Note: Descriptions are shown in the official language in which they were submitted.
CA 02269215 1999-07-19
11137/03903
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.
CA 02269215 1999-07-19
11137103903
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
2
CA 02269215 1999-07-19
11137/03903
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,
3
CA 02269215 1999-07-19
11137/03903
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.
4
CA 02269215 1999-07-19
11137/03903
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.
5
CA 02269215 1999-07-19
11137/03903
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.
6
CA 02269215 1999-07-19
11137/03903
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).
7
CA 02269215 1999-07-19
11137/03903
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$gion ilhistrsting two (Z) random
amplified
polyrnorphic DNA (RAPD) mariloers (RAPD271 and Tgmr), seventeen ( 17)
amplified
fiagment length polymorpl>isms (AFLP) marlaers) and Fourteen (14) restriction
fiagment
length polymorphisms (RFiP) marloers originating from duplicated sequenczss.
Genetic
ZS distances in centiimigaria are presented to the left of the map. AFLP
markers AA3 and CC3
are repetitive sequences that ale not polymorphic between resistant and
susceptible parents.
Therefore, the map positions of these two (2) marlaers were determined only by
the AFLP
8
CA 02269215 1999-07-19
11137/03903
mapping of sixteen (16) recombii~~a (rpsl-k rpsl-1~ RAPD271 [+/ ]~ whereas TC
1 and
CGl were mapped by RFLP analysis from a large segregating p~pulabon.
CA 02269215 1999-07-19
11137ro3903
DETAILED DESCRZPT>iDN
Markers to a locus for the Rpsl gene, which is associated with Phytophthora
root and
stem mt (PRST) resistance were genetically identified and mapped. The markers
include
OPRK15, Tgmr, TC 1, CGl, RAPD 217 and RAPD 271, which pmvide new a~ vahiable
tools to soybean breeders For selecting and developing future soybean
cultivars having PRST
resistarxx. The described herein additionally provide the ~nundation for the
initiation
of a chromosorre landing or walking experimesrt, thereby enabling the
isolation and
ident>scation of the agranomically important soybeaa resistant 8ene, R,p~!-k.
The cbning of
this Rps gene can contribute toward the elucidation ofthe recognition proces$
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).
CA 02269215 1999-07-19
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).
11
CA 02269215 1999-07-19
11137/03903
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
12
CA 02269215 1999-07-19
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.
13
CA 02269215 1999-07-19
11137/03903
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.
14
CA 02269215 1999-07-19
11137/03903
TSOLATION AND 11ZAPPING OF OPRK 15
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~
CA 02269215 1999-07-19
11137/03903
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
16
CA 02269215 1999-07-19
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
OPRK15-
specific sequences. One pool was screenod and cosmid 31) wh~h contains the
OPRK15
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
17
CA 02269215 1999-07-19
11137/03903
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
18
CA 02269215 1999-07-19
11137/03903
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
19
CA 02269215 1999-07-19
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.
CA 02269215 1999-07-19
11137/03903
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.
21
CA 02269215 1999-07-19
11137/03903
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
22
CA 02269215 1999-07-19
11137103903
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.
23
CA 02269215 1999-07-19
11137/03903
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.
24
CA 02269215 1999-07-19
11137/03903
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.
CA 02269215 1999-07-19
11137/03903
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.
26
CA 02269215 1999-07-19
11137103903
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.
M 1997-
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
Z7
CA 02269215 1999-07-19
11137/03903
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
RAPD271
and RAPD217 ma,rlaers arse diB'erent fiom these two previously reported RFLP
markers (Dier$
et al. 1992). Table 2 sutnmari~es these difi$.
28
CA 02269215 1999-07-19
11137/03903
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
29
CA 02269215 1999-07-19
11137103903
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
CA 02269215 1999-07-19
11137/03903
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
AC3 RD CD ATT/CGT
AT 1 RD dd AGAfTAG
AT2 RD dd AGA/TAG
AT3 RD dd AGA~TGG
CAl RD mono CAT/ACT
CC1 RD RD CGAJCCT
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
31
CA 02269215 1999-07-19
11137/03903
GA3 CD'' dd GCAIAGG
GT1 RD SD GCA/TCA
TAl RD dd TAC/AGT
TC 1 SD CD TGT/CCG
TC2 SD SD TGTICCT
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-
32
CA 02269215 1999-07-19
11137/03903
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
33
CA 02269215 1999-07-19
11137/03903
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
AT 1 RD ND ND
AT2 RD ND ND
AT3 RD ND ND
CAl RD Mono 0.$0
34
CA 02269215 1999-07-19
11137/03903
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
TC2 SD SD ND
TC3 SD Mono ND
TGl RD ND ND
TG2 RD ND ND
TT3 RD ND ND
TT4 SD 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.
CA 02269215 1999-07-19
11137/03903
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
TC1-BAC
36
CA 02269215 1999-07-19
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
82-
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
37
CA 02269215 1999-07-19
11137/03903
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.
38
CA 02269215 1999-07-19
11137/03903
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
39
CA 02269215 1999-07-19
11137/03903
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.
CA 02269215 1999-07-19
SEQUENCE LISTING
GENERAL INFORMATION
(i) APPLICANT: The Samuel Roberts Noble Foundation, Inc.
(ii) TITLE OF INVENTION: Genetic Markers for Rpsl-k Gene and
Methods of Use
(iii) NUMBER OF SEQUENCES: 53
(iv) CORRESPONDENCE ADDRESS: Rirby Eades Gale Baker
Box 3432, Station D
Ottawa, Ontario
K1P 6N9
CANADA
(v) COMPUTER RIsADABLE FORM:
(A) MEDIUM TYPE:. Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS -
(D) SOFTWARFs: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,269,215
(B) FILING :DATE: April 29, 1999
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/083,439
(B) FILING DATE: 29-APR-1998
(C) CLASSIFICATION:
(viii) PATENT AGENT INFORMATION:
(A) NAME: K:imberley Lachaine
(C) REFERENCE NUMBER: 43193
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 237-6900
(B) TELEFAX: (613) 237-0045
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL:
(iv) ANTISENSE:
41
CA 02269215 1999-07-19
y 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: misc_feature
(B) LOCATION: 1..21
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at
position 293-313 of SEQ ID NO:10
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) I~~SUE:
(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:1:
taaaaggata gggtcttagt c 21
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 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) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MA.P POSITION:
(C) UNITS:
42
CA 02269215 1999-07-19
' ' 11137!03903
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LQCATION: 1..20
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at
position 557-576 of SEQ ID N0:10
(x) PUBLICATION INFORMATION:
(A) A(1THORS
(B) T7:TLE:
(C) JOURNAL:
(D) VOLUME:
(E) I:iSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
( I ) FI:LING DATE
(J) P(1BLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO.:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
tattaatatg gtatc:agagc 20
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENC;E CHARACTERISTICS:
(A) LENGTH: 18 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) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..18
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at
position 5013-5030 of SEQ ID NO:10
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TLTLE:
(C) JOURNAL:
(D) VOLUME:
43
CA 02269215 1999-07-19
11137/03903
(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 N0:3:
gagggggggc tcttagca 18
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 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) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..21
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note= Corresponds to nucleotides at
position 5262-5283 of SEQ ID NO:10
(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 N0:4:
tattaatata gggaaaagag g 21
44
CA 02269215 1999-07-19
' ' 11137/03903
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(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:
CA 02269215 1999-07-19
' ' 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:
46
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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:
47
CA 02269215 1999-07-19
' ~ 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
1 5 10
(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
1 5 10
48
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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:
49
48
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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:
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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:
51
CA 02269215 1999-07-19
' 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
1 5 10 15
Arg Lys Gly Ile Ile His Gln Thr Thr Cys Val Glu Thr Pro Glu Gln
20 25 30
Asn Gly Ile Ala Glu Arg Lys His Gln His Leu Leu Asn
35 40 45
(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.:
52
CA 02269215 1999-07-19
~ ~ 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
1 5 10 15
Tyr Cys Ser Ser His Gly Ile Arg His Glu Lys Thr Val Pro Gly Thr
20 25 30
Pro Gln His Asn Gly Val Ala Glu Arg Met Asn Arg Thr Ile Val Glu
35 40 45
(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
10 15
Tyr Cys Lys Glu His Gly Ile Glu Arg His Lys Thr Cys Thr Tyr Thr
20 25 30
53
CA 02269215 1999-07-19
11137/03903
Pro Gln Gln Asn Gly Val Ala Glu Arg Met Asn Arg Thr Ile Met Glu
35 40 45
(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
1 5 10 15
Phe Cys Val Lys Lys Gly Ile Ser Tyr His Leu Thr Val Pro His Thr
20 25 30
Pro Gln Leu Asn Gly Val Ser Glu Arg Met Ile Arg Thr Ile Thr Glu
35 40 45
54
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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:
CA 02269215 1999-07-19
11137103903
(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
1 5 10
(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:
56
CA 02269215 1999-07-19
' ~ 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
1 5 10
(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
CA 02269215 1999-07-19
11137/03903
(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
1 5 10
(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
1 5 10
58
CA 02269215 1999-07-19
11137/03903
(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
1 5 10 15
Pro Pro Gly Leu Val Val Asp Ile Leu Val Tyr Val Asp Asp Ile Ile
20 25 30
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
59
CA 02269215 1999-07-19
11137/03903
(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
1 5 10 15
Pro Glu Gly Phe Glu Val Ala Leu Leu Leu Tyr Val Asp Asp Met Leu
20 25 30
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:
CA 02269215 1999-07-19
- 11137/03903
(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
1 5 10 15
Pro Glu Gly Cys Ile Ser Glu Leu Leu Leu Tyr Val Asp Asp Met Leu
20 25 30
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:
61
CA 02269215 1999-07-19
' 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
Z 5 10 15
Pro Gln Gly Ile Ser Cys Asn Val Leu Leu Tyr Val Asp Asp Val Val
20 25 30
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:
62
CA 02269215 1999-07-19
11137/03903
(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:
63
CA 02269215 1999-07-19
' ' 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
CA 02269215 1999-07-19
11137/03903
(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:
CA 02269215 1999-07-19
' ' 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:
66
CA 02269215 1999-07-19
11137/03903
(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
67
CA 02269215 1999-07-19
11137/03903
(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
68
CA 02269215 1999-07-19
11137/03903
(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.:
69
CA 02269215 1999-07-19
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
CA 02269215 1999-07-19
11137/03903
(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:
71
CA 02269215 1999-07-19
11137/03903
(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:
72
CA 02269215 1999-07-19
11137/03903
(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
73
CA 02269215 1999-07-19
11137/03903
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
74
CA 02269215 1999-07-19
11137/03903
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
CA 02269215 1999-07-19
11137/03903
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
76
atttttgggt ccctt.ttctt caacctgcca cactctat
CA 02269215 1999-07-19
11137/03903
(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
77
CA 02269215 1999-07-19
11137/03903
(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:
78
CA 02269215 1999-07-19
11137/03903
( 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:
79
CA 02269215 1999-07-19
11137/03903
(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:
CA 02269215 1999-07-19
11137/03903
(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
81
CA 02269215 1999-07-19
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:
82
CA 02269215 1999-07-19
' 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:
83
CA 02269215 1999-07-19
11137/03903
(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:
84
CA 02269215 1999-07-19
11137/03903
(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
8S
CA 02269215 1999-07-19
' 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:
86
CA 02269215 1999-07-19
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 ) 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:
87
CA 02269215 1999-07-19
11137/03903
(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
88
