First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Fi s TILLING Pla o m in Cucu bi a pepo : A New Mu an Resou ce o Gene Func ion and C op Imp o emen Nelly Vicen e-Do ´le a 1 , Ch is elle T oadec 2 , Manuel Moya 1 , Me cedes del Rı ´o-Celes ino 1 , Te esa Poma es-Viciana 1 , Abdelha id Bendahmane 2 , Bele ´n Pico ´ 3 , Bele ´n Roma ´n 4 , Ped o Go ´mez 1 * 1IFAPA Cen o La Mojone a, Camino de San Nicola ´s, 1, 04745 La Mojone a, Alme ı ´a, Spain, 2INRA-URGV, UMR1165, Uni e ´de Reche che en Ge ´nomique Ve ´ge ´ ale, Saclay Plan Sciences, E y, F ance, 3Ins i u e o he Conse a ion and B eeding o Ag icul u al Biodi e si y (COMAV-UPV), Uni e si a Poli e ´cnica de Valencia, Camino de Ve a s/ n, 46022 Valencia, Spain, 4IFAPA Cen o Alameda del Obispo, A d. Mene ´ndez Pidal s/n, 14004, Co ´ doba, Spain Abs ac Al hough he a ailabili y o gene ic and genomic esou ces o Cucu bi a pepo has inc eased signi ican ly, unc ional genomic esou ces a e s ill limi ed o his c op. In his di ec ion, we ha e de eloped a high h oughpu e e se gene ic ool: he i s TILLING (Ta ge ing Induced Local Lesions IN Genomes) esou ce o his species. Addi ionally, we ha e used his esou ce o demons a e ha he p e ious EMS mu an popula ion we de eloped has he highes mu a ion densi y compa ed wi h o he cucu bi s mu an popula ions. The o e all mu a ion densi y in his i s C. pepo TILLING pla o m was es ima ed o be 1/133 Kb by sc eening i e addi ional genes. In o al, 58 mu a ions con i med by sequencing we e iden i ied in he i e a ge ed genes, hi een o which we e p edic ed o ha e an impac on he unc ion o he p o ein. The geno ype/pheno ype co ela ion was s udied in a pe oxidase gene, e ealing ha he pheno ype o seedling homozygous o one o he isola ed mu an alleles was albino. These esul s indica e ha he TILLING app oach in his species was success ul a p o iding new mu a ions and can add ess he majo challenge o linking sequence in o ma ion o biological unc ion and also he iden i ica ion o no el a ia ion o c op b eeding. Ci a ion: Vicen e-Do ´le a N, T oadec C, Moya M, del Rı ´o-Celes ino M, Poma es-Viciana T, e al. (2014) Fi s TILLING Pla o m in Cucu bi a pepo: A New Mu an Resou ce o Gene Func ion and C op Imp o emen . PLoS ONE 9(11): e112743. doi:10.1371/jou nal.pone.0112743 Edi o : Heping Cao, USDA-ARS, Uni ed S a es o Ame ica Recei ed June 11, 2014; Accep ed Oc obe 14, 2014; Published No embe 11, 2014 Copy igh : ß2014 Vicen e-Do ´le a e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: Financial suppo was p o ided by he Spanish P ojec INIA (Ins i u o Nacional de In es igacio ´n y Tecnologı ´a Ag a ı ´a y Almen a ı ´a) RTA2011-00044- C02-01, he ANR MELODY (ANR-11-BSV7-0024), he Eu opean Resea ch Council (ERCSEXYPARTH), FEDER, and FSE unds. NVD has been awa ded a g an by he Andalusian Ins i u e o Ag onomy Resea ch IFAPA. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * Email: ped o.gomez.j@jun adeandalucia.es In oduc ion Cucu bi a pepo is he main species o he genus Cucu bi a and ep esen s one o he mos impo an ege able c ops wo ldwide in e ms o ood consump ion. O he eigh exis ing mo pho ypes in his species, he mo pho ype Zucchini is he mos economically aluable. I was ecen ly de eloped and displays low le els o gene ic a ia ion [1] bu ne e heless i is commonly cul i a ed. Nowadays, in his species he de elopmen o new cul i a s is based on he a ailabili y o a iable ge mplasm. Howe e , apa om he in aspeci ic esou ces, g ouped in h ee subespecies, only a ew c ossable Cucu bi a species can be used as sou ces o ag icul u ally use ul alleles and new geno ypes. In his way, he de elopmen o new gene ic esou ces along wi h he applica ion o new molecula echniques in b eeding would acili a e he imp o emen o cha ac e s, mainly hose wi h mo e complex egula ion, allowing b eede s o ace new challenges in his species. The high compe i i eness o plan b eeding has inc eased he in e es in he de elopmen o new sou ces o gene ic a ia ion using e e se gene ics app oaches. The silencing o in e up ion o indi idual a ge genes p o ide he oppo uni y o in es iga e gene unc ion, linking sequence a ia ion o speci ic pheno ypes [2], [3]. Thus, TILLING (Ta ge ing Induced Local Lesions IN Genomes), which combines adi ional chemical mu agenesis wi h high- h oughpu genome-wide sc eening o poin mu a ions in desi ed genes, has been de eloped in esponse. In a TILLING p ojec , chemical mu agenesis based on an alkyla ing agen like EMS (e hyl me hane sulphona e) [4] ha causes andom poin mu a ions a high densi y, p o ides an easy and cos -e ec i e way o es ablish a se ies o allelic mu a ions in he whole gene se o any species. The sc een o he mu an popula ion wi h an e icien me hod o iden i y poin mu a ions, o example a misma ch speci ic diges ion ollowed by a Li-COR sepa a ion o he esul ing agmen s, may allow he iden i ica ion o mul iple alleles o a speci ic gene ega dless o he gene size [5], [6]. I he mu a ion equency is high enough mos , i no all genes, will p esen mu a ed alleles in he popula ion. This me hod may be p e e able o o he e e se gene ic app oaches o a ious easons, o example because EMS gene a es a la ge spec um o mu a ions, including missense and unca ion mu a ions, allowing mo e lexibili y han inse ional mu agenesis o ansgenesis [5]. The newly iden i ied alleles in TILLING could be used o elucida ing gene unc ion. This s a egy is in aluable because i gene a es allelic se ies o en including null alleles, al hough a low equency [7]. TILLING also inds applica ion in c op imp o emen , as he iden i ied PLOS ONE | www.plosone.o g 1 No embe 2014 | Volume 9 | Issue 11 | e112743 mu a ions can be eadily u ilized in adi ional b eeding p og ams since i is non ansgenic and he no el a ia ions a e s ably inhe i ed [8–10]. Howe e , o da e he e a e no TILLING-de i ed c op a ie ies ha ha e ye been eleased hough he e ha e been many success ul examples o TILLING app oaches in basic plan science. I has p o ed o be use ul in a la ge numbe o ag onomically impo an c ops [11] and has allowed o selec mu an s wi h allelic a ian s in i us esis ance genes [12], [13], o in genes in ol ed in abio ic s ess [14], nu i ional p ope ies [15] o shel li e in Cucumis melo [16]. Also, some ag icul u al bio echnology companies ha e di ec ed his echnology owa ds he enhancemen o he oma o shel li e and he educ ion o glu en con en o whea o celiacs [2]. Al hough his e e se gene ic app oach has been epo ed in many species, ega ding he Cucu bi aceae amily, only se e al epo s ha e been published on Cucumis melo [13], [16] and ecen ly on Cucumis sa i us [17]. No TILLING pla o ms ha e been epo ed o o he cucu bi s species such as Cucu bi a pepo. The de elopmen o a TILLING pla o m o his species combined wi h he ecen sequence o he genome (a i s e sion a ailable a cucu bigene.ne ) and o he genomic ools such as he i s SNP-based gene ic map and he i s ansc ip ome [18], [19] could be o g ea in e es , because i could be a way o induce new gene ic a ia ion ha complemen s he na u al ex an a ia ion in his species. Based on ou p e iously epo ed me hod o ob ain mu an s in C. pepo ha o igina ed a e e ence EMS mu an popula ion, wi h he p esen s udy we ha e es ablished he i s TILLING pla o m de eloped o his species which could ep esen an impo an ad ance o c op imp o emen in Zucchini. Ma e ials and Me hods Mu agenized plan ma e ial and g owing condi ions Field s udies we e ca ied ou a he IFAPA esea ch cen e in Alme ia (Spain) (GPS coo dina es: 36.78881667, 22.703297222) and no speci ic pe missions we e equi ed o hese loca ions/ ac i i ies. The ield s udies did no in ol e endange ed o p o ec ed species. Fou o nine seeds o each o he 1464 M2 amilies om a mu an popula ion p e iously gene a ed wi h h ee di e en EMS doses: 80 mM, 65 mM and 40 mM [20] we e sown and g own in g eenhouses ollowing s anda d local comme cial p ac ices o bo h plan nu i ion and pes and disease con ol. Ge mina ion and s e ili y a es o he M2 amilies we e e alua ed and con olled sel -pollina ions we e a emp ed on each plan . Female lowe s we e p o ec ed he day be o e an hesis o p e en he ans e o pollen by insec s, and ea ly in he mo ning each plan was sel - pollina ed by hand. The plan s o each amily we e sel -pollina ed un il one ui wi h seeds pe each M2 amily was ob ained. A 60– 80 days a e sel -pollina ion, M3 seeds we e ha es ed, ai ed and s o ed a 4uC. Pheno ypic al e a ions in M2 plan s also we e egula ly moni o ed and compa ed wi h un ea ed plan s ha we e g own a he same ime. Genomic DNA ex ac ion and pooling Lea ma e ial om only one plan o each M2 amily was collec ed o DNA isola ion. The plan was selec ed i i p oduced a leas 30 iable seeds. Th ee lea discs (diame e 10 mm) we e collec ed and DNA was ex ac ed acco ding o Ma in e al. 2009 [21]. Genomic DNAs we e quan i ied on 1% aga ose gel using l DNA (P omega Bio ech Ibe ica) as a concen a ion e e ence. DNA concen a ion was no malized o 3 ng/ml and pooled eigh old in a 96-well o ma . M3 and M2 seeds o all pooled M2 amilies a e main ained a he genebank o he IFAPA esea ch cen e . PCR ampli ica ion, mu a ion de ec ion and alida ion P ime se s we e designed o i e genes (Table S1) and DNA ampli ica ion was based on nes ed-PCR and uni e sal p ime s o imp o e he speci ici y o he ampli ica ion [22], [23]. Ca o enoid gene s uc u e was es ablished based on he i s e sion o he C. pepo, mo pho ype Zucchini, genomic sequence (a ailable a h p://cucu bigene.ne ) while pe oxidase gene s uc u e was de i ed om NCBI da abases. Sequences o he e hylene ecep o s (CpETR1 and CpERS1) we e p e iously de e mined in ou labo a o y om he homology o o he cucu bi s. P ime s we e designed o ampli y ,1 kb segmen s based on he selec ed C. pepo genomic sequences. The i s PCR ampli ica ion was a s anda d PCR eac ion pe o med in a 25 ml olume consis ing o dH2O, 106PCR bu e , 2.5 mM MgCl2, 5 mM dNTPs, 1 U Taq polyme ase, 10 mM o wa d and e e se a ge -speci ic p ime s and 4.5 ng o genomic DNA. The he mocycling condi ions we e 95uC o i e minu es o ini ial dena u ing, ollowed by 35 cycles o 94uC o 15 seconds, 60uC o 20 seconds, 72uC om one o wo minu es and one cycle o 72uC o i e minu es. One mic oli e dilu ed en imes o he i s PCR p oduc was employed o he second PCR wi h a combina ion o speci ic p ime s ca ying M13 ail and M13 uni e sal p ime s, M13F700 (59-CACGACGTTGTAAAACGAC-39) and M13R800 (59-GGA- TAACATTTCACACAGG-39), labelled a he 59end wi h in a- ed dyes IRD700 and IRD800 (LI-COR, Lincoln, Neb aska, USA) espec i ely. This PCR was ca ied ou using 0.125 o 0.25 mM o each p ime [23], using he ollowing wo s ep cycling p og am: 94uC o 5 minu es, 10 cycles a 94uC o 15 seconds, p ime -speci ic annealing empe a u e o 30 seconds and 72uC o 2 minu es, ollowed by 25 cycles a 94uC o 15 seconds, 50uC o 30 seconds and 72uC o 1 minu e, hen a inal ex ension o 5 minu es a 72uC. PCR p oduc s we e sepa a ed in 2% aga ose gels. Mu a ions we e de ec ed in he ampli ied a ge s using he misma ch-speci ic endonuclease ENDO1 as p e iously desc ibed T iques e al. 2007 [24]. Elec opho esis was pe o med on a Table 1. Summa y o he Cucu bi a pepo mu an collec ion de elopmen . EMS concen a ion Ob ained M1 plan s M2 seed amilies F ui s wi h less han 30 seeds (%) M3 seed amilies 80 mM 4500 259 63 95 65 mM 2700 231 56 52 40 mM 2900 974 36 621 doi:10.1371/jou nal.pone.0112743. 001 Fi s TILLING Pla o m in Cucu bi a pepo PLOS ONE | www.plosone.o g 2 No embe 2014 | Volume 9 | Issue 11 | e112743 LICOR 4300 (LI-COR, Lincoln, NE, USA) and gel images we e analysed using Adobe Pho oshop so wa e (Adobe Sys ems Inc., San Jose´, CA, USA). A e disco e y, mu a ions we e alida ed by sequence analysis. The mu a ion equency o each amplicon was calcula ed as p e iously desc ibed by Dalmais e al. 2008 [23]. Sequence Analysis Tools The PARSESNP so wa e (P ojec Aligned Rela ed Sequences and E alua e SNPs) [25] was used o illus a e he dis ibu ion o mu a ions wi hin he gene and o indica e he na u e o each single mu a ion. The SIFT so wa e (So ing In ole an om Tole an , h p://si .bii.a-s a .edu.sg/) [26], was used o p edic he impac o he mu a ion on he p o ein. Sco es below 0.05 a e p edic ed o a ec p o ein unc ion. Mul iple sequence alignmen o ull-leng h p o ein sequences was pe o med wi h Clus al Omega so wa e (h ps://www.ebi.ac.uk/Tools/msa/clus alo/) Mu an p o ein s uc u e modelling and geno ype de e mina ion The APRX h ee-dimensional s uc u e was gene a ed using he Geno3D se e (h p://geno3d-pbil.ibcp. ). Supe posi ion o he wild ype APRX s uc u e and he mu an APRX p o ein was ca ied ou and isualized using Chime a (h p://www.cgl.ucs . edu/chime a). Fo each APRX mu an line, homozygous and he e ozygous M3 plan s we e de e mined by sequencing APRX gene. How o access he TILLING popula ion Sc eening o he C. pepo TILLING pla o m is no es ablished as a se ice ye bu we a e cu en ly wo king on de eloping a websi e. Fo u he in o ma ion abou how o access o he pla o m please con ac o: [email p o ec ed] Resul s Valida ion o he Cucu bi a pepo TILLING pla o m ma e ial A popula ion o 1464 M2 lines om a p e ious mu agenesis expe imen pe o med wi h h ee di e en EMS dosages (80 mM, 65 mM and 40 mM) was used and he e ec on seed ge mina ion, plan de elopmen and M3 seed p oduc ion was e alua ed. All un ea ed plan s used as con ols ge mina ed. Ne e heless, ge mina ion a e was poo in M2 amilies ob ained wi h 80 mM and 65 mM dosages (52.6%, and 56.37% espec i ely) and signi ican ly be e in amilies ob ained wi h 40 mM (70.83%). Addi ionally, g ow h o M2 plan s was delayed and ook longe o ge mina e a he wo highe doses. Mo eo e , inc eased EMS concen a ion led o an inc eased pe cen age o M2 ui s in which seeds we e non- iable o in which he numbe o iable seeds was oo low o pe o m TILLING analysis, 63% and 56% o he ea men wi h 80 mM and 65 mM espec i ely and 36% o 40 mM ea men . In o al 95 and 52 M3 amilies we e collec ed om he 80 and 65 mM ea men s, espec i ely, and 621 M3 amilies om he 40 mM ea men (Table 1). Figu e 1. Examples o mo phological mu an s obse ed wi hin he C. pepo TILLING popula ion. (a) Plan a ec ed in co yledon colou , albino. (b) Plan a ec ed in co yledon colou , chlo o ic. (c) Plan a ec ed in co yledon numbe . (d–e) Female lowe s wi h abno mal s igma. ( –h) Di e en colou ed ui s. (i–k) Semi-dwa plan s wi h bushy and hype compac a chi ec u e. (l) Albino dwa plan . (m) Di e en size and colou lea es. doi:10.1371/jou nal.pone.0112743.g001 Fi s TILLING Pla o m in Cucu bi a pepo PLOS ONE | www.plosone.o g 3 No embe 2014 | Volume 9 | Issue 11 | e112743 Compa ed o un ea ed plan s, each amily was e alua ed o isual mu a ions in he pheno ypic analysis and a ange o di e en pheno ypes was obse ed in all M2 plan s. Albinism and chlo ophyll de iciency occu ed in 1.3% o he M2 amilies. Abou 1.84% showed al e a ions in co yledons wi h a ia ion in hei numbe o shape and 2.2% o he M2 plan s seg ega ed indi iduals displaying ‘‘dwa ’’ o ‘‘semidwa ’’ pheno ypes. A lowe ing and ui s ages he mos commonly obse ed pheno- ypes we e ela ed o he lea colou and mo phology, he g owing habi , he plan size o ui ai s. In Figu e 1 examples o C. pepo mu an pheno ypes a key de elopmen al s ages a e shown con i ming he high le el o he mu agenesis. Iden i ica ion o mu a ions in a ge genes by TILLING: mu a ion e iciency To se up he C. pepo TILLING pla o m, DNA samples we e p epa ed om 768 M2 lines, each ep esen ing an independen amily and o ganized in pools o 8 M2 amilies. The selec ion o M2 amilies was based on he M3 seed disposabili y. To alida e he TILLING me hod and o es ima e he mu a ion densi y o he popula ion, a se o i e genes o C. pepo in ol ed in di e se me abolic ou es we e chosen. Two genes we e chosen om he e hylene signaling pa hway, in pa icula o he e hylene esponse such as ERS1 (e hylene esponse senso 1) and ETR1 (e hylene ecep o 1). LCYb (lycopene be a cyclase) and PSY (phy oene syn hase) we e also chosen as impo an enzymes in ol ed in he ca o enoid biosyn hesis pa hway [27]. The las one was he APRX gene, a gene encoding a pe oxidase and ha seems o ha e an impo an unc ion in he g ow h and de elopmen o he plan [28]. PCR p oduc s used o TILLING ha e a maximum size o abou 1200 bp and, he e o e, longe genes we e di ided in o se e al amplicons. We ocused ou e o s on iden i ying o each candida e gene he mos p omising egions o TILLING analysis. In his way, new p ime pai s lanking his egion could be a ge ed o he in onic sequences, wi h he aim o imp o ing he sc eening e iciency on he coding egions in he pilo assay (Figu e 2). In o al 10 Mb o C. pepo genomic sequence we e sc eened in 768 Figu e 2. Gene s uc u e o he a ge genes sc eened in he C. pepo popula ion (PSY, LCYb, ETR1, ERS1, and APRX). This d awing was made using he PARSESNP p og am, which maps he mu a ion on a gene model o illus a e he dis ibu ion o mu a ions. O ange boxes ep esen exons and o ange lines in ons, he size (pb) is indica ed in blue. Dashed lines in ed and black indica e amplicons analyzed by TILLING. Pu ple iangles ep esen silen mu a ions and black and ed iangles ep esen missense and unca ion mu a ions, espec i ely. Mu a ions be o e ATG and a e STOP codon a e no shown. doi:10.1371/jou nal.pone.0112743.g002 Fi s TILLING Pla o m in Cucu bi a pepo PLOS ONE | www.plosone.o g 4 No embe 2014 | Volume 9 | Issue 11 | e112743 M2 plan s and 58 induced poin mu a ions we e iden i ied o which ou and eigh mu a ions we e de i ed om he 65 mM and 80 mM EMS ea men espec i ely, and 46 mu a ions om he 40 mM ea men popula ion (Table 2). Among he 58 iden i ied mu a ions, we ha e iden i ied 59% non-synonymous mu a ions (including nonsense and splicing mu a ions). Ex apola ing om his small se o genes, we calcula ed an a e age mu a ion a e o one in a ound 133 kb o 6300 mu a ions pe genome o a genome size o 845 Mb [29]. We iden i ied 57 lines, co esponding o 58 di e en mu a ions, among which eigh missense mu a ions cause changes in he PSY amino acid sequence, se en in he LCYb amino acid sequence, ou and nine in he ETR1 and ERS1 amino acid sequence and ou in he APRX amino acid sequence (Table 3). The exonic mu a ions we e mos ly p esen as he e ozygo es (36 ou o 45 mu a ions), bu 9 lines we e homozygous o he mu a ions. As expec ed wi h EMS mu agenesis, hese mu a ions we e dis ibu ed ela i ely e enly wi hin he sc eened amplicons (Figu e 2). The exonic mu a ions we e mos ly p esen as he e ozygo es (36 ou o 45 mu a ions) and 9 lines we e homozygous o he mu a ions. Thus, e en hough hese esul s a e highe han expec ed, he de ia ion be ween da a is no s a is ically signi ican (P.0.05). Fu he mo e, as expec ed wi h EMS mu agenesis, hese mu a ions we e dis ibu ed ela i ely e enly wi hin he sc eened amplicons (Figu e 2). All he EMS-induced mu a ions we e de ec ed in single plan s, excep o he APRX gene, whe e he same mu a ion was ound in wo di e en plan s. Only one induced s op codon mu a ion was iden i ied o one o he i e genes, APRX, also an splice mu an junc ion was iden i ied in he ETR1 amino acid sequence. Because o he high numbe o alleles iden i ied, he possible impac o missense mu a ions on he unc ion o he p o ein was assessed be o e sys ema ic pheno yping o he mu an plan s using SIFT (So ing In ole an F om Tole an ). Ou o he 32 missense mu a ions, a o al o 14 mu a ions (44%) we e p edic ed dele e ious o he p o ein’s ac i i y (p,0.05): h ee and wo missense mu a ions o ERS1 and ETR1 espec i ely, ou o each o he wo genes in he ca o enoid syn hesis and one missense mu a ion o APRX (Table S2). Geno ype/pheno ype associa ion: cha ac e iza ion o he APRX mu an s Plan pe oxidases a e encoded by mul igenic amilies and in ol ed in se e al impo an physiological and de elopmen p ocesses, al hough hei a ied unc ions a e no clea ly de e mined [30]. The appea ance o nonsense mu an s in hese genes has no been desc ibed p e iously o his species so based on he geno ype/pheno ype ela ionship we decided o s udy he unc ion o he only p e iously pe oxidase s udied in his species, APRX. APRX is a globula p o ein o 330 aa whose s uc u e is highly conse ed be ween dis an plan amilies. Along conse ed esidues, he e a e eigh cys eine esidues bounded by disulphide b idges and essen ial o s abilise he s uc u e o he p o ein. The p o ein also shows hi een alpha helices wi h an heme pocke and wo dis al Ca 2+ binding si es disposed symme ically in he cen e o he heme pocke . Th oughou i s s uc u e o ou exons and h ee in ons we ound nine amilies wi h mu a ions ha a ec ed di e se s uc u al mo i s. Fo each APRX mu an amily, he geno ype/pheno ype associa ion was ca ied ou using 18 M3 seeds ha we e ge mina ed and geno yped by sequencing he APRX gene. No associa ed pheno ype was obse ed in Cp-34 and Cp-773 amilies, which ag ees wi h he posi ion o he mu a ion in an in onic and non-coding egion, espec i ely. Cp-675 and Cp-879 amilies, which showed a silen mu a ion, also did no show di e ences wi h he wild ype. Among he ou mu an amilies ha p esen ed missense mu a ions, only he Cp-86 did no ge mina e, so i could no be e alua ed. Cp-661 ha p esen ed an amino acid change a posi ion R215 and Cp-359 and Cp-579 amilies wi h he same mu a ion a posi ion S14F showed di e en pheno ypes as dwa plan s o plan s wi h de o med lea es. These pheno ypes we e caused by o he mu a ions since, a e geno yping o he lines, we could no associa e hese geno ypes o a pa icula pheno ype. The induced s op codon mu a ion inden i ied in amily Cp-161 (W132*) was he only mu a ion p edic ed o a ec he p o ein unc ion by SIFT. The nine ge mina ed plan s ou o he 18 seeds sown we e geno yped by sequence he APRX gene and six homozygous mu an s, wo he e ozygous and one homozygous wild ype we e iden i ied. The pheno ype o seedling homozygous o he mu an allele was albino (Figu e 3c). The emaining h ee M3 plan s, co esponding o homozygous o he e ozygous o he wild ype allele, showed no mal pheno ypes. Alignmen o he homologous p o eins showed ha he W132* nonsense mu a ion occu ed in he middle o a highly conse ed p o ein mo i . Based on he Geno3D de i ed s uc u e o APRX and X- ay c ys allog aphy s udies [31] (Figu e 3b), he unca ed mu an p o ein is p edic ed o miss helices A o D and he connec ed abili y o bind heme and Ca ++ . Consis en wi h he mu an pheno ype, he loss o mo e han hal o he p o ein should be highly dele e ious (Figu e 3a). Discussion The TILLING pla o m alida ed he e is he i s de eloped in Cucu bi a pepo, p obably because he low e ili y ha he mu an s o his species ha e shown in p e ious a emp s, e en using di e en a ie ies [32], has hampe ed he pe pe ua ion o he popula ions. The de elopmen o a me hod o selec ing M1 lines wi h highe e ili y used in he i s s eps o he gene a ion o his Table 2. E ec o EMS dose on mu a ion equency in C. pepo mu an collec ion. EMS dose M2 plan s Induced mu a ions* Mu a ion equency 80 mM 95 8 1/119 Kb 65 mM 52 4 1/130 Kb 40 mM 621 46 1/135 Kb TOTAL 768 58 1/133 Kb *Numbe o iden i ied mu a ions in all he amilies o his EMS dose a e sc eening i e genes. doi:10.1371/jou nal.pone.0112743. 002 Fi s TILLING Pla o m in Cucu bi a pepo PLOS ONE | www.plosone.o g 5 No embe 2014 | Volume 9 | Issue 11 | e112743 cu en pla o m [20] was success ul. Despi e he mu agen’s oxici y, he analysis o M2 and M3 lines pe o med in his s udy demons a e ha his TILLING pla o m consis s o e ile lines wi h mu a ions a ec ing mos phenological s ages o de elopmen . A p ima y objec i e in a mu agenesis p ojec is o gene a e a sa u a ed esou ce, whe e e e y locus is mu a ed and ep esen ed by mul iple alleles. In he C. pepo TILLING pla o m mo e han one mu a ion was iden i ied pe analysed gene. We sc eened 768 M2 amilies o ob ain a sui able allelic se ies which a e ages o 1 mu a ion pe 133 Kb, so we can conclude ha ou mu an popula ion is su icien ly sa u a ed. Fu he mo e, by compa ing ou esul s wi h hose epo ed in o he TILLING p ojec s wi h di e en plan species we can also a i m ha ou popula ion is sui able o use in high- h oughpu mu a ion disco e y. The densi y o mu a ions ha we disco e ed in his assay appea s o be much highe han he ob ained o o he cucu bi species such as Cucumis melo [13], [16] o Cucumis sa i us [17] and i we compa e his wi h o he species, we ind only i e popula ions o diploid species wi h highe mu a ion densi ies: A abidopsis haliana [21], B assica apa [33], O yza sa i a [34], Linum usi a issimum [35] and a diploid whea (T i icum monococcum) [36]. The high mu a ion a io ha we desc ibe he e comes om he analysis o one indi idual plan o each o he 768 M2 amilies. This mu a ion a io is likely o be unde es ima ed as M1 plan s ha a e he e ozygous o a new mu a ion will yield M2 amilies seg ega ing 1:2:1 o he mu an homozygous, he e ozygous and wild ype homozygous. The sampling o only one plan pe M2, e en assuming good iabili y and e ili y o he mu an allele, a e likely o cause a bias in his seg ega ion. The e o e, ou nex s ep will be o expand he popula ion by gene a ing DNA pools o a leas ou indi iduals pe M2 amily. This ac , maybe, could inc ease ou mu a ion densi y. We also ound ha we had unde epo ed mu a ions owa ds bo h ends o agmen s and we a ibu e hese losses p ima ily o a poo luo escence signal [4]. A o al o 58 mu a ions pe 9 Mb sc eened we e iden i ied in 768 M2 plan s and as expec ed o EMS mu agenesis, single nucleo ide subs i u ions we e iden i ied bo h in coding and non- coding egions [4] and all he induced nucleo ide changes by EMS we e G/A and C/T subs i u ions (Table S2) [37]. All he EMS-induced mu a ions we e de ec ed in single amilies, excep o one in he APRX gene, whe e he same change was ound in wo di e en amilies; his ci cums ance is expec ed and obse ed o occu 4% o he ime in A abidopsis based on andom dis ibu ion o induced mu a ions, GC con en o he genome and dis ibu ion o loca ion o mu a ions disco e ed in agmen s [4]. Finding one coincidence among 58 mu a ions is no signi ican ly di e en om ou expec a ions. Fu he mo e, inding ha all 58 mu a ions a e G/C- o-A/T ansi ions e ec i ely ules ou he possibili y ha hey a e na u ally occu ing polymo phisms and al hough G/C o A/T a e he expec ed ansi ions, hese changes a e no always ob ained [38]. This indica es ha he EMS has had he p edic ed e ec on he backg ound and demons a es no p oblems o c ossing showing a popula ion wi h a high homoge- nei y and inb eeding le el. Despi e his highe mu a ion a e in he popula ion, i we conside ha abou hal o missense mu a ions a e expec ed o be damaging o a ypical p o ein [39], we expec ed o ha e abou 16 dele e ious mu a ions in he sequences s udied. This ac ag ees wi h he esul s based on SIFT algo i hm which p edic s dele e ious missense mu a ions wi h ,75% o e all accu acy. Acco ding o SIFT, 44% ou o he 32 missense mu a ions we e p edic ed dele e ious o he p o ein’s ac i i y (p,0.05) ep esen - ing an ex ensi e allelic se ies o add ess u u e s uc u e- unc ion s udies in his species. Pheno ypic analyses con i m he pheno ype/geno ype co ela- ion in APRX mu an s. The anionic pe oxidase analyzed [40], [41] is one o a b oad amily o Class III Pe oxidases wi h up o 72 membe s iden i ied in A abidopsis genome [42]. The conse ed s uc u e and he low subs a e speci ici y o hese p o eins ha e cons i u ed a handicap o de e mine i s speci ici y o unc ion [28] because edundancy o unc ion p e en s pheno ypes o o e ex- p essed and an isense s a egies. The unc ional analysis o his C. pepo pe oxidase has been es ic ed o he e ologous complemen- a ion in A. haliana, whe e o e exp ession and silencing seems o indica e a ole o APRX in auxin ca abolism, bu conclusi e analysis is necessa y o assess i s speci ic unc ion [28]. E alua ion o allelic se ies ob ained by TILLING in C. pepo, which includes missense, nonsense and null alleles, cons i u es an impo an esou ce o analyze he unc ion o his gene. Some mu a ions we e si ua ed in signi ican s uc u es o he p o ein. One in he signal pep ide, and o he s in he alpha helix F, which con o ma ion could change he posi ion o one cys eine espon- sible o he s uc u al con o ma ion by co alen o ces, howe e and acco ding o SIFT, hese mu a ions we e ole a ed and did no show any isual pheno ype di e en om he wild ype. Finally, he unca ed p o ein o line Cp-161, which showed a s op mu a ion iden i ied a he conse ed W132 esidue, yielded Table 3. Tilled genes and mu a ion equency in he C. pepo mu an collec ion wi h 768 M2 amilies sc eened. Ta ge names Amplicon size (bp) GC con en (%) Iden i ied mu an s Missense a Nonsense b Splicing c Silen d Non coding e PSY 2263 41 8 0 0 3 5 ERS1 2663 42 9 0 0 3 4 ETR1 1620 39 4 0 1 1 1 APRX 1398 47 4 1 0 2 2 LCYb 2035 42 7 0 0 2 1 TOTAL 9979 42 32 1 1 11 13 a nucleic acid ansi ion is a non-synonymous mu a ion and induce amino acid change in he ansla ed p o ein. b nucleic acid ansi ion p oduces a s op codon and may induce a unca ed p o ein. c nucleic acid ansi ion is loca ed in splicing mo i . d nucleic acid ansi ion induces a synonymous mu a ion and hen no change in he ansla ed p o ein. e nucleid acid ansi ion is loca ed in an in onic o p omo o egion. doi:10.1371/jou nal.pone.0112743. 003 Fi s TILLING Pla o m in Cucu bi a pepo PLOS ONE | www.plosone.o g 6 No embe 2014 | Volume 9 | Issue 11 | e112743 albino seedlings ha died in ea ly s ages when he mu an allele was homozygous, as was con i med wi h he M3 geno yped plan s. No di e ences om he wild ype we e obse ed when he mu an allele was he e ozygous. Those esul s p o ide he i s p edic ed nullimo phic mu a ion o APRX and a pheno ype which is much mo e se e e han ha o p e iously epo ed mu an s [28]. This pheno ype could indica e he main ole o his pe oxidase a ea ly s ages o he plan ’s de elopmen and i could be ela ed wi h he accumula ion o hyd ogen pe oxide ha becomes oxic and p e en s he de elopmen o essen ial o ganelles o he cell such as chlo oplas s. Al hough se e al s udies a e equi ed o con i m his hypo hesis, hese mu an s cons i u e a new sou ce o ob ain new insigh s o impo an and mul i unc ional p o eins as pe oxidases. Conclusions The es ablishmen o he EMS-mu agenized TILLING popu- la ion desc ibed he e ep esen s an impo an ad ance in he gene a ion o new gene ic a ia ion in C. pepo. The high mu a ion densi y ob ained in his species makes i an a ac i e gene ic sys em o he iden i ica ion o new alleles ha may be o alue o c op imp o emen . Mo eo e , TILLING me hodology becomes a easible goal making possible he in es iga ion o he ole o key genes o his species bypassing he p oblems o o he unc ional genomic ools. Wi h he a ailabili y o his new esou ce we hope o ul ill he expec a ions o bo h, c op b eede s and scien is s, who a e using his species as hei model o s udy. Suppo ing In o ma ion Table S1 P ime pai s used o ampli y he i e genes illed in C. pepo mu an popula ion. As e isk indica e ha he p ime ca ied M13 ail and M13 uni e sal p ime s, M13F700 (59-CACGACGTTGTAAAACGAC-39) and M13R800 (59GGA- TAACATTTCACACAGG-39), labelled a he 59end wi h in a- ed dyes IRD700 ( o wa d p ime s) and IRD800 ( e e se p ime s). (DOC) Table S2 Allelic se ies o mu a ions iden i ied in coding egions by TILLING. (DOC) Figu e 3. Sequence and s uc u al analysis o APRX. (A) Amino acid alignmen o Cucu bi a pepo APRX and homologous p o eins om A abidopsis haliana (AAM66044.1), Capsicum annun (AFU51540.1), Cucumis sa i us (AAA33128.1), Phaseolus ulga is (AAD37427.1) and Py um sa i us (BAD97438.1). Numbe s abo e he alignmen indica e he amino acid posi ions along he APRX p o ein. The EMS-induced s op mu a ion in W132 is shown abo e he alignmen in ed. (B) Supe posi ion o he p edic ed 3D s uc u e model o APRX WT p o ein indica ed in g ey and he mu an p o ein indica ed in blue. The p oximal heme pocke is ep esen ed in ed and he wo calcium ions in g een. The posi ion o he induced s op codon mu a ion inden i ied in Cp-161 amily (W132*) is indica ed in ed. The APRX model was de e mined using he Geno3D se e (h p://geno3d-pbil.ibcp. ). 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