Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into 'Piel de Sapo' Melon (Cucucumis melo L.)

Mapping and In og ession o QTL In ol ed in F ui Shape T ansg essi e Seg ega ion in o ‘Piel de Sapo’ Melon ( Cucucumis melo L.) Au o a Dı ´az 1 *, Belkacem Za ou i 2 , Mohamed Fe gany 3¤ , Iban Edua do 3 , Jose ´M. A ´l a ez 4 , Bele ´n Pico ´ 5 , An onio J. Mon o e 1 * 1Ins i u o de Biologı ´a Molecula y Celula de Plan as (IBMCP). Uni e sidad Poli e ´cnica de Valencia (UPV)-Consejo Supe io de In es igaciones Cien ı ´ icas (CSIC), Valencia, Spain, 2Labo a o io de Biologı ´a Molecula . Dp o. In es igacio ´n Ag oalimen a ia. Ins i u o Mad ilen ˜o de In es igacio ´n y Desa ollo Ru al, Ag a io y Alimen a io (IMIDRA), Alcala ´de Hena es, Mad id, Spain, 3Cen e de Rece ca en Ag igeno `mica (CRAG), IRTA-CSIC-UAB, Bella e a, Ce danyola del Valle `s, Ba celona, Spain, 4Cen o de In es igacio ´n y Tecnologı ´a Ag oalimen a ia de A ago ´n (CITA), Za agoza, Spain, 5COMAV-UPV, Ins i u e o he Conse a ion and B eeding o Ag icul u al Biodi e si y, Uni e si ad Poli e ´cnica de Valencia, Valencia, Spain Abs ac A mapping F 2 popula ion om he c oss ‘Piel de Sapo’ 6PI124112 was selec i ely geno yped o s udy he gene ic con ol o mo phological ui ai s by QTL (Quan i a i e T ai Loci) analysis. Ten QTL we e iden i ied, i e o FL (F ui Leng h), wo o FD (F ui Diame e ) and h ee o FS (F ui Shape). A leas one obus QTL pe cha ac e was ound, lqs8.1 (LOD = 16.85, R 2 = 34%), dqs12.1 (LOD = 3.47, R 2 = 11%) and sqs8.1 (LOD = 14.85, R 2 = 41%). lqs2.1 and sqs2.1 coseg ega e wi h gene a (and omonoecious), esponsible o lowe sex de e mina ion and wi h pleio opic e ec s on FS. They display a posi i e addi i e e ec (a) alue, so he PI124112 allele causes an inc ease in FL and FS, p oducing mo e elonga ed ui s. Con e sely, he nega i e a alue o lqs8.1 and sqs8.1 indica es a dec ease in FL and FS, wha esul s in ounde ui s, e en i PI124112 p oduces e y elonga ed melons. This is explained by a signi ican epis a ic in e ac ion be ween sqs2.1 and sqs8.1, whe e he e ec s o he alleles a locus aa e a enua ed by he addi i e PI124112 allele a sqs8.1. Roundes ui s a e p oduced by homozygous o PI124112 a sqs8.1 ha do no ca y any dominan A allele a locus a(PiPiaa). A signi ican in e ac ion be ween sqs8.1 and sqs12.1 was also de ec ed, wi h he alleles a sqs12.1 p oducing mo e elonga ed ui s. sqs8.1 seems o be allelic o QTL disco e ed in o he popula ions whe e he exo ic alleles p oduce elonga ed ui s. This model has been alida ed in assays wi h backc oss lines along 3 yea s and ul ima ely ob aining a sqs8.1-NIL (Nea Isogenic Line) in ‘Piel de Sapo’ backg ound which yields ound melons. Ci a ion: Dı ´az A, Za ou i B, Fe gany M, Edua do I, A ´l a ez JM, e al. (2014) Mapping and In og ession o QTL In ol ed in F ui Shape T ansg essi e Seg ega ion in o ‘Piel de Sapo’ Melon (Cucucumis melo L.). PLoS ONE 9(8): e104188. doi:10.1371/jou nal.pone.0104188 Edi o : Rongling Wu, Pennsyl ania S a e Uni e si y, Uni ed S a es o Ame ica Recei ed Feb ua y 4, 2014; Accep ed July 10, 2014; Published Augus 15, 2014 Copy igh : ß2014 Dı ´az 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. Funding: This wo k was suppo ed by g an s AGL2009-12698-C02-02 and AGL2012-40130-C02-02 om he Spanish Minis y o Economy and Compe i i eness o AJM. AD was suppo ed by a JAE-Doc con ac om CSIC, MF by a Pos doc o al con ac om GRAG, IE by a ellowship om he o me Spanish Minis y o Educa ion and BZ by a ellowship om Ins i u o Ag ono ´mico Medi e a ´neo de Za agoza (IAMZ), Spain. 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: [email p o ec ed]s (AD); amon o [email p o ec ed] (AJM) ¤ Cu en add ess: Ag onomy Depa men . Facul y o Ag icul u e Ain Shams Uni e si y, Cai o, Egyp In oduc ion Melon (Cucumis melo L., 2n = 2x = 24) is one o he mos impo an ho icul u al c ops, anking in he nin h posi ion o he wo ld p oduc ion o ege ables, wi h mo e han 27 million ons in 2012 [1]. In he las wo decades, melon p oduc ion has expe ienced a 29% ise, wha has been accompanied by an inc ease o 8% in he a ea ha es ed. I does no seem easible o achie e he same gain in p oduc ion in he nex decades by inc easing he land dedica ed o i s cul i a ion, so he e o s o plan b eede s should be di ec ed o imp o e ai s ha ing a g ea po en ial o inc ease yield and ui quali y. In his sense, ui mo phology can be conside ed a quali y ai as i is one o he i s ea u es o be pe cei ed. Al hough consume s associa e pa icula sizes and o ms o ce ain ma ke classes (i.e. medium size and ound can aloupes o la ge and o al inodo us), a whole wo ld o possibili ies is being opened up be o e us wi h he de elopmen o p oduc s wi h o iginal appea ance, ei he by adi ional b eeding, like he ZESPRI GOLD kiwi ui cul i a [2], o by gene ic enginee ing, like he pu ple oma o [3]. Mode n ma ke ends demand smalle melons, as he amily size is p og essi ely alling; and wi h ounde shapes, wha minimizes cos s and mechanical damages du ing s o age and anspo . Melon ge mplasm shows an imp essi e a ay o ui sizes and mo phologies [4–6], anging om a ew ens o g ams up o 10 Kg and om sligh ly la o ex emely elonga ed shapes. Mos o his di e si y is ound in adi ional Cen al Asian land aces and wild melons [7–8]. This gene pool is conside ed ‘‘exo ic’’ when compa ed o he popula Occiden al a ie ies (i. e. Galias, honeydews, wes e n shippe s, can aloups, and inodo us like ‘Piel de Sapo’, om he e on e e ed o as ‘PS’) al hough i ep esen s a aluable sou ce o a iabili y wi h a huge po en ial in b eeding p og ams. In his sense, he accession PI124112 (C. melo ssp. PLOS ONE | www.plosone.o g 1 Augus 2014 | Volume 9 | Issue 8 | e104188 ag es is a . momo dica) collec ed in 1937 in India is gene ically e y dis an om comme cial cul i a s, as e ealed by s udies ca ied ou wi h SSR (Simple Sequence Repea ) [5], [9], and AFPL (Ampli ied F agmen Leng h Polymo phism) and SNP (Single Nucleo ide Polymo phism) [10] ma ke s, and shows esis ance o se e al ungal and i al diseases ( e iewed in [7]). A ew majo genes wi h pleio opic e ec s on melon ui shape (FS) a e cu en ly known, like and omonoecious (a), ha has been ecen ly cloned [11] and is esponsible o sex de e mina ion oge he wi h gynoecious (g), and like pen ame ous (p), ha con ols ca pel numbe . Howe e , mos o he pheno ypic a ia ion obse ed in FS seems o be caused by o he Quan i a i e T ai Loci (QTL), as demons a ed by a numbe o QTL mapping s udies ha ha e been unde aken o gene ically dissec i s molecula basis [12–18]. Geno yping echnologies ha e expe ienced an au hen ic e o- lu ion in he las ew yea s [19–20], wha has led o a enewed in e es in QTL mapping. The a ailabili y o a o dable high- h oughpu geno yping pla o ms will enable esea che s o anno a e mo e p ecisely he genomic egions o in e es and speed up d as ically he o me ly ime-consuming p ocess o ob aining aluable plan ma e ial. All his will make possible o alida e QTL and s udy pu a i e in e ac ions among hem, a he same ime ha a o able alleles a e ans e ed in o eli e a ie ies wi h a minimal d ag o non- a ge ed agmen s wi h po en ial de imen al e ec s. Consequen ly, i is expec ed ha in a nea u u e, new QTL will be added o hose al eady cloned in impo an c ops, like he QTL o ui suga con en B ix9-2-5 [21], o ui size w2.2 [22], and hose o ui shape o a e [23], ascia ed [24] and sun [25] in oma o; a QTL o g ain sha e ing sh4 [26] and hose o lowe ing da e Hd1 [27], Hd6 [28], Hd3a [29], and Ehd1 [30] in ice; and he QTL o plan a chi ec u e Tb1 [31] and lowe ing ime Dwa 8 [32] and Vg 1 [33] in maize, among o he s ( e iewed in [34–35]). In mos cases, he s a egy ollowed was he posi ional cloning, o wha he a ailabili y o Nea -Isogenic Lines (NILs) was essen ial, highligh ing he impo ance o gene a ing he app op i- a e gene ic ma e ial o be success ul. In his sense, he de elopmen o a collec ion o NILs in melon be ween he Ko ean cul i a ‘Songwhan Cha mi’ and ‘PS’ [36] has allowed o dissec in single Mendelian ac o s economically impo an ai s as ui mo phol- ogy [14], [17], climac e ic ipening [37] and i us esis ance [38]. In he p esen wo k, we iden i y and localize se e al QTL associa ed wi h melon ui mo phological ai s in a c oss be ween he Spanish cul i a ‘PS’ and he Indian accession PI124112 and s udy he in e ac ion be ween hose wi h he s onges and mos obus e ec s. Fu he mo e, we ha e alida ed he QTL o FS Figu e 1. F ui Shape (FS) polymo phism a each s age o he c ossing p og am o ob ain he sqs8.1 -NIL. F ui s o (A) ‘PS’; (B) PI124112; (C) 2M158-3 (F 1 ); (D) di e en F 2 plan s o show he ansg essi e seg ega ion o FS; (E) 3M70-47 (F 2 ); (F) 5M113-1 6‘PS’ (BC1); (G) 6M59-13 6‘PS’ (BC2); (H) 7M36-1 6‘PS’ (BC3); (I) 8M42-24 6‘PS’ (BC4); (J) 9M7-15 : (BC4S1); (K) 10M2-30 6‘PS’ (BC5); (L) 11M27 : (BC5S1), (L.1) 11M27-20: he e ozygous a sqs8.1 and (L.2) 11M27-11: homozygous a sqs8.1; (M) he sqs8.1-NIL 12M57-3 OP (BC5S2). doi:10.1371/jou nal.pone.0104188.g001 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 2 Augus 2014 | Volume 9 | Issue 8 | e104188 sqs8.1 in a QTL-NIL de eloped in he gene ic backg ound o he cul i a ‘PS’, wha p oduces o ally ound melons ins ead o he ypical o al ones. Ma e ials and Me hods All expe imen s we e conduc ed in compliance wi h he cu en Spanish laws. No speci ic pe missions we e equi ed o he expe imen s ca ied ou in hese wo loca ions, Cab ils and Za agoza, as hey a e esea ch ins i u es, Ins i u de Rece ca i Tecnologia Ag oalimen a ia (IRTA) and Cen o de In es igacio´n y Tecnologı ´a Ag oalimen a ia de A ago´n (CITA), espec i ely. Mo eo e , au ho s IE and JMA belong o IRTA and CITA, espec i ely. Plan ma e ial and pheno ypic e alua ion The F 2 plan s o he mapping popula ion we e de i ed om a single c oss be ween he monoecious Indian land ace accession PI124112 (momo dica g oup) and he and omonoecious Spanish cul i a ‘PS’ (inodo us g oup) (Figu e 1). PI124112 ypically p oduces medium size, elonga ed and non-swee ui s, whe eas ui s coming om ‘PS’ a e big, o al-shaped and e y swee [4–5]. A mul i-loca ion assay was conduc ed in summe o 2003 wi h a o al o app oxima ely 400 F 2 plan s. On one hand, 200 o hem we e andomly dis ibu ed in a g eenhouse a IRTA in Cab ils (Ba celona, Spain), g own in d ip-i iga ed pea bags and sel - pollina ed by hand. The emaining 200 plan s we e andomized in an open ield wi hou i iga ion a CITA, loca ed in Za agoza (Spain) and allowed o open-pollina e. The e iliza ion me hod became i ele an as all he ui excep he seed con en is de i ed om he ma e nal geni o . Ten plan s o e e y geni o plus he F 1 we e included in bo h ials. F ui leng h (FL) and maximum ui diame e (FD) we e measu ed on longi udinal sec ions o collec ed ui s and hen, FS was calcula ed as he a io o FL o e FD. The absence o s a is ically signi ican geno ype-by-en i onmen (G6E) in e ac ions was es ed by calcula ing he leas squa e means o FS in a wo-way ANOVA wi h he da a coming om six o nine eplica es o F1, PI124112 and ‘PS’ in Cab ils and Za agoza as a necessa y condi ion o me ge he da a coming om he wo loca ions. Pai -wise Pea son’s co ela ion coe icien was calcula ed be ween he h ee ai s wi h he da a coming om he whole F 2 popula ion and also wi h hose ob ained in he wo loca ion assays (Cab ils and Za agoza) independen ly. Pe cen mid-pa en al he e osis (MPH) was calcula ed using he da a de i ed om a o al o 15 eplica es o each o he geno ypes, F 1 and he bo h pa en s, as: MPH~2F1{P1{P2  =P1zP2  |100 whe e F1,P1and P2a e he mean alues o he F 1 , ‘PS’ and PI124112, espec i ely, o he ai unde s udy. One-way ANOVA and -S uden mean compa ison es s we e ca ied ou wi h he da a coming om he whole F 2 popula ion and also wi h hose ob ained in he wo loca ion assays independen ly. All s a is ical analyses we e pe o med using JMP 5.1.2 so wa e o Windows (SAS Ins i u e Inc., Ca y, NC). DNA ma ke analysis in he mapping popula ion A selec i e geno yping s a egy [39], which exploi s maximum pheno ypic di e ences wi h a minimal labo and ime in es men , was app oached o s udy he FS ai . The plan s selec ed o be geno yped we e hose wi h alues mo e ex eme han he mean o he F 2 popula ion 61SD om each loca ion, esul ing in 84 indi iduals om bo h loca ions (File S1). DNA was isola ed om young lea es acco ding o [40], wi h sligh modi ica ions and geno yped wi h a o al o 133 ma ke s (66 SSRs, 65 SNPs and 2 Clea ed Ampli ied Polymo phic Si es, CAPS) chosen o co e all he genome among hose a ailable in he li e a u e (File S2). SSR ma ke s we e ampli ied ollowing he p o ocol wi h M13- ailed o wa d p ime s plus dye labeled M13 p ime s desc ibed by [41] and subsequen ly analyzed on a 3130 Gene ic Analyze (Applied Biosys ems). Geno yping wi h SNP ma ke s was ca ied ou using he Sequenom MassA ay iPLEX sys em (Illumina), ollowed by MALDI-TOF mass spec ome y a Se icio de In es igaciones Biome´dicas, Unidad Cen al de In es igacio´n (Uni e si y o Valencia, Valencia, Spain). Ampli i- ca ions o he CAPS ma ke s, AluICAPS [11] and PSI_25-H03 [42], we e pe o med in 25-ml olume solu ions con aining 20 ng o genomic DNA, 7.5 mM T is HCl (pH 9.0), 5 mM KCl, 2 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 , 0.2 mM o each dNTP, 0.2 mMo e e se and o wa d p ime s and 2.5 U o DNA polyme ase (Bio ools, Mad id, Spain). PCRs we e ca ied ou on a TC-512 he mocycle (Techne, UK) p og ammed wi h an ini ial dena u - a ion s ep a 94uC o 5 min, ollowed by 35 cycles o 94uC o 30 s, 62uC o 45 s and 72uC o 1 min, plus a inal elonga ion s ep a 72uC o 5 min. A 10-ml olume o he AluICAPS and PSI_25-H03 PCR p oduc s we e diges ed wi h he es ic ion enzymes AluI and Bsh1236I (Fe men as, Fishe Scien i ic, Mad id, Spain), espec i ely, ollowing he supplie ’s ecommenda ions and sepa a ed by aga ose gel-elec opho esis. Linkage map cons uc ion and QTL analysis The linkage map was cons uc ed using MAPMAKER 3.0 [43] and he dis ances we e calcula ed wi h he Kosambi unc ion [44]. Ma ke s we e associa ed wi h he g oup command wi h LOD. 3.0. When possible, hey we e pu in o de wi hin each g oup using he o de command wi h LOD.3.0, ollowed by a e i ica ion wi h he ipple command and he building o he amewo k map. I necessa y, emaining ma ke s in he g oup we e included wi h he place command. I he p e ious app oach did no ende any map, he compa e unc ion was used o de e mine he bes possible o de o i e s a ing ma ke s, adding he emaining ones wi h he y command. In he case o g oups wi h a low numbe o ma ke s, he o de and dis ances we e es ima ed using he g oup command, ollowed by compa e, wha gene a ed he amewo k map. Finally, maps we e d awn wi h MapCha e sion 2.2 [45]. QTL analysis was pe o med o each loca ion independen ly and o da a om bo h loca ions me ged oge he using Windows QTL Ca og aphe 2.5 [46] wi h he composi e in e al mapping (CIM, [47]) p ocedu e. The LOD sco e h eshold o a signi ican le el p,0.05 was calcula ed by a pe mu a ion es wi h 1000 esamplings, gi ing a alue o LOD.2.5 o he whole popula ion o all ai s and la ge numbe s in he indi idual popula ions. Addi ionally, he h eshold LOD.2.5 was also es ablished o he single loca ion analyses. Ma ke s associa ed o he mos obus QTL (6 ou o he 10 QTL as se e al o hem sha e he same ma ke as he s onges linked one, i.e. lqs8.1 and sqs8.1) we e geno yped in all F 2 plan s in o de o ob ain unbiased es ima es o he gene ac ion (d/[a]). The QTL we e named acco ding o he nomencla u e sys em adop ed by [48], whe e he i s le e s Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 3 Augus 2014 | Volume 9 | Issue 8 | e104188 designa e he ai abb e ia ion, ollowed by a ‘‘Q’’ ha s ands o QTL, hen a le e e e ed o he mapping expe imen (‘‘s’’, in his case), ollowed by a digi ep esen ing he linkage g oup (LG) o which he QTL maps, and hen ollowed by a do and a inal digi ha dis inguishes di e en QTL om he same expe imen on he same LG. In hose cases in which wo o e lapping QTL we e iden i ied in a limi ed egion and o he same ai , hey we e e ec i ely conside ed as only one QTL. S udy o QTL in e ac ions Epis a ic in e ac ions be ween he FS QTL de ec ed we e in es iga ed by wo-way ANOVA wi h he JMP 5.1.2 so wa e o Windows and using he geno ypic da a om he linked ma ke s in all F 2 plan s (no only he subse employed o he selec i e geno yping). These ma ke s we e AluICAPS in he case o sqs2.1, CMAT141 o sqs8.1, and ECM 67 o sqs12.1. In e ac ions we e conside ed o be s a is ically signi ican when p#0.05. Sum o squa es alues we e used o calcula e he pe cen age o he pheno ypic a ia ion explained by he in e ac ion be ween wo QTL (R 2 ). QTL alida ion by de eloping a QTL-NIL The s a egy ollowed o alida e one o he FS QTL de ec ed and o in og ess i in o he ‘PS’ eli e cul i a consis ed in selec ing a single F 2 plan om he ial loca ed in Cab ils, ha ca ied he PI124112 alleles a he QTL egion and also showed ound ui , and backc oss i o he pa en al ‘PS’ (Figu e S1). Fi e ounds o backc ossing guided by a doubled selec ion, pheno ypic ( a o ing ound-shaped ui s) and assis ed by ma ke s linked o he a ge QTL ( o moni o he in og essions ha bo ing PI124112 alleles), we e ca ied ou . Simul aneously, backg ound ma ke s co e ing all he genome we e also employed o g adually inc ease he pe cen age o he ecu en pa en genome by selec ing agains he dono (PI124112) genome. An ad anced backc oss selec ion, 10M2-30, was geno yped wi h 548 SNPs using he Golden-Ga e assay (Illumina, San Diego, CA) desc ibed in [10] in o de o e i y in og ession size and in eg i y, and he composi ion o he genomic backg ound. A e wo cycles o sel - e iliza ion, he de ini i e in og ession line in he BC5S2 gene a ion was ob ained (Figu e 1M). Pheno ypical e alua ions o he ui s we e conduc ed in he g eenhouse a se e al s ages o he backc oss scheme. In 2008, 42 ui s collec ed om 37 plan s belonging o h ee ull-siblings BC4 amilies (8M42-37, 41 and 42) ca ying he PI124112 allele o he FS QTL in LG VIII (homozygous and he e ozygous o wo linked ma ke s placed 23.3 cM apa in he gene ic map) we e e alua ed in Cab ils. In 2009, BC4S1 plan s (Figu e S1) we e geno yped wi h he ma ke s linked o he QTL and 30 o hei ui s we e pheno yped in a g eenhouse a Poly echnic Uni e si y o Valencia. The coseg ega ion o ma ke s and FS pheno ypes was assessed by ANOVA. Finally, in 2012, melon ui s om he BC5S2 amily 12M57 and he pa en al cul i a ‘PS’ we e analyzed in Paipo a (Valencia). 12M57, de i ed om a homozygous plan o he closes ma ke s o he FS QTL sqs8.1 (11M27-11, Figu e S1), con ained he in og ession o he PI124112 allele a hose same ma ke s linked o he QTL. Means o he ui s p oduced by 10 12M57 plan s we e compa ed wi h means o he ui s collec ed om 15 ‘PS’ plan s by a - es . Resul s Pheno ypic a ia ion in mo phological ai s Cul i a ‘PS’ p oduces o al-shaped ui s (FS = 1.460.1; Table 1 and Figu e 1A), while PI124112 displays elonga ed ui s (FS = 1.760.2; Table 1 and Figu e 1B). The F 1 exhibi ed highe FS alues (FS = 2.060.2, Table 1 and Figu e 1C), in acco dance wi h bes -pa en he e osis o his cha ac e p e iously obse ed in melon [16], pa icula ly in hyb ids be ween exo ic accessions and ‘PS’ (inodo us g oup), caused mainly by an inc ease in FL [5]. No signi ican G6E in e ac ion was obse ed o FS, he e o e he pa en al mean alues we e calcula ed wi h he da a coming om bo h loca ions. In Cab ils, he MPH alues we e 77%, 10% and 60% o FL, FD and FS, espec i ely; in he assay loca ed in Za agoza, 120%, 34% and 68%, espec i ely; and o he whole se o da a, 100%, 24% and 64%, espec i ely. In all cases, he e osis was con i med and, hough he e a e sligh di e ences in he MPH alues o each cha ac e among assays, he anking emains unal e ed. A con inuous dis ibu ion o alues was obse ed in he F 2 (Figu e 2), as expec ed in a quan i a i e ai like FS, wi h alues anging om 0.86 (obla e ui s) o 3.23 (ex emely elonga ed ui s), making clea a ansg essi e seg ega ion o his cha ac e in his popula ion (Figu e 1D and Figu e 2C). T ansg essi e seg ega ion was also obse ed o FL. In bo h cases, i was obse ed in bo h di ec ions in he whole F 2 popula ion (Table 1 and Figu e 2), al hough he pheno ypes whe e mo e ex eme o he highe alues, ha means ui s wi h a mo e elonga ed shape. In all cases, indi iduals wi h ui s exceeding he mid-pa en alue 63.84SD we e ound. The ai showing a b oade ange o alues was FL ( om 7.1 o 38 cm), displaying ex eme alues co e- sponding o 26.56 and +13.52 imes he SD o he mid-pa en al FL. Con e sely, FD showed he lowes a ia ion ( om 7.30 o 17.70 cm), wha ep esen s he mid-pa en FD alue 23.84SD and +5.55SD, espec i ely. In be ween hese wo cases, he FS alues anged om 24.31SD and +11.23SD he mid-pa en alues, espec i ely. FL and FS showed a highly signi ican (p,0.001) and s ong co ela ion wi h alues o he Pea son’s co ela ion coe icien o 0.84 o he whole popula ion, and 0.86 and 0.83 o he subpopula ions o Cab ils and Za agoza, espec i ely. In con as , FD and FS we e no signi ican ly co ela ed in any case. Linkage map cons uc ion One hund ed and wen y- wo ou o he 133 o al ma ke s (63 SNPs, 57 SSRs and 2 CAPS) used o geno ype he F 2 mapping popula ion could be inco po a ed o a gene ic map consis ing in 18 LGs ha span 1437.3 cM (Figu e 3), wha ende s a densi y o 1 ma ke e e y 11.8 cM. By compa ing wi h he melon consensus map [48], each LG could be iden i ied, inding ha i e o hem we e pa ial LGs o he in eg a ed map. The map shows an a e age ma ke gap o 13.8 cM. QTL associa ed wi h ui mo phology ai s Gi en ha no G6E in e ac ion was ound among loca ions, da a om bo h loca ions we e me ged o subsequen QTL analysis. A o al o en QTL o ui mo phology- ela ed ai s we e posi ioned along he melon gene ic map (Table 2 and Figu e 3) wi h he selec i e geno yping s a egy, i e unde lying FL (in LGs II, IIIb, VIa, VIII and Xb), wo esponsible o FD (in LGs IIIa and XII), and h ee con olling FS (in LGs II, VIII and XII). LOD sco e alues anged om 2.80 o 16.85, in he case o dqs3a.1 and lqs8.1, espec i ely. The QTL wi h he highes e ec o each o he h ee cha ac e s s udied we e lqs8.1 (LOD = 16.85, R 2 = 34%), dqs12.1 (LOD = 3.47, R 2 = 11%) and sqs8.1 (LOD = 14.85, R 2 = 41%). The emaining QTL accoun ed o 4 o 17% o he pheno ypic a ia ion. LOD peaks o he wo majo QTL loca ed in LG II, lqs2.1 and sqs2.1, and in LG VIII, lqs8.1 and sqs8.1, o e lapped comple ely (Figu e S2). Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 4 Augus 2014 | Volume 9 | Issue 8 | e104188 The in e al de ined by he ma ke s lanking he QTL anged om 12.9 ( lqs3b.1) o 69.1 ( lqs6a.1) cM, being less han 26 cM o i e ou o he en QTL (Table 2). The QTL on LG VIII ( lqs8.1 and sqs8.1) we e consis en ly de ec ed in he h ee analyses ( ha is, wi h he me ged da a and wi h hose coming om indi idual loca ions, File S3). The QTL sqs2.1 was also de ec ed in he h ee analyses wi h a LOD = 2.5.The es s o he QTL we e iden i ied in wo o he analyses, excep o lqs10b.1. In all cases, he addi i e e ec s sign emained in a iable ac oss he di e en en i onmen s. Since sqs2.1, sqs8.1 and sqs12.1 we e he QTL mos consis en ly de ec ed among analyses, all plan s we e geno yped wi h ma ke s AluICAPS, CMAT141, and ECM67, loca ed in he espec i e QTL egion, in o de o ob ain unbiased es ima es o hei e ec s (Table 2). Same di ec ion e ec s o he exo ic allele (PI124112) o FL and FS QTL wi h he same ch omosomal loca ion we e obse ed. In his sense, lqs2.1 and sqs2.1 displayed a posi i e alue o he addi i e e ec (a), 3.06 and 0.27, espec i ely, wha means ha he PI124112 allele causes an inc ease in FL and FS, p oducing mo e elonga ed ui s. Con e sely, he nega i e alue o a o lqs8.1 and sqs8.1 (24.95 and 20.50, espec i ely) is indica i e o a dec ease in bo h cha ac e s, FL and FS, wha esul s in ounde ui s, e en i PI124112 i sel p oduces e y elonga ed melons. In all he emaining QTL, excep o dqs12.1, de ec ed wi h he da a coming om Cab ils and wi h he whole se (a=21.03 and a= 21.10, espec i ely), he PI124112 allele con ibu ed o inc ease he alue o he pa icula mo phological ai (Table 2 and File S3). So, he PI124122 allele a dqs12.1 will ende elonga ed ui s by dec easing hei diame e s, whe eas in he case o lqs2.1 and sqs2.1, a simila pheno ype will be ob ained by inc easing he leng h o he ui . Gene ac ion alues anged om 20.35 and 20.32 ( lqs8.1 and sqs8.1, espec i ely) o 0.75 ( lqs2.1 and sqs2.1; Table 2). In mos cases, he alleles a he QTL loci showed an addi i e e ec , excep o lqs2.1 and sqs2.1, whe e he inhe i ance mode is nea comple e dominance. In gene al, he gene ac ion alues ob ained o he same QTL using he subse s o da a coming om bo h loca ions (File S3) ag ee wi h he ones calcula ed when all he da a we e pooled oge he , wi h sligh di e ences ha can be a ibu ed o he smalle sample size o he i s analysis. The addi i e e ec a sqs8.1 is suppo ed by he pheno ypic da a, as he he e ozygous NIL- sqs8.1 displays a FS alue (i.e. 1.24) in e media e be ween he wo homozygo es, he homozygous line o he PI124112 allele (i.e. 1.06) and he ‘PS’ pa en (a e age FS = 1.40). QTL 6QTL in e ac ion In o de o de ec he pu a i e e ec s o o he genomic egions on each o he FS QTL, all he possible digenic in e ac ions be ween hem we e s udied al hough only hose ha we e s a is ically signi ican when p#0.05 a e shown (Table 3 and Figu e 4). Table 1. Mean and S anda d De ia ion (SD) alues o he ai s o he pa en s, he F 1 and he whole F 2 . FL FD FS Mean SD Mean SD Mean SD ‘PS’ 19.5 1.3 14.0 1.1 1.4 0.1 PI124112 14.8 1.8 9.2 1.1 1.6 0.2 F 1 25.1 3.5 12.7 1.8 2.0 0.2 F 2 19.5 5.7 11.7 2.0 1.7 0.5 FL: F ui Leng h; FD: F ui Diame e ; FS: F ui Shape. doi:10.1371/jou nal.pone.0104188. 001 Figu e 2. F equency dis ibu ion o he ai s ac oss he F 2 popula ion de i ed om he c oss ‘PS’ 6PI124112. (A) FL (F ui Leng h), (B) FD (F ui Diame e ) and (C) FS (F ui Shape). Bo h pa en s and F 1 alues a e ma ked. In he uppe box, 25 h ,50 h and 75 h qua iles a e displayed; he sample mean and he 95% con idence in e al a e ep esen ed by a diamond, and he ou lie alues as do s. The b acke along he edge o he box s ands o he pa o he g aph in which 50% o he obse a ions a e ga he ed oge he . Mean and s anda d de ia ion es ima es o a no mal dis ibu ion a e also shown. doi:10.1371/jou nal.pone.0104188.g002 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 5 Augus 2014 | Volume 9 | Issue 8 | e104188 sqs2.1 6 sqs8.1 A signi ican in e ac ion was iden i ied be ween sqs2.1 and sqs8.1 (p = 0.013, Figu e 4A, Table 3), ha explains a 4% o he gene ic a iance o FS. lqs2.1 has been p e iously iden i ied as gene a[12], [49] and he ma ke linked o i (AluICAPS) is ac ually he causal mu a ion o and omonoecy [11]. Fo ha eason and om now on, he alleles a his locus will be e e ed o as A and a. I is well known ha he dominan allele A causes elonga ed shapes in some gene ic backg ounds [12], as becomes clea in he cases o homozygous o PS allele a locus sqs8.1 (PsPs). Fo each o he h ee possible geno ypes a locus a (AA, Aa, aa), he lowes Figu e 3. Loca ion o 10 QTL esponsible o mo phological ui ai s mapped in a F 2 popula ion de i ed om he c oss ‘PS’ 6 PI124112. FL: F ui leng h, open ba s; FD: F ui Diame e , ha ched ba s; and FS: F ui Shape, solid ba s. Leng h o QTL ba s co esponds o he wo- LOD suppo in e als ( om peak ma ke ) based on he esul s ob ained by CIM using Windows QTL Ca og aphe 2.5. Numbe s on he le side co espond o he dis ance in cM (acco ding o [44]) om he op o each ch omosome. doi:10.1371/jou nal.pone.0104188.g003 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 6 Augus 2014 | Volume 9 | Issue 8 | e104188 Table 2. QTL de ec ed o FL, FD and FS using he me ged da a om wo loca ions o he F 2 popula ion om he c oss ‘PS’ 6PI124112. T ai LG QTL name a Flanking ma ke s In e al size (cM) LOD sco e a b d b d/[a] b R 2c Homologue QTL ID a Homologue published QTL d Re e ence e FL II lqs2.1 CMPSNP431-ECM61 38.4 8.44 3.06 2.29 0.75 0.16 lqn2.1 l2.1 [54] FL IIIb lqs3b.1 ECM205-CMPSNP998 12.9 2.92 +0.05 lqc3.5 3.5 [14] FL VIa lqs6a.1 ECM52-CMTCN41 69.1 9.56 +0.11 lqc6.4 6.4 [14] FL VIII lqs8.1 GCM241-PSI_25-H03 23.3 16.85 24.95 21.71 20.35 0.34 lqc8.3 8.3 [14] lqn8.1 l8.1 [54] FL Xb lqs10b.1 CMPSNP671-CMPSNP665 31.6 3.02 – 0.04 – – – FD IIIa dqs3a.1 CMBR100-AI_18-E05 25.6 2.80 – 0.11 – – – FD XII dqs12.1 ECM67-CMTCN14 52 3.47 21.03 20.17 20.17 0.11 dqi12.1 w6.10 [65] FS II sqs2.1 CMPSNP431-AluICAPS 16.9 6.74 0.27 0.20 0.75 0.17 sqj2.1 s2.3 [15] sqq2.1 s2.2 [12] sqn2.1 sh2.1 [54] FS VIII sqs8.1 GCM241-PSI_25-H03 23.3 14.85 20.50 20.16 20.32 0.41 sqc8.3 8.1 in [14] sqn8.1 sh8.1 [54] FS XII sqs12.1 AI_35-A08-CMPSNP361 53.1 3.61 0.15 0.03 0.21 0.03 sqa12.1 s11.1 [13] sqp12.1 s12.1 [12] sqc12.1 12.1 [14] FL: F ui Leng h; FD: F ui Diame e ; FS: F ui Shape. a Nomencla u e acco ding o [48]. b Only calcula ed o QTL in which he whole popula ion was geno yped wi h he s onges linked ma ke (s); o he es , only he sign is shown. c When possible, calcula ed using he da a coming he whole popula ion geno yped wi h he s onges linked ma ke (s); o he es , he alue ob ained by he QTL analysis using he selec i e geno yping subse o samples is shown. d Nomencla u e acco ding o he au ho s ha o iginally iden i ied he QTL. e Re iewed in [48] and, he FS QTL, in [53]. doi:10.1371/jou nal.pone.0104188. 002 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 7 Augus 2014 | Volume 9 | Issue 8 | e104188 alues o FS index ( ounde ui s) we e always obse ed in he homozygous o he PI124112 allele a locus sqs8.1 (PiPi) (Figu e 4A). So, when he PI124112 allele is p esen a sqs8.1 and he dominan A allele is absen a locus a(PsPiaa and PiPiaa), he e is a conside able dec ease o FS. In he homozygous PiPi, he di e ences among he h ee di e en geno ypes a locus aa e he sligh es . I seems ha homozygous PiPi a sqs8.1 diminish o , in some way, a enua es he e ec s o he dominan A allele o gene aon ui elonga ion. In he case o he e ozygous geno ypes a sqs8.1 (PsPi), some di e ences can be obse ed depending on he alleles p esen a locus a(A_ a e expec ed o p oduce mode a ely elonga ed ui s while aa will ende ed mo e ounded ones). So, we can only see ma ked di e ences in he h ee possible geno ypes a gene awhen he PI124112 allele is no p esen a QTL sqs8.1 (no masking e ec ) o i is in he e ozygosis, PsPs o PsPi (mode a e a enua ion o he pheno ype caused by A), wi h FS showing nea ly pa allel inc emen s when hese wo scena ios a e compa ed. In bo h cases, he elonga ed pheno ype caused by he a o emen ioned dominan A allele becomes clea , as homozygous AA p oduce he mos elonga ed ui s, ollowed by he he e ozygous (Aa). sqs8.1 6 sqs12.1 A signi ican in e ac ion was iden i ied be ween sqs8.1 and sqs12.1 (p = 0.014, Figu e 4B, able 3), explaining 7% o he gene ic a iance o FS. Simila ly o wha can be obse ed in he in e ac ion sqs2.1 6 sqs8.1, he lowes alues o FS ( ounde ui s) a e ob ained when he PI124112 allele is in homozygosis a sqs8.1 QTL (PiPi) (Figu e 4B). In such a case, he pheno ypic a ia ions due o he di e en allelic composi ion a sqs12.1 locus a e p ac ically impe cep ible. Those di e ences in he FS alue become mo e e iden wi h he p esence o he PS allele a sqs8.1, speci ically when i is in homozygosis (PsPs). In e es ingly, he highes alue o FS ( he mos elonga ed ui s) a e eached o he PS homozygous a sqs8.1 locus ha a e homozygous o he PI124112 a sqs12.1 (PsPsPiPi), wha is o ally in line wi h he posi i e addi i e alue o sqs12.1 QTL ( he Pi allele causes an inc ease in he FS index). He e, unlike wha happens in he p e ious in e ac ion, wi hin he same geno ype o QTL sqs8.1, he inc ease in FS due o he PI124112 allele a sqs12.1 becomes clea when he PI124112 allele is absen a locus sqs8.1. QTL alida ion in he sqs8.1-NIL The iden i ica ion and mapping o he sqs8.1 QTL in he F 2 popula ion has allowed us o use ma ke -assis ed selec ion o p ecisely in og ess he PI124112 allele esponsible o he ound shape in o he eli e cul i a ‘PS’ as he esul o a backc oss p og am (Figu e S1). To ensu e he in eg i y and main enance o he QTL e ec s, wo ounds o QTL e i ica ion we e ca ied ou in in e media e gene a ions. Figu e 4. Digenic in e ac ions s udied by wo-way ANOVA be ween he FS QTL using he ma ke s signi ican ly linked o hem. (A) sqs8.1 and sqs2.1 ( sqs2.1 6 sqs8.1); (B) sqs12.1 ( sqs8.1 6 sqs12.1). Alleles a locus lqs2.1 a e named A and a since his QTL has been p e iously iden i ied as gene a[12], [49]. PsPs: homozygous o he allele PS (solid line); PsPi: he e ozygous (dashed line); PiPi: homozygous o he allele PI124112 (do ed line). doi:10.1371/jou nal.pone.0104188.g004 Table 3. Signi ican digenic in e ac ions be ween all he FS QTL. QTL pai Sou ce DF Sum o Squa es F Ra io P ob . F sqs2.1- sqs8.1 sqs2.1 (AluICAPS) 2 7.83 35.72 ,0.0001 sqs8.1 (CMAT141) 2 26.79 122.21 ,0.0001 sqs2.1 (AluICAPS) 6 sqs8.1 (CMAT141) 4 1.42 3.24 0.0132 * sqs8.1- sqs12.1 sqs8.1 (CMAT141) 2 23.02 82.76 ,0.0001 sqs12.1 (ECM67) 2 1.59 5.71 0.0038 sqs8.1 (CMAT141) 6 sqs12.1 (ECM67) 4 1.78 3.19 0.0142 * The in e ac ions we e explo ed by wo-way ANOVA using he geno ypes o he closes ma ke s in he whole F 2 popula ion de i ed om he c oss ‘PS’ 6PI124112. * : In e ac ions we e conside ed o be s a is ically signi ican when p#0.05. doi:10.1371/jou nal.pone.0104188. 003 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 8 Augus 2014 | Volume 9 | Issue 8 | e104188 In 2008, he e ec o he sqs8.1 was moni o ed wi h ma ke s CMAT141 and PSI_25-H03 in BC4 plan s (Table 4, 8M-codes in Figu e S1). Plan s ca ying he PI124112 allele in he e ozygosis a sqs8.1 p oduced signi ican ly ounde melons han plan s ca ying he PS allele in homozygosis (F = 21.10 and F = 25.94 o CMAT141 and PS_25-H03, espec i ely, p,0.001). In 2009, he h ee geno ypes a sqs8.1 (moni o ed wi h ma ke CMAT141 and PSI_25-H03) seg ega ed in a BC4S1 popula ion (9M7, Figu e S1), e-con i ming he coseg ega ion o PI124112 alleles a sqs8.1 wi h he ound ui pheno ype (Table 4; F = 13.75 and p,0.001 o CMAT141; F = 5.71 and p,0.01 o PSI_25-H03). A he nex s age, whole-genome geno yping was pe o med in a selec ed BC5 plan (10M2-30) in o de o check he in eg i y o he in og essed egion and he composi ion o he gene ic back- g ound. The a ge in og ession ha bo ed 8.5 Mb in he LG VIII, and he e we e only se en addi ional in og essions anging om 250 kb o 86.8 Mb. Finally, he de ini i e in og ession line was ixed in a BC5S2 amily (12M57, Figu e S1). The amily 12M57 showed highly signi ican sho e and ounde ui s, ha is, lowe alues o FL and FS, espec i ely (Table 4; F = 10.27 and F = 105.71, o FL and FS, espec i ely, and p,0.001). Addi ionally, signi ican e ec s on FD we e also obse ed (F = 10.90, p,0.001, Table 4). Discussion The F 2 popula ion be ween ‘PS’ and PI124112 showed an imp essi e a iabili y o ui mo phology. T ansg essi e seg e- ga ion in bo h di ec ions was ound o FL and FS, indica ing ha a leas one o he pa en s con ibu ed alleles wi h opposi e e ec s and making possible o ob ain geno ypes ha could ende ound ui s. In ac , he exis ence o an allele inducing ound-shaped melons was iden i ied in he geni o PI124112 and e i ied wi h an in og ession line in he ‘PS’ backg ound. The e ec s o his allele seem o be masked by epis a ic in e ac ions wi h o he gene(s) in he PI124112 backg ound, and hose e ec s we e un eiled in he ‘PS’ backg ound. A simila disco e y has been epo ed ecen ly wi h he ob aining o a climac e ic line de i ed om he c oss be ween wo non-clima e ic melon accessions, ‘PS’ and ‘Shong- wan Cha mi’ [37]. This is a u he indica ion o he ichness o he melon gene pool ha emains s ill la gely unexplo ed. The seg ega ing popula ion coming om pa en s gene ically dis an and wi h mo phological di e ences used in his wo k has e ealed i sel as sui able o he gene ic dissec ion o mo pholog- ical ai s and mapping QTL. Fu he mo e, he mos obus FS (and he highly co ela ed FL) QTL de ec ed ( lqs2.1, sqs2.1, lqs8.1 and sqs8.1) we e consis en ac oss he loca ions es ed, in ag eemen wi h o he esul s published [16], in which QTL a ec ing FS displayed a high he i abili y, wha explained hei ep oducibili y in assays ca ied ou in di e en loca ions and along se e al yea s. The a ailabili y o a melon consensus map including he posi ion o QTL con olling a panoply o impo an ai s [48] made easie he compa ison o he QTL iden i ied he e wi h hose p e iously desc ibed. In his sense, mos QTL con olling he same cha ac e s s udied in his wo k ha e been de ec ed p e iously and mapped o simila genomic posi ions (Table 2). In ac , homolo- gous QTL ob ained in di e en popula ions can be assigned o all he QTL desc ibed he e, excep in he case o lqs10b.1 (only de ec ed wi h he ull se o da a) and dqs3a.1 (no ound in Cab ils). Only a QTL associa ed o FL was ecen ly epo ed in he LG II o he ela ed cucu bi Zucchini (Cucu bi a pepo L.) in a egion syn enic o he LG III o melon a which dqs3a.1 maps [50]. Howe e , his QTL could no be con i med in u he backc oss gene a ions. The QTL in LG II ( lqs2.1 and/o sqs2.1) was p e iously iden i ied as a pleio opic e ec o he gene a[12], [49], whose alleles a e esponsible o sex de e mina ion in melon lowe s [51] as i has been epo ed in o he plan species like Vi is ssp. [52]. The dominan inhe i ance mode obse ed o he alleles a lqs2.1 and sqs2.1 loci also suppo s i . The cloning o he gene has un eiled ha he causal mu a ion o he and omonoecious melon ypes is in he ac i e si e o 1-aminocyclop opane-1-ca boxylic acid syn hase [11]. The map posi ion o sqs8.1 (and lqs8.1, as hey o e lap) happened o be simila o hose epo ed o o he FS QTL (Table 2; e iewed in [48], [53]), sqc8.3 (8.1 in [14]) and sqn8.1 ( sh8.1 in [54]). Those QTL we e de ec ed in a NIL popula ion de i ed om he c oss ‘PS’ (C. melo a . inodu us)6PI161375 ‘Shongwan Cha mi’ (C. melo a . chinensis), and a Recombinan Inb ed Line (RIL) popula ion coming om he c oss PI414723 (C. melo a . momo dica)6‘Dulce’ (C. melo a . e icula us), espec i ely. So, sqs8.1 seems o be allelic o hose majo QTL unde lying ui mo phology in a b oad a ay o melon ge mplasm. The PI161375 and PI414723 alleles p oduced elonga ed ui s. On he con a y, PI124112 allele (in a ce ain allelic combina ion in ans wi h gene a a ian s) induced ound ui s, wha may be showing he exis ence o an allelic se ies a his QTL when di e en ge mplasm sou ces a e in es iga ed. Th ee mo e QTL ha e been epo ed in he su ounding egion ( sqa8.1, sqn8.1 and sqc8.1, compiled in [48]) and wo addi ional ones u he away in he same ch omosome ( sqc8.1 and sqp8.2, e iewed in [48]). These esul s sugges ha an impo an pa o he loci con olling FS in melon could be concen a ed in he LG VIII. I is also possible ha some o hem a e he same QTL ha he one desc ibed he e (ha bo ing common and/o new alleles). To disce n be ween hese wo cases, highe esolu ion in he mapping and QTL s udies is equi ed. Simila QTL clus e s ha e Table 4. F- alues ob ained om ANOVA o alida e he e ec s o QTL sqs8.1 on FS. 2008 2009 2012 CMAT141 PSI_25-H03 CMAT141 PSI_25-H03 CMAT141 FL 5.58 * 8.76 ** 10.10 *** 4.82 * 10.27 *** FD 3.46 ns 2.49 ns 0.53 ns 0.38 ns 10.90 ** FS 21.10 *** 25.94 *** 13.75 *** 5.71 ** 105.71 *** Da a coming om he assays ca ied ou along 3 yea s we e used. Plan s we e geno yped wi h linked ma ke s (CMAT141 and/o PSI_25-H03). FL: F ui Leng h; FD: F ui Diame e ; FS: F ui Shape. *** P,0.001; ** P,0.01; * P,0.05; ns P.0.05. doi:10.1371/jou nal.pone.0104188. 004 Mapping and In og ession o Melon F ui Shape QTL PLOS ONE | www.plosone.o g 9 Augus 2014 | Volume 9 | Issue 8 | e104188