ÿWPC4 F‚¤)ý!Tz5È«Hù©XlêGâ`gðZmM<]‰Â˜¢‡7ø‰—YsßJ¶ÓR2w­{N¬Tã‰óS¡tö- @®ÉºÐgªaÚÒ—òôE°â E7&à4Ô” ·@fiÊÛoá>;ÀZ©jL-ZF'ÜGÕ~$gSE$ î}\-W,dE.ç1évM\R ² ÿFÅCñ Tƒ;`!ü2±³|Øž­¹ý;'ëÞ•lðG÷ªÜN­°J7#Â{TcÉ ‹[eåž?@öH 0‰ð 0ƒy 0vü 0vr 0¥è 0‰ 0Ü 0ïò 0±á 0’ 0” 0)© 0<Ò 0N 0a\ 0Ú½ 0¯— 0¯F 0Îõ 0Çà 0ÆŠ 0äP 0®4 0®â 0Ê 0ÝZ 0ð7 0' 0* 0)@ 0<i 0O¥ U@ô! %4" 0z:" 0®´" 1b# 0¹g$  % 0=1% 0Fn% 0P´% 1“& 0·—&–N' 0Cä*E'+ 0})+ 0x¦+ 0, 0³«, 0Ð^- 0¦..-Ô. C/f/a/ B*1/ B[/U:x/w²/ 1§¶/ 1§]01n1#!3 0(84N`44b4v4 mx4V1 ×ß8Document[8]Document Styleà0..àà8` ..` àÛ  ÛÌÌV$‡›8Document[4]Document Styleà..àò òòòÛ  Û.óó€€ó óV äå8Document[6]Document Styleà8..àÛ  ÛÌÌV Äã8Document[5]Document Styleà0..àÛ  ÛÌÌV:ä8Document[2]Document StyleÌò òÛ  ÛÔ2ÔA.Ô3  Ô€òòÔ  ÔÔ  ÔÌÌó óóóV1 ·Ý8Document[7]Document Styleà0..àà0` ..` àÛ  ÛÌÌz` :Right Par[1]Right-Aligned Paragraph NumbersàÐ.. àÛ  ÛÔ2ÔI.Ô3  Ô€ˆà°..°àà0..àÌÌzs BÇ:Right Par[2]Right-Aligned Paragraph Numbersà..ààÐ` ..øàÛ  ÛÔ2ÔA.Ô3  Ô€ˆà..àà0` ..` àÌÌVJ8Document[3]Document StyleÌà..àò òÛ  ÛÔ2Ô1.Ô3  Ô€Ô  ÔÔ  ÔÌó óz† Çû:Right Par[3]Right-Aligned Paragraph Numbersà..àà` ..` ààи ..P àÛ  ÛÔ2Ô1.Ô3  Ô€ˆà` ..` àà0¸ ..¸ àÌÌz™ &ò:Right Par[4]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ ààÐ..¨ àÛ  ÛÔ2Ôa.Ô3  Ô€ˆà¸ ..¸ àà0..àÌÌz­ {j:Right Par[5]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààÐh..ˆàÛ  ÛÔ2Ô(1)Ô3  Ô€ˆà..àà0h..hàÌÌzÀ Ä´:Right Par[6]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÐÀ..ààÛ  ÛÔ2Ô(a)Ô3  Ô€ˆàh..hàà0À..ÀàÌÌzÒ Ž:Right Par[7]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÀ..ÀààÐ..°àÛ  ÛÔ2Ôi)Ô3  Ô€ˆàÀ..Ààà0..àÌÌzå :Right Par[8]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÀ..Ààà..ààÐp..àÛ  ÛÔ2Ôa)Ô3  Ô€ˆà..àà0p..pàÌÌVcÍp8Document[1]Document StyleÌÔ  Ôà@ì..8àòòò òÔ  ÔÛ  ÛÔ2ÔI.Ô3  Ô€Ô  ÔÔ  ÔÔ  ÔˆÌÌóóó ól= g:Technical[5]Technical Document Styleà..àò òÛ  ÛÔ2Ô(1)Ô3  Ô€.€€ó ól= …:Technical[6]Technical Document Styleà..àò òÛ  ÛÔ2Ô(a)Ô3  Ô€.€€ó ól:%ÊÀ:Technical[2]Technical Document StyleÌò òÛ  ÛÔ2ÔA.Ô3  Ô€òòÔ  ÔÔ  ÔóóÌÌà..àó ól7!›:Technical[3]Technical Document StyleÌò òÛ  ÛÔ2Ô1.Ô3  Ô€Ô  ÔÔ  ÔÌà..àó ól6! ):Technical[4]Technical Document Styleò òÛ  ÛÔ2Ôa.Ô3  Ô€Ô  ÔÔ  ÔÌà..àó ólE0 D:Technical[1]Technical Document StyleÌòòò òÔ  ÔÛ  ÛÔ2ÔI.Ô3  Ô€Ô  ÔÔ  ÔÔ  ÔÌÌóóà..àó ól<a~:Technical[7]Technical Document Styleà..àò òÛ  ÛÔ2Ôi)Ô3  Ô€.€€ó ól<a…:Technical[8]Technical Document Styleà..àò òÛ  ÛÔ2Ôa)Ô3  Ô€.€€ó ój^ ëJ*1[1]Right-Aligned Paragraph NumbersàÐ..àÛ  ÛÔ2ÔÔ3  Ô€ˆà°..°àà0..àÌÌjq 4ç*1[2]Right-Aligned Paragraph Numbersà..ààÐ` ..` àÛ  ÛÔ2ÔÔ3  Ô€ˆà..àà0` ..` àÌÌj„ @*1[3]Right-Aligned Paragraph Numbersà..àà` ..` ààи ..¸ àÛ  ÛÔ2ÔÔ3  Ô€ˆà` ..` àà0¸ ..¸ àÌÌj— áQ*1[4]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ ààÐ..àÛ  ÛÔ2ÔÔ3  Ô€ˆà¸ ..¸ àà0..àÌÌjª é*1[5]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààÐh..hàÛ  ÛÔ2ÔÔ3  Ô€ˆà..àà0h..hàÌÌj½ ,Ø*1[6]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÐÀ..ÀàÛ  ÛÔ2ÔÔ3  Ô€ˆàh..hàà0À..ÀàÌÌjÐ W¹*1[7]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÀ..ÀààÐ..àÛ  ÛÔ2ÔÔ3  Ô€ˆàÀ..Ààà0..àÌÌjã N¸*1[8]Right-Aligned Paragraph Numbersà..àà` ..` àภ..¸ àà..ààh..hààÀ..Ààà..ààÐp..pàÛ  ÛÔ2ÔÔ3  Ô€ˆà..àà0p..pàÌÌ*Ͼ¦ `(CG TimesScalable!XR& ÿÿ8BibliogrphyBibliographyà0..àà°..°àÌÌjDÿÿ4Tech InitInitialize Technical StyleÓ(€ % (ÓØØ ÿÿCuy±ÉåTechnicalTechnical Document Style11.11.1.11.1.1.11.1.1.1.11.1.1.1.1.11.1.1.1.1.1.11.1.1.1.1.1.1.1fEÿÿ2Doc InitInitialize Document StyleÓ€'ÓÈÓ(€ +   (ÓØØ()*(ÿÿ$˜˜Ìà0 àà à(ÿÿ$™™à0 àà0 àà à((ÿÿ$ššà0 àà0 àà0 àà à  ÿÿA_ekqw‡DocumentDocument StyleI.1.A.a.(1)(a)i)a)x?u2PleadingHeader for Numbered Pleading PaperÓÈÓÑ °ÑÑè°ÑÖ€-ÿÿÖ ŒÝ ƒ.‚(ÝÝ  ÝÔ€!cÛXX!XÔÓÈÓÒX°ÒÒ°°ÒÓÿÿÀ°œXÓß?€;/'ü°ø*–dEü°ø*?ßß?€;/'°ø*–dE°ø*?ßÌÌà\\ Àà1ˆÌÌà\\ Àà2ˆÌÌà\\ Àà3ˆÌÌà\\ Àà4ˆÌÌà\\ Àà5ˆÌÌà\\ Àà6ˆÌÌà\\ Àà7ˆÌÌà\\ Àà8ˆÌÌà\\ Àà9ˆÌÌàøø Àà10ˆÌÌàøø Àà11ˆÌÌàøø Àà12ˆÌÌàøø Àà13ˆÌÌàøø Àà14ˆÌÌàøø Àà15ˆÌÌàøø Àà16ˆÌÌàøø Àà17ˆÌÌàøø Àà18ˆÌÌàøø Àà19ˆÌÌàøø Àà20ˆÌÌàøø Àà21ˆÌÌàøø Àà22ˆÌÌàøø Àà23ˆÌÌàøø Àà24ˆÌÌàøø Àà25ˆÌÌàøø Àà26ˆÌÌàøø Àà27ˆÌÌàøø Àà28Ô  à.À+Ô(2‚$¨¨Ý ƒC!ÝÝ  Ýpÿÿ4Heading 2Underlined Heading Flush LeftòòòòÌóóóóVÿÿ4Heading 1Centered Headingà@ì..ìàòòò òˆÌÌóóó ób* ÿÿ8Bullet ListIndented Bullet Listà0..àððà0` ..` àÌPMÿÿ0HeadingChapter HeadingÈà@ì.. àòòò òÛ  ÛÔ2ÔI.Ô3  Ô€Ô  ÔÔ  ÔˆÌÌóóó ótZ ÿÿ4Right ParRight-Aligned Paragraph NumbersàÐ.. àÛ  ÛÔ2ÔI.Ô3  Ô€ˆÔ úÿ Ôà0..àÌÌLHÿÿ6SubheadingSubheadingò òÛ  ÛÔ2ÔA.Ô3  Ôà.. àÔ  ÔÔ  ÔÌÌó ó #Ñ#€!!Xd#Ñ ÿÿÿdTable_A&ÿÿ0 d d d<þ6X9`("Courier 10cpi ÿÿ3sy…‹“›¡1Right-Aligned Paragraph NumbersI.A.1.a.(1)(a)i)a)  ÿÿ3sy…‹“›¡2Right-Aligned Paragraph NumbersI.A.1.a.(1)(a)i)a)#|S«*Ͼ¦ `(CG TimesScalableXXwϾ¦ P쉊7ûXP<þ6X9`("Courier 10cpileXôxþ6X@É“8Ç;X@)Ͼ¦ `(CG TimesScalableôôcϾ¦ P쉊7ûôP˜HP LaserJet 4,ü,,,,,ü0($¡¡ëU+&ê:i¢Ø+00 ÿU‹ÿÀÀÀÝ ƒC!ÝÝ  ÝÔ_ÔÓ  ÓÓ@ÓÒX°ÒSouthern€Illinois€University,ÌDepartment€of€Plant€and€Soil€Science,ÌMolecular€Science€Program,ÌCarbondale,€IL€62901.ÌÌò òòòLoci€underlying€resistance€to€sudden€death€syndrome€and€òòFusarium€solanióó€in€field€and€greenhouseÐ Œ Ü Ðassays€do€not€correspond.Ìó óóóà ° àÌò òÑ€6Ñó ó€€€€€€In€1971,€a€new€fungal€disease,€soybean€Sudden€Death€Syndrome€(SDS)€was€documented€in€ArkansasÐ `  Ð(Roy€et€al.,€1989;€Ô_ÔRupeÔ_Ô€1989).€This€disease€has€now€been€observed€in€most€of€the€major€producing€areasÏin€the€United€States.€Sudden€death€syndrome€in€soybean€(òòGlycine€maxóó€(L.)€is€caused€by€òòFusarium€solanióóÐ h¸  ÐÔ_Ôf.spÔ_Ô.€òòÔ_ÔphaseoliÔ_Ôóó€(Ô_ÔO'DonellÔ_Ô€and€Gray,€1995).€Leaf€symptoms€include€a€mosaic„like€coloration€of€the€leavesÐ ”ä  Ðfollowed€by€Ô_ÔinterveinalÔ_Ô€Ô_ÔchlorosisÔ_Ô€and€necrosis€(Roy€et€al.,€1989;€Ô_ÔRupeÔ_Ô,€1989).€However,€the€fungus€isÏrestricted€to€the€roots€where€infection€causes€rotting€and€discoloration€in€the€tap€root€and€a€loss€of€lateralÏroots€(Roy€et€al.,€1989;€Ô_ÔRupeÔ_Ô,€1989;€Ô_ÔO'DonellÔ_Ô€and€Gray,€1995).Ìà ° àResistant€or€tolerant€varieties€are€being€sought€to€withstand€the€pressure€of€SDS.€Field€trials€haveÏbeen€conducted€over€several€years€to€select€for€resistant€and€tolerant€varieties,€but€this€is€time€consumingÏand€expensive€(Ô_ÔHnetkovskyÔ_Ô€et€al.,€1996).€To€offset€some€of€these€problems,€screening€in€the€greenhouseÏ(Stephens€et€al.,€1993)€and€marker€assisted€selection€may€be€used€as€complementary€techniques€to€developÏimproved€varieties€(Ô_ÔHnetkovskyÔ_Ô€et€al.,€1996).Ì€€€€€€€Two€greenhouse€assays€have€been€developed.€The€first€assay€is€based€on€òòÔ_ÔF.solaniÔ_Ôóó€infested€oat€orÐ  p Ðsorghum€seed€placed€close€to€the€taproot€of€a€seedling€(Stephens€et€al.,€1993).€Plants€are€grown€toÏmaturity€(until€maximum€contrast€in€the€Ô_ÔAUDPCÔ_Ô€for€controls),€DS€is€recorded€every€48€h€and€plantÏweight€is€determined€at€the€end€of€the€experiment.€The€second€assay€is€based€on€òòÔ_ÔF.solaniÔ_Ôóó€infestedÐ ¤ô Ðcornmeal€and€sand€distributed€throughout€the€soil€(Ô_ÔO'DonellÔ_Ô€and€Gray,€1995).€Plants€are€grown€for€onlyÏthree€weeks€before€data€is€recorded.€DS€and€plant€weights€are€recorded.€Our€goal€was€to€compare€theÏmethods€for€accuracy€in€predicting€SDS€in€the€field€using€Ô_ÔRILsÔ_Ô€from€Essex€x€Forrest€(Ô_ÔExFÔ_Ô)€whichÏcontrasted€in€their€field€SDS€resistance€(Ô_ÔHnetkovskyÔ_Ô€et€al.,€1996;€Chang€et€al.,€1996).Ìò òÌòòMaterials€and€Methodsóóó óÐ ¬&ü! Ѐ€€€€€òòFusarium€solanióó€strain€ST90,€maintained€on€5X€Ô_ÔBilaysÔ_Ô€medium€at€19€C,€was€transferred€onto€Ô_ÔPDAÔ_ÔÐ Ø'(# Ðplates€at€28€C€for€Ô_ÔinoculumÔ_Ô€preparation.€The€40€Ô_ÔExFÔ_Ô€Ô_ÔRILsÔ_Ô€used€were€the€20€most€resistant€to€SDS€in€theÏfield€and€the€20€most€susceptible€to€SDS€in€the€field.Ìò òInfested€Seed€Assay:ó ó€Plants€were€inoculated€employing€the€infested€oat€technique€(Stephens€et€al.,€1993).€Ð \+¬&! ÐOats€were€soaked€in€tap€water€overnight,€then€excess€water€was€strained€away.€The€150€ml€portions€wereÏput€into€250€ml€Erlenmeyer€flasks,€capped,€and€autoclaved€for€20€minutes.€€Oats€were€then€cooled€andÐ ´-)# ÐÑ6Ñâ 6âÑ8€!cÛ!XXdðXXdð8Ñâ 6âÑ  ÑÔ_Ôthree€1€cmòò2óó€agar€plugs€from€the€colony€borders€of€òòF.€solanióó€petri€plates€were€aseptically€added€to€theÐ ° Ðsterilized€oats.€€The€inoculated€oats€were€incubated€in€the€dark€at€room€temperature€(24€C)€for€threeÏweeks.€€Flasks€were€constantly€shaken€for€uniform€growth.€Plants€were€grown€in€a€soil€mediumÏcontaining€a€1:1€(v/v)€mixture€of€sterilized€sand€and€sterilized€soil€taken€from€Ridgway.€€Inoculation€ofÏthe€plants€was€done€at€V2„V3€stage€of€growth€by€placing€five€infested€oats€next€to€the€taproot€below€theÏsoil€surface.€€Plants€were€then€irrigated€to€seal€the€soil,€and€soil€was€kept€saturated€to€ensure€theÏpathogenicity€of€the€fungus.€€Ìò òCornmeal€Sand€Assayó ó:€The€cornmeal€method€of€inoculation€€was€used€with€a€few€modifications.€TwoÐ ä 4 Ð1€cm€X€1€cm€square€pieces€of€the€infested€agar€was€transferred€into€a€50€ml€volume€of€cornmeal€and€silicaÏmix€(1:1)€and€moistened€with€sterile€water.€€This€was€kept€in€an€incubator€for€14€days.€At€the€same€timeÏseeds€were€sown€on€steamed€sand€.€After€14€days,€the€inoculum€was€mixed€with€steamed€sand/soil€(1:1Ïv/v)€in€a€1:€40€proportion.€Styrofoam€cups€were€filled€and€14€day€old€seedlings€were€transferred€into€theÏfilled€cups.€These€were€set€in€water€filled€basins€with€enough€water€to€keep€them€moist.€The€experimentalÏdesign€was€a€2„€factor€randomized€complete€block€with€treatment€as€a€split€on€genotype.€After€21€days,Ïdisease€severity€based€on€a€scale€of€1„9€(1€=€plants€with€no€leaf€chlorosis€and€9€=€dead€plant).€wasÏscored.€Other€trait€data€scored€were€root€weight,€shoot€weight€and€plant€height.€Data€analysis€used€a€one„¼way€analysis€of€variance€(ANOVA).Ìò òÌòòResults€and€Discussionóóó óÐ È Ѐ€ò òGreenhouse€traitsó ó:€There€was€a€high€significant€difference€between€treatments€for€EXF€€and€the€RILsÐ ôD Ð(Table€1).€€This€significant€dry€root€weight€and€shoot€weight€confirm€the€pathogenicity€of€the€strain€ofÐ  p ÐòòFusarium€solanióó€used€and€the€effectiveness€of€the€assays€as€a€whole.€As€seen€on€the€same€table,€there€wasÐ Lœ Ðsignificance€among€the€lines€for€all€the€traits€measured€except€disease€severity.€The€frequency€distributionÏof€disease€severity€for€the€EXF€RILs€was€normal€(òòPóó€>€0.16)€not€bimodal.€The€field€scores€andÐ ¤ô Ðgreenhouse€scores€were€not€significantly€correlated€for€either€assay.ÌÌTable€1:€Probabilities€of€F€for€variance€among€lines€and€treatments€€ÌÔ*qƒ89 ddd Xdd Xdd XX(#X(#qÔÔ,°”d ÔÔ,Àtd ÔÔ,Àtd ÔÔ,Àtd ÔÔ,Àtd ÔÔ,t” ÔÔ+  ÔÐ 1€::" T$¤ €Ë€‚¨‚ƒƒ„„1Ðò òSourceÐ 2€:;#ü$L :: „„2Ðà@yyðà€dfˆÐ 2€:;#ü$L :; „„2Ѐ€àH° ° $° àˆàH° ° $° àˆàH    !° à€€€€€DSˆÐ 2€:;#ü$L :; „„2Ðà@ee0pàRWT€ˆÐ 2€:;#ü$L :; „„2Ðà@<0àSHWTˆÐ 6€:;:#ü$L :; „„6Ѐ€€HTó óÐ H€;::7#ü$L ! ¨ Ë :;: €!€‚ˆ‚„„HÐà@ÕÕ Èàò òLINEˆÐ t&Ä!" Ðà@rr ÈàTREATó óˆÐ 4€;;:# 'ð"#;:: „„4Ѐà@ŒŒðà41ˆÐ t&Ä!$ Ѐ€€€1ÌÐ 4€;;:#Ì($&;;: „„4Ðà@W W &° ànsˆÌà@Ç Ç %° à0.001ˆÐ 4€;;:# 'ð"(;;: „„4Ðà@‡‡0pà0.001ˆÌà@‡‡0pà0.001ˆÐ 4€;;:# 'ð"*;;: „„4Ðà@yy<0à0.03ˆÌà@GG<0à0.001ˆÐ 6€;;::# 'ð",;;: „„6Ðà@ËËI‚à0.05ˆÌà@™™I‚à0.001ˆÐ3)' 'ð". ˆ0 !  ;;:: 3Ѐ€DS„€Disease€Severity.€RWT„€Dry€root€weight.€SHWT„€Dry€Shoot€Weight.€Ht„€Height̀Р+h&/ Ðò òMarker€associationsó ó:€Five€markers€were€associated€with€the€traits€measured€for€the€forty€lines€tested€inÐ ° Ðthe€cornmeal€sand€assay€(Table€2).€OO19òò400óó€was€strongly€associated€with€both€plant€height€and€shoot€dryÐ Ü, Ðweight.€The€beneficial€allele€was€derived€from€Essex.€The€other€markers€were€only€marginally€significantÏ(P€>€0.01)€and€none€corresponded€to€the€QTL€underlying€field€SDS€resistance€(Hnetkovsky€et€al.,€1996;ÏChang€et€al.,€1996).€Ì€€€€€€€Further€analysis€using€the€extreme€twenty€lines€of€EXF€(10€best€and€10€worst€for€field€SDS€score)Ïshowed€three€markers€previously€identified€as€being€significantly€associated€with€field€disease€resistanceÏto€be€associated€with€some€traits€in€the€greenhouse.€The€two€greenhouse€assays€detected€distinct€loci€asÏassociated€with€SDS.€However,€for€OE02òò1000óó€and€OC01òò650óó€the€beneficial€allele€in€the€greenhouse€wasÐ `  Ðopposite€to€that€in€the€field.€€€ÌÔ€<X¯-XX!XÔÌTable€2:€Marker€association€with€traits€in€the€cornmeal€sand€assay€usingÏthe€extreme€40€ExF€lines€for€field€SDS.Ìòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ð V¦  Ðóó€€€€MARKER€€€€€€€€LG€€€€€€€€TRAIT€€€€€€€€P€€€€€€€€Ròò2óó€€€€€€ALLELIC€MEANSÐ P  Ѐ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€E€€€€€€€€FÌòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€óóÐ D” Ѐ€€€OO19òò400óó€€€€€€€?€€€€€€€€€HT€€€€€€€€€€€0.0001€€€0.348€€€€68.2€€€€€88.9Ð >Ž Ѐ€€€OO19òò400óó€€€€€€€?€€€€€€€€€SHWT€€€€€€€€€0.005€€€€0.203€€€€48.8€€€€€61.2Ð 8ˆ Ѐ€€€OE01òò450óó€€€€€€€?€€€€€€€€€RWT€€€€€€€€€€0.034€€€€0.188€€€€38.7€€€€€58.1Ð 2‚ Ѐ€€€OC11òò300óó€€€€€€€N€€€€€€€€€SHWT€€€€€€€€€0.034€€€€0.125€€€€59.3€€€€€49.6Ð ,| Ѐ€€€A063€€€€€€€€€€C1€€€€€€€€DS€€€€€€€€€€€0.028€€€€0.429€€€€€5.0€€€€€€3.0Ìòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ð  p ÐóóÔ€!XcÛXX<X¯-ÔDS„€Disease€Severity.€RWT„€Dry€root€weight.€SHWT„€Dry€Shoot€Weight.€HT„€HeightÐ j ÐÔ€<X¯-XX!XcÛÔÌÌTable€3:€Markers€showing€significant€association€with€field€diseaseÏparameters€as€well€as€greenhouse€traits.Ìòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€óóÐ ."~ ÐMARKER€€€€€€LG€€€€€€P(field)€€€€€€TRAIT€€€€€€P€€€€€€Ròò2óó€€€€€€ALLELIC€MEANS€Ð (#x Ѐ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€E€€€€€€€€FÌòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€óóÐ %l  Ða,€Cornmeal€AssayÌOR10òò380óó€€€€€Anon1€€€0.036€€€€€€€€€€DS€€€€€€€€0.010€€€0.440€€€3.4€€€€€€Ð '`"! Ð5.1ÌOR10òò400óó€€€€€Anon1€€€0.036€€€€€€€€€€DS€€€€€€€€0.010€€€0.440€€€3.4€€€€€€Ð )T$# Ð5.1ÌOE02òò1000óóà  àG€€€€€€€0.003€€€€€€€€€€RWT€€€€€€€0.006€€€0.809€€90.4€€€€€€39.0Ð ø*H&% ÐOE02òò1000óóà  àG€€€€€€€0.003€€€€€€€€€€SHWT€€€€€€0.017€€€0.707€€70.3€€€€€€48.0Ð ò+B'& Ðb,€Infested€Seed€AssayÌOC01òò650óó€€€€€N€€€€€€€0.001€€€€€€€€€€SHWT€€€€€€0.004€€0.44€€òòóó109.0€€€€€€70.6Ð ªú Ðòò€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ð ¤ô ÐóóÔ€!XcÛXX<X¯-ÔDS„€Disease€Severity.€RWT„€Dry€root€weight.€SHWT„€Dry€Shoot€Weight.€HT„€HeightÐ žî ÐÌ€€€€€€€None€of€the€markers€that€showed€the€expected€association€between€òòF.solanióó€resistance€in€theÐ ö F Ðgreenhouse€and€SDS€resistance€in€the€field.€The€use€of€the€most€extreme€genotypes€does€not€increase€theÏaccuracy€of€detection€of€the€field€QTL.€It€is€possible€the€greenhouse€assays€will€detect€SDS€resistanceÏQTL€for€which€the€ExF€population€do€not€contrast€or€for€traits€that€are€not€measured€in€field€assays€(rootÏweight).€However,€we€conclude€that€further€refinement€of€the€greenhouse€assays€are€necessary€before€theÏassays€can€make€any€meaningful€contribution€to€the€improvement€of€soybean€SDS€resistance.€WeÏencourage€researchers€developing€greenhouse€assays€for€SDS€to€use€the€immortal€population€of€ExF€RILsÏand€molecular€map€as€material€for€validating€"improved"€assays€of€SDS.€€Until€an€effective€greenhouseÏassay€is€developed€and€proven€effective€(see€Kilo€and€Lightfoot,€1996),€soybean€improvement€by€fieldÏevaluation€and€marker€assisted€selection€will€be€pursued.ÌÔ€<X¯-XX!XcÛÔÌÔ€!XcÛXX<X¯-Ôò òòòReferencesóóó óÐ ¨ø Ðà , àChang€S.J.C.,€T.W.€Doubler,€V.€Kilo,€R.€Suttner,€J.€Klein,€M.E.€Schmidt,€P.T.€Gibson€and€D.A.€Lightfootòòóó.Ð Ô$ Ð1996.€Two€additional€Loci€underlying€Durable€Field€Resistance€to€Soybean€Sudden€Death€Syndrome€€Ï(SDS).€Crop€Science€36(5):(in€press).Ìà , àHnetkovsky,€N.,€S.J.C.€Chang,€T.W.€Doubler,€P.T.€Gibson€and€D.A.€Lightfootòòóó.€1996.€Genetic€MappingÐ X¨ Ðof€Loci€Underlying€Field€Resistance€to€Soybean€Sudden€Death€Syndrome€(SDS).€Crop€SciÌ36(2):(in€press).Ìà , àKilo,€V.€and€D.A.€Lightfoot.€1996.€Genetic€€analysis€of€resistance€to€manganese€toxicity€in€soybean.€SoybeanÏGenetic€Newsletter.€(this€issue).Ìà , àO'Donell€K.€and€L.E.€Gray€1995.€Phylogenetic€Relationships€of€the€Soybean€Sudden€Death€SyndromeÏPathogen€òòFusarium€solanióó€f.sp.€òòphaseolióó€Inferred€from€rDNA€Sequence€Data€and€PCR€Primers€for€ItsÐ `"° ÐIdentification.€Molecular€Plant€Microbe€Interactions€.€8:709„716.Ìà , àRoy,€K.W.,€G.W.€Lawrence,€H.H.€Hodges,€K.S.€Mclean,€and€J.F.€Killebrew.€1989€Sudden€Death€ofÏSoybean:€òòFusarium€solanióó€as€Incitant€and€Relation€of€òòHeterodera€glycinesóó€to€Disease€Severity.Ð ä%4! ÐPhytopathology.€79:191„197€Ìà , àRupe,€J.C.€1989.€Frequency€and€Pathogenicity€of€òòFusarium€solanióó€Recovered€from€Soybeans€with€SuddenÐ <(Œ# ÐDeath€Syndrome.€Plant€Disease€73:581„584.à À àà  àÐ h)¸$  Ðà , àStephens,€P.A.,€C.D.€Nickell,€C.K.€Moots€and€S.M.€Lim.€1993.€Relationship€between€Field€and€GreenhouseÏReaction€to€òòFusarium€solanióó€in€Soybean.€Crop€Sci.33:929„930.Ð À+'" ЇÌÒÀXÒG.A.€TORTOòò*óó,Ð Ü, ÐV.€NJITI,€ÌD.A.€LIGHTFOOTÌÔ€<X¯-XX!XcÛÔÓ@ÓÌÒXÀÒò òIntroductionÐ ( x ÐÌà ° àó óSudden€Death€Syndrome€(SDS),€a€disease€found€in€soybeans,€is€causedÐ  X Ðby€soilborne€fungus,€òòFusarium€solanióó.€€SDS€first€appears€as€mosaicÐ Ð  Ðmottling€which€then€progresses€to€chlorosis€and€necrosis€on€leaf€tissue.€ÏThe€major€leaflet€veins€remain€dark€green€(Rupe€et€al.,€1991).Ìà ° àDifferences€in€SDS€susceptibility€among€soybean€lines€were€observedÏin€field€evaluations€(Hershman€et€al.,€1990;€Rupe€et€al.,€1991).€ÏHowever,€due€to€environmental€differences,€greenhouse€assays€were€set€upÏto€evaluate€disease€progression€on€a€controlled€basis.€€The€objective€ofÏthis€research€was€to€screen€varieties€(Douglas,€Ripley,€Lee,€andÏPyramid)€against€two€different€strains€ofòò€F.€solanióó€and€to€studyÐ 8ˆ Ðresistance€of€òòF.€solanióó€strains€in€progeny€lines€from€Pyramid€x€DouglasÐ (x Ðand€Essex€x€Forrest.ÌÐ  à0 ÐÌò òMaterials€and€Methodsó óÐ xÈ ÐÌà ° àTwo€separate€assays€were€conducted€in€the€greenhouse.€€The€firstÏassay€examined€disease€severity€between€òòF.€solanióó€strains€269€and€ST90„Ð øH Ð1.€€Both€strains€were€tested€on€four€varieties,€Ripley,€Pyramid,ÏDouglas,€and€Lee.€€The€experiment€consisted€of€five€replications€perÏvariety,€per€strain.€€The€second€assay€analyzed€disease€severity€onÏprogeny€lines€of€Pyramid€x€Douglas€and€Essex€x€Forrest.€€In€concurrenceÏwith€field€data,€(P.€Gibson,€personal€communication)€the€populationÏconsisted€of€the€ten€most€susceptible€lines€to€SDS€and€the€ten€leastÏsusceptible€lines€to€SDS.€€There€were€five€replications€per€line.€ÏPlants€were€inoculated€with€òòF.€solanióó€strain€ST90„1.€€Ð `°  Ðà ° àòòF.€solanióó€isolates€were€maintained€on€1/3€strength€PDA€plus€50ppmÐ P   Ðtetracycline.€€For€both€assays,€plants€were€inoculated€employing€theÏinfested€oat€technique€(Lim,€1991).€€Oats€were€soaked€in€tap€waterÏovernight,€then€excess€water€was€strained€away.€€150ml€portions€were€putÏinto€250ml€Erlenmeyer€flasks,€capped,€and€autoclaved€for€20€minutes.€ÏOats€were€then€cooled€and€three€1cmòò2óó€agar€plugs€from€the€colony€bordersÐ `° Ðof€òòF.€solanióó€petri€plates€were€aseptically€added€to€the€sterilized€oats.€Ð (x ÐThe€inoculated€oats€were€incubated€in€the€dark€at€room€temperature€(24C)Ïfor€three€weeks.€€Flasks€were€constantly€shaken€for€uniform€growth.€€¼à ° àPlants€were€grown€in€a€soil€medium€containing€a€1:1€(v/v)€mixtureÏof€sterilized€sand€and€sterilized€soil€taken€from€Ridgeway.€€InoculationÏof€the€plants€was€done€at€V2„V3€stage€of€growth€by€placing€five€infestedÏoats€next€to€the€taproot€below€the€soil€surface.€€Plants€were€thenÏirrigated€to€seal€the€soil,€and€soil€was€kept€saturated€to€ensure€theÏpathogenicity€of€the€fungus.€€ÌÐ  X¨ ÐÌò òResults€and€Discussionó óÐ xÈ ÐÌà ° àIn€both€assays,€characteristic€SDS€symptoms€first€appeared€at€theÏR2€stage€of€growth.€€Symptoms€included€chlorosis,€and€in€some€casesÏleaves€were€fasciated.€€Chlorosis€then€progressed€into€necrosis.€PlantsÏdeveloped€until€the€R6€stage€when€final€data€was€taken.Ìà ° àIn€the€first€assay,€comparisons€showed€that€the€mean€symptomaticÏpercentage€of€infection€(the€number€of€leaves€infected€divided€by€theÏtotal€number€of€leaves€on€each€plant)€was€less€in€strain€ST90„1€than€inÏstrain€269€for€varieties€Douglas,€Ripley,€and€Lee€(Table€1).€€However,ÏPyramid€showed€reverse€effects.€€Further€analysis€of€data€showed€thatÏRipley,€Lee,€and€Pyramid,€which€were€inoculated€with€ST90„1,€had€aÏgreater€mean€height€than€those€same€varieties€infected€with€269.€ÏHowever,€Ripley,€Lee,€and€Pyramid€that€were€inoculated€with€269€had€aÏhigher€mean€weight€than€those€infected€by€ST90„1.€€Results€show€thatÏshoot€height€and€weight€may€not€be€an€adequate€way€to€score€diseaseÏseverity.€€€€Ìà ° àIn€the€second€assay,€analysis€of€progeny€from€Essex€x€Forrest€andÏPyramid€x€Douglas€revealed€that€disease€progression€in€the€greenhouse€isÏnot€related€to€the€severity€shown€in€field€data.€€In€the€greenhouseÏassay,€disease€severity€scores€tended€to€be€lower€(Table€2).€€PlantsÏshowed€mostly€chlorotic€effects€and€curling€of€leaves.€€Few€replicationsÏper€line€showed€necrotic€symptoms.€€In€some€cases,€no€disease€symptomsÏwere€evident.€€Resistance€of€the€progeny€lines€to€òòF.€solanióó€suggestsÐ ˜è Ðthat€òòF.€solanióó€may€be€only€one€component€of€SDS€severity.€€AnotherÐ ˆØ Ðcomponent€to€SDS€severity€is€the€soybean€cyst€nematode,€òòHeteroderaÐ xÈ Ðglycines.€€óóPerhaps€the€combination€of€the€two€elements€may€explain€theÐ h¸ Ðmagnified€severity€of€SDS€found€in€field€assays.Ð  X¨ Ðà@ðð3Ààò òBibliographyó óˆÐ ° ÐÌà0 ° àà X àHershman,€D.E.,€J.W.€Hendrix,€R.E.€Stuckey,€and€P.R.€Bachi.€€1990.€ÏInfluence€of€planting€date€and€cultivar€on€soybean€sudden€deathÏsyndrome€in€Kentucky.€€Plant€Dis.€€74:761„766.Ð °(#°(# ÐÌà0 ° àà X àLim,€S.M.€€1991.€€A€technique€for€inoculating€soybeans€in€the€greenhouseÏwith€òòFusariumóó€òòsolani.€€óóPhytopathology€81:1238€(abstr).ÐP  °(#°(# ÐÌà0 ° àà X àRupe,€J.C.,€€E.E.€Gbur,€and€D.M.€Marx.€€1991.€€Cultivar€responses€toÏsudden€death€syndrome€of€soybean.€€Plant€Dis.€75:47„50.Ð Ð  °(#°(# Ð