Popultion Biology Mting-Type Distriution n Geneti Diversity of Cerospor sojin Popultions on Soyen from Arknss: Eviene for Potentil Sexul Reproution Hun Kim, Annky D. Newell, Royn G. Cot-Siekmeyer, John C. Rupe, Ahm M. Fkhoury, n Burton H. Bluhm First, seon, thir, fourth, n sixth uthors: Deprtment of Plnt Pthology, University of Arknss, Fyetteville 72701; n fifth uthor: Deprtment of Plnt, Soil n Agriulturl Systems, Southern Illinois University Cronle, Cronle 62901. Aepte for pulition 12 April 2013. ABSTRACT Kim, H., Newell, A. D., Cot-Siekmeyer, R. G., Rupe, J. C., Fkhoury, A. M., n Bluhm, B. H. 2013. Mting-type istriution n geneti iversity of Cerospor sojin popultions on soyen from Arknss: Eviene for potentil sexul reproution. Phytopthology 103:1045-1051. Cerospor sojin uses frogeye lef spot of soyen, whih n use serious eonomi losses in the Unite Sttes. In this stuy, 132 C. sojin isoltes were ollete from six fiels (from two ounties, Cross n Crwfor) in Arknss. To etermine mting type, multiplex polymerse hin retion ssy ws evelope with primers speifi for C. sojin. Of the 132 isoltes, 68 isoltes h the MAT1-1-1 iiomorph n 64 isoltes h the MAT1-2 iiomorph; no isoltes possesse oth iiomorphs. Both mting types were present in vriety of sptil sles, inluing seprte lesions on iniviul leves. Clone-orrete t from eight mirostellites inite tht mting-type loi were present in pproximtely equl proportions in ll popultions nlyze, whih suggests tht Arknss popultions of C. sojin re unergoing rypti sexul reproution. All six popultions evlute h high genotypi iversity of 26 to 79%. In ition, mong strins isolte from single lef, multiple n istint hplotypes were ssoite with oth mting types, supporting the hypothesis tht sexul reproution ours within the popultions. Most popultions showe signifint gmeti isequilirium ut levels of isequilirium were reltively low, prtiulrly in popultions from Crwfor County. A low ifferentition inex (G ST ) ws oserve for ll simple-sequene repet mrkers ross ll popultions. Furthermore, the vlue of G sttistis etween popultions suggests tht signifint geneti exhnge exists mong the popultions. Tken together, these results emonstrte tht C. sojin popultions from Arknss re genetilly iverse n most likely unergoing sexul reproution. Frogeye lef spot (FLS), use y Cerospor sojin Hr, is n eonomilly importnt isese of soyen in the Unite Sttes (27). FLS ws first esrie in Jpn in 1915 (25), n the first report of C. sojin in the Unite Sttes ws in South Crolin in 1924 (25). For mny ees, FLS hs een prevlent in southern U.S. sttes (37,38) n, more reently, hs eome enemi throughout the U.S. Miwest n upper Miwest (26,42). Beyon the Unite Sttes, C. sojin hs een reporte in t lest 27 ountries spnning North n South Ameri, Europe, Afri, n Asi (6). Symptoms of FLS inlue irulr or ngulr lesions of 1 to 5 mm in imeter tht initilly resemle rk, wter-soke spots (13). As the isese progresses, lesions enlrge into istintive rown spots with reish-rown mrgins, whih n olese n use severe lef lighting or efolition. C. sojin overwinters in infeste soyen resiue n n survive in hevily infete see (27). The isese yle of FLS is initite y spores originting from infete lef eris or lesions on otyleons resulting from plnting hevily infete see (27). Lesions my not pper until 2 weeks fter invsion of tissue n, therefore, re not typilly oserve on young leves (27). However, onii my pper s erly s 48 h fter inoultion on plnts expose to fvorle tempertures (25 to 30 C) n high reltive humiity (>90%). Conii re rrie y ir urrents n rin splsh n use Corresponing uthor: B. H. Bluhm, E-mil ress: luhm@urk.eu http://x.oi.org/10.1094/ PHYTO-09-12-0229-R 2013 The Amerin Phytopthologil Soiety seonry infetions throughout the growing seson when environmentl onitions re fvorle. Beuse FLS is polyyli isese, symptoms n inrese ontinuously throughout the growing seson n reh epiemi proportions when environmentl onitions re fvorle or suitle ontrol mesures re not employe. Regring the ltter, serious onern regring FLS mngement is the reent emergene of resistne to stroilurin fungiies mong U.S. popultions of C. sojin (45). The rte n mehnism of spre of fungiie resistne throughout popultions of C. sojin hve onsierle implitions for FLS mngement, yet re poorly unerstoo. The potentil role of sexul reproution mong popultions of C. sojin in FLS epiemiology n mngement hs not een estlishe. In mny plnt-pthogeni fungi, sexul reproution is key mehnism through whih geneti iversity is proue, n often llows fungl pthogens to overome host resistne y generting more fit genotypes through reomintion (11). However, for most Cerospor spp., inluing C. sojin, sexul stge hs not een oserve in either fiel or lortory onitions. Bse on moleulr nlyses, Cerospor spp. form monophyleti group within the teleomorphi genus Myospherell (6 8,12), n Myospherell teleomorphs hve een foun for few Cerospor spp. (9,39). Like mny other heterothlli somyetes, ompletion of the sexul yle in Cerospor spp. is preite on the presene n intertion of iniviuls with ifferent iiomorphs, nmely MAT1-1 n MAT1-2, t single mting-type lous (5,14). Although the istriution of mtingtype iiomorphs throughout nturl popultions of given fungus neither proves nor isproves the existene of sexul stge, equl Vol. 103, No. 10, 2013 1045
proportions of mting type iiomorphs re expete in popultions unergoing sexul reproution (28). In the sene of known sexul stge, severl pprohes n provie eviene of rypti sexul reproution, inluing mesurements of geneti iversity, popultion ifferentition, n mting-type frequenies (28). Popultions unergoing sexul reproution typilly hve high geneti iversity n equl mting-type frequenies ompre with popultions solely or preominntly reprouing sexully (28). Reent reports y Groenewl et l. (15,17) showe tht t lest some popultions of C. zee-myis, C. zein, n C. etiol hve 1:1 istriutions of mting type iiomorphs, n tht popultions of C. etiol hve high levels of geneti iversity. Bse on these results, the uthors postulte tht C. etiol popultions unergo sexul reomintion even though no teleomorph hs yet een oserve, n tht other speies of Cerospor my lso unergo rypti sexul reproution. In the present stuy, our overll gol ws to ssess whether popultions of C. sojin isolte from Arknss unergo sexul reproution. The speifi ojetives of this stuy were to (i) evelop multiplex polymerse hin retion (PCR) onitions for rpi ientifition of mting types in C. sojin n use this ssy to etermine the frequenies n istriutions of the two mting types, (ii) etermine the level of geneti iversity n popultion ifferentition etween n within popultions se on mirostellite nlyses, n (iii) ssess the possiility of sexul reproution in fiel popultions with multilous nlyses of popultion struture. To this en, 132 isoltes of C. sojin were ollete from two lotions in Arknss, n the mting type n hplotype of eh isolte ws efine n nlyze. From this work, more etile unerstning of the popultion struture of C. sojin hs emerge, inluing eviene for rypti sexul reproution. MATERIALS AND METHODS Soyen smpling n fungl isoltion. To isolte C. sojin from symptomti soyen tissue, leves with visile lesions were inute in moist hmer ( petri plte ontining moist filter pper) t room temperture for 2 to 3 ys with 12-h photoperio to inue sporultion. Leves were heke ily for the onset of sporultion with isseting mirosope. Conii were ollete with flme-sterilize neele n trnsferre onto V8 gr meium ontining mpiillin (100 µg/ml). The ultures were inute t room temperture until the olonies were lrge enough to trnsfer onto new V8 gr pltes. For single-spore isoltion, olonies were srpe with flme loop n streke onto fresh V8 gr pltes n, susequently, single isolte olonies were trnsferre to new V8 gr pltes. The pltes were inute for 5 to 7 ys fter olonies grew visily. All isoltes were store t 80 C in 20% glyerol. Soyen plnts showing typil FLS isese symptoms were ollete from six fiels in two ounties (Cross n Crwfor) in Arknss on 11 or 30 Septemer 2009, respetively; these two ounties re seprte y 300 km. Four popultions (popw1, n = 24; popw2, n = 23; popw3, n = 20; n popw4, n = 20) were ollete from four iniviul fiels ner Wynne, AR, in Cross County; eh fiel ws seprte y rowys whih were 10 m wie, n five to six isese plnts were ollete from eh fiel for C. sojin isoltion. Aitionlly, two popultions (popk1, n = 21 n popk2, n = 24) were isolte from two fiels of the University of Arknss Vegetle Reserh Sttion ner Kiler in Crwfor County, n smpling methos were performe s esrie ove. Ultimtely, from these six fiels, 132 C. sojin isoltes were ollete for further nlysis. DNA extrtion. A 0.5-y-0.5-m segment of myelium from eh single-spore isolte ws ollete from ultures grown on V8 gr meium n ple into 1.5-ml miroentrifuge tue ontining 100 µl of Tris-EDTA uffer (10 mm Tris-Cl n 1 mm EDTA, ph 7.5). The tissue ws groun in the tue with sterile miropestle followe y inution in oiling wter for 10 min. After entrifugtion, the onentrtion of genomi DNA in the superntnt ws quntifie with ND-1000 spetrophotometer (NnoDrop Tehnologies, Wilmington, DE). Multiplex PCR ssy for etermintion of mting types. Degenerte primer pirs MgMfSpMt1-1f1/1r2 n MgMfSpmt1-2f2/2r1 n Cerospor-speifi primer pirs CerosporMt1f/1r n CerosporMt2f/2r (Tle 1) (14) were use to mplify onserve regions of the MAT1-1-1 n MAT1-2 genes of C. sojin isoltes J2L2 n 07-IN. All PCR onitions were followe s esrie y Groenewl et l. (14). Retion prouts were nlyze y eletrophoresis in 1% (wt/vol) grose gel with ethiium romie t 0.1 µg/ml in 1 Tris-orte-EDTA (TBE) uffer, visulize on UV trnsillumintor, n purifie with the QIAquik gel extrtion kit (Qigen, Vleni, CA). PCR prouts were then sequene with the originl primers, n the sequene informtion ws eposite t GenBnk. From these sequenes, three sets of primers were esigne for multiplex PCR ssy: Csojin18Sf/28Sr, CsMt1f/1r, n CsMt2f/2r. Primers were synthesize y Integrte DNA Tehnologies (Corlville, IA). For eh PCR, the totl retion of 50 µl ontine 2 µl of genomi DNA, 10 µl of 10 PCR retion uffer, 2 µl of 10 mm NTPs, 10 pmol of primer sets CsMt1f/1r n CsMt2f/2r, 5 pmol of primer set Csojin18Sf/28Sr, 5 µl of 25 mm MgCl 2, n 0.7 units of Tq polymerse. The PCR retions were performe with 2720 Therml Cyler (Applie Biosystems, Crls, CA). The initil enturtion step of 94 C for 2 min ws followe y 40 yles of mplifition (94 C for 30 s, 58.5 C for 30 s, n 72 C for 1 min), followe y finl elongtion step of 72 C for 5 min. All PCR prouts were visulize uner UV light on 1% (wt/vol) grose gel ontining ethiium romie t 0.1 µg/ml in 1 TBE uffer fter eletrophoreti seprtion t 95 V for 1 h. TABLE 1. Primers use in multiplex polymerse hin retion (PCR) for ientifition of mting types Primer Sequene (5 3 ) Note Soure MgMfSpMt1-1f1 CATTNGCNCATCCCTTTG Degenerte primer 14 MgMfSpMt1-1r2 GGCTTNGANACCATGGTGAG Degenerte primer 14 MgMfSpmt1-2f2 CAAAGAANGCNTTCNTGATCT Degenerte primer 14 MgMfSpmt1-2r1 TTCTTCTCNGATGGCTTGC Degenerte primer 14 CerosporMt1f CTTGCAGTGAGGACATGG Degenerte primer 14 CerosporMt1r GAGGCCATGGTGAGTGAG Degenerte primer 14 CerosporMt2f GATNTACCNTCTCGA Degenerte primer 14 CerosporMt2r CTGTGGAGCAGTG Degenerte primer 14 Csojin18Sf TCTCCGTAGGTGAACCTGCG Multiplex PCR primer This stuy Csojin28Sr TATCCCTACCTGATCCGAGGTCAA Multiplex PCR primer This stuy CsMt1f TGAGGACATGGCCACCCAAATA Multiplex PCR primer This stuy CsMt1r AAGAGCCCTGTCAAGTGTCAGT Multiplex PCR primer This stuy CsMt2f TGTTGTAGAGCTCGTTGTTCGCA Multiplex PCR primer This stuy CsMt2r TCAGACCTTATGAGCTTGAAAGTGCT Multiplex PCR primer This stuy 1046 PHYTOPATHOLOGY
Anlysis of geneti iversity. To nlyze geneti iversity in eh popultion, we use eight mirostellite (simple sequene repet [SSR]) mrkers (Tle 2) tht showe high levels of polymorphism mong wie rnge of C. sojin isoltes (29). SSR loi were mplifie vi PCR in 25-µl retions ontining 100 ng of DNA, 30 nm forwr n reverse primers, 0.5 mm NTPs, 2.5 mm MgCl 2, n 0.7 units of Tq polymerse. Cyling prmeters onsiste of n initil enturtion t 95 C for 5 min; followe y 30 yles of 94 C for 30 s, 56 C for 30 s, 72 C for 60 s; n extension t 72 C for 7 min. Retion prouts were seprte y eletrophoresis in 3% Metphor grose gels n etete y stining with ethiium romie. A 10-p DNA ler (Invitrogen, Crls, CA) ws use s size mrker. Consistent with similr stuies, this pproh llowe resolution of 3 to 4 p mong PCR prouts (30). Polymorphi DNA ns were sore mnully with GenAlex 6 (35) to etermine the numer of hplotypes n rete input file formts require for other nlysis tools. The totl numer of lleles, numer of effetive lleles, n geneti iversity t eh SSR lous were estimte with POPGENE, version 1.32 (43), whih lso estimtes vlues of Nei s unise geneti ientity n istne for popultions. In ition, POPGENE (43) n GenoDive (24) were use to etermine inies of geneti ifferentition suh s G ST n G ST. Genotypi iversity ws estimte y the metho of Stort n Tylor (41), n normlize y iviing y the numer of smples in eh popultion, s esrie y Grunwl et l. (17). Multilous v1.3 (1) ws use to etermine the vlue of rbrd, n lterntive mesure of the inex of ssoition for isequilirium, n the numer of lous pirs in linkge isequiliri ws estimte with POPGENE, version 1.32 (43). RESULTS Optimiztion of multiplex PCR onitions. Although sequenes of eh mting-type iiomorph re firly well onserve mong mny Cerospor spp., primers previously esigne from other speies inonsistently mplifie C. sojin mting-type genes. Thus, the mting-type loi of C. sojin were sequene n new primers were esigne for rpi, high-throughput ssessment of mting types. Degenerte primers (Tle 1) were use to mplify portions of the mting-type genes MAT1-1-1 n MAT1-2, n PCR-se genome wlking ws susequently use to otin the full-length sequenes of oth iiomorphs. Sequenes of mting-type genes were eposite in the Ntionl Center for Biotehnology Informtion tse (MAT1-1-1, GenBnk numer JX047865 n MAT1-2, GenBnk numer JX047866), n primers tht speifilly mplifie the mting-type lleles of C. sojin were rete (CsMt1f/r n CsMt2f/r) (Tle 1). Also, primer pir tht mplifies the internl trnsrie sper (ITS) region of rdna (GenBnk ession numer AY266156) of C. sojin ws esigne to provie n internl positive ontrol to onfirm the presene of mplifile DNA (Tle 1). Multiplex PCR onitions suh s nneling tempertures n primer onentrtions were optimize. To etermine the optimum temperture for PCR, the primers were teste t nneling tempertures of 57.5, 58, 58.5, 58.8, n 59 C. Consequently, 58.5 C ws etermine to e the optimum nneling temperture for mplifition se on the intensity of PCR prouts. Inresing the onentrtion of primer pirs CsMt1f/r n CsMt2f/r reltive to the primers mplifying the ITS region of rdna provie more equl mplifition of the two single-opy loi reltive to the multiopy rdna sequene. After evluting severl rtios of primer onentrtions, 10 pmol of CsMt1f/r n CsMt2f/r primer pirs with 5 pmol of primer pirs for rdna provie the most onsistent, even mplifition of ll three prouts. With this multiplex PCR ssy, the mting type lous of 132 C. sojin isoltes ws ssesse, n single mplion of either 406 p (MAT1-1-1) or 298 p (MAT1-2) ws otine from eh isolte (Fig. 1). None of the isoltes ontine oth iiomorphs. All retions proue n mplion of 500 p, onfirming the presene of mplifile DNA from the ITS region (Fig. 1). Thus, the ssy provie roust n relile tool to sore lleles t the mting type lous of C. sojin. Distriution of mting types within popultions. Among the 132 C. sojin isoltes ollete from Arknss, 68 isoltes possesse MAT1-1-1 n 64 isoltes possesse MAT1-2, frequeny tht oes not evite signifintly from 1:1 rtio (χ 2 = 0.12) (Tle 3). At the level of iniviul fiels, popultion popw3 n popk2 signifintly evite from the null hypothesis of 1:1 Fig. 1. Multiplex polymerse hin retion for MAT genes of 10 representtive isoltes. Lnes 1 (07-IN) n 2 (J2L2) re stnr strins for MAT1-2 n MAT1-1-1, respetively; lnes 3, 5, 7, 9, n 10 show isoltes tht hve MAT1-1-1; n lnes 4, 6, n 8 represent isoltes tht possess MAT1-2. ITS = internl trnsrie sper. TABLE 2. Simple-sequene repet (SSR) mrkers use to nlyze geneti iversity of Cerospor sojin Lous Primer sequene (5 3 ) Repet motif Size rnge (p) N e SSRCs1 F: GGTAATCGCCCAGATGAAGA (AAC) 16 160 185 (179) 6 R: CGAGGAGGTTGGTTGTTGTT SSRCs2 F: CGCAAAATTTCAGTTCAGCA (GAA) 13 170 205 (178) 5 R: CACGATTGAAGAATGCATGG SSRCs3 F: GACGGAATACGGGCTTATGA (TTC) 19 165 190 (171) 4 R: TCAGAGACTTGTCGCCTCAG SSRCs4 F: CCATATTGTCGCCAGGTCTT (AGA) 12 190 200 (189) 3 R: GCTCGCATCTGACATTTCCT SSRCs5 F: GCCCATGCTAATGACTGGAT (CGCTGTAT) 13 225 240 (228) 3 R: GGCACGACACACCCTCTATG SSRCs6 F: TGGCGAAGACGTCTGATATG (TTG) 13 215 220 (219) 3 R: TTCTTTCGCATTGCATCAAC SSRCs7 F: AAGTATCGGAGCGGACACAG (TTCT) 14 205 240 (235) 3 R: GTCCAGGCGTTTTAGACGAG SSRCs8 F: CCCCATATGCTGCAGAGCTA (ATG) 10 170 185 (171) 3 R: TCCCTTCTTTGTGCATCATC Originlly esrie y the Moleulr Eology Resoures Primer Development Consortium (29). F, forwr primer; R, reverse primer. Numers inite how mny times the repet ourre in the originl sequene. Numers in prentheses re the lengths of the lleles erive from the originl sequene. e Oserve numer of lleles. Vol. 103, No. 10, 2013 1047
rtio for the two mting type lleles (χ 2 = 7.2 n 8.17, respetively) (Tle 3). Upon initil onsiertion, this ppere to inite high mount of lonl propgtion, espeilly when onsiering tht the other popultions i not evite signifintly from 1:1 rtio of mting type lleles (Tle 3). At ll lotions, we oserve tht oth mting types were frequently etete mong isoltes ollete from istint lesions on iniviul leves. For exmple, popultions popw1, popw2, popw3, n popw4, eh of whih onsiste of 10 iniviuls isolte from single lef, h mting type rtios of 5:5, 5:5, 0:10, n 4:6, respetively (Tle 4). However, when lone-orrete t erive from nlyses of SSRs were use for the nlysis of mting-type istriution, none of the six popultions evite signifintly from 1:1 rtio of mting types (Tle 3). Thus, the presene of oth mting types mong popultions in lose physil proximity (e.g., iniviul leves) n the lone-orrete 1:1 mting type frequeny throughout sptilly istriute popultions re onsistent with the hypothesis tht sexul reproution is ourring in C. sojin popultions in Arknss. Genotypi iversity within popultions. Vrious moleulr mrkers suh s rnom mplifie polymorphi DNA, mplifie frgment length polymorphism, restrition frgment length polymorphism, n mirostellites (SSRs) hve een use to nlyze geneti iversity of Cerospor spp. (4,10,15,20,34). In popultions of C. etiol, Groenewl et l. showe the potentil of sexul reproution se on high genotypi iversity n pproximtely equl proportions of mting-type lleles mong popultions (15). In ition, the existene of oth mting types n ifferent genotypes, s etermine y SSR nlyses, in iniviul leves supports preitions of sexul reproution in C. etiol popultions (2). Reently, useful SSR mrkers were hrterize from C. sojin (Tle 2) (29). In this stuy, eight SSR loi were nlyze to test the hypothesis tht sexul reproution ws ourring in C. sojin popultions within Arknss. Among the 132 isoltes, 29 lleles were sore t eight SSR loi, n 71 hplotypes were etete. Also, we oserve hplotypes tht were unique to single popultion (12 in popw1, 11 in popw2, 7 in popw3, 6 in popw4, 11 in popk1, n 8 in popk2) n foun tht few hplotypes were present in multiple popultions. For exmple, only 11 hplotypes were foun in t lest two popultions, n just one hplotype ws present in five of the six popultions. Vlues of gene iversity (H) were 0.42 to 0.58, whih were high in ll popultions (Tle 3). The popk2 popultion h the lowest H n the popw1 n popw2 popultions h the highest vlues (Tle 4). Normlize genotypi iversity ( /N) ws estimte t vrious sptil sles: iniviul leves, fiels, n lotions (Tle 4). Bse on nlyses of eight polymorphi SSRs, genotypi iversity ws oserve in vrious rnges through ll popultions smple. Moreover, multiple hplotypes n oth mting types were oserve mong isoltes from iniviul leves, whih suggests tht none of the popultions were solutely lonl. The TABLE 3. Distriution of mting-type genes of Cerospor sojin popultions from Arknss Frequenies Popultion Numer of smples Clonl frtion MAT1-1-1 MAT1-2 Unorrete t Clone-orrete t P popw1 24 0.29 0.50 0.50 0 0.06 0.81 popw2 23 0.13 0.52 0.48 0.04 0.20 0.66 popw3 20 0.40 0.20 0.80 7.2* 1.33 0.25 popw4 20 0.35 0.40 0.60 2.98 0.08 0.78 popk1 21 0.24 0.62 0.38 1.19 1.00 0.32 popk2 24 0.50 0.79 0.21 8.17* 0.34 0.56 Totl 132 0.46 0.52 0.48 0.12 0.01 0.92 Estimte s 1 [(numer of ifferent genotypes)/(totl numer of isoltes)] (44). Frequenies were lulte from unorrete smples. Asterisk (*) inites mting-type frequenies tht re signifintly ifferent t P < 0.05. Proility (P) for χ 2 of lone-orrete t with 1 egree of freeom. χ 2 test TABLE 4. Genotypi n geneti iversity within popultions t ifferent sptil sles Sptil sle, popultion N N H H /N (%) e rbrd f LD (%) g Within lef popw1 10 9 8.3 83 popw2 10 8 7.1 71 popw3 10 4 1.9 19 popw4 10 6 4.5 45 Within fiel popw1 24 17 0.58 12.5 52 0.204* 26/271 (9) popw2 23 20 0.58 18.2 79 0.190* 24/279 (9) popw3 20 12 0.51 5.1 26 0.170* 17/191 (9) popw4 20 13 0.56 10.0 50 0.301* 39/228 (17) popk1 21 16 0.43 12.6 60 0.048** 12/190 (6) popk2 24 12 0.42 6.4 27 0.137** 7/207 (3) Within lotion popw 87 49 23.8 27 0.198* popk 45 26 15.2 34 0.088* Numer of smples in popultion. Numer of hplotypes. Nei s gene iversity (H) within popultions (31); inites not lulte. Genotypi iversity (Ĝ) ws lulte for eh popultion oring to the metho of Stort n Tylor (41). Ĝ = 1/Σpi 2, where pi = the oserve frequeny of the ith multilous genotype in popultion. e Normlize genotypi iversity ( /N) ws lulte y iviing y the numer of smples in eh popultion (17). f Stnrize inex of ssoition (rbrd) ws estimte from lone-orrete t (1), n the null hypothesis of multilous equilirium ws rejete if P < 0.01 (*) or 0.05 (**); inites not lulte. g Numer of lous pirs in linkge isequiliri (LD) t P < 0.01 (21). Vlues re isplye s the numer of lous pirs in isequiliri/totl numer of linkge pirs, with the perentge given in prentheses; inites not lulte. 1048 PHYTOPATHOLOGY
TABLE 5. Nei s gene iversity (33) of Cerospor sojin t eight mirostellite (simple sequene repet [SSR]) loi from lone-orrete smples Lous N 0 N E H T H S H e ST SSRCs1 5 2.2 0.639 0.550 0.139 SSRCs2 5 2.2 0.587 0.549 0.065 SSRCs3 4 2.1 0.581 0.524 0.097 SSRCs4 3 2.0 0.529 0.488 0.079 SSRCs5 3 1.9 0.521 0.479 0.081 SSRCs6 2 1.7 0.461 0.410 0.112 SSRCs7 4 3.2 0.719 0.691 0.040 SSRCs8 3 1.7 0.445 0.417 0.062 Men 3.6 2.1 0.560 0.514 0.084 Numer of lleles in the totl smples. Effetive numer of lleles. Gene iversity in the totl smples over ll popultions. Men gene iversity verge over ll popultions. e Nei s oeffiient of geneti ifferentition (32); lulte y H T H S /H T. All vlues were signifint t P < 0.05. normlize genotypi iversity vrie etween popultions: 20 to 80% t the level of iniviul leves n fiels (Tle 4) s well s t the level of lotion (popw, Wynne n popk, Kiler). Tests for multilous gmeti isequilirium y inex of ssoition (rbrd) showe tht ll popultions were in signifint isequilirium (Tle 4). The lowest rbrd vlue ourre in popk1 (0.048), n oth popultions from Kiler h reltively low vlues ompre with popultions from Wynne, t 0.170 to 0.301 (P < 0.01). In ition, the perentge of lous pirs in signifint linkge isequilirium (χ 2 test, P < 0.01) in the six popultions rnge from 3% (7 of 207 pirs) in the popk2 popultion to 17% (39 of 228 pirs) in the popw4 popultion (Tle 4); t the P < 0.05 signifine level, the perentge ws 12 to 24% (t not shown). Geneti iversity n ifferentition of popultions. The ontriution of eh SSR lous to mesurements of geneti iversity vrie sustntilly (Tle 5). All eight loi evlute were polymorphi, with 2 to 5 lleles etete per lous (verge = 3.6 lleles), n the effetive numer of lleles ws 1.6 to 3.2 (Tle 5). Among the SSR loi nlyze, SSRCs7 h the highest H T n H S vlues, n lous SSRCs8 h the lowest (Tle 5). The SSR lous SSRCs1 h the highest fixtion inex (G ST, 0.139) n SSRCs7 h the lowest fixtion inex (G ST, 0.040); the verge G ST of ll loi ws 0.084 (Tle 5). The low vlue of G ST oserve in this stuy ws onsistent with previous report of Myospherell grminiol popultions retining sexul reproution (18), n inites reltively high migrtion rte. Furthermore, when other prmeters suh s G ST n G ST were use, the vlues were lso reltively low (t not shown). Pirwise lultions of Nei s unise geneti ientity etween six popultions of C. sojin in Arknss were uniformly high (0.851 to 0.991) (Tle 6). Intriguingly, the vlues mong the popultions ollete t Wynne (0.945 to 0.991) were reltively high ompre with popultions ollete t Kiler (0.857) (Tle 6). In some ses, the geneti ientity etween popultions from ifferent lotions ws s high s tht oserve etween popultions from the sme lotion (e.g., popw2/popk2 versus popw2/popw4). The lowest geneti ientity (0.851) ws foun etween popw3 n popk1. These results inite tht the mjority of geneti iversity is istriute on smll sptil sle in Arknss. Relte to popultion ifferentition, G ST vlues etween popultions inite signifint geneti ifferentition etween pproximtely two-thirs of the pirwise omprisons; the remining omprisons were not signifint (Tle 6). Other prmeters suh s G ST, G ST, n Jost s D inite either low levels of ifferentition or nonifferentition. Overll, these vlues of ifferentition reflet the orreltion in geneti ientity etween popultions. Tken together, these results suggest tht there hs een signifint geneti exhnge mong the popultions. DISCUSSION In this stuy, we evelope multiplex PCR ssy to rpily sore mting type loi in C. sojin, n investigte geneti iversity n struture of Arknss popultions with polymorphi SSR mrkers. The popultions i not evite signifintly from 1:1 rtio of mting types, n the nlysis of SSRs supporte the hypothesis tht sexul reproution ours mong popultions of C. sojin. Aitionlly, y proviing the first moleulr-level perspetive of the geneti iversity mong popultions of C. sojin, this stuy provies importnt informtion to guie FLS mngement strtegies, prtiulrly in the ontext of emerging resistne to stroilurin fungiies, s well s ongoing efforts to improve FLS resistne vi onventionl reeing pprohes. Anlyses of multilous gmeti isequilirium y inex of ssoition (rbrd) showe tht ll popultions were in signifint isequilirium. However, it is importnt to note tht the presene of gmeti isequilirium within popultions of C. sojin is not surprising given the urrent unerstning of the FLS isese yle. The popultions nlyze in this stuy were otine from soyen fiels experiening high levels of FLS reltively lte in the growing seson n, thus, sexul reproution ws likely to preominte. Thus, rnom mting t speifi point in the isese yle (e.g., the initil or finl stge) is the most likely explntion for the simultneous etetion of high levels of genotypi iversity n signifint levels of gmeti isequilirium. Consistent with this onept, low inex of ssoition vlues hve een oserve in fungl popultions in whih sexul reproution is postulte to our t low levels (e.g., C. etiol n Pyrenophor teres f. sp. teres) (15,36). Aitionlly, popultions unerlying isese outreks my e ominte y losely relte iniviuls, euse ertin genotypes my e fvore y seletion pressure ssoite with environment or previling host genotypes (40). Host effets on pthogen iversity re prtiulrly relevnt for FLS in the Unite Sttes, in whih monoultures of reltively limite numer of soyen ultivrs my ominte wie geogrphil re. Thus, uring FLS outreks, ertin genotypes of the pthogen my inrese in frequeny n generte isequilirium until sexul reomintion hs h time to rnomize the geneti kgroun. TABLE 6. Nei s unise geneti ientity (32) n orrete stnrize fixtion inex (19,23) y pirwise omprisons of fiel popultions Geneti ientity/fixtion inex Popultion popw1 popw2 popw3 popw4 popk1 popk2 popw1 0.991 0.950 0.979 0.882 0.863 popw2 ns 0.945 0.984 0.885 0.933 popw3 0.001 0.008 0.991 0.970 0.851 popw4 ns ns ns 0.918 0.917 popk1 0.077 0.077 ns 0.045 0.857 popk2 0.087 0.035 0.100 0.041 0.119 Geneti ientity (ove the igonl) n orrete stnrize fixtion inex (G ST, elow the igonl) vlues were signifint t P < 0.05; ns = nonsignifint. Vol. 103, No. 10, 2013 1049
Regring the linkge isequilirium oserve mong lous pirs, vriety of phenomen oul explin the levels of isequilirium etete in this stuy, inluing popultion strtifition, geneti rift, n linkge mong mrkers. However, in the sene of physil or geneti mp of the C. sojin genome, it is not urrently possile to exmine the uses of the linkge isequilirium oserve mong SSR mrkers in this stuy. Overll, the levels of genotypi iversity oserve within eh popultion were onsistent with levels of genotypi iversity typilly oserve in sexully reprouing popultions of fungi (28). Prtiulrly in popk1, the low lonl frtion, low gmeti isequilirium, high genotypi iversity, n equl frequenies of the two mting types re onsistent with reent or ongoing sexul reproution. Other popultions, in ontrst, h intermeite levels of isequilirium n moerte lonl frtions, most likely resulting from higher levels of sexul reproution or less outrossing. The ifferenes oserve etween popultions might e ue, in prt, to smll smple sizes or smpling methos rther thn popultion strutures. However, these ftors o not prelue the possiility of sexul reproution, espeilly onsiering tht smll mount of sexul reomintion my e suffiient to sustin high levels of genotypi iversity (22). The level of geneti iversity oserve mong popultions of C. sojin in Arknss inites tht sexul reproution my filitte rpi pttion in the pthogen. This fining hs signifint implitions for the mngement of FLS. First, sexul reproution presumly provies C. sojin mehnism to overome host resistne. Consistent with this ie, multiple res of C. sojin hve een esrie se on virulene on ifferentil soyen ultivrs (25), eh of whih presumly possesses istint set of resistne genes. The high level of geneti iversity ientifie in this stuy suggests tht mny more res of C. sojin re likely to exist or re ple of rising in response to seletion pressure n, thus, onventionl strtegies to ree for resistne shoul tke high levels of iversity into ount. For exmple, sexul reomintion is likely to e ourring outsie of Arknss popultions of C. sojin n, thus, resistne tht is roust in one geogrphil re my not e effetive or urle ginst regionlly istint popultions of the pthogen. Seon, rypti sexul reomintion in C. sojin my elerte the spre of resistne to stroilurin fungiies. For exmple, if the initil emergene of stroilurin resistne ours t reltively low frequeny, resistne woul e present in limite numer of hplotypes (n presumly only few res) in the sene of sexul reomintion. Thus, the spre of resistne oul e limite y nturl onstrints on the ility of those speifi hplotypes to spre. Sexul reomintion, however, oul rpily elerte the istriution of fungiie resistne throughout popultions n into iverse geneti kgrouns of the pthogen. In this senrio, lolize emergene of resistne oul oneivly e istriute throughout mny res within few sexul yles. Resistne to stroilurin fungiies ontinues to e reporte in new lotions throughout the Unite Sttes; therefore, popultions of resistnt isoltes oul e evlute s esrie in this stuy to ssess the role tht sexul reproution plys in issemintion of resistne. The results of this stuy ll for itionl experimenttion to ress the potentil role of sospores in the epiemiology of FLS. The C. sojin popultions nlyze in this stuy were ollete from fiels in whih plnts were pprohing mturity n exhiiting lrge numers of lesions per lef, whih more urtely pproximtes the pek of n outrek rther thn its initition. Bse on the levels of geneti iversity etete in this stuy n the lk of oservtions of sexul fruiting oies or sospores uring folir isese evelopment, it is possile tht FLS is initite, t lest in some yers or irumstnes, y sospores forme in resiul lef eris from the previous growing seson; in this senrio, sospores oul e isperse lolly or over onsierle istnes, s hs een oserve for relte Myospherell spp. (3). Although the popultions nlyze in this stuy h outlessly unergone severl yles of sexul reproution efore smpling ourre, the lone-orrete results still inite tht sexul reproution ws likely to e riving fore unerlying the oserve popultion iversity. The extent to whih rypti sexul reproution is preite to e ourring mong glol popultions of C. sojin oul e etermine y nlyzing popultions ollete from itionl, geogrphilly iverse lotions. Moreover, temporl nlyses of geneti iversity of C. sojin popultions throughout the initition, pex, n onlusion of isese outrek oul potentilly illuminte the timing n importne of sexul reproution within growing seson. In this stuy, eight polymorphi SSR mrkers, with repet motifs of three to eight ses (Tle 2), were use to nlyze popultions of C. sojin. These mrkers were suffiiently polymorphi to sore on high-resolution grose gels with onventionl eletrophoresis rther thn more sophistite nlysis pltforms, suh s pillry eletrophoresis. Anlyses of SSR mrkers on grose gels hs the istint vntges of eing inexpensive n wiely essile (30); thus, this pproh ws selete with the hope of filitting follow-up stuies mong memers of the U.S. n interntionl FLS reserh ommunity, in whih wie rnge of geogrphilly istint C. sojin popultions oul potentilly e nlyze. However, it is possile tht ertin level of geneti iversity will e lost with grose-se nlyses ue to less resolution of PCR prout sizes ompre with other methos. We elieve tht SSR mrkers with lrger repet motifs, s use in this stuy, ovite this onern to onsierle extent, eviene of whih is the ft tht grosese nlyses of the SSR mrkers use in this stuy ientifie suffiient iversity to strongly support preitions of rypti sexul reproution within Arknss popultions of C. sojin. As itionl genome sequene resoures eome ville for C. sojin, the ientifition n eployment of new SSR mrkers will outlessly ontriute to future efforts to stuy the geneti iversity of the pthogen. Thus, in the future, refine methos of SSR nlysis my e require to fully ress questions of geneti iversity in C. sojin. Bse on the high levels of geneti iversity oserve within Arknss popultions of C. sojin, new hypotheses regring the geneti sis of host speifiity n e formulte n teste. Mny speies of Cerospor, inluing C. sojin, hve nrrowly restrite host rnge (6). The geneti n moleulr sis of host speifiity mong Cerospor spp. is fsinting yet poorly stuie phenomenon, n hs ro potentil implitions for the mngement of Cerospor iseses s well s the refinement of txonomi reltionships within the genus. Historilly, host rnge ws key omponent of the speies onept mong Cerospor spp., lthough reent refinements to the txonomy of Cerospor spp., inluing C. sojin, hve rwn most hevily on morphologil hrteristis n DNA sequene informtion t selete loi (16). To te, C. sojin hs een reporte s pthogeni on hnful of speies within the Leguminose fmily, preominntly of the gener Glyine n Muun (6). Given this host rnge, future experiments oul e evise to test whether reltionships exist etween selete C. sojin genotypes n pthogeniity on vrious plnt speies reporte to e omptile hosts for the pthogen, thus potentilly lrifying the importne of host rnge s omponent of the speies onept for the orgnism. ACKNOWLEDGMENTS This reserh ws supporte y the Unite Soyen Bor (wr numer 2262), the Arknss Soyen Promotion Bor, n the University of Arknss Division of Agriulture. We thnk S. Goowin for guine uring the preprtion of the mnusript. 1050 PHYTOPATHOLOGY
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