFLP gel as a template, the TC1 fragment and CGl fragment were isolated and cloned into the vector pRG51 (Bhattacharyya et al., 1997). Sequence analysiss using the dideoxy method, was separately performed on both the TC 1 specific clone and the CG 1 specific cbne.
Corr6rmation of the TC 1 and CG 1 sequences was estabfiahed by using each sequence as a DNA probe in RIFLP mapping.
An analysis ofthe physical distance between TCl and CGl was uaderralaen in order to fmd out the approximate size of the DNA fiagmetrt cormlining Rpsl-k High molecular weight DNA was digested separately with the following rare cxrtting enzymes:
Bsu3fil;
BssH>; B,st~d, N~l; Nru>; Mlu>; Salt) Sinai, XmaIQ and ~oI. The enzyme ,Salt produced an approximately 145-kb DNA fisg<ne~, which hybridized to both TC1 and CGl probes in cvs.
W>'i>>am~s and Williama 82. This fragment was common for boththe TC1 and CGl probes is cva. Wi111ama and Willisms 82. These findings suggest that the "to be"
analyzed DNA
fisgment ~or the cloning of Rpsl-k is around 145kb in length when using either TC 1 or CGl as a 8erretic marker.
USE OF TSE NEWLY IDENTIF~'.D lIZARi~'.~4 IN PLANT BREEDING
Coaventiorral methods for improving the resistance of soybean to the P. sojae pathogen includes the transfer ofRps genes to cultivated varieties through successive back crossing. In performing the back crossing process, it is necessary to evahrate beck-cased progenie$ is every generation ~or the expression of resistance. Such a progeny testing process Z0 is extremely time consuming and requires the expertise of a plant pathologist. Therefore, an alternative approach for improving the transfer of Rps ,genes to cultivated variieties is needed.
As previously discu~d, the Rps1 locus soc (6) fu~tional disease red geu~es that oo~'er resistance to di~arent P. sojae races. Several DNA
soquarcxs have been iderrdged and ripped tightly to the Rpsl gene. Additionally, conventional genetic analysis ZS has slrowa that all s~ (6) Rpsl genes (Rpsl-a, b, c~ d, a and k) map to the same soyb~a genonric location or locus. Therafone, the identification of the Rpsl-8anxing sequencxa should allow us to develop very useful molecular marloers for all Rpa 1 genes.
These sequetrxs, tlre<efote, should be ap~icable in developing triarher-assisted selection for all Rpal alleles with limited effort.

For example, the OPRKI 5 marker can be used for the screerring of biding populations containing the Rpsl-k 8ene, which provides for resjstance against the P. s~ojae pathogen. Thus, in a pedigree breeding fvr the improvement of yield, yield attributes, or other desirable quantitative traits, such Rpsl-specific markers can assist soybean breeders in selecting the desirable genotypes carrying the Rps gene (and its various alleles). Such a selection can be perfon~ed by using an advance generation of the cross produced from two elite lines, in which) one of the lines carry the ~Sl gene.
The perfon~ance of this screening eliminates the need of a plant physiologist.
Most importantly, since we can study the genotype of an individual using these markers, there is no need to perform any pmgerry testsng; thus saving a generation time of approximately six (6) months.
USE OF THE NEWLY IDENTIFIED 11ZARIOaRS FOR
INCREASING R~psl REPRESENTATION IN THE SOYBEAN
GENOME AND MAP BASED CLONING OF THE Rpsl GENE
Development ofa soybean clritivar carrying more than one Rpsl gene has not been achieved because all snc (6) alleles of the Rpsl sane are mapped to the same chromosomal region or locus. T6er$fore, isolation of one of these and application of plant ttans~onnation techniques wdl allow for the develop~nt ofa line of soybean having an 2D additional R,psl gene. For e~le, the isolated Rpsl gene can be transferred to the soybean genome by using an Ag~robacterium-mediated traaa~n~tion procedure. As o~ Rpsl gene can confer resistance to a selective number ofP. so, jae rsces~ a line carrying two Rpsl genes will have increased resistance against wider range of P. sofae races.
Furthern~ore, the cbning of the Rps1-k or other Rpsl alleles allows for tire devdop~m oftn~nsgenic elite lines) which can preclude the linlrage-drag of deleterious genes that commonly nesuh in coanal back-cmssea breeding.
We have also applied the previously discussed map-based arethod for cloning the Rpsl gene. By isolating large DNA fia~merrts from soybean bacterial artificial chromosome (BAC) hbn~ries for CGl and TC1 markers, which are mapped react to the Rpsl locus, a bridge between these two s can be constructed using BAC clones. This DNA bride or °contig, ~~ once constructed, should carry the Rpsl-k gene thereby facilitating the processes for ide~ifying and isolating l~rl-k.

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(i) APPLICANT: The Samuel Roberts Noble Foundation, Inc.
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tattaatata gggaaaagag g 21 ' ' 11137/03903 (2) INFORMATION FOR SEQ ID N0:5:
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(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITIQN IN GENOME:
(A) CfiROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LC)CATION: 1..13 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at position 293-305 of SEQ ID NO:10 (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JC1URNAL
( D ) VC1LUME
(E) I~~SUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE:
(J) PCfBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENC:E DESCRIPTION: SEQ ID N0:5:
taaaaggata ggg 13 (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL:

' ' 11137/03903 (iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..12 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at position 565-576 of SEQ ID NO:10 (x) PUBLICATION INFORMATION:
(A) AC1THORS
(B) T7:TLE:
(C) JUURNAL: .
( D ) VOLUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE:
(J) PC1BLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
accauagucu cg 12 (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENC:E CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITIQN IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UD1ITS:

(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
( G ) DATE
(H) DOCUMENT NUMBER:
( I ) F7:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Cys Ala Tyr Cys Arg Lys Leu Gly His Thr Ile Asp Val Cys (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UrIITS:
( ix ) FEATURE.:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
( D ) VC1LUME
(E) ISSUE:
(F) PAGES:

' ~ 11137/03903 (G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENC;E DESCRIPTION: SEQ ID N0:8:
Cys His Gly Cys Glu Gly Tyr Gly His Ile Lys Ala Glu Cys (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) T7i'PE: amino acid (D) TC>POLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MP,P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
( G ) DA.TE
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Cys His Tyr Cys Gly Lys Tyr Gly His Ile Lys Pro Phe Cys (2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA.P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x).PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
( F) PAGES
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Cys Tyr Asn Cys .Asn Gln Pro Gly His Phe Lys Arg Asp Cys (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:

(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICP,TION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Cys Trp Tyr Cys Lys Lys Glu Gly His Val Lys Lys Asp Tyr (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:.
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:

(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) T7:TLE:
(C) JOURNAL:
( D ) VOLUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) F7:LING DATE:
(J) PC)BLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENC;E DESCRIPTION: SEQ ID N0:12:
Cys His His Cys Gly Arg Glu Gly His Ile Lys Lys Asp Cys (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (H) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UrIITS:
(ix) FEATURE::
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) T7:TLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:

' 11137/03903 (H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PCIBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENC:E DESCRIPTION: SEQ ID N0:13:
Leu Gln Ser Asp Asn Gly Ala Glu Phe Leu Met His Asp Phe Tyr Ala Arg Lys Gly Ile Ile His Gln Thr Thr Cys Val Glu Thr Pro Glu Gln Asn Gly Ile Ala Glu Arg Lys His Gln His Leu Leu Asn (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:

~ ~ 11137/03903 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Leu Arg Ser Asp Asn Gly Gly Glu Tyr Thr Ser Arg Glu Phe Glu Glu Tyr Cys Ser Ser His Gly Ile Arg His Glu Lys Thr Val Pro Gly Thr Pro Gln His Asn Gly Val Ala Glu Arg Met Asn Arg Thr Ile Val Glu (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECUhE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA.P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL: .
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Leu Arg Thr Asp Asn Gly Leu Glu Phe Cys Asn Leu Lys Phe Asp Ala Tyr Cys Lys Glu His Gly Ile Glu Arg His Lys Thr Cys Thr Tyr Thr Pro Gln Gln Asn Gly Val Ala Glu Arg Met Asn Arg Thr Ile Met Glu (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA:P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICA'PION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JO(7RNAL
(D) VOLUME:
(E) IS:iUE:
( F ) PAGES
(G) DA'.PE:
H ) DO(:UMENT NUMBER
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Leu Tyr Ile Asp Asn Gly Arg Glu Tyr Leu Ser Asn Glu Met Arg Gln Phe Cys Val Lys Lys Gly Ile Ser Tyr His Leu Thr Val Pro His Thr Pro Gln Leu Asn Gly Val Ser Glu Arg Met Ile Arg Thr Ile Thr Glu (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
( D ) VO:LUME
(E) ISSUE:
(F) PAGES:
(G) DA'PE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) REl'~EVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Ile Leu Asp Ser Gly Ala Thr Asp His Val (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:

(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA.P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Tyr Leu Asp Ser Gly Cys Ser Arg His Met (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN7:TS:

' ~ 11137/03903 (ix) FEATURE:
(A) NAME/KEY:
( B ) LOCAT I ON
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
( D ) VC1LUME
(E) I~~SUE:
(F) PAGES:
( G ) DP.~TE
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Val Val Asp Thr Ala Ala Ser His His Ala (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
{A) ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA:P POSITION:
(C) UNITS:
(ix) FEATURE:
{A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
{D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) IS:iUE:
(F) PAGES:
$7 (G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Val Leu Asp Ser Gly Cys Thr Ser His Met (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
( D ) VO:LUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Val Leu Asp Ser Gly Ala Ser Asp His Leu (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Arg Gln Leu Asp Val Asn Asn Ala Phe Leu His Gly Tyr Met Lys Leu Pro Pro Gly Leu Val Val Asp Ile Leu Val Tyr Val Asp Asp Ile Ile Leu Ala Gly Asp (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Nicotiana tabacum (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TI'.PLE:
( C ) JO1JRNAL
( D) VOLUME
(E) IS:iUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FILING DATE
(J) PUBLICATION DATE:
(K) REhEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Glu Gln Leu Asp Val Lys Thr Ala Phe Leu His Gly Tyr Met Glu Gln Pro Glu Gly Phe Glu Val Ala Leu Leu Leu Tyr Val Asp Asp Met Leu Ile Val Gly Lys (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOURNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE: , (H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Glu Gln Met Asp 'Jal Lys Thr Ala Phe Leu His Gly Tyr Met Glu Gln Pro Glu Gly Cys Ile Ser Glu Leu Leu Leu Tyr Val Asp Asp Met Leu Ile Ala Gly Lys (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYI?E: amino acid (D) T01?OLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:

' 11137/03903 (B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
His Gln Met Asp Val Lys Thr Ala Phe Leu Asn Gly Tyr Met Arg Leu Pro Gln Gly Ile Ser Cys Asn Val Leu Leu Tyr Val Asp Asp Val Val Ile Ala Thr Gly (2) INFORMATION :EOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHE'PICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:

(C) IDENTIFICATION METHOb:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENC:E DESCRIPTION: SEQ ID N0:26:
ccataatgaa ggtatataaa aggatagggt agggaaaaga gggggtgggg aatagaaaaa 60 (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHE'PICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max {vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
{C) UN:LTS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TI'PLE:
(C) JOURNAL:
{D) VOLUME:
(E) ISSUE:
(F) PAGES:
{ G ) DA'rE
(H) DOCUMENT NUMBER:
(I) FILLING DATE:

' ' 11137/03903 (J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
ccataatgaa ggtatataaa aggataagga aaagaggggg aggggaatag aaaaa 55 (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OT',HER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TI'PLE:
C ) JOrJRNAL
( D ) VO:LUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
{ I ) FI:LING DATE
{J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
ccataatgaa ggtatataaa aggataggga aaagagggag agcggaatag aaaaa 55 (2) INFORMATION :EOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..10 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= RAPD primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JO'URNAL
( D ) VO:LUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ctcctgccaa 10 (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHE'PICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:

' ' 11137/03903 (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1..23 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at position 97-119 of SEQ ID NO:10 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..23 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= PCR primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
gtaatctctt tatagtatgc atg 23 (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
° (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:

(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Reverse complement to nucleotides at position 5861-5880 of SEQ ID NO:10 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= PCR primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
cattgtacca ataatgattg 20 (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) ST~tANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..25 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at :position 202-226 of SEQ ID N0:10 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..25 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= PCR primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
gatttgacat catgtgaatt tggag 25 (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature.
(B) LOCATION: 1..26 (C) IDENTIFICATION METHOD:
(D) OTIiER INFORMATION: /note= Reverse complement to nucleotides at position 5363-5388 of SEQ ID NO:10 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LO(:ATION: 1..26 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= PCR primer (x) PUBLICA'PION INFORMATION:
(A) AUTHORS:
(B) TI'PLE:
(C) JOURNAL:
( D ) VO:LUME
(E) ISSUE:
{F) PAGES:
(G) DA'PE:
{H) DOCUMENT NUMBER:
( I ) FI:LING DATE
{J) PU13LICATION DATE:
(K) RE1LEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
ccacttcagc tagtgr_aact tgtatg 26 (2) INFORMATION 1?OR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TO1?OLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORCiANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNJ:TS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..33 (C) IDENTIFICATION METHOD:
(D) OTFIER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOLiRNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOC;UMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:

y 11137/03903 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
ggccgctcta gaactagtcc cacgcgtgcc atg 33 (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) ST:RANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CH:ROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..33 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
D ) VO:LUME
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
gatccatggc acgcgtggga ctagttctag agc 33 (2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENC:E CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..43 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
agcttcccat gcgaggcctg gatggccatc gataccgtcg acc 43 (2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (H) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:

(viii) POSITION LN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..43 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
tcgaggtcga cggtatcgat ggccatccag gcctcgcatg gga 43 (2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5889 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITIGN IN GENOME:
(A) CH.ROMOSOME/SEGMENT:
(B) MP,P POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NRME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) ACITHORS:

(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
tcattctctt gaaaaagtat gtcactatat ggagtcaagg taattattgt gttcacatga 60 acatttctcc catgaaaata tatattttct gactttgtaa tctctttata gtatgcatgg 120 aaaaataatt ttctgatttt tttctctgta gtgttatata ttatttttaa tcacattttc 180 ttatttattt agtttgtttc tgatttgaca tcatgtgaat ttggagattt gggttagaaa 240 tgtttttgga attttcctgg ttagaacttg ataggtccat aatgaaggta tataaaagga 300 tagggtctta gtcgaagctt attaatcagt tagaaatctg tttagtagtt agttagttag 360 ttaaaagatg ttagagttag tttcctaaat gtaactgact aaactactca aagcgcttct 420 ctttcctcag caactctgca aagatataaa tgcataataa atgcaacaat aaaaaaaagc 480 tctcatgcag tatttggtca tcttcacgtg ttttacgtag cttactacat tttctctctc 540 tatggcacaa tatgcttatt aatatggtat cagagctctt cttgtgaaga gttctgttgt 600 gccaccgtct ttacgctcac gagtttgcct ttttcatcgt tttctcacca tgaactagtt 660 actactaaac tcaaa.aagct atctctatct ccacccaagt gagaacccag ctgttgcact 720 agtttctcca gcttt.agatt ccagcaatta ccattcatgg agtaggtaca tgataacggc 780 attgagcgcc aagaacaaag tagaattcgt aaacggaaaa gcacccgagc cattgaagtc 840 tgatagaact tacggggcat ggcgtcgctg caacaacatg gtggtatcct ggttagttca 900 ttcagtatcc atctc:catta gacaaagtgt cttgtggatg gacaaagcgg aagaaatttg 960 gaatgacttg aaatca cgat acgcacaagg ggaccttttg agagtttctg aactccaaca 1020 agaagcttca tccat:caagc aaggatctct ttctattacg gagtatttta. caaagctgcg 1080 agtcatatgg gacgagatcg aaaacttcat acctgatccc atatgttcat gcactgtcaa 1140 gtgtacttgc ttagt:actca ccaccatagc tcaacgaaag cgagaagacc gagctatgca 1200 gttcctgcaa gggttgaacg aacagtatag caatatatgt tctcatgtgt tgctcatgga 1260 cccaataccc acaat:accga aaatcttctc gtacgtggca cagcaggaaa gacaacttac 1320 aggtaacaac tctttatcaa gctttaatct cgaaactaaa gagggacctt ccattaacgc 1380 tgtcaaaagt gtttgtgaat tcggtggacg cattgaccat aatgaaagcg tttgttataa 1440 gaagcacgga ctacctcaga attacgatgg gaaagggaaa agatacaaca caagaaagac 1500 atgtgcctac tgcagaaaac ttggacacac aattgatgtt tgctacaaga aacaagggta 1560 tcccccagga ttcaaattca acaatggcaa agcaatagct aacaatgtag tggcagtaga 1620 aggaaaagcc acagatgacc agatactacc ccaagaatct caagaactgg tttgtttctc 1680 accggagcaa tacaaggcac tgctagcttt aatacaacag ccatcggccg gaaactcagc 1740 acccatcaag ccata~ggtcg cctttatttc atcttgttcc aataacgatg caacaggtat 1800 aattctatct tgcga~aaaag ccaattctac ctcctggatc ttagattcag gagccactga 1860 tcatgtttcc tcctctctaa caaattttca ctcatatcat caaattaatc ccatcacagt 1920 taaactacca aatggt catc ttgtctatgc tacccactca ggcacaatac aactttctgc 1980 attcattaca ctaaatgatg tcttatacgt gccatctttt actttcaact taatatccat 2040 atcaaagctt gtgtcttcta ctaattgcaa attaatattt tcatcaaata tttgtattct 2100 acaggatacc aataccaagg tgaggattgg tacagctgaa gtgagtcgcg gtctctatca 2160 attcaccccc gaagcataaa aaacacacac catatgttcc actattacac acccaaaatg 2220 taaagtcctt cctataaatc tgtggcactt tcgtatgggc catccttccc tcgaaagatt 2280 acaagccatg cagtcctatt atccgttttt gaacaacaac aaaaacttca tatgtaatac 2340 atgtcattat gcaaagcata agagattact tttctcttct agcacctttc atgcatcaaa 2400 caaatttgag cttttacata ttgatatttg ggggccatgc tccataacaa ccctttatgg 2460 gcaccgatat ttcttaacca tcatagatga cttctcccga tatacatgga ctcatctcat 2520 gcacacaaaa gccgaaacac gtaaaatcat tactgatttc attgcatatg ttgaaactca 2580 atttgatagt.aaagttaaaa ttctacaaag cgataatgga gctgaatttc tcatgcatga 2640 tttctatgct cgaaagggta taatccatca aactacttgt gtcgaaacgc ctgagcaaaa 2700 tggcattgcc gagagaaagc atcaacatct cttgaatgtc acacgagcac tcttatatca 2760 agcacaactt ccactaaatt tctggtgttt tgcattgctt catgctgcat acctcataag 2820 tcgtatacct accccttttt tgaaagatat atctccttat gaaaaattat atgtgcaacc 2880 ctgcgatatc tctaatctcc gtgtttttgg gtgcttatgt tacgttagta cccttcagaa 2940 tcatcgacaa aaacttgatc ctcgagctca tccttgcata tttcttggtt gtaaaccaca 3000 tacaaaaggg tatctcgttt ataatctcca ttctcataac ataactgctt ctcgtaatat 3060 tgttttttta tgaggatcac tttccaatat tacatgaaac ccaacacttc gataataccg 3120 atacgcatat ctcgtcaatt cccttctcta gcaacaccca aattcctgac actatgatga 3180 cacacacagc caacccaaac aatcctaccc acctcacgat acctcctgcc aactccccta 3240 cttcttctac tcaggacaac tcatcttcct cacctccttg gacacccagc ttacgtcgtt 3300 ctacgagaac gagacatcca cccacatact tacaggactt tcatcgcgtt ctcacttcac 3360 atgctgatac ctcct.cgacc aaggttaagt accctctcca ctttgtctta tcttattctc 3420 atttgtctca ttccc:ataaa cacttcatca tgtctataac cgcgatagct aaacccaatt 3480 catatgctga ggctt.ctagc tatgactgtt ggattaaagc catgcaggtc gagttaaagg 3540 ctcttcaaca gaaccacact tggattctaa ctccacttcc cccacacaaa aaggccattg 3600 gatgtcgctg ggttt.acaaa gtcaagcaca atgcggatgg aaccattgaa agatacaaag 3660 cgtgactagt agccaaagga tacactcaac aggaaggttt ggattttctc gatacttttt 3720 cccccgtggc caagctcacc actgttcaaa tgcttctagc ccttgctgct ctttgtaact 3780 ggcaccttag acaactcgac gtcaacaatg cttttcttca tggggatctt aacgaagaag 3840 tttatatgaa gcttc:cacca ggactggttg tggataaccc caacctcatt tgtcaccttc 3900 aacgttcctt atatggactt aaacaagcaa gtcggaaatg gttcacacgg ctttcgtcat 3960 tccttttctc ccaaggcttt cgtcaatctt cagttgacca ctcacttttc ttatactcta 4020 acaatgataa tactgtaaca gctattcttg tttatgttga cgacatcatc ttggcaggag 4080 ataacctcaa gtctatcact cactttacca agtttctcga tcaaactttc agcattaagg 4140 atctcggtat tctgaagttc ttcctcggac tcgaggttgc tcgttcaagt cacggcatac 4200 acttatgtca gggtaaatat gccctggata ttctatcaga ttcaggtatg cttggttccc 9260 gtcctacctc tactc:ccatg gattactcta cacgtttgag tgcttccatg ggaacgccat 4320 tgtcagaaac ttctgcttct tcctacagat gattaatcga tcgcctcata tatcttacga 4380 acacccgacc caatattact catgcagttc aacaactaag tcaatatatg gccaatccca 4440 cttctacaca ctctc:aggct actttttgaa tcttacgcta cctcaaagga tctcctggtt 4500 cgggcatatt ctttgctgct cacagtacac tcactctcaa ggcattcagt gactccgatt 4560 ggacaggctg tcgcgacaca cgatgctcca tcactggctt ctcagtttat ctgggtgatt 4620 atctcatttc ctggc:gatcc aagaagcaat ctacagtctc tcgcagctcc tctgaggcag 4680 agtatcgagc cttggcctcc actacatgtg aacttcaatg gctaagctac cttcttcacg 4740 atttttgggt ccctt.ttctt caacctgcca cactctattg tgacaatcaa tccgctattc 4800 aaatagcttc aaacccagtt ttccacgaac gtactaaaca catcgagatc gactgccata 4860 tcgtgcgaga caaagtgaac gcaggcttgt taaaacttct tccagtttct tcttcaatgc 4920 agcttgccga catct.tcaca aagcctctca cccctgctat ttttcaaggt ttatgttcca 4980 agctgggaat gatgaacatc cattcccagc ttgagggggg gctcttagca gaagcttatt 5040 aatcagttag aaatcagttt agtagttagt tagttagtta aaagatgtta gagttagttt 5100 cctaaatgta actgactaaa ctactcaaag cgcttctctt tcctctgcaa ctctgcaaag 5160 atataaatgc aacaataaaa aaaagctctc atgcagtatt tggtcatctt cacgtgtttt 5220 acgtatctta ctacattttc tctctctatg gcacaatacg cttattaata tagggaaaag 5280 agggggtggg gaatagaaaa agaaaggaca aatgagcctt agaaacaaga cctgcttgtg 5340 tatgacagca gaaagaatcc gccatacaag ttgcactagc tgaagtggaa aaagaacata 5400 ggttggcata atggaaagga tagcttgtaa ggagagtgta gaggctgaga ataagttcat 5460 ccttaggaaa gggagggggt ctgggttcct tttttcctaa ttaccattct caagatctgg 5520 cttgtatccg ccactcccct tctctctgag tcctattttt catacctgga agcctatttt 5580 ttttaatata aatattaaat tatggggata tctacctgtc aaaactttta atatatattt 5640 ttattggttt tatttagtaa aaatcactca cttttctttc cacatacatc tttgacatat 5700 atgtttttat atttaattca ttgaaatctt taattcatta atattatgtt tttagggatc 5760 aattaacatg tgttctttct ttaattctat gcctaaaagt taatcaaaat tcaaattcta 5820 gatcatttat ttaagaaata caagtcatta ctacttgtgt caatcattat tggtacaatg 5880 cacaagttt 5889 (2) INFORMATION :FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISEN:iE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITIOIJ IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA1? POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature ( B ) LO(:AT I ON : 1. .12 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at position 565-576 of SEQ ID NO:10 (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOURNAL
( D ) VOLUME

atttttgggt ccctt.ttctt caacctgcca cactctat (E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
tggtatcaga gc 12 (2) INFORMATION FOR SEQ TD N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISEN,SE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:CTS:
(ix) FEATURE:.
(A) NAME/KEY: misc_feature (B) LOCATION: 1..16 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOURNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
( H ) DOC;UMENT NUMBER
(I) FILING DATE:
(J) PUEiLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE; DESCRIPTION: SEQ ID N0:40:
gacgatgagt cctgag 16 (2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..14 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DA'.PE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
tactcaggac tcat 14 (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE: TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:

( v ) FRAGMENT TYPE
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIA'PE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1..17 (C) IDENTIFICATION METHOD:
(D) OTIiER INFORMATION: /note= adaptor (x) PUBLICA'PION INFORMATION:
(A) AUTHORS:
(B) TI'~LE:
( C ) JOI:JRNAL
( D ) VOILUME
(E) IS;iUE:
(F) PAGES:
(G) DA'.PE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
ctcgtagact gcgtac:c 17 (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYF?E: nucleic acid (C) STFtANDEDNESS:
(D) TO~?OLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN7:TS:
(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1..15 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= adaptor (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
( F) PP,GES
(G) DF,TE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) Rf~LEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
ctgacgcatg gtcga 15 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= HindIII primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TI'.PLE:
( C ) JOfJRNAL
( D ) VOLUME
(E) ISSUE:
(F) PAGES:
(G) DA'.PE:

(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) Rf~LEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
agactgcgta ccagcttnnn 20 (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LO(:ATION: 1..22 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Mse primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOtJRNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DA7.'E:
( H ) DOC;UMENT NUMBER
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE: DESCRIPTION: SEQ ID N0:45:
gacgatgagt cctgagtaan nn 22 i 11137/03903 (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIA'PE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:LTS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOURNAL
(D) VOLUME:
(E) IS:iUE:
(F) PAGES:
(G) DA7.'E:
( H ) DOC;UMENT NUMBER
(I) FILING DATE:
(J) PUF3LICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE: DESCRIPTION: SEQ ID N0:46:
gccatcaaga 10 (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STP,ANDEDNESS:
(D) TOF~OLOGY: linear (ii) MOLECULE. TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:

' 11137/03903 (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
( C ) JOURNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DA'PE:
{H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
acaggtagac 10 (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LEIJGTH: 20 base pairs (B) TY1?E: nucleic acid (C) STItANDEDNESS:
(D) TOI?OLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAE' POSITION:
(C) UNITS:

(ix) FEATURE:
(A) NRME/KEY: misc_feature (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= HindIII primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
agactgcgta ccagcttaca 20 (2) INFORMATION FOR SEQ ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIA'PE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA1? POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LO(:ATION: 1..22 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= MseI primer (x) PUBLICA'.PION INFORMATION:
(A) AU'PHORS
(B) TITLE:
( C ) JOL7RNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:

(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
gacgatgagt cctgagtaaa ga 22 (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: mist feature (B) LOCATION: 1..20 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= HindIII primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE; DESCRIPTION: SEQ ID N0:50:
agactgcgta ccagctatac 20 ' 11137/03903 (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(vii) IMMEDIA'PE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA1? POSITION:
(C) UN:CTS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LO(:ATION: 1..22 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= MseI primer (x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TI7.'LE:
( C ) JOLJRNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
( H ) DOC:UMENT NUMBER:
(I) FILING DATE:
(J) PUE~LICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE. DESCRIPTION: SEQ ID N0:51:
gacgatgagt cctgagtaat gt 22 (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:

(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS
(B) TITLE:
( C ) JO'URNAL
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
tgtcacattg accaaaaaaa ccaaggtgac attgaccaaa aaatagtcct gacaagatgt 60 tggtaaaaaa atataatcgg 80 (2) INFORMATION.1?OR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 209 base pairs (B) TY1?E: nucleic acid (C) STFtANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Glycine max (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAF' POSITION:
(C) UN7:TS:

(ix) FEATURE
(A) NAME/KEY:
(B) LQCATION:
(C) IC>ENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AL;fTHORS
(B) TITLE:
(C) JOURNAL:
( D ) VOLUME
(E) ISSUE:
(F) PAGES:
(G) DP,TE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
aagcttcgat gctagatgag gtgatccaat atatgaagca attgcaagcg caagtgcaaa 60 tgatgaattg aagcttcgat gctagatgag gtgatccaat atatgaagca attgcaagcg 120 caagtgcaaa tgatgaattg gatgaaaatg tacacctcca tgatgctgcc aataaccatg 180 cagcagcagc agcaacaaca acaacttaa 209

Claims (83)

1. A nucleic acid molecule comprising a series of nucleotides ranging from about nucleotides 3,223 to about 4,080 as depicted in SEQ ID NO:38, said nucleic acid molecule assists in the selection and development of the soybean plant carrying the Rps1-k allele.

2. An isolated DNA molecule functioning as a genetic marker for the Rps1-k allele of the soybean plant, said Rps1-k gene provides resistance against Phytophthora root and stem rot.

3. An isolated DNA molecule according to Claim 2 wherein said molecule is the genetic marker OPRK15 comprising a series of nucleotides from about nucleotides 3,223 to about

4,080 as depicted in SEQ ID NO:38.
4. An isolated DNA molecule according to Claim 2 wherein said molecule is the genetic marker Tgmr comprising a series of nucleotides from about nucleotides 306 to about 5,270 as depicted in SEQ ID NO:38.

5. An isolated DNA molecule according to Claim 2 wherein said molecule is the genetic marker TC1, which is depicted in SEQ ID NO:52.

6. An isolated DNA molecule according to Claim 2 wherein said molecule is the genetic marker CG1, which is depicted in SEQ ID NO:53.

7. A nucleic acid molecule comprising a series of nucleotides ranging from about nucleotides 306 to about 5,270 as depicted in SEQ ID NO:38, said nucleic acid molecule assists in the selection and development of the soybean plant carrying the Rps1-k allele.

8. A acid molecule according to Claim 7 wherein said nucleic acid molecule further comprises a 5' end long terminal repeat ranging from about nucleotides 300 to 546 of SEQ ID NO:38 and a 3' end long terminal repeat ranging from about nucleotides 5,023 to about 5,270 of SEQ ID NO:38.

9. A DNA molecule functioning as a genetic marker for the Rpsl gene of the soybean plant, wherein said genetic marker has a map position of 0.07 centimorgans from the Rpsl locus.

10. A DNA molecule according to Claim 9 wherein said DNA molecule is the genetic marker TC1.

11. A DNA molecule capable of functioning as a genetic marker for the Rpsl gene of the soybean plant, wherein said Genetic has a map position of 0.06 centimorgans from the Rpsl locus.

12. A DNA molecule according to Claim 11 wherein said DNA molecule is the genetic marker CG1.

13. A nucleic acid molecule for selecting and developing soybean cultivars comprising a series of nucleotides depicted in SEQ ID NO:52, said nucleic acid molecule assists is the selection and development of the soybean plant carrying the Rpsl-kallele.

14. A nucleic acid molecule for selecting and developing soybean cultivars comprising a series of nucleotides depicted in SEQ ID NO:53, said nucleic acid molecule assists in the selection and development of the soybean plant carrying the Rps1-k allele.

15. A method for detng which allele of the Rpal gene a particular soybean comprises by use of a genetic marker comprising a series of nucleotides ranging from about nucleotides 3,223 to about 4,080 as depicted in SEQ ID NO:38.

16. A method for determining which allele of the Rpsl gene a particular soybean comprises by use of a genetic marker comprising a series of nucleotides ranging from about nucleotides 306 to about 5,270 as depicted in SEQ ID NO:38.

17. A method for determining which allele of the Rps1 gene a particular soybean comprises by use of a genetic marker comprising a series of nucleotides as depicted in SEQ ID
NO:52.

18. A method for determining which allele of the Rpsl gene a particular soybean comprises by use of a genetic marker comprising a series of nucleotides as depicted in SEQ ID
NO:53.

19. A method for selecting a desirable genotype of soybean carrying the Rpsl gene by using a genetic marker comprising a series of nucleotides ranging from about nucleotides 3,223 to about 4,080 as depicted in SEQ ID NO:38.

20. A method for selecting a desirable genotype of soybean according to Claim 19 wherein said method selects for the Rps 1-k allele.

21. A method for selecting a desirable genotype of soybean carrying the Rps1 gene by using a genetic marker comprising a series of nucleotides ranging from about nucleotides 306 to about 5,270 as depicted in SEQ ID NO:38.

22. A method for selecting a desirable genotype of soybean according to Claim 21 wherein said method selects for the Rps1-k allele.

23. A method for selecting a desirable genotype of soybean comprising the Rpsl gene by using a genetic marker comprising a series of nucleotides as depicted in SEQ
ID NO:52.

24. A method for selecting a desirable genotype of soybean according to Claim 23 wherein said method selects for the Rps1-k allele.

25. A method for selecting a desirable genotype of soybean seed comprising the Rpsl gene by using a genetic marker comprising a series of nucleotides as depicted in SEQ ID
NO:53.

26. A method for selecting a desirable genotype of soybean according to Claim 25 wherein said method selects for the Rpsl-k allele.

27. A method for identifying generic markers for the Rps-1 gene by using a nucleic acid molecule comprising a series of nucleotides as depicted in SEQ ID NO:38.

28. A method for identifying genetic markers for the Rps-1 gene according to Claus 27 wherein said nucleic acid molecule depicted in SEQ ID NO:38 identifies the Tgmr a marker.

29. A method for identifying genetic markers for the Rps-1 gene according to Claim 27 wherein said nucleic acid molecule depicted in SEQ ID NO:38 identifies the Tgmr f market.

30. A method for identifying genetic markers for the Rps-1 gene according to Claim 27 wherein said nucleic acid molecule depicted in SEQ ID NO:38 identifies the Tgmr b marker.

31. A method for identifying genetic markers for the Rps-1 gene according to Claim 27 wherein said nucleic acid molecule depicted in SEQ ID NO:38 identifies the Tgmr c markers.

32. A method for identifying genetic markers for the Rps-1 gene according to claim 27 wherein said nucleic acid molecule depicted in SEQ ID NO:38 identifies the OPRK markers, which comprises from about nucleotides 3,223 to about 4080 of said SEQ ID
N0:38.

33. A method for cloning the Rpsl gene by isolating large DNA fragments from a soybean bacterial artificial chromosome and using said DNA fragments with a nucleic acid molecule as depicted in SEQ ID NO:52 and a nucleic acid molecule as depicted in SEQ ID
NO:53 to form a DNA bridge between said nucleic acid molecules, which includes the Rpsl locus.

34. A nucleic acid molecule comprising a series of nucleotides as depicted in SEQ ID
NO:44, said nucleic acid molecule assists in the development of other nucleic acid molecules that serve as genetic markers for the Rps locus in the soybean genome.

35. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN-3' of said SEQ ID NO:44 is 5'-AAC-3'.

36. A nucleic acid molecule according to Claim 35 wherein said nucleic acid molecule assists in the development of a genetic marker, AC 1, specific to the Rps locus.

37. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN 3' of said SEQ ID NO:44 is 5'-ATT-3'.

38. A nucleic acid molecule according to Claim 37 wherein said nucleic acid molecule assists in the development of a genetic marker, AA3, specific to the Rps locus.

39. A nucleic acid molecule according to Claim 37 wherein said nucleic acid molecule assists in the development of a genetic marker, AA4, specific to the Rps locus .

40. A nucleic acid molecule according to Claim 37 wherein said nucleic acid molecule assists in the development of a genetic marker, AC3, specific to the Rps locus.

41. A nucleic acid molecule according to Claim 37 wherein said nucleic acid molecule assists is the development of a generic marker, AC3, specific to the Rps locus.

42. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN 3' of said SEQ ID NO:44 is 5'-CAT-3'.

43. A nucleic acid molecule according to Claim 42 wherein said nucleic acid molecule assists in the development of a genetic marker, CAI, specific to the Rpa locus.

44. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN 3' of said SEQ ID NO:44 is 5'-CGA 3'.

45. A nucleic acid molecule according to Claim 44 wherein said tic acid molecule assists in the development of a genetic marker, CC1, specific to the Rps locus.

46. A nucleic acid molecule according to Claim 44 wherein said nucleic acid molecule assists in the development of a genetic marker, CGl, specific to the Rps locus.

47. A nucleic acid molecule according to Claim 44 wherein said nucleic acid molecule assists in the development of a genetic marker, CT2, specific to the Rps locus.

48. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN-3' of said SEQ ID NO:44 is 5'-CCA-3'.

49. A nucleic acid molecule according to Claim 48 wherein said nucleic acid molecule assists in the development of a genetic marker, CC3, specific to the Rps locus.

50. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5' NNN-3' of said SEQ ID NO:44 is 5'-GCA-3'.

51. A nucleic acid molecule according to Claim 50 wherein said nucleic acid molecule assists in the development of a genetic marker, GT1, specific to the Rps locus.

52. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'-NNN 3' of said SEQ ID NO:44 is 5'-TAC-3'.

53. A nucleic acid molecule according to Claim 52 wherein said nucleic acid molecule assists in the development of a genetic marker, TA1, specific to the Rps locus.

54. A nucleic acid molecule comprising the SEQ ID NO:44 according to Claim 34 wherein said 5'=NNN-3' of said SEQ ID NO:44 is 5'-TGT-3'.

55. A nucleic acid molecule according to Claim 54 wherein said nucleic acid molecule assists in the development of a genetic marker, TC1, specific to the Rps locus.

56. A nucleic acid molecule comprising a series of nucleotides as depicted in SEQ ID
NO:45, said nucleic acid molecule assists in the development of other nucleic acid molecules that serve as genetic markers for the Rps locus in the soybean genome.

57. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-AAG-3'.

58. A nucleic acid molecule according to Claim 57 wherein said acid molecule assists in the development of a genetic marker, AA3, specify to the Rps locus.

59. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-ACT-3'.

60. A nucleic acid molecule according to Claim 59 wherein said nucleic acid molecule assists in the development of a genetic marker, AA4, specify to the Rps locus.

61. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN-3' of said SEQ ID NO:45 is 5'-ATC-3'.

62. A nucleic acid molecule according to Claim 61 wherein said nucleic acid molecule assists in the development of a genetic marker, AA5, specific to the Rps locus.

63. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-CTG-3'.

64. A nucleic acid molecule according to Claim 63 wherein said nucleic acid molecule assists in the development of a genetic marker, AC1, specific to the Rps locus.

65. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-CGT-3'.

66. A nucleic acid molecule according to Claim 65 wherein said nucleic acid molecule assists in the development of a genetic marker, AC3, specific to the Rps locus.

67. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-ACT-3'.

68. A nucleic acid molecule according to Claim 67 wherein said nucleic acid molecule assists in the development of a genetic marker, CA1, specific to the Rps locus.

69. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN-3' of said SEQ ID NO:45 is 5'-CCT-3'.

70. A nucleic acid molecule according to Claim 69 said nucleic acid molecule assists in the development of a genetic marker specific to the Rps locus.

71. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-CAT-3'.

72. A nucleic acid molecule according to Claim 71 wherein said nucleic acid molecule assists in tire development of a genetic marker, CC3, specific to the Rps locus.

73. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN 3' of said SEQ ID NO:45 is 5'-GTT-3'.

74. A nucleic acid molecule according to Claim 73 wherein said nucleic acid molecule assists in the development of a genetic marker, CG1, specific to the Rps locus.

75. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5;=NNN-3' of said SEQ ID NO:45 is 5'-TCA 3'.

76. A nucleic acid molecule according to Claim 75 wherein said nucleic acid molecule assists in the development of a genetic marker, CT2, specific to the Rps locus.

77. A nucleic acid molecule seconding to Claim 75 wherein said nucleic acid molecule assists in the development of a generic marker, GT1, specific to the Rps locus.

78. A acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein sand 5'-NNN-3' of said SEQ ID NO:45 is 5'-AGT-3'.

79. A nucleic acid molecule according to Claim 78 wherein said nucleic acid molecule assists in the development of a genetic marker, TA1, specific to the Rps locus.

80. A nucleic acid molecule comprising the SEQ ID NO:45 according to Claim 56 wherein said 5'-NNN-3' of said SEQ ID NO:45 is 5'-CCG-3'.

81. A nucleic acid molecule according to Claim 80 wherein said nucleic acid molecule assists in the development of a genetic marker, TC1, Specific to the Rps locus.

82. A nucleic acid molecule comprising a series of nucleotides as depicted in SEQ ID
NO:46, said nucleic acid molecule assists in the selection and development of the soybean plant carrying the Rpsl-k allele.

83. A nucleic acid molecule comprising a series of nucleotides as depicted is SEQ ID
NO:47, said nucleic acid molecule assists in the selection and development of the soybean plant carrying the Rpsl-k allele.