genome wide gene expression patterns in dikaryon of the basidiomycete fungus pleurotus ostreatus

Themycelial growth rate ofMK3 was fasterthanMK13onpotatodextroseagarPDAplatesFig.1 DikaryonDK13×3wasfromMK13andMK3throughA1B1and A2B2mating,asidentifiedusingmatingtests.28DK13×3grew faste

h tt p : / / w w w b j m i c r o b i o l c o m b r /

ostreatus

Henan Agricultural University, College of Life Sciences, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Zhengzhou, China

a r t i c l e i n f o

Article history:

Received14June2015

Accepted20September2016

Availableonlinexxx

AssociateEditor:GiseleMonteirode

Souza

Keywords:

Differentialgeneexpression

Monoallelicexpression

Monokaryon

RNAediting

RNA-Seq

a b s t r a c t

Dikaryais asubkingdom offungithatincludes AscomycotaandBasidiomycota.The gene expressionpatternsofdikaryonarepoorlyunderstood.Inthisstudy,webredadikaryon DK13×3bymatingmonokaryonsMK13andMK3,whichwerefromthebasidiosporesof Pleu-rotus ostreatusTD300.UsingRNA-Seq,weobtainedthetranscriptomesofthethreestrains

Wefoundthatthetotaltranscriptnumbersinthetranscriptomesofthethreestrainswere allmorethantenthousand,andtheexpressionprofileinDK13×3wasmoresimilarto MK13thanMK3.However,thegenesinvolvedinmacromoleculeutilization,cellular mate-rialsynthesis,stress-resistanceandsignaltransductionweremuchmoreup-regulatedin thedikaryonthanitsconstituentmonokaryons.Allpossiblemodesofdifferential gene expression,whencomparedtoconstituentmonokaryons,includingthepresence/absence variation,andadditivity/nonadditivitygeneexpressioninthedikaryonmaycontributeto heterosis.BysequencingtheureasegenepouresequencesandmRNAsequences,we iden-tifiedthemonoallelicexpressionofthepouregeneinthedikaryon,anditstranscriptwas fromtheparentalmonokaryonMK13.Furthermore,wediscoveredRNAeditinginthepoure

genemRNAofthethreestrains.Theseresultssuggestthatthegeneexpressionpatternsin dikaryonsshouldbesimilartothatofdiploidsduringvegetativegrowth

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/)

Introduction

Dikaryonisauniqueorganisminwhicheachcompartmentof

ahyphacontainstwohaploidnuclei,eachderivedfroma

dif-ferentparent.ItconsistsofasubkingdomoffungiDikarya,

∗ Corresponding author.

E-mail:qliyou@henau.edu.cn(L.Qiu)

including Ascomycota and Basidiomycota. A dikaryon strain

is formed by mating two compatible monokaryon strains, resulting in plasmogamy but not karyogamy in the fused compartment.Whennewhyphaegrow,thetwonuclei syn-chronously divide, and each new compartment keeps two nuclei1;karyogamyonlyoccursbeforetheinitiationofsexual

http://dx.doi.org/10.1016/j.bjm.2016.12.005

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Pleasecitethisarticleinpressas:LiuT,etal.Genome-widegeneexpressionpatternsindikaryonofthebasidiomycetefungusPleurotus ostreatus.

reproduction.Thissexualreproductionmodewasdistinctly

differentfromthatindiploids.Theinteractionbetweenthe

geneticmaterialsofthetwonucleiindikaryonshasnotbeen

wellcharacterized.Arethemodesofgeneactionindikaryons

thesameasthatindiploidsduringvegetativegrowth?

The major types of gene expression patterns found in

diploids during vegetative growth are mitotic crossover or

mitoticrecombination,2,3 DNAmethylationandgene

silenc-ing by RNAi,4 monoallelic expression (sex chromosome

inactivation, imprintedgeneexpression, orautosomal

ran-dommonoallelicexpression),5RNA-editing,6anddifferential

alleleexpressioninhybridsandparentsthatcontributesto

heterosis,7etc.Mitoticrecombination(alsonamed

parasexu-alityinfungi),DNAmethylationandgenesilencingbyRNAi

werealsofoundindikaryons,8–10 whilemonoallelic

expres-sionandRNA-editinghavenotbeenidentifiedinthedikaryon

Althoughnotstrictlytrueforallreportedspecies,intermsof

thegrowthrate,enzymeactivityandpathogenicity,diploids

have a significant advantage over their parental haploids,

which is similar to what is exhibited when dikaryons are

comparedto their parental monokaryons Itwas proposed

thatthe heterosisindiploidsresultedfrom the allelegene

differentialexpressioninhybridsandtheirparents,suchas

presence/absencevariationandadditive/non-additive

(high-andlow-parentdominance,underdominance,and

overdom-inance)geneexpression.11–14Themechanismofheterosisin

dikaryonsremainsobscure

Aneffectiveapproachforexploringtheallelegene

differ-entialexpressionindikaryonsisthecomparisonofsoluble

proteinprofiles or isoenzyme patterns betweena dikaryon

anditsconstituentmonokaryons.Thesolubleproteinprofile

ofSchizophyllum communedikaryonwasdramaticallydifferent

fromthatofitsparentalmonokaryons,and therearemany

newbandsinthedikaryon15;furtherstudiesshowedthat14

outof15isoenzymepatternschangedbetweenthedikaryon

andtwomonokaryons.16 Similarresultswere alsoreported

inotherbasidiomycetes,suchasCoprinus congregatus17 and

Coprinopsis cinerea.18Thosestudiesindicatedthatalleleshad

differentexpressionpatternsindikaryonsandmonokaryons

However, subsequent studies found no such difference in

higher basidiomycetes and suggested that those reported

differenceswereprobablycausedbygrowthconditionsand

the electrophoresis procedure.19,20 Since then, many other

observationshaveconfirmedsuchfindings.Forexample,

com-paringS commune monokaryonsand the dikaryon, protein

two-dimensionalgel electrophoresisshowedonly6.6%and

7.7%differences,21andthesequencecomplexitiesandcoding

propertiesofpolysomalRNAandtotalRNAhadnodetectable

difference.22,23Nevertheless,usinggeneexpressionprofiling,

therelativedifferencesinthetranscriptionquantityofthe12

laccasegenesinthePleurotus ostreatusdikaryonanditstwo

parentalmonokaryonsshowedthatthedikaryotic

superior-ityinlaccaseactivitywasduetonon-additivetranscriptional

increasesintwogenes.24Genome-widegeneexpression

pat-ternanalysisofdikaryonsand theirparental monokaryons

hasnotbeenreported

OystermushroomP ostreatus(Jacq.Fr)Kumm.isawhite

rotbasidiomycetethat isan importantedible and medical

mushroom,25–27 and ithasbeen studiedasamodel

organ-ismforbasidiomycetegeneticsandgenomicstudies.24Inthis

study,wecomparedthegenome-widetranscriptionalprofiles amongthedikaryonanditstwoconstituentmonokaryonsof

P ostreatusbySolexa-basedRNA-Seqwithafocusonthe trans-criptomicprofilingdifferenceanalysisbetweenthedikaryon and monokaryons, investigation ofthe mechanisms ofthe advantagesofsexual reproduction, monoallelicexpression, andRNA-editingindikarya

Materials and methods

Monokaryons MK13 and MK3 were from the basidiospores

of P ostreatus TD300, which is a commercial cultivation straininChinaandwasobtainedfromZhengzhou Compos-iteExperimentstation,ChinaEdibleFungiResearchSystem (Zhengzhou, China) Themycelial growth rate ofMK3 was fasterthanMK13onpotatodextroseagar(PDA)plates(Fig.1 DikaryonDK13×3wasfromMK13andMK3throughA1B1and

A2B2mating,asidentifiedusingmatingtests.28DK13×3grew fasterthanitsconstituentmonokaryonsinPDAandformed normalfruitingbodies withabiological efficiencythatwas similartoTD300incottonseedhullmedium(Fig.2 Thethree strains wereculturedinpotatodextrosebroth(150mLina

TD300

MK13

6.0

5.0 4.0 3.0

2.0 1.0 0.0

TD300

a

a

c

b

DK13 ×3 MK13 MK3

MK3 DK13×3

Fig 1 – Mycelial growth of the monokaryons and reconstituted dikaryon ofPleurotus ostreatuson PDA plates MK13, monokaryon; MK3, monokaryon; DK13 × 3, dikaryon; TD300, dikaryon and the two monokaryons’ parent; MGR, mycelial growth rate Data are given as the means and SE of four replicates Data with the same lower case letter do not significantly differ from other data atp< 0.05.

TD300

Biological efficiency, %

120 150

DK13×3

DK13×3

Fig 2 – Fruiting body morphology and biological efficiency

of TD300 and DK13 × 3 in cottonseed hull medium.

Biological efficiency indicates the percentage of the fresh

weight of harvested 1st and 2nd flush mushrooms over the

dry weight of inoculated substrates.

500-mLflask)at25◦Cunder150rpmshaking;myceliawere

harvestedinthelateexponentialphase(10and25daysof

cul-turingfordikaryonandmonokaryons,respectively)forDNAor

totalRNAextraction

Mycelia were isolated from culturebroth bycentrifugation

at5000×g for10min; 100goffresh myceliawas

homoge-nizedinliquidnitrogen;andtotalRNAwasextractedusingan

RNApuretotalRNAfastisolationkit(Bioteke,Beijing,China)

ThetotalRNAwasusedforRT-PCRorenrichmentofmRNA

(poly(A)+RNA)withaDynabeadsmRNAPurificationKit

(Invi-trogen,GrandIsland,NY),andmRNAwasthenbrokeninto

shortfragments.Using theseshortfragmentsastemplates,

first-and second-strand cDNA were synthesized

Sequenc-ing adapters, which also served as sample markers, were

ligatedtoshortfragmentsafterpurificationwithaQiaQuick

PCRExtractionKit(Qiagen,Hilden,Germany).Fragmentsthat

were 200–700bp were then separated by agarose gel

elec-trophoresisandselectedforPCRamplificationassequencing

templates.Thethreestrain librarieswere sequencedusing

IlluminaHiSeqTM2000bytheBeijingGenomeInstitute(BGI)

(Shenzhen,China)

Rawreadscontainedlow-quality,adaptor-pollutedandhigh

contentsofunknownbase(N)reads,andthesenoisereads

should be removed before downstream analyses Weused

internalsoftwaretofilterreads.Afterfiltering,theremaining

readswerecalled“CleanReads”andstoredintheFASTQ

for-mat

Contigs were assembled from clean readsusing a denovo assemblerTrinity29;then,non-redundantunigenesetsforall threestrainswereconstructedusingtheESTassembly pro-gramTGICL.30Anall-unigenesetwasproducedfromthethree contigdatasetsbyfurthersequenceoverlapsplicingand non-redundancies

Cleanreadsweremappedtothereferencegenomesequence

of Pleurotus ostreatus PC15 (http://genome.jgi-psf.org/ PleosPC152/PleosPC152.home.html) using Bowtie231; then, thegeneexpressionlevelwascalculatedusingRSEM.32

TheunigeneexpressionlevelswerecalculatedusingtheReads per kb per Million reads(RPKM) method.33 Under the null hypothesisofequalexpressionbetweentwosamples,the fol-lowing test gives the p-values for identifying differentially expressedgenes(DEGs)betweentwosamples.34

P(y|x)=N2

N1

x!y!(1+(N2/N1))(x+y+1)

N1isthetotalnumberofcleantagsinMK3orMK13;N2is thenumberinDK13×3;xisthenumberofthecleantagsofthe targetgeneinMK3orMK13,andyisthenumberinDK13×3

p≤0.001and|log2Ratio|≥1wereusedasthethresholdtofilter DEGs

TheDEGsexpressedinallthreestrainswereusedto esti-matethemid-parentexpressionvalue(MPV).TheMPVwas calculatedbyaveragingtheexpressionleveloftheparental monokaryons,assumingan(MK3:MK13)ratioofRNA abun-danceinthenucleusofDikaryonDK13×3of1:1,asdescribed elsewhere.35

To validate the gene expression profiles obtained by RNA-seq,ureasegenepoureofthemonokaryonsanddikaryonwas cloned,amplified,andsequenced.Cloningwasperformedby colony direct PCR36 using primers POU1

conditionsof94◦Cfor30s,51◦Cfor40s,and72◦Cfor3min, whichwererepeated31times.mRNAswereamplifiedusing RT-PCRwithprimersPOU3(TTACCGAGGGAAGAAGCGAA)and POU4 (GGTGGTGACAGAAACGGGAGTA), and PCR conditions were setat94◦Cfor30s, 52◦Cfor40s,and72◦Cfor2min, whichwasrepeated31times.ThePCRproductsofDNAand mRNAwere purifiedand were thencloned intothe

pGEM-T Vector (Promega, Madison, WI, USA) The vectors were transformed intoE coliDH5␣,and fivetransformantswere randomly selected and sequenced by the Beijing Genome Institute(BGI)(Shenzhen,China)

Pleasecitethisarticleinpressas:LiuT,etal.Genome-widegeneexpressionpatternsindikaryonofthebasidiomycetefungusPleurotus ostreatus.

Table 1 – Throughput and quality of RNA-Seq of the dikaryon and its constituent monokaryons ofPleurotus ostreatus.

reads(Mb)

Totalclean reads(Mb)

Totalclean bases(Gb)

Cleanreads Q20(%)

Cleanreads ratio(%)

Total mapping ratio(%)

Uniquely mapping ratioa(%)

a Uniquemapping:readsthatmaptoonlyonelocationofthereference,calleduniquemapping

Results

Table1liststhestatisticsofthereads.TheRNA-seqreadswere

ofhighquality;almostallmRNAfragmentsweresequenced,

and97%ofthereadshadaPhredqualityscoregreaterthan

20.Wemappedcleanreadstothereferencegenomesequence

of Pleurotus ostreatus PC15 (http://genome.jgi-psf.org/

PleosPC152/PleosPC152.home.html) using HISAT.37 On

average,60.44%ofreadsaremapped,andtheuniformityof

themappingresultforeachsamplesuggeststhatthesamples

are comparable The GenBank accession number for the

RNA-seqdatasetsofthethreestrainsisBioProjectAccession:

PRJNA326297

Aftergenome mapping,weused StringTie38 toreconstruct

transcripts,andwithgenomeannotationinformation,wecan

identifynoveltranscriptsinoursamplesusingcuffcompare,

atoolofcufflinks.39Intotal,weidentified4261novel

trans-cripts.Then, wemerged novelcoding transcripts withthe

referencetranscripttoobtainacompletereference,mapped

cleanreadsusingBowtie2,40andcalculatedthegene

expres-sionlevelforeachsamplewithRSEM.41Thereupon,thetotal

mappingratiosofthecleanreadsinthetranscriptomesofthe

threestrainswereincreased.Totaltranscriptnumberswere

allmorethantenthousand(Table2

We then calculated the read coverage and read

distri-butiononeachdetectedtranscript.ThePearsoncorrelation

betweenthetranscriptomesofthethreestrainswasobtained

ThePearsoncorrelationsofthedikaryonDK13×3toits

con-stituentmonokaryons,MK13andMK3,were0.8523and0.8100,

respectively,whilethePearsoncorrelationbetweenthe two

monokaryonswas0.8124,indicatingthattheexpression

pro-fileinDK13×3wasmoresimilartoMK13thanMK3(Fig.3

ThetotalRPKMsoftheunigenesinMK13,MK3andDK13×3

were559494,550716,and586583.ThetotalRPKMsofthe

uni-genesinDK13×3were4.8%and6.5%higherthan thosein

MK13andMK3(p<0.05)(Fig.4 Amongtheunigenesbetween

DK13×3andMK13orMK3,thecommonunigenesofthethree

strainswere27.6%,the commonunigenesforDK13×3and

MK13were 10.8%,and the common unigenesfor DK13×3

andMK3were11.3%.ThespecialunigenesinDK13×3,MK13

MK13

MK13

1 0.95 0.9 0.85 0.8 0.75

MK13×3

MK13×3

MK3

MK3

Fig 3 – Heatmap of Pearson correlations between the dikaryon and its constituent monokaryons ofPleurotus ostreatus.

and MK3 were 13.5%,17.6%,and15.5%,respectively Upto 38%ofunigenesinDK13×3were derivedfrom itsparental monokaryons(Fig.5 indicatingthatthegeneexpression pat-tern of present/absentvariation occurred amongthe three strains,andmorethanone-thirdoftheDEGsinthedikaryon weremonoallelicexpressiongenes

Usingp≤0.001and|log2Ratio|≥1asthestandardtoscreen the differentiallyexpressedgenes (DEGs)betweenDK13×3 andMK13orMK3,comparedtoMK13,thenumberofgenes whoseexpressionlevelswereup-regulated inDK13×3was 11323;7953wereup-regulatedmorethan3-fold,and114were up-regulatedmorethan15-fold.Additionally,8421geneswere down-regulated;2573weredown-regulatedmorethan3-fold, whilenoneweredown-regulatedmorethan15-fold(Fig.6A) ComparedtoMK3,thenumberofgeneswhoseexpressionwas up-regulatedinDK13×3was11578;7787wereup-regulated morethan3-fold,and116wereup-regulatedmorethan 15-fold.Furthermore,7425genesweredown-regulated;2176were down-regulatedmorethan3-fold,and1wasdown-regulated morethan15-fold(Fig.6B).Theresultssuggestthatthe num-berofup-regulatedgenesinthedikaryonwasmuchhigher thanthatofdown-regulatedgenes,especiallycomparedtothe constituentmonokaryons

Thegenesinthedikaryonthatwere15-foldup-or down-regulated comparedwiththemonokaryonswere examined with anNCBI onlineBLASTP homology analyzer Addition-ally,28and21up-regulatedgeneswerefoundtohaverelated

Table 2 – Summary of gene expression in the dikaryon and its constituent monokaryons ofPleurotus ostreatus.

mapping

ratio(%)

Uniquely mapping ratio(%)

Totalgene number

Knowngene number

Novelgene number

Total transcript number

Known transcript number

Novel transcript number

–4 –4 –3 –2 –1 0 1

Log 10 (DK13×3 RPKM) Log 10 (DK13×3 RPKM)

2 3 4 5 6 –4 –3 –2 –1 0 1 2 3 4 5 6 –3

–2 –1 0 1 2 3 4 5 6

FDR≤0.001 and |log2ratio|≥1 Up-regulated genes Down-regulated Not DEGs

–4 –3 –2 –1 0 1 2 3 4 5 6

Fig 4 – Comparison of the unigene expression levels between MK3 or MK13 and DK13 × 3 Up-regulated genes,

down-regulated genes, and NOT DEGs were determined using a threshold ofp≤ 0.001 and |log2Ratio| ≥ 1 A, MK3 vs DK13 × 3; B, MK13 vs DK13 × 3; NOT DEGs, Unigenes were not obviously changed upon MK3 or MK13 to DK13 × 3.

Unigenes of MK13 13.5%

10.8%

27.6%

11.3%

15.5%

3.7%

17.6%

Unigenes of MK3 Unigenes of DK13×3 Specific genes of DK13×3 Specific genes of MK13 Specific genes of MK3 The common genes of three strains The common genes of MK13 and MK3 The common genes of DK13×3 and MK3 The common genes of DK13×3 and MK13

Fig 5 – Distribution diagram of DEGs between MK3 or MK13 and DK13 × 3 DEGs were screened by a threshold ofp≤ 0.001 and |log2Ratio| ≥ 1.

(Tables3and4 Thesefindingshaveprovidedevidenceforthe

growthadvantagethatthedikaryonhasovertheconstituent

monokaryons

AmongthecommonDEGsofthethreestrains,whenthe

DK13×3levelswerecomparedtoMPVadditivemodelvalues,

approximately63.0%(878/2027)oftranscriptswereidentified

tobeengagedinnon-additivegeneexpression(thresholdof

greaterthantwo-foldhigher/lower).Asmallpluralityofgenes,

36.8%,hadlowerexpressionlevelsinDK13×3thanexpected,

while 26.2% were higher and potentially upregulated (Fig.7

Forexample,weobtainedthetranscriptionprofilingfrom the RNA-seq of the 17 laccase genes in the three strains The gene action modes of the 17 laccase genes could be dividedintothefollowingthreepatterns:genesexpressedin bothparental monokaryonsbut notinthedikaryon; genes expressedinoneparentalmonokaryonanddikaryonbutnot

in another parental monokaryon; and genes expressed in parentalmonokaryonsandthedikaryon.However,thetotal RPKMsoftheselaccasegenesinDK13×3didnotpresent sig-nificantdifferencescomparedtotheparentalmonokaryons (Table5

Pleasecitethisarticleinpressas:LiuT,etal.Genome-widegeneexpressionpatternsindikaryonofthebasidiomycetefungusPleurotus ostreatus.

1 10 100 1000 10000

12–15 9–12 6–9 3–6

Up-regulation Down-regulation

1 10 100 1000 10000

12–15 9–12 6–9 3–6 1–3

|log2 ratio of RPKM| |log2 ratio of RPKM|

Fig 6 – Differentially expressed genes in dikaryon DK13 × 3 compared to parental monokaryons MK13 (A) or MK3 (B) RPKM, reads per kb per million reads.

Table 3 – Function annotation of differentially expressed genes in dikaryon DK13×3 compared to its parental

monokaryon MK13.

poure monoallelic expression in the dikaryon

Thepouregeneofthe twomonokaryonsand mRNAofthe

(http://genome.jgi-psf.org/PleosPC152/PleosPC152.home.html;

http://genome.jgi-psf.org/PleosPC91/PleosPC91.home.html);

those for MK3 (GenBank access number:KF312590.1) were

96%and95%identical.Thedifferentbasesbetweenthepoure

geneCDSofMK13andMK3were93(Table6 ThepouremRNA

sequencesofMK13,MK3andDK13×3wereall100%identical

tothe RNA-seqresults.However,themRNAsequencesand

geneCDSofpouredifferedby4basesinMK13and12inMK3

InMK13,thedifferencesweretwoTstoCsandtwoGstoAs

InMK3,thedifferenceswereoneCchangingtoG,fourCsto

Ts,fourAstoGs,andthreeGstoAs(Table7 Thisrevealed that P ostreatussimultaneously occurred innumerousRNA editing types Furthermore, the poure mRNA sequences of DK13×3 were more identical to that of MK13 than MK3, withonlytwodifferentbasesandonepredictedaminoacid

to MK13, whilethere were 89 differentbases comparedto MK3 As with MK13, the mRNA sequence and gene CDS

ofPoureinDK13×3involved4bases,oneTtoC,oneCto

T, andtwo GstoAs(Tables6and 7 Urease catalyzedthe hydrolysisofureaintocarbondioxideandammonia.Urease was the first enzyme to be crystallized from jack beans, and it was the first protein whose enzymatic properties weredemonstratedbySumnerin1926.42Ureaseshavebeen

Table 4 – Functional annotation of differentially expressed genes in dikaryon DK13×3 compared to its parental MK3 monokaryon.

Table 5 – Laccase gene expression profile inPleurotus ostreatusdikaryon DK13×3 and its parental monokaryons MK13 and MK3.

Group1

Group2

Group3

a Group1,genesexpressedinbothparentalmonokaryonsbutnotinthedikaryon;Group2,genesexpressedinoneparentalmonokaryonand thedikaryonbutnotinanotherparentalmonokaryon;Group3,genesexpressedinparentalmonokaryonsandthedikaryon

foundinnumerousbacteria, fungi, algae,plantsand some

invertebrates, and they have been found to help

microor-ganismsandplantsuseendogenousandexogenousureaas

anitrogensource.Theammonia produced issubsequently

utilizedto synthesizeproteins.43 Ureases ofbacteria, fungi

and higher plantsare highly conserved.44 In higher plants

andfungi,theenzymeisencodedbyasinglegene.45,46Thus,

ourresultsshowedthatthepouretranscriptofDK13×3was

fromtheMK13pouregeneandthatRNAeditingalsooccurred

(Table6

Discussion

Our resultsshowed thatthe global geneexpressionprofile

ofdikaryonwasdistinctfrom itsconstituentmonokaryons, andtherewasanexpressiondifferenceinnearlytwo-thirds

of the genes This change was also confirmed by RT-PCR cloningandsequencingofthepouremRNAofthethreestrains Theseresultsarenotinagreementwithpreviousreports,22,23

which is probably due to the different gene expression

Pleasecitethisarticleinpressas:LiuT,etal.Genome-widegeneexpressionpatternsindikaryonofthebasidiomycetefungusPleurotus ostreatus.

Table 6 – Sequence alignment of thepouregene CDS between the two monokaryons ofPleurotus ostreatus.

CDSa Thepositionofmismatchedbasesfromthe5endofthePoureCDS

924 966 975 978 1005 1061 1074 1083 1170 1200 1209 1236 1290 1311 1326 1335

1383 1521 1523 1527 1536 1587 1605 1628 1641 1680 1689 1707 1709 1713 1764 1767

1769 1782 1788 1808 1848 1857 1876 1917 1992 2061 2067 2070 2076 2079 2158 2208

2224 2229 2268 2317 2325 2364 2409 2450 2451 2469 2475 2478 2480

a TheaccessionnumbersinGeneBankofthepouregeneCDSofPleurotus ostreatusMK13andMK3areKF312589.1andKF312590.1

Table 7 – Sequence alignment of thepouregene CDS, mRNA and predicted AAs between the three strains ofP ostreatus.

Strain Thepositionofmismatchedbasesfromthe5endof

AAresidues

Based on the gene transcriptional quantity, heterosis

indiploids was considered toresultfrom differential gene

expression,includingthefollowingfivegeneexpression

pat-terns:(i)genesexpressedinbothparentsbutnotinhybrids,(ii)

genesexpressedinoneparentandhybridbutnotinanother

parent,(iii)genesexpressedinoneparentbutnotinanother

parentorhybrid,(iv)genesexpressedonlyinahybridbutnot

inbothparents,and(v)genesexpressedinbothparentsand

thehybrid.Thefirstfourpatternsarethepresence/absence

variations (PAV)48; the fifth could be divided into additive

andnon-additivegeneexpressionpatternsforwhichhybrids

showedatranscriptlevelequaltoordeviatingfromthe

mid-parentvalue(averageofthetwoparents).49–51Inthisstudy,the

mycelialgrowthrateofP ostreatusdikaryonDK13×3was

sig-nificantlyhigherthanthatofthetwoparentalmonokaryons,

indicating the advantage of sexual reproduction or

heterosisinthedikaryon.Thetotalgeneexpressionquantity

inthedikaryonwas4.8%and6.5%higherthanitsconstituent monokaryons, and all possible modes of differential gene expressionthatwerepresentinthedikaryonwhencompared

toitsconstituentmonokaryons,includingpresence/absence variation and additive/non-additive gene expression, may

becontributingtoheterosis.Thiswasconfirmedinprevious studies.24

Monoallelicexpressiongeneshavebeenfoundinanumber

oforganisms,includinghumans,rodents,corn,andyeast.52

They are on theX chromosome infemale placental mam-malsoronautosomes,5 andtheselectionoftheexpressed allele may depend on the parental origin or be random.53

However, this phenomenon has not been reported in the dikaryon Those DEGsin the dikaryon can bedivided into fourgroups.Themaingroupwassimultaneouslyexpressedin bothofthemonokaryons.Theothertwosmallergroupswere expressedinonlyoneoftwomonokaryons.Thefourthgroup wasexpressedinthedikaryonalone DEGsinthedikaryon

Log 10 (DK13×3 RPKM)

–1

0

1

2

3

4

5

Fig 7 – Scatter plots showing the expression levels of the

differentially expressed genes in dikaryon DK13 × 3 vs.

mid-parent expression value model estimates RPKM, reads

per kb per million reads and MPV, mid-parent expression

values.

onlyexpressingMK3orMK13mightberegardedas

monoal-lelicexpressiongenes, asevidencedbyRT-PCRcloningand

sequencingresults.Forexample,thepouretranscriptinthe

dikaryonwasfromtheMK13nucleusgenebutnotMK3.More

than10%ofthemonoallelicexpressiongenesinthedikaryon

were from each parental monokaryon However, we could

notdetermine whether theydemonstrated autosomal

ran-dommonoallelicexpression,sexchromosomeinactivation,

orimprintedgeneexpression.Infungi,thechromosome

con-tainingmatinggenesmaybedeemedasthesexchromosome

Inmiceandhumans,morethan10%ofthegeneshave

autoso-malrandommonoallelicexpression.54,55 Theisozymebands

thatareonlypresentintheS communedikaryonwere

demon-stratedtodependontheexpressionofmatinggenesAand

B.16 Accordingly,therelationshipbetweenthefourthgroup

andthematinggenesmeritsfurtherstudy

RNA-editing by base deamination has been reported

in plant mitochondria and plastids (C-to-U editing)56 and

mammals (A-to-I editing)57; U-to-Cand guanosine(G)-to-A

changes, which are probably by trans-amination, are also

reportedinmammals.58,59Nosimilarcaseshavebeenfoundin

higherfungi.Inthisstudy,ourresultsshowedthatnumerous

typesofRNAeditingexistedinthepouremRNAinP ostreatus,

includingC-T,A-G,andC-Gbasesubstitution

Takentogether,ourresultssuggestthatthegene

expres-sionpatternsindikaryonsshouldbesimilartodiploid.Finally,

we strongly propose that the fungal dikaryon is a perfect

experimentalmodelforstudyingsexevolutionand

monoal-lelicexpressionduetoits uniquebiology.Thetwoparental

monokaryons can independently live with asexual

repro-duction It was proposed that the monokaryons were the

temporarystageofdikaryonsandhadlesscombativeability

than dikaryons,60 but several speciesmodels have

demon-stratedthatmonokaryonshaveasimilarormorecombative

phenotype compared to dikaryons.61,62 Therefore, it was

suggestedthat monokaryonswith greater adaptive genetic

potentialmayimprovethecombativeabilitytodikaryons.63

In dikaryons, the two monokaryon nuclei do not fuse to

karyogamy,andthetwochromosomalsetsonlyoccasionally

recombineduringvegetativegrowth63;therefore,itiseasyto determinetheoriginsofallelesinadikaryon.Althoughthere

isnopaternalandmaternaldistinctioninthematingoftwo compatiblemonokaryons,aswithothersexualreproduction, themitochondrioninalmostalldikaryonsisfromonlyone monokaryon.64 Theexampledonor canberegarded as the femaleparent

Conflicts of interest

Theauthorsdeclarenoconflictsofinterest

Acknowledgments

This work was funded by a grant from the Natural Sci-ence FoundationofHenanProvince(112300410115)and the programforInnovativeResearchTeam(inScienceand Tech-nology)inUniversityofHenanProvince(15IRTSTHN014)

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