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Applica ion o high esolu ion mel ing assay (HRM) o s udy empe a u e-dependen
in aspeci ic compe i ion in a pa hogenic bac e ium
Ash a i, Roghaieh; B uneaux, Ma hieu; Sundbe g, Lo a-Riina; Pulkkinen, Ka ja;
Ke ola, Ta mo
Ash a i, R., B uneaux, M., Sundbe g, L.-R., Pulkkinen, K., & Ke ola, T. (2017).
Applica ion o high esolu ion mel ing assay (HRM) o s udy empe a u e-dependen
in aspeci ic compe i ion in a pa hogenic bac e ium. Scien i ic Repo s, 7, A icle 980.
h ps://doi.o g/10.1038/s41598-017-01074-y
2017
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Scien i ic RepoR s | 7: 980 | DOI:10.1038/s41598-017-01074-y
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Applica ion o high esolu ion
mel ing assay (HRM) o s udy
empe a u e-dependen
in aspeci ic compe i ion in a
pa hogenic bac e ium
Roghaieh Ash a i 1, Ma hieu B uneaux1, Lo a-Riina Sundbe g1, Ka ja Pulkkinen2 & Ta mo
Ke ola1
S udies on species’ esponses o clima e change ha e ocused la gely on he di ec e ec o abio ic
ac o s and in pa icula empe a u e, neglec ing he e ec s o bio ic in e ac ions in de e mining
he ou come o clima e change p ojec ions. Many mic obes ely on s ong in e e ence compe i ion;
hence he i ness o many pa hogenic bac e ia could be a unc ion o bo h hei g ow h p ope ies
and in aspeci ic compe i ion. Howe e , due o echnical challenges in dis inguishing and acking
indi idual s ains, expe imen al e idence on in aspeci ic compe i ion has been limi ed so a . He e,
we de eloped a obus applica ion o he high- esolu ion mel ing (HRM) assay o s udy head- o-
head compe i ion be ween mixed geno ype co-cul u es o a wa e bo ne bac e ial pa hogen o ish,
Fla obac e ium columna e, a wo di e en empe a u es. We ound ha compe i ion ou come in liquid
cul u es seemed o be well p edic ed by g ow h yield o isola ed s ains, bu was mos ly inconsis en
wi h in e e ence compe i ion esul s measu ed in inhibi ion es s on solid aga , especially as no
g ow h inhibi ion be ween s ain pai s was de ec ed a he highe empe a u e. These esul s sugges
ha , o a gi en empe a u e, he ac o s d i ing compe i ion ou come di e be ween liquid and solid
en i onmen s.
Clima e models p edic ha clima e change will lead o inc eased empe a u es and la ge he mal luc ua ions in
he u u e1. As a consequence o clima e-induced ange shi s and expanded human ac i i ies leading o he ans-
po a ion o species, we expec o see p e iously sepa a ed popula ions come in o con ac wi h one ano he , p o-
iding new oppo uni ies o in e ac ions. Hence, i is o he u mos impo an o no e ha he e ec s o clima e
change can in oke ac o s o he han empe a u e, pa icula ly changes in bio ic in e ac ions wi hin and among
species2–8. Fo example, winne s judged by he sui abili y o habi a can be compe i i ely excluded by supe io
compe i o s9, 10. Gi en he global signi icance o pa hogens, p edic ing how clima e wa ming changes he ou come
o in aspeci ic in e ac ions in pa hogens, which in u n would a ec hei popula ion dynamics such as g ow h
and i ulence, is ex emely impo an o he p e en ion o pa hogen sp ead and he diseases caused by hem.
Empi ical in es iga ions o in aspeci ic in e ac ions using mul iple s ains om a single species ha e limi-
a ions in acking indi idual s ains in mixed cul u es. T adi ionally, geno yping o pheno ypic ma ke s based
on LacZ, luo escence o an ibio ic esis ance ha e been used disc imina e s ains and de e mine he ela i e
abundance o mul iple s ains ha in e ac wi hin a mixed cul u e11, 12. Howe e , ma ke s based on gene ic engi-
nee ing may p o ide misleading esul s, as hey may incu i ness cos s o bene i s in bac e ia13–15. To add ess
hese p oblems, and in o de o pe o m ou s udy, we applied a me hod o he quan i ica ion o compe i ion
ou come o ou s udy species, a bac e ial ish pa hogen, Fla obac e ium columna e. We implemen ed an assay
based on high- esolu ion mel ing cu e (HRM) which has been used o a ious applica ions, mos commonly
1Depa men o Biological and En i onmen al Science (and Nanoscience Cen e ), Uni e si y o Jy askyla, Cen e
o Excellence in Biological In e ac ions, P.O. Box 35, FI-40014, Jy askyla, Finland. 2Depa men o Biological and
En i onmen al Science, Uni e si y o Jy askyla, P.O. Box 35, FI-40014, Jy askyla, Finland. Co espondence and
eques s o ma e ials should be add essed o R.A. (email: [email p o ec ed])
Recei ed: 10 No embe 2016
Accep ed: 22 Ma ch 2017
Published: xx xx xxxx
OPEN
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he de e mina ion o DNA me hyla ion s a us and geno yping16–18. Using genomic DNA con aining a na u ally
occu ing mu a ion in he yp ophan syn hase gene pB enabled us o quan i a i ely dis inguish se e al F.
columna e s ains belonging o wo geno ypes o pB om mixed-geno ype compe i ion cul u es. To ou knowl-
edge, his is he i s s udy ha has used a HRM app oach o moni o and quan i y e y closely ela ed geno ypes
o one bac e ial species in a single ube, and he me hod is ex endable o mul iple geno ypes by mul iplexing, as is
exempli ied in AppendixS1 (See also Fig.S4).
F. columna e se es as an excellen model sys em o examine whe he empe a u e-dependen bio ic in e -
ac ions, such as compe i ion, could al e popula ion dynamics and clima e wa ming p ojec ions o a ew ea-
sons. Fi s , i has been shown ha high summe empe a u es co ela e wi h inc eased F. columna e ou b eaks19.
Second, mul iple geno ypes a e commonly p esen in a single egion20, 21 implying he po en ial o in aspeci ic
compe i ion among geno ypes. Thi d, compe i ion among F. columna e s ains, especially be ween he ones cho-
sen o his expe imen , is no es ic ed o esou ce compe i ion and s ains can di ec ly in e e e wi h compe i-
o s ia g ow h inhibi ion21, 22.
The aim o ou s udy was o es i head o head compe i ion, in e e ence compe i ion, and g ow h pa ame-
e s in wo empe a u es will lead o he same o di e en conclusions. I o he majo i y o he clone pai s he
compe i ion ou comes in di e en empe a u es can be p edic ed by g ow h assays o in e e ence assays ha will
acili a e p edic ions o u u e clima e change on his impo an ish pa hogen. Howe e , i p edic ions be ween
g ow h assays, in e e ence, and head o head compe i ion d as ically di e , i will cause challenges in p edic ing
which o he F. columna e geno ypes will excel unde p og essing clima e change. Al hough we use he e a species
wi h applied impo ance, he same ques ion applies o all a emp s o unde s and he a e o species unde chang-
ing condi ions in he wild, whe e in aspeci ic compe i i e in e ac ions a e he no m a he han he excep ion.
Resul s
Quan i ica ion o geno ype p opo ions in con ol mix u es using HRM. A single SNP (T > G
subs i u ion) a posi ion 222 o he pB gene was used o implemen he high- esolu ion mel ing analysis (HRM)
o F. columna e geno ypes, G and A, ci cula ing in Finnish ish a ms. The amplicon subjec ed o high esolu ion
mel ing analysis could be used o dis inguish be ween he wo SNP alleles based on he mel ing cu e shape. The
mel peak o he geno ype A was cen e ed a 80 °C, while ha o he geno ype G was cen e ed a 80.60 °C (Fig.1).
Sequencing o he ampli ied p oduc s in bo h di ec ions con i med he HRM esul s (Fig.S2).
Compa ison o he mel ing empe a u e alues o he e e ence samples ac oss he HRM uns showed ha
he e we e some a ia ions be ween he uns. Fo his eason, i is impo an o use wi hin-pla e e e ence samples
o he calib a ion o each un. The wi hin-pla e calib a ion cu es showed a good linea i be ween mel ing em-
pe a u es and geno ype p opo ions o he calib a ion samples (see R2 alues on Fig.S3). Fo compe i ion g oup
C, he e was a echnical issue du ing he p epa a ion o he e e ence samples o mixes om 20% o 60%, and
hose calib a ion samples we e disca ded om he analysis. Ou es ima e o geno ype p opo ions we e consis en
wi h he clus e ing esul s ob ained using he P ecision Mel Analysis so wa e (Bio- ad); howe e , hey enabled
us o ob ain a mo e quan i a i e es ima e o geno ype p opo ions in he expe imen al samples compa ed o he
clus e classi ica ion ob ained wi h P ecision Mel Analysis so wa e (Fig.2).
Compe i ion and g ow h. The empo al change o geno ype p opo ions as es ima ed by HRM du ing
he compe i ion in liquid medium is p esen ed in Fig.3. O e all, he i e eplica es wi hin each compe i ion
g oup showed consis en esul s. G oup A was he only compe i ion g oup o which he ou come o compe i ion
di e ed be ween 26 °C and 31 °C (Table1). The s ains wi h he as e g ow h and highe yield we e hose able
o u ilize esou ces as e and won in all es ed compe i ion pai s a bo h empe a u es, excep in one g oup (C)
whe e he losing compe i o had a highe yield han he winne a 26 °C. Howe e , he esul s o in e e ence
Figu e 1. High- esolu ion DNA mel ing cu e analysis esul s o a 97-bp amplicon con aining one SNP in
pB gene o Fla obac e ium columna e. (Righ ) No malized mel ing cu es, (Le ) de i a i e plo s. The mel ing
cu es depic pu e geno ype A, pu e geno ype G, and 50/50 a io o bo h geno ypes. Solid and dashed lines
ep esen eplica e measu emen s.
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Scien i ic RepoR s | 7: 980 | DOI:10.1038/s41598-017-01074-y
compe i ion on pla es wi h solid aga ma ched he esul s o he compe i ion in liquid media in only hal o he
cases (Table1). A 31 °C no inhibi ion be ween s ains was de ec ed (Table1).
Discussion
While empe a u e change can a ec he mal sensi i i ies, ole ance ange, and op imal pe o mance o popula-
ions, p edic ing how his change will ul ima ely a ec he s uc u e and dis ibu ion o species depends also on
indi ec e ec s media ed by bio ic in e ac ions5. Howe e , due o echnical limi a ions, such as acking indi idual
isola es in co-cul u es, ela i ely ew s udies ha e empi ically examined how join changes in in aspeci ic in e -
ac ions and empe a u e a ec ecological esponses o inc easing empe a u es12. Based on a diagnos ic SNP, we
success ully es ablished a me hod based on eal- ime PCR and HRM analysis o he apid di e en ia ion and
quan i ica ion o wo closely ela ed geno ypes o F. columna e in mixed cul u e. Using his me hod, we could
Figu e 2. (Righ ) No malized mel ing cu es and (Le ) di e ence plo s o e e ence samples consis ing o
pu i ied DNA om geno ypes G and A pooled in known p opo ions (13 di e en p opo ions o he wo
geno ypes: 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% and 0%). Solid and dashed lines
ep esen eplica e measu emen s.
Figu e 3. Geno ype p opo ions es ima ed by HRM uns a e 1, 2, 4, 6, 7 and 15 days o compe i ion in
liquid medium. Fo each day, e ical ba s show he ela i e p opo ions o each compe ing geno ype in i e
expe imen al eplica es wi hin each compe i ion g oup (A, B, C and D) and black lines indica es he 90%
con idence in e al o each p opo ion es ima e based on he iplica e HRM samples. The o de ing o
expe imen al eplica es in he ba plo s a e he same om day 1 o day 15 wi hin each g oup × empe a u e
ea men . The names o he compe ing geno ypes a e indica ed on he e ical axes. Ha ched ba s
indica e eplica es o which HRM da a could no be used o es ima e geno ype p opo ions. Fo each day
wi hin each compe i ion g oup, symbols be ween he 26 °C and 31 °C ba plo s indica e he p- alues o
a Welch’s - es compa ing he geno ype p opo ions o he 26 °C eplica es and o he 31 °C eplica es
(*** < 0.001 < ** < 0.01 < * < 0.05).
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esol e he ou come o head- o-head compe i ion o bac e ial s ains in liquid cul u e. Since his me hod does no
equi e any al e a ion in he geno ype o he wild- ype s ain, i is a sui able me hod o s udy ecological i ness and
compe i i e in e ac ions be ween na u al isola es. O e all, ou esul s con i m ha he HRM me hod can be used
o accu a ely quan i y he ela i e abundance o di e en s ains in co-cul u e samples, and sugges ha HRM
o e s a simple and cos e ec i e means o de ec ion o hese s ains in moni o ing in aspeci ic compe i ion in
a single cul u e ube.
We obse ed ha he compe i ion ou comes o wo s ains in mixed cul u e a wo empe a u es we e asso-
cia ed wi h changes in bac e ial g ow h a e in he gi en empe a u e, a he han wi h hei capaci y o in e -
e ence (Table1). This gi es a posi i e indica ion ha g ow h measu emen s could be e ec i e in p edic ing
he success o he clones in liquid cul u es and in di e en empe a u es, despi e he in e e ence compe i ion
be ween geno ypes o his species. This e i ica ion o he close ma ch be ween g ow h pa ame e s and compe-
i ion is impo an o model possible ou b eak scena ios unde changing en i onmen al condi ions in en i on-
men s whe e mul iple pa hogen s ains co-occu .
Bac e ia o en p oduce oxins ha kill o inhibi he g ow h o he s ains23–25. Inconsis ency be ween compe-
i ion ou comes in liquid cul u es and on pla e cul u e obse ed in ou s udy sugges s ha bene i s o in e e ence
on a su ace is elaxed in liquid cul u e, whe e he bac e ial cells a e mo e likely o ha e less equen con ac
wi h compe i o cells, and he oxins a e subjec ed o dilu ion. This indica es di e en ajec o ies o compe i ion
s a egies depending whe he he cells li e in bio ilm o in a plank onic s a e26, 27. Toxins a e, indeed, commonly
eleased in su aces (such as bio ilm) in which bac e ial popula ions a e dense and consequen ly nu ien lim-
i ed28. Fu he mo e, limi ed dispe sal can a ou oxin p oduc ion, sugges ing ha his mechanism will p o i
bac e ia g owing on su aces a he han he plank onic cells in ee wa e s and liquid cul u es29.
Inc eased global empe a u es can s ongly dec ease he e iciency o ood p oduc ion by in luencing pa hogen
species30, such as F. columna e19, 21, 31. P olonged in ec i e seasons p o ided by he highe mean empe a u es
allow o mo e bac e ium-bac e ium and bac e ium-hos in e ac ions, inc easing he bene i s o in e e ence.
Inc eased equency o high and maximum empe a u es, on he o he hand, may selec o bac e ial s ains wi h
a wide empe a u e op imum, and change he s ain composi ion o bac e ial popula ions. In e es ingly, how-
e e , we did no ind any in e e ence a high empe a u es (31 °C). Technical p oblems can be sa ely uled ou as
in e e ence es s a bo h empe a u es we e conduc ed a he same ime, om he same o iginal bac e ial s ocks.
P e ious s udies ha e shown ha bac e iocin (g ow h-inhibi ing subs ance needed o in e e ence) p oduc ion
is sensi i e o high empe a u e in lac ic acid bac e ia and in Ye sinia32–34. In empe a e egions, he empe a u e
o he aqua ic en i onmen a ely exceeds he +25 °C used as he lowe empe a u e in ou in e e ence expe i-
men s, which could main ain di e si y and compe i i e in e ac ions in bac e ial popula ions. Fo example, du ing
ecen yea s he p opensi y o F. columna e s ains isola ed om disease epidemics om ish a ms o inhibi he
g ow h o compe ing s ains has inc eased, sugges ing inc eased in aspeci ic compe i ion in he ish a ming
en i onmen 21. The bene i s o in e e ence in F. columna e migh be ealized in he ield condi ions du ing bio-
ilm o ma ion and mul iple in ec ions, which a e likely o occu when se e al s ains a e p esen .
Mos o he cu en esea ch on he e ec s o clima e change concen a es on single geno ypes, o species in
isola ion, bu in he wild he si ua ion is no so simple. Al hough labo a o y expe imen s, such as ou s, can shed
ligh on he pai wise in e ac ions o bac e ial s ains and imp o e ou unde s anding on ai s ha make ce ain
s ains mo e success ul, condi ions in he en i onmen a e mo e complex. In eal li e, species and geno ypes in e -
ac and may ha e addi i e e ec s o ac o s such as i ulence35, hus p oxies o i ness, such as indi idual g ow h
a es, migh no be p edic i e enough o which o he geno ypes will excel in compe i ion. The use o he HRM
me hod wi h high sensi i i y and speci ici y will likely allow us mimic mo e na u al sys ems, which will in u n
help in unde s anding he ci cums ances ha a ec pa hogenici y o his en i onmen ally g owing oppo unis ic
bac e ium. Mo eo e , as shown in AppendixS1 (See also Fig.S4), his me hod is ex endable o simul aneous
quan i a i e sepa a ion o mul iple geno ypes. We expec his me hod o be use ul in disen angling how global
wa ming and o he abio ic and bio ic ac o s con ibu e o bac e ial in e ac ions and impac he dynamics and
unc ionali y o bac e ial popula ions.
S ains Geno ype Yea O igin Compe i ion
G oup
26 °C
G ow h Yield Inhibi ion
31 °C
G ow h Yield Inhibi ionComp. Comp.
B185 G 2009 Fish a m A+0.199 ± 0.020 (5)0.788 ± 0.088 (5)− − 0.199 ± 0.012 (4) 0.198 ± 0.006 (4) No inhibi ion
B396 A 2010 Fish a m −0.158 ± 0.028 (5) 0.699 ± 0.052 (5) + + 0.200 ± 0.010 (4)0.298 ± 0.038 (4)No inhibi ion
B407 G 2010 Wild B−0.138 ± 0.038 (5) 0.717 ± 0.054 (5) − − 0.184 ± 0.044 (5) 0.137 ± 0.022 (5) No inhibi ion
TULO1 A 2010 Fish a m +0.220 ± 0.022 (3)0.779 ± 0.106 (3)+ + 0.208 ± 0.022 (5)0.285 ± 0.068 (5)No inhibi ion
B407 G 2010 Wild C−0.138 ± 0.038 (5) 0.717 ± 0.054 (5)− − 0.184 ± 0.044 (5) 0.137 ± 0.022 (5) No inhibi ion
B396 A 2010 Fish a m +0.158 ± 0.028 (5) 0.699 ± 0.052 (5) + + 0.200 ± 0.010 (4)0.298 ± 0.038 (4)No inhibi ion
B393 G 2010 Wild D−0.189 ± 0.030 (5) 0.622 ± 0.046 (5) + − 0.190 ± 0.022 (5) 0.166 ± 0.006 (5) No inhibi ion
B067 A 2007 Fish a m +0.189 ± 0.010 (5) 0.773 ± 0.064 (5)− + 0.216 ± 0.016 (5)0.347 ± 0.070 (5)No inhibi ion
Table 1. De ails o Fla obac e ium columna e s ains used in his s udy, and hei pe o mance in compe i ion
pai s (A–D, + indica es winne s ain in compe i ion). G ow h and yield equals g ow h a e and yield measu ed
in liquid medium, and inhibi ion (+) e e s o which s ain inhibi ed which in compe i ion pai s. In bold:
indica es which s ains o a gi en pai has he as es g ow h a e o highes yield a a gi en empe a u e.

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Ma e ial and Me hods
Bac e ial s ain cha ac e is ics and cul u e condi ions. Six F. columna e isola es, p e iously assigned
o wo gene ic g oups (geno ypes G o A) wi h he MLSA me hod20, we e used in his s udy. Be o e g ow h
yield measu emen , inhibi ion and compe i ion, he s ains we e e i ed om he ozen s ocks o 3 ml o Shieh
medium36 and incuba ed o 24 h in a shake (200 pm) a 26 °C. A e 24 hou s, cul u es we e dilu ed in o esh
modi ied Shieh medium (1:10) and allowed o eg ow a 26 °C on he shake o ano he 24 hou s. The six F.
columna e isola es we e dis ibu ed in o ou compe i ion pai s, each con aining one s ain om he G- and one
s ain om he A- geno ype, and all di e en g ow h and compe i ion assays we e pe o med on hese s ains
(Table1).
Compe i ion. The op ical densi y (OD, a 570 nm) o he bac e ial cul u es was measu ed wi h a spec opho-
ome e adjus ed o an app oxima e alue o 0.140 (~7.6 × 106 cells/ml) using he Shieh medium. Then, eplica es
made om 250 µl o p e-cul u e om a geno ype A s ain and 250 µl o p e-cul u e om a geno ype G s ain
mixed in 9.5 ml o Shieh medium we e pu in wo di e en empe a u es (26 °C and 31 °C, 5 eplica es pe em-
pe a u e). A e 24 hou s, he o e nigh compe i ion mix u e was dilu ed 100- old (0.1 ml in o 9.9 ml) in o esh
medium, and his was epea ed e e y 24 hou s o 14 days. To al DNA was ex ac ed immedia ely a e mixing
he p e-cul u es a he s a o he expe imen (day 0) and om o e nigh cul u es p io o dilu ion in o esh
medium on day 1, 2, 4, 6, 7, and 15. The ex ac ed DNA was s o ed a −20 °C un il u he analysis wi h HRM
assay (below).
HRM assay de elopmen . HRM analysis uses a sa u a ing, double-s anded DNA-binding dye, which spe-
ci ically in e cala es wi h double s anded DNA. In he pos -PCR s ep, when he dsDNA dissocia es in o single
s ands, he e is no longe any double s anded DNA p esen and hus luo escence is educed. The change in
luo escence is plo ed agains he empe a u e, gene a ing a mel ing cu e cha ac e is ic o he amplicon16, 17. The
six s ains used in his s udy (Table1) se ed as con ols o de eloping he HRM assay based on eal- ime PCR
associa ed wi h mel ing cu e analysis in o de o quan i y he p opo ions o s ains o G geno ype and s ains
o A geno ype in a mixed cul u e. These wo geno ypes exhibi ed h ee single-nucleo ide polymo phisms (SNPs)
in he pB gene, in posi ions 222, 587, and 590 o he ampli ied DNA agmen s o he gene (NCBI Re e ence
Sequence: LN624115, LN624122)20. HRM assay was no able o co ec ly disc imina e be ween a ge ed geno-
ypes and amplicons con aining wo o mo e SNPs. In his s udy, he e o e, we desc ibe he de elopmen o a
eal- ime PCR-HRM echnique a ge ing only one SNP (posi ion 222) o apid disc imina ion be ween he wo
geno ypes, as desc ibed below. Ex ension o his me hod o disc imina e be ween se e al geno ypes, by mul iplex-
ing, is desc ibed in AppendixS1 (See also Fig.S4).
DNA ex ac ion. Bac e ial cells we e ha es ed om o e nigh cul u es om he compe i ion expe imen
by cen i uga ion a 6000 g o 10 min a oom empe a u e, and genomic DNA was ex ac ed using he Wiza d
Genomic DNA Pu i ica ion Ki (P omega, USA). The DNA concen a ion and pu i y a e ex ac ion was es i-
ma ed using he Qubi ® Fluo ome e (Li e Technologies, Ca lsbad, USA). DNA quali y was assessed on an aga-
ose gel and he inal concen a ion was adjus ed o 5 ng µl−1.
P ime design and PCR ampli ica ion. Fo wa d (5′-CGCAGAAGTCCGTCCTG-3′) and e e se
(5′-AAAGGTATCTAGCGGATTGTT-3′) p ime s we e designed o a ge 97 bp o he pB gene (bp 171–267),
spanning a single SNP (T > G subs i u ion) a posi ion 222 o he gene. This p ime was designed using he
P ime -BLAST so wa e (h p://www.ncbi.nlm.nih.go / ools/p ime -blas /) (Fig.S1). Op imal p ime annealing
empe a u es we e es ablished by empe a u e g adien PCR and HRM.
PCR ampli ica ion and HRM analysis we e pe o med using CFX Real Time PCR De ec ion Sys em (Bio-Rad
Labo a o ies, USA). The PCR ampli ica ions we e pe o med and moni o ed using he CFX Manage So wa e,
and he HRM da a was analyzed wi h he Bio-Rad P ecision Mel Analysis. PCR ampli ica ion was pe o med in a
o al olume o 10 μL, con aining 5 μL o 1x P ecision Mel Supe mix (Bio-Rad Labo a o ies, USA), 1 μL 200 nM
o each designed p ime , and 4 μL 5 ng µl-1 o he DNA empla e. All samples we e ampli ied in iplica e. The
PCR eac ion s a ed a 95 °C o 1 minu e o ini ial dena u a ion, ollowed by 40 cycles o 30 seconds a 95 °C o
dena u a ion, 30 seconds a 63 °C o annealing and ano he 30 seconds a 72 °C o ex ension. The PCR ampli i-
ca ion was hen ollowed by he e oduplex o ma ion by hea ing a 95 °C o 30 seconds and subsequen cooling a
60 °C o 1 minu e. The high- esolu ion mel ing analysis was pe o med immedia ely a e wa ds by inc easing he
empe a u e om 65 °C o 95 °C by s eps o 0.2 °C main ained o 10 seconds each.
P ime e iciency. The p ime e iciency was assessed using bo h G and A geno ypes using 10- old se ial
dilu ions o DNA o each geno ype. The slopes o he s anda d cu es (an indica o o PCR e iciency) we e
simila , anging om −3.45 (geno ype A) o −3.56 (geno ype G), demons a ing ha he assays should be able
o de ec each geno ype wi h simila high e iciencies anging om 94.8% (geno ype A) o 90.8% (geno ype G).
Sequencing o a ge s. Real- ime PCR p oduc s we e subsequen ly sequenced o e i y he iden i y o he
ampli ied sequences. Sange sequencing was pe o med on he same amplicons as used o HRM analysis. 5 μl
o PCR p oduc s we e pu i ied wi h exonuclease I and Fas -AP (The mo Fishe Scien i ic, Wal ham, USA) o
15 min a 37 °C and 15 min by 80 °C. A sequencing eac ion was se up wi h 1 μl o pu i ied PCR p oduc s and
he BigDye® Te mina o 1.1 Cycle Sequencing Ki (Li e Technologies). B ie ly, each 20 μl sequencing eac ion
mix u e con ained 1 μl o PCR amplicon, 0.16 μM o ei he o wa d o e e se PCR p ime , 0.5 μl o BigDye
Ready Reac ion Mix, and 1 X sequencing bu e . The sequencing eac ion condi ions we e as ollows: 30 cycles
o dena u ing a 96 °C o 10 seconds, annealing a 50 °C o 5 seconds, and ex ension a 60 °C o 4 minu es.
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Scien i ic RepoR s | 7: 980 | DOI:10.1038/s41598-017-01074-y
The sequencing p oduc s we e pu i ied using e hanol/EDTA/sodium ace a e p ecipi a ion. Sequencing was pe -
o med on an ABI 3130xl 16-capilla y au oma ed gene ic analyse .
Quan i ica ion o mixed geno ypes in compe i ion cul u es. Fo each HRM un, e e ence samples
consis ing o pu i ied DNA om geno ypes G and A pooled in known p opo ions (13 di e en p opo ions
o he wo geno ypes: 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% and 0%) we e included
in duplica e in he pla e, and a no empla e con ol (NTC) was included in iplica e in he pla e. Samples om
he compe i ion expe imen o which geno ype p opo ions we e o be de e mined we e un in iplica e. Fo
each well, mel ing empe a u e was de ined as he empe a u e o he in lexion poin o he mel ing cu e, using
no malized ela i e luo escen uni s (RFU). This poin was de e mined by sea ching o he minimum alue o
he i s -o de de i a i e o he mel ing cu e. Wi hin each pla e, he e e ence samples we e used o calib a e he
ela ionship be ween geno ype p opo ion and mel ing empe a u e and o es ima e he geno ype p opo ions in
he expe imen al samples. A linea model o he o m [mel ingTemp = a + b * geno ypeP op] was i ed o he cali-
b a ion da a. The geno ype p opo ions in he expe imen al samples we e es ima ed by using a piecewise-de ined
unc ion using he ela ion [geno ypeP op = 1/b * (mel ingTemp − a)] and se ing he es ima ed alues o 0% i
hey we e nega i e and 100% i hey we e g ea e han 100% (Fig.S3). P io o geno ype p opo ion es ima ion
expe imen al sample, wells o which he mel ing empe a u e was ou side he ange o alues obse ed o cali-
b a ion samples by mo e han 10% he calib a ion ange span, we e disca ded. Con idence in e als we e calcu-
la ed acco ding o La agnini e al.37.
G ow h. The g ow h a e and yield o isola es we e measu ed in liquid cul u e using a empe a u e-con olled
spec opho ome e (Biosc een C, G ow h Cu es L d, Helsinki, Finland)21. B ie ly, a e isola e e i al a 26 °C
o 24 h on a shake (120 pm.), he op ical densi y (OD, a 570 nm) o he bac e ial cul u es was measu ed wi h
a spec opho ome e adjus ed o same le el (0.10–0.20). A e OD adjus men , 40 µl o each isola e was inoc-
ula ed in o 400 µL o esh Shieh liquid medium on a Biosc een C 100 wells pla es in i e eplica es and and-
omized o de . Pla es we e placed in Biosc een a 26 °C and 31 °C o 4 days un il g ow h in all wells s opped. To
ind he maximum g ow h a e (OD460–580 nm h−1) and maximum popula ion size (yield), we es ima ed he
biomass wi h a MATLAB ( e sion 2008b; Ma hWo ks Inc., Na ick, MA) sc ip ha i s linea eg essions in o
ln- ans o med popula ion g ow h da a consis ing o 30 da apoin s’ sliding ime window. The MATLAB code o
pe o m hese analyses is u he desc ibed Ke ola e al.38.
Inhibi ion assays. Wi hin each o he abo e men ioned compe i ion g oups, he inhibi o y ac i i y o F.
columna e s ains was es ed ecip ocally using an inhibi ion zone me hod wi h ou eplica es pe assay, and a
double laye me hod21. The op ical densi y (OD, a 570 nm) o he bac e ial cul u es was i s adjus ed o same
le el (0.250–0.290). F esh o e nigh -g own ‘ ecipien ’ bac e ial cul u es in abou 300 µl we e mixed wi h 3 ml o
mol en so Shieh aga (47 °C), which was hen pou ed on o he su ace o d ied Shieh aga . Fi e mic oli e s o
he un il e ed supe na an o he ‘dono ’ cul u es, which has been cen i uged a 17 000 g o 3 min in oom em-
pe a u e, we e spo ed on he su ace o he op aga . Pla es we e incuba ed o 48 h ei he a 26 °C o 31 °C; hen,
he pla es we e checked o de e mine whe he he ‘dono ’ s ain had caused a g ow h inhibi ion o he unde lying
‘ ecipien ’ bac e ial lawn.
Da a Accessibili y. DNA sequences used o de elop he p ime s: NCBI Re e ence Sequence: LN624115,
LN624122. Mel ing empe a u es and calib a ion cu e o HRM uns in supplemen a y ma e ial: Fig.S3. Da a
om HRM measu emen s (mel ing cu es om con olled mixes o geno ypes and mel ing empe a u es o
expe imen al and calib a ion samples om he compe i ion expe imen ) we e deposi ed in JYx and a e publicly
a ailable a h ps://jyx.jyu. i/dspace/handle/123456789/53541.
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Acknowledgemen s
We would like o hank D . Ilkka K onholm and D . Elina Laan o o p o iding cons uc i e commen s and help
in imp o ing he con en s o his pape ; and Do i Hämäläinen o help wi h he in e e ence assays. This wo k
was suppo ed by KONE ounda ion (Roghaieh Ash a i ia p ojec “Cons ain s o e olu iona y adap a ion o
clima e change” o Ta mo Ke ola), OLVI ounda ion (Roghaieh Ash a i #201620393), he Jane and Aa os E kko
Founda ion (Lo a-Riina Sundbe g), Finnish Cul u al Founda ion (Ka ja Pulkkinen) and Academy o Finland
(Lo a-Riina Sundbe g #272995, Ta mo Ke ola #278751, Jouni Taskinen #260704 o Ka ja Pulkkinen) and Cen e
o Excellence in Biological In e ac ions (#252411, P o . Johanna Mappes) o Roghaieh Ash a i, Lo a-Riina
Sundbe g, and Ta mo Ke ola.
Au ho Con ibu ions
R.A., T.K. concei ed and designed he expe imen . R.A., M.B., L.S., K.P., T.K. w o e he pape . R.A., M.B. analysed
he da a.
Addi ional In o ma ion
Supplemen a y in o ma ion accompanies his pape a doi:10.1038/s41598-017-01074-y
Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s.
Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
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