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
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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
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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.
). (C) Wild ype and albino pheno ypes obse ed in line Cp-161.
doi:10.1371/jou nal.pone.0112743.g003
Fi s TILLING Pla o m in Cucu bi a pepo
PLOS ONE | www.plosone.o g 7 No embe 2014 | Volume 9 | Issue 11 | e112743
Acknowledgmen s
The au ho s hank Nicholas Da ies o his help in g amma ical e ision o
he manusc ip and Sa a Va´zquez o helping wi h c ea ing igu es.
Au ho Con ibu ions
Concei ed and designed he expe imen s: PG NVD CT AB. Pe o med
he expe imen s: NVD MM TPV CT. Analyzed he da a: NVD CT PG.
Con ibu ed eagen s/ma e ials/analysis ools: MRC BR BP CT AB.
W o e he pape : NVD PG.
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