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Mapping and Validation of qHD7b: Major Heading-Date QTL Functions Mainly under Long-Day Conditions

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Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2,997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.
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Plants2022,11,2288.https://doi.org/10.3390/plants11172288www.mdpi.com/journal/plants
Article
MappingandValidationofqHD7b:MajorHeadingDateQTL
FunctionsMainlyUnderLongDayConditions
AmirSohail
1
,LiaqatShah
1,2
,LingLiu
1
,AnowerulIslam
1,3
,ZhengfuYang
1,4
,QinqinYang
1
,GalalBakrAnis
1,5
,
PengXu
1
,RiazMuhammadKhan
1,6
,JiaxinLi
1
,XihongShen
1
,ShihuaCheng
1
,LiyongCao
1,7
,YingxinZhang
1,
*
andWeixunWu
1,
*
1
StateKeyLaboratoryofRiceBiology,ChinaNationalRiceResearchInstitute,Hangzhou310006,China
2
DepartmentofAgriculture,MirChakarKhanRindUniversity,Sibi82000,Pakistan
3
DepartmentofAgriculturalExtension,MinistryofAgriculture,Dhaka1215,Bangladesh
4
StateKeyLaboratoryofSubtropicalSilviculture,ZhejiangA&FUniversity,Hangzhou311300,China
5
RiceResearchandTrainingCenter,FieldCropsResearchInstitute,AgricultureResearchCenter,
Kafrelsheikh33717,Egypt
6
CerealCropsResearchInstitute(CCRI)PirsabakNowshera,AgricultureResearchSystemKhyber
Pakhtunkhwa24100,Pakistan
7
NorthernCenterofChinaNationalRiceResearchInstitute,Shuangyashan155600,China
*Correspondence:wuweixun@caas.cn(W.W.);zhangyingxin@caas.cn(Y.Z.)
Abstract:Headingdate(HD)isoneoftheagronomictraitsthatinfluencematurity,regional
adaptability,andgrainyield.Thepresentstudywasafollowupofapreviousquantitativetraitloci
(QTL)mappingstudyconductedonthreepopulations,whichuncoveredatotalof62QTLs
associatedwith10agronomictraits.TwooftheQTLsforHDonchromosome7(qHD7aandqHD7b)
hadacommonflankingmarker(RM3670)thatmaybeduetotightlinkage,and/orweaknessofthe
statisticalmethod.TheobjectivesofthepresentstudyweretomapQTLsassociatedwithHDina
setof76chromosomesegmentsubstitutionlines(CSSLs),finemapandvalidateoneoftheQTLs
(qHD7b)using2,997BC
5
F
2:3
plants,andidentifycandidategenesusingsequencingandexpression
analysis.UsingtheCSSLsgenotypedwith120markersandevaluatedundertwoshortdayandtwo
longdaygrowingconditions,weuncoveredatotaloffourteenQTLs(qHD2a,qHD4a,qHD4b,
qHD5a,qHD6a,qHD6b,qHD7b,qHD7c,qHD8a,qHD10a,qHD10b,qHD11a,qHD12a,andqHD12b).
However,onlyqHD6aandqHD7bwereconsistentlydetectedinallfourenvironments.The
phenotypicvarianceexplainedbyqHD6aandqHD7bvariedfrom10.1%to36.1%(mean23.1%)and
from8.1%to32.8%(mean20.5%),respectively.OneoftheCSSLlines(CSSL52),whichharboreda
segmentfromtheearlyheadingXieqingzaoB(XQZB)parentattheqHD7blocus,wasthenusedto
developaBC
5
F
2:3
populationforfinemappingandvalidation.Usingabackcrosspopulation
evaluatedforfourseasonsunderdifferentdaylengthsandtemperatures,theqHD7bintervalwas
delimitedtoa912.7kbregion,whichislocatedbetweenRM5436andRM5499.Sequencingand
expressionanalysisrevealedatotalof29candidategenes,ofwhichGhd7(Os07g0261200)isawell
knowngenethataffectsheadingdate,plantheight,andgrainyieldinrice.Theghd7mutants
generatedthroughCRISPR/Cas9geneeditingexhibitedearlyheading.Takentogether,theresults
fromboththepreviousandpresentstudyrevealedaconsistentQTLforheadingdateon
chromosome7,whichcoincidednotonlywiththephysicalpositionofaknowngene,butalsowith
twomajoreffectQTLsthatcontrolledthestigmaexertionrateandthenumberofspikeletsinrice.
Theresultsprovidecontributionstothebroaderadaptabilityofmarkerassistedbreedingto
develophighyieldricevarieties.
Keywords:rice(OryzasativaL.);quantitativetraitlocus;chromosomesegmentsubstitutionlines;
qHD7b;finemapping
Citation:Sohail,A.;Shah,L.;Liu,L.;
Islam,A.;Yang,Z.;Yang,Q.;Anis,
G.B.;Xu,P.;Khan,R.M.;Li,J.;etal.
MappingandValidationofqHD7b:
MajorHeadingDateQTLFunctions
MainlyUnderLongDayConditions.
Plants2022,11,2288.https://doi.org/
10.3390/plants11172288
AcademicEditors:KassaSemagn
andTikaAdhikari
Received:28June2022
Accepted:30August2022
Published:1September2022
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2022bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(https://creativecommons.org/license
s/by/4.0/).
Plants2022,11,22882of15
1.Introduction
Riceisastaplefoodformorethan50%oftheworld’spopulation,withitsproduction
expectedtoincreasebyabout25%in2030tokeeppacewithpopulationgrowth.Riceisa
facultative,shortdaycropthatflowersearlierundershortday(SD)conditionsandlater
underlongday(LD)conditions[1].Headingdate(HD)isacrucialtraitaffectingrice
adaptiontodiversecultivationareas,croppingseasons,maturity,andgrainyield[2].The
developmentofearly‐orlatematuringcultivarsdependsonecologicalconditions.Inthe
regionswheregrowingseasonsareshort,theaimistodevelopearlymaturingvarieties
toescapefrostdamage,buttheremaybeayieldpenalty.However,intheregionswhere
growingseasonsarelong,theaimistodeveloplatematuringvarietieswithallofthe
assimilatesefficientlytransmittedtothegrains,therebyenhancinggrainweightandyield.
Generally,therewasatradeoffbetweenfloweringtimeandyield,whichaimedto
maximizeproduction[3].
Numerousstudieshavebeenconductedtomapandcharacterize712HDgenesand
quantitativetraitloci(QTLs)thathavebeendocumentedintheGramenedatabase
(http://archive.gramene.org/qtl/(accessedon29August2022)).TheE1/Ghd7wasthefirst
HDQTLreportedinrice,whichpossessesafunctionaldominantE1alleleandanon
functionale1allele[4].TheallelicvariationofGhd7contributestothegeographic
distributionofcultivatedrice[5],whichhasbeeninvestigatedforphotoperiodsensitivity
andregionaladaptability[6].ThefunctionalGhd7alleles(e.g.,Ghd71,Ghd72,andGhd7
3)delayheading,whilethenonfunctionalalleles(e.g.,Ghd70andGhd70a)shorten
headingdateinthedifferentgeneticbackgroundsofrice.BoththeGhd71andGhd73
alleleswerefoundinricevarietiesgrowninthetropics,subtropics,andareaswithhot
summersandlonggrowingseasonsinChinaandSoutheastAsia.TheGhd72allelewas
foundintemperatejaponicavarietiesfromJapanandnorthernChinaandhadasmaller
phenotypiceffectthanGhd71[7].Se1/Hd1wasthefirstclonedheadingdateQTL,an
orthologofArabidopsisCONSTANSthatpromotesandsuppressesfloweringundershort‐
andlongdaygrowingconditions,respectively[8].Headingdate6(Hd6)[9],Headingdate
3a(Hd3a)[10],Earlyheadingdate1(Ehd1)[11],Daystoheading8(DTH8)/Ghd8[12],Heading
date17(Hd17)[13],RICEFLOWERINGLOCUST1(RFT1)[14],andDaystoheading2
(DTH2)[3]areotherHDQTLsinricethathavebeenclonedusingamapbasedapproach.
Theanalysisofthesegenesexhibitedtwomainphotoperiodicfloweringpathwaysinrice:
Hd1Hd3aandGhd7Ehd1Hd3a/RFT1.MajorQTLsassociatedwiththelateheading,such
asGhd7,Hd1,DTH8/Ghd8,andDTH7/Ghd7.1[5,15],showedastrongcorrelationwithan
increaseingrainyield,whichsuggeststhattheuseofsuchtypesofHDQTLscan
significantlyinfluencerice’sproductivityandadaptabilitytospecificgrowingconditions.
Recentprogressinmoleculartechnologyandstatisticalmethodologyhasprovided
researchersanopportunitytomapandcharacterizeHDQTLsindiversetypesof
populations,includingF2,recombinantinbredlines(RILs),anddoubledhaploidlines
(DHLs)inrice[16,17].However,thesepopulationsmaynotbeidealfortheprecise
mappingofQTLsduetothesimultaneoussegregationofmultiplelocioriginatingfrom
thetwoparents.Moreover,itwouldbemorechallengingtodeterminetheactualgenetic
actionsoftheQTLsanddifferentiatetheQTLeffectsfrombackgroundnoise[18,19].
Chromosomesegmentsubstitutionlines(CSSLs)aregeneticstocksthatconsistof
overlappingsegmentsofthecompletegenomeofanygenotype.CSSLshavebeenwidely
usedtomapQTLsaccurately,evaluategeneinteractions,discovernewalleles,and
comparethephenotypiceffectofgenesorQTLs[19,20].FurtherfinemappingofQTLsof
interestcanbedonebyconstructingsegregatingpopulationsobtainedfromcrossingone
oftheCSSLsandtheirrecurrentparent[21].
Inapreviousstudy,ourgroupidentified9HDQTLsinaRILpopulation(FigureS1)
and2BCF1populationsderivedfromacrossbetweenXieqingzaoB(XQZB)and
Zhonghui9308(ZH9308),whichindividuallyaccountedfor2.6–18.6%ofthephenotypic
variance[22].ThreeofthenineHDQTLsweremappedonchromosome7between
RM3670andRM2markers(qHD7a),betweenRM5436andRM3670(qHD7b),andbetween
Plants2022,11,22883of15
RM118andRM3555(qHD7c),explaining18.6%,12.1%,and5.6%,respectively.TheqHD7a
andtheqHD7bQTLwerephysicallylocatedbetween13439924–16022676bpand9075636–
13439924bp,respectively.RM3670,locatedat13439924bp,wasacommonflanking
markerinbothqHD7aandqHD7b,suggestingthatthetwoQTLsareeithertightlylinked
orthestatisticalmethodwronglyidentifiedthemastwoindependentQTLs.Bothissues
mayberesolvedusingCSSLs,whichformthebasisofthepresentstudy.Therefore,the
objectivesofthepresentstudyweretounderstandthephenotypicvariationoftheCSSLs
forheadingdate,finemaptheHDQTLonchromosome7,andidentifycandidategenes
associatedwithHDundershort‐andlongdayricegrowingconditions.Furthermore,we
werealsointerestedindeterminingtheproportionofphenotypicvarianceexplainedby
oneoftheQTLsonchromosome7,validatingandfinemappingitspositionusingthe
BC5F2:3populationderivedfromacrossbetweenoneoftheCSSLsandtherecurrent
parent,identifyingcandidategenesnearthetargetQTL,anddeterminingitsactualeffect
inmutantsgeneratedthroughCRISPR/Cas9geneediting.
2.Results
2.1.PhenotypicandGenotypicAnalysisofCSSLs
SeventysixCSSLsandthetwoparentswereevaluatedforHDundernaturalSD
(NSD)atHainanin20152016andundernaturalLD(NLD)atHangzhouin20142015for
twoseasons.TheXQZBmaturedabout32and20daysearlierthantheZH9308inthe
HangzhouandHainangrowingconditions,respectively(Figure1A,B;Table1).Thedays
toheadingof76CSSLsexhibited59to122daysinNLDsandfrom93to124daysinNSDs
(Figure1C–F;Table1).Overall,HDshowedcontinuousvariationinbothgrowing
conditionsbutskeweddistribution(Table1,Figure1).Thebroadsenseheritabilitywas
computedfromallfourenvironments,Hainan,andHangzhou,andwere0.83,0.82,and
0.79,respectively(Table1).PC1andPC2fromtheprincipalcomponentanalysis(PCA)
accountedfor75.7%and15.5%,respectively(FigureS2),withmostCSSLsshowingan
averageheadingdateclusteredtogetherattheorigin.Highlysignificantpositive
correlationswereobservedamongthetestedenvironmentsforheadingdate(FigureS2).
Table1.Summaryofheadingdatesofparentsand76chromosomesegmentsubstitutionlines
(CSSLs)evaluatedundertwonaturalshortdayconditionsatHainanandlongdayconditionsat
Hangzhou.
Parentsb76CSSLsa
Year/LocationZH9308XQZBMinMaxMeanSDKurtosisSkewnessH
2014Hangzhou91.83±1.11**60.17±1.1060.40119.2287.128.895.21−0.230.79
2015Hangzhou88.33±0.50**56.67±0.5457.67124.2888.219.424.45–0.33
2015Hainan110.99±1.52**91.00±1.3196.57124.94110.873.944.330.630.82
2016Hainan109.67±1.24**91.44±1.6790.00122.80103.596.101.540.36
Combined
TraitMinMaxMeanσ2Gσ2GxEσ2ECVH2
HD80.77119.7897.4534.8624.918.983.080.83
aValuesforCSSLsareminimum(Min),maximum(Max),mean,standarddeviation(SD),and
repeatability(H2).bDataofparentsarepresentedasmean±SDwith**indicatingsignificant
differencesbetweenZH9308andXQZBatthep<0.01.
Plants2022,11,22884of15
Figure1.ComparisonofZH9308andXQZBparentswith**indicatingsignificantdifferencesatp<
0.01(AB)andheadingdatedistributionofthe76chromosomesegmentsubstitutionlines(CSSLs)
basedonthebestlinearunbiasedprediction(BLUP)evaluatedattheHangzhouandHainan
growingconditions(CF).
2.2.QTLAnalysisoftheCSSLs
ThegeneticlinkagemapoftheCSSLpopulationwasconstructedusing87simple
sequencerepeats(SSRs)and33insertion/deletion(InDel)markersthatfollowedthe1:1
Mendeliansegregationpattern(FigureS3).Atotalof120markersweredistributedacross
thewholegenomewithtotalcoverageof1311.02cmusingtheKosambifunctionof
IciMappingsoftware(FigureS3;TableS1).Inclusivecompositeintervalmapping
conductedontheBLUPHDdataofthefourenvironmentsusingaLODthresholdvalue
≥2.5identified14QTLsassociatedwithheadingdateonChr2(qHD2a),Chr4(qHD4aand
qHD4b),Chr5(qHD5a),Chr6(qHD6aandqHD6b),Chr7(qHD7bandqHD7c),Chr8
(qHD8a),Chr10(qHD10aandqHD10b),Chr11(qHD11),andChr12(qHD12aandqHD12b)
Plants2022,11,22885of15
(Table2).Theproportionofphenotypicvariance(PVE)explainedbyeachQTLranged
from0.4%to36.1%,andtheadditiveeffectfrom15.4to18.2.OfthefourteenQTLs,only
qHD6aandqHD7bwereconsistentlydetectedinallfourenvironments,from10.1%to
36.1%(mean23.14%)andfrom8.1%to32.8%(mean20.5%),respectively.Theremaining
12QTLsweredetectedonlyin1or2environments(Table2).WethenfocusedonqHD7b
forfurtherresearchduetoitsconsistentdetectionnotonlyinallfourenvironmentsinthe
presentstudy,butalsointwotypesofmappingpopulationsinourpreviousstudy[23].
ThefavorableallelesofallQTLwithanegativeadditiveeffectoriginatedfromtheXQZB,
whilethosewithapositivealleleoriginatedfromZH9308.TheXQZBalleleattheqHD7b
leadstoearlyheadingunderbothNSDandNLDconditions(Figure1A,B;Table2).
Table2.ChromosomallocationsofputativeHDQTLsunderfourdifferentenvironmentsusing76
CSSLpopulations.
QTLsYear/locationaChr.Region(cM)FlankingmarkersLODbPVE(%)cAddd
qHD2a2015HZ219.77RM6424InD312.802.542.37
qHD4a2014HZ46.58InD62RM120538.899.8713.13
2015HZ410.54RM1205RM59796.464.501.75
qHD4b2015HZ421.08RM3839RM24111.339.148.79
qHD5a2014HZ525.20RM3638RM68412.590.45–0.47
2016HN514.52InD79RM36382.917.78–3.91
qHD6a2014HZ66.58RM5754RM596349.6719.0318.21
2015HN65.27RM510RM575411.1236.146.78
2015HZ66.58RM5754RM596313.1410.139.90
2016HN65.27RM510RM57548.8325.018.67
qHD6b2014HZ611.85RM20069InD943.770.330.36
qHD7b2014HZ727.69RM3859RM587554.4226.54–15.44
2015HN727.69RM3859RM58753.248.10–2.61
2015HZ727.69RM3859RM587526.5632.84–12.90
2016HN727.69RM3859RM58757.5520.33–6.76
qHD7c2015HZ721.13RM1132RM4552.562.790.88
qHD8a2014HZ87.91RM5556RM2252946.4016.12–12.03
2015HZ87.91RM5556RM2252915.9113.30–8.22
qHD10a2015HZ102.63InD133InD13512.279.14–13.28
2015HN102.63InD133InD1356.6718.74–7.37
qHD10b2016HN106.58RM6142RM56203.608.89–5.96
qHD11a2014HZ1110.54RM7463RM2665218.141.94–7.76
2015HZ1117.16RM26652InD1512.513.10–2.72
qHD12a2014HZ122.63InD156RM700315.422.21–6.10
qHD12b2014HZ1223.82InD165RM13002.850.43–0.44
aYear/Locationreferstotheyearoftheexperiment(2014,2015,and2016)followedbythelocation
(Hangzhou—HZ/Hainan—HN).bLogarithmofodd,ctheproportionofthephenotypicvariance
explainedbytheQTLeffect,dthesignoftheadditiveeffectsshowstheparentaloriginofthe
favorablealleles(negative=XQZBandpositive=ZH9308).
2.3.PhotoperiodicResponseofqHD7b
TheCSSL52isoneofthechromosomesegmentsubstitutionlines,whichcontainstwo
segmentsfromXQZBintheZH9308backgroundandharborstheearlyheadingalleleat
theqHD7bregionflankedbyRM3859andRM5875markers(FigureS4).ZH9308and
CSSL52wereevaluatedforfiveconsecutiveseasonsunderNLDandNSDconditionswith
differentdaylengthsandtemperatures(Figure2A–D).ThereweresignificantHD
differences(p<0.01)betweentheparentsinallthestudiedenvironments(Figure2E–G).
ZH9308headed24.6–26.4dayslaterthanCSSL52atHangzhouNLDconditions(Figure
Plants2022,11,22886of15
2E,G).AtHainanNSDconditions,ZH9308headed6.2–10.4dayslaterthanCSSL52(Figure
2F,G).Similarly,ZH9308hadahigherplantheightthanCSSL52underallfive
environments(Figure2H).Takentogether,ZH9308headedlaterthanCSSL52underboth
SDandLDconditions,andthephenotypicdifferencesofdaystoheadingweremore
significantunderLDconditions.Duetolongerdaystoheading,ZH9308alsoexhibiteda
tallerPH,longerpaniclelength,morenumbersofinternodes,longerinternodelength,a
greaternumberofpaniclesperplant,andamoresignificantnumberofgrainsinthemain
paniclethanCSSL52(FiguresS5andS6).
Figure2.ComparisonofheadingdateandplantheightofZH9308andCSSL52underfive
environmentalconditions.(AD)Dailyphotoperiod(A,B)andmeantemperature(C,D)under
HangzhouandHainanconditionsin2020duringthericegrowingseason.(E,F)Thephenotypeof
ZH9308andCSSL52underHangzhou(E)andHainan(F).ThephotowastakenattheCSSL52
headingstagein(E)andtheCSSL52maturationstagein(F).(G,H)Daystoheading(G)andplant
Plants2022,11,22887of15
height(H)comparisonbetweenparentsfrom2019–2021underNLDconditionsinHangzhouand
NSDconditionsinHainan.Theasterisks**indicatesignificancebetweentheparentsatthep<0.01,
accordingtoStudent’sttest.
HeadingdateshowedahighlysignificantpositivePearsoncorrelationwithplant
height(r=0.83,p<0.01),paniclelength(r=0.86,p<0.01),andnumberofgrainsinthe
mainpanicle(r=0.79,p<0.01)(TableS2).Plantheightshowedhighpositivecorrelations
withthepaniclelength(r=0.83,p<0.01)andnumberofgrainsinthemainpanicle(r=
0.80,p<0.01).Similarly,therewasalsoasignificantpositivecorrelationbetweenpanicle
lengthandnumberofgrainsinthemainpanicle(r=0.76,p<0.01)(TableS2).
2.4.QTLMappinginBC5F2:3Population
TheqHD7bwasinitiallydelimitedtothe7.1MbregionbetweentheRM3859and
RM5875markers(Table1).WeusedaBC5F2populationtovalidateandfinemapthe
qHD7bQTL,whichwasdevelopedbycrossingCSSL52thathastheearlyheadingallele
withtheZH9308parentandthenbackcrossingtheprogeniesfivetimeswiththeZH9308
todevelopasecondaryF2(BC5F2)populationforfinemappingqHD7b(FigureS7).
Usingalinkagemapof9markersneartheqHD7bQTLandheadingdataofasubset
of501BC5F2plants(Figure3)evaluatedfortwoconsecutiveseasonsunderHangzhou
NLDandtwoseasonsunderHainanNSDconditions(FigureS8),wemappedtheqHD7b
QTLbetweentheInDel4373andInDel3markers.TheQTLwasdetectedinallfour
environments(seasons)andwasbetween10.8%and41.1%ineachenvironment(mean=
26.0%),andhadaLODscorerangingfrom4.9to62.7(mean=33.8).UnderHangzhou
conditions,however,theqHD7bhadaverylargeeffect(31.5–41.1%)andmoresignificant
LODscores(59.8–62.7)thanintheHainanconditions(PVE=10.8–21.2%,LOD=4.9–7.5),
whichwasabouttwofoldgreaterinthemeanphenotypiceffectandnearlytenfoldlarger
inthemeanLODscore(TableS3).Wethenclassifiedthe501BC5F2aslateandearly
headingandcalculatedthemeandifferenceinheadingdate.Theaveragedifference
betweentheearlyandlatesegregatingprogenieswas9.6and25.5daysunderNSDand
NLD,respectively(FigureS8).AChisquareanalysisperformedontheBC5F2population
fittheexpected3(late):1(early)ratio(386:115;χ2=0.52,p=0.47),indicatingthatqHD7b
behavesasasingledominantgenethatismorefunctionalunderLDconditionsthanSD
conditions.
2.5.FineMappingofqHD7b
TonarrowdowntheconfidenceintervaloftheqHD7bidentifiedusingthe501BC5F2
plants,weusedatotalof2997BC5F2:3individualsgenotypedwith7markersthatwere
polymorphicbetweentheZH9308andCSSSL52,aswellasthetwoflankingInDels
(InDel4373andInDel3)identifiedduringtheinitialmapping.Thegenotypedatarevealed
14homozygousrecombinants(Figure3D)thatbelongto4groups(G1=3plants,G2=5
plants,G3=4plants,andG4=2plants).TherecombinantplantsinG1hadthesame
headingdate(86.0d)astheZH9308parent(85.2d),whiletheremainingthree
recombinantgroupshadnearlythesameheadingdate(56.2–58.0d)astheCSSL52parent
(58.8d).Inthefinemapping,InDel4477,locatedat9075693bp,wastheclosestmarkerto
qHD7b,whileRM5436andRM5499weretheflankingmarkerslocatedat9075636bpand
9988139bponchromosome7.IncontrasttotheG1recombinantsthathadtheZH9308
parentgenomebetweenRM5436andRM5499attheqHD7binterval,theremainingthree
groupsofrecombinants(G2toG4)allinheritedtheCSSL52genome.Thephysicalinterval
betweenRM5436andRM5499markersspans912.7kb(Figure3D),whichwasconfirmed
using20BC5F3:4progenyfromeachrecombinantgroup.
Plants2022,11,22888of15
Figure3.CoarsemappingandfinemappingofqHD7bonchromosome7.(A)ThepositionofqHD7b
basedon76CSSLsgenotypedwith12molecularmarkersonchromosome7.(B)Thepositionof
qHD7bbasedon501BC
5
F
2
plantsgenotypedwith7markerslocatedbetweenRM3859andRM5875
(theflankingmarkersidentifiedusingtheCSSLs).(C)FinemappingofqHD7busingBC
5
F
2:3
plants
genotypedwith7markersthatmappedbetweenInDel4373andInDel3(theflankingmarkers
identifiedusingthe501BC
5
F
2
plants).(D)Genotypesandphenotypesofthetwoparents(ZH9308
andCSSL52)and14homozygousrecombinantlinesusedforfinemappingofqHD7b.TheZH9308
andCSSL52genotypicmarkersarerepresentedbywhiteandblackbars,respectively.The14
homozygousrecombinantsbelongtofourgroups(G1=3plants,G2=5plants,G3=4plants,and
G4=2plants).Thesuperscriptedletters(aandb)indicatestatisticallysignificantdifferencesinthe
headingdatesofrecombinantsrelativetotheparents.(E)Approximately29openreadingframes
(ORFs)werelocatedbetweenthetwoflankingmarkersidentifiedduringthefinemapping(RM5436
andRM5499),whicharesummarizedinTableS4.(F)SequencecomparisonbetweenZH9308and
CSSL52with5.984kbdeletioninCSSL52atOs07g0261200using7markers(M1toM7).Thedeleted
regioninCSSL52issignificant,andsevenmolecularmarkers(Ghd7M1toGhd7M7)linkedwith
qHD7bareamplifiedinZH9308.
2.6.CandidateGeneAnalysisofqHD7bandValidationUsingCRISPR/Cas9
Acandidategenesearchusingthephysicalpositionofthetwoflankingmarkers
identifiedduringthefinemapping(RM5436andRM5499)intheGramenedatabase
(https://www.gramene.org/(accessedon29August2022))usingtheOryzasativajaponica
groupreferencegenomeidentified29predictedgenes,ofwhich18hadOryzaindica
homologuesthatfellwithinthe912.7kbregion(7:90756369988139)ofqHD7b(Figure3E
andTableS4).ORF4(Os07g0261200)isphysicallylocatedat9,152,377bponchromosome
7,encodestheCCTmotiffamilyprotein,andhasbeenannotatedasGhd7(Os07g0261200).
WethensequencedOs07g0261200inthetwoparentsusingsevenmarkers(M1toM7),
whichrevealeda5.984kbdeletionintheORF4regionintheCSSL52parentbutnotin
Plants2022,11,22889of15
ZH9308(Figure3F).TheexpressionlevelsofOs07g0261200inCSSL52werealsonearly
zeroascomparedwithZH9308,whichalsosuggeststhatGhd7isaprobableputative
candidategeneforqHD7b(FigureS9).
Tovalidatethemutantphenotype,weknockedoutGhd7intheNipponbaregenetic
backgroundutilizingtheCRISPR/Cas9system(Figure4A).TheHDofthewildtypewas
9.4dayslaterthantheghd7mutantunderNLDconditionsinHangzhou(Figure4B,C)and
2.0dayslaterunderNSDconditionsinHainan(Figure4C).Thewildtypeshowed
significantdifferenceswiththeghd7mutantforHD,plantheight,andthenumberof
grainsperpanicle(Figure4D,E).
Figure4.Headingdateofwildtype(WT)andghd7mutantintheNipponbaregeneticbackground.
(A)SchematicoftheGhd7genewiththesgRNA:Cas9targets(green)andcorresponding
protospaceradjacentmotifsequences(underlined).Theinsertionnucleotideisshownasaredletter.
(B)ThephenotypeofWTandmutantghd7attheheadingstageunderHangzhouconditions.(C)
DaystoheadingofWTandghd7undernaturallongdayHangzhouandnaturalshortdayHainan
conditions.(DandE)ComparisonoftheWTandghd7mutantforplantheight(D)andthenumber
ofgrainsinthemainpanicle(E)undernaturallongdayconditions.Thedataareexpressedasmean
values±SD.Theasterisks**indicatesignificancebetweenWTandghd7mutantatthep<0.01,as
determinedbyStudent’sttest.
3.Discussion
Headingdateisoneofthemostimportantagronomictraitsandvarieswidelyinrice
dependingonthegeneticdifferencesamonggenotypes,environmentalconditions,day
length,temperature,andtheirinteractions[23].Aclearunderstandingofthegenetic
driversofheadingdatesisessentialforcultivatingriceindifferentgeographicalregions
andseasons[24].Numerousgeneticmappingstudieswereconductedtoidentifygenes
andQTLsassociatedwithheadingusingbiparentalpopulations,suchasF2,backcross,
doubledhaploidlines,RILs,andCSSLs[25,26].Hundredsofgenomicregionsrelatedto
HDhavebeenreportedinrice;however,fewhavebeenmappedandcloned[27].qHD7b
wasidentifiedusingtwotypesofpopulationsthatwere76CSSLsandaBC5F2population.
Using76CSSLlines,thepresentstudyconfirmedtheqHD7bQTLthatwepreviously
reportedbetweenmarkersRM5436,locatedat9075636bp,andRM3670,locatedat
13439924bp[22].However,thephysicalintervaloftheQTLwasaboutdoubleinthe
Plants2022,11,228810of15
CSSLs(7120.8kb)ascomparedwiththepreviousstudy(4,364.3kb),whichislikelydue
tothesmallpopulationsizeoftheCSSLs.Theuseof501BC5F2plantsreducedthephysical
confidenceintervaloftheQTLto1245.9kb,followedby912.7kbwhenitwasfinemapped
using2997BC5F2:3plants(Figure3).RM5436andRM5499weretheflankingmarkersof
theqHD7bQTLafterfinemapping,whichwerealsopreviouslyreportedasflanking
markersforamajorQTL(qSSP7),therebycontrollingthenumberofspikeletsperpanicle
[28],andanothermajorQTL(qSE7),therebyinfluencingthestigmaexertionrate[29]in
rice.AcandidategenesearchconductedusingthephysicalintervaloftheqHD7b
(7:90756369988139)andtheOryzasativajaponicagroupreferencegenomeinGramene
identifiedatotalof29candidategenes.Ghd7(Os07g0261200)isoneofthe29candidate
genes,whichhasbeenextensivelystudiedforitseffectoninfluencingheadingdate,grain
yield,plantheight,andotheragronomictraitsinrice[7,30].However,othermultiple
genesshouldalsobeexploredinthefuture.
OneofthechallengesinQTLmappingisidentifyingQTLsthatareconsistently
detectedacrossdifferentenvironments,whichwasnotthecaseforqHD7b.ThisQTLwas
consistentlydetectedinallenvironmentsregardlessofthetypeofmappingpopulations,
butitseffectandLODscorestendtobeveryerratic.TheLODscorefortheqHD7bvaried
from3.2to54.4inthe76CSSLsandfrom4.9to62.7inthe501BC5F2plants,whilethe
phenotypicvarianceexplainedbytheQTLvariedfrom8.1%to32.8%intheCSSLsand
from10.8%to41.1%intheBC5F2plants(Table2andTableS3).Apreviousstudyshowed
thattheQTLeffectdetectedbythedifferentmappingpopulationsandenvironmental
conditionsisnotnecessarilythesame[31].Inourcase,thediscrepanciesmaybedueto
differencesinthepopulationsizeand/orthetypeofmappingpopulations.CSSLsare
powerfulforQTLdiscoverystudiesduetothepresenceofmultipleoverlappingsegments
andseveralrecombinationevents[32].Still,thesmallsizeofthepopulationinthecurrent
studymayaffecttheQTLresults.TheBC5F2populationsize,ontheotherhand,wasideal
forQTLdiscoverystudiesintermsofpopulationsizebuthaslimitedrecombination
frequency,whichmakesitinferiorintermsofmappingresolutions.Thetwophenotyping
locationswerealsodifferentinbothtemperatureanddaylength,withHangzhou’s
showingahighertemperatureandlongerdaylengththanHainan(Figure2A–D),which
resultedinacleardifferenceinphenotypebetweentheparentsinHangzhou’sthan
Hainan(Figure2E–H)[33].Asaresult,theproportionofphenotypicvarianceexplained
byqHD7bwasverylowinHainaninboththeCSSLandBC5F2populations(Table2and
TableS3).Liuetal.[23]alsoreportedrelativelyloweffectsforqHD1binHainanthanin
Hangzhou.Thedifferencesinheadingdatesbetweenthewildtypeandtheghd7mutant
werealsosmallerinHainanthanintheHangzhougrowingconditions,andrecentstudies
alsosupportthisresult[34].TheseresultssuggestqHD7basamajorHDQTLfunction,
mainlyunderLDconditions.
OursequencingresultrevealedthatattheGhd7locus,theqHD7bXQZBallelebelongs
toGhd70,whichisnonfunctional,andtheqHD7bZH9308allelebelongstoGhd74,whichis
functionaltogetherwithGhd71,Ghd72,andGhd73[35].ThenonfunctionalMinghui63
alleleofGhd7showednonsignificantphenotypedifferencesunderSDconditions[7].
However,underSDconditions,theqHD7bXQZBallelefloweredearlierthantheqHD7bZH9308
allele(Figure2G).Thesephenotypicdifferencesmaybecausedbybackground
differences,indicatingqHD7balsofunctionsinSDconditions,buttheeffectissmallerthan
thatinLDconditions.ThetwoflankingmolecularmarkersforqHD7b(RM5436and
RM5499)andthesevenmolecularmarkersthatmappedwithintheQTLconfidence
interval(Ghd7M1locatedat9,150,263toGhd7M7locatedat9,155,572bp)cancontribute
towardstheeffortinthebreedingofricevarietiesusingmarkerassistedselection(MAS).
Forexample,qHD7bZH9308couldbeusefulinbreedinglatematuringcultivars.Whenthe
ricecultivarswithGhd70a,Ghd70,andGhd72allelesoriginatingfromnorthernChina
wereintroducedtosouthernChina,theirheadingdatewouldbesignificantlyearlier.The
qHD7bZH9308allelecouldbeintroducedintothesecultivarstoprolongtheirheadingdate
tomakethemhavedelayedheadingandincreasetheiryield.Onthecontrary,qHD7bXQZB
Plants2022,11,228811of15
couldbeusefulinbreedingearlymaturingcultivars.WhenthericecultivarswithGhd7
1,Ghd73,andGhd74allelesoriginatingfromthetropicalandsubtropicalregionswere
introducedtonorthernChina,theirgrainscouldn’treachmaturityduetolaterheading.
TheqHD7bXQZBallelecouldbeintroducedintothesecultivarstomakeearlyheadingto
matureandharvestintime.
4.MaterialsandMethods
4.1.PopulationDevelopmentandPhenotyping
Thepresentstudywasconductedusingthreepopulations.Oneofthepopulations
wasdevelopedbycrossing134RILswithZhonghui9308(ZH9308),whichwasthen
backcrossedthreetimes,andselfedsixtimestoformBC4F6generation.TheRILswere
initiallydevelopedfromacrossbetweenanearlyheadingXieqingzaoB(XQZB)donor
parentandalateheadingZH9308recipientparentandparentallinesofXieyou9308(an
indicajaponicasubspeciessuperhybridricewith87.5%indicaand12.5%japonicagenome)
[22].Seventysixofthe134BC4F6lineswereselectedtorepresentCSSLsforQTLmapping,
andthegenotypesofthe76CSSLswerealsoinvestigated[36].Thesecondpopulationwas
developedbycrossingoneoftheCSSLs(CSSL52)thatexhibitedearlyheadingwiththe
ZH9308parentandthenbackcrossingtheprogeniesfourtimeswiththeZH9308to
developasecondaryBC5F2populationforvalidationandaBC5F2:3populationforthefine
mappingoftheqHD7bQTL(FigureS1).
Asetof76CSSLsalongwithparentallines,ZH9308andXQZB,wereevaluatedfor
headingdatethroughaRandomizedCompleteBlock(RCB)designconsistingof3
replications,with4rows×8plantsforeachlineatHangzhouandHainanfor4seasons.
Eachplotconsistedof4rowsof1.32squaremeterswith16.5cm×26.5cmspacingbetween
plantsandrows.TheBC5F2population,alongwithZH9308andCSSL52parents,was
evaluatedfortwoyearsunderNLD(daylength>14h)conditionsinHangzhou,Zhejiang
Province(120.0°E,30.15°N),andNSD(daylength<12h)conditionsinLingshui,Hainan
Province(110.0°E,18.5°N).HDwasrecordedasthenumberofdaysfromthesowingdate
totheemergenceofthefirstheading.Plantheight(PH)wasmeasuredfromtheground
leveltothetipofthepanicleatfullphysiologicalmaturity.Internodelengthwasrecorded
asthelengthbetweentwonodes.Numberofpaniclesperplantwasrecordedasthe
numberofallpaniclesperplant.Paniclelength(PL)wasrecordedfromthenecktothe
apexofthepanicle.Thenumberofgrainsinthemainpaniclewasrecordedasthefilled
grainnumbersofthemainpanicle.Paddyfieldmanagementfollowedconventional
practices[37].
4.2.TestofDayLengthResponseinGrowthChambers
Toinvestigatethephotoperiodicresponse,ZH9308andCSSL52plantsweregrown
underCLD(14hlight,30°C/10hdarkness,25°C)andCSD(10hlight,30°C/14h
darkness,25°C).Thehumiditywas75%,andthelightintensitywas300μmolm2s1[38].
4.3.MolecularMarkersDevelopmentandDNAExtraction
PolymorphismsbetweenZH9308andCSSL52werescreenedusing
Insertion/Deletion(InDel)andSimpleSequenceRepeat(SSR)markers[39].Todetermine
thecandidategenesofqHD7bQTL,thesequencebetweenRM3859andRM5875was
downloadedfromtheEnsemblePlants(http://plants.ensembl.org/index.html(accessedon
29August2022))andGramenewebsite(https://www.gramene.org/(accessedon29
August2022))usingtheOryzasativajaponicagroupreferencegenomebyablastsearchof
theprimersequences.NewInDelmarkersintheQTLregionweredesignedbasedon
differencesingenomicsequencebetweenIndicaandJaponicausingthePrimerPremier
5.0software(PREMIERBiosoftInternational,CA,USA).ThemarkersarelistedinTables
S5andS6.Themarkersthatamplifiedthemutatedregionbetweenparentsarelistedin
TableS7.
Plants2022,11,228812of15
GenomicDNAwasextractedfromthefreshleavesofparentsandthesecondaryF2
(BC5F2)populationusingthecetyltrimethylammoniumbromide(CTAB)method
describedbyLuoetal.[40].Apolymerasechainreaction(PCR)wasperformedina12μL
volumethatconsistedof5μLofmastermix,3μLofddH2O,2μLof10pmolμL1primers
(1μLforwardprimerandreverseprimers),and2μLoftemplategenomicDNA(200ng).
ThePCRamplificationprotocolconsistedofapredenaturationstep(95°Cfor3min),
followedby32cycles(denaturationat95°Cfor15s,annealingat55°Cfor15s,and
extensionat72°Cfor5s),andfinalextension(72°Cfor10min).ThePCRproductswere
separatedusinggelelectrophoresison8%nondenaturingpolyacrylamidegeland
visualizedwithsilvernitratestainingusingaformaldehydesolution[41].
4.4.RNAExtractionandqRTPCR
ThetotalRNAwasextractedfromleavesofZH9308andCSSL52usinganRNAprep
PurePlantkit(TiangenBiotechCo.Ltd.,Beijing,China).Atotalof50μLof
complementaryDNA(cDNA)wassynthesizedusing5μgofRNAwithReverTraAce®
qPCRRTMasterMixwithgDNARemover(ToyoboCo.Ltd.,Osaka,Japan).Realtime
quantitativeRTPCR(RTqPCR,20μLreactionvolume)wasperformedusing0.5μLof
cDNA,0.2μMofeachgenespecificprimer,andTBGreenPremixExTaqII(TakaraBio,
Inc.,Kusatsu,Shiga,Japan)inaLightCycler®480II(Roche).ThericeUbqgene
(Os03g0234350)wasexploitedastheendogenouscontrol.TheprimersforqRTPCRwere
designedusingGeneScript(https://www.genscript.com/tools/realtimepcrtaqman
primerdesigntool)andaredisplayedinTableS7.
4.5.Generationofghd7MutantUsingCRISPR/Cas9System
TheCRISPR/Cas9systemwasusedtoknockoutGhd7accordingtothemethodsas
describedpreviously[42].The18bpsgRNA:Cas9targetsequenceofGhd7wasintroduced
intothepCas9sgRNAvectorattheAarIsite.Thefinalvectorwastransformedinto
NipponbareusingAgrobacteriummediatedtransformation[43].Theprimersusedare
displayedinTableS7.
4.6.StatisticalAnalysis
Theexperimentaldesignineachenvironmentwasarandomizedcompleteblock
designwiththreereplicationsperenvironment/location.Thebestlinearunbiased
prediction(BLUP)valueswereobtainedthroughMETARv6.03[44],usingthelinear
model:
Yik=μ+Repi+Genk+εik(withintheenvironment)
Yijk=μ+Repi(Envj)+Envj×Genk+Genk+Envj+εijk(acrossenvironments)
whereYikisthetraitofinterest,μisthemeaneffect,Repiistheeffectoftheithreplicate,
Genkistheeffectofthekthgenotype,εikistheerrorassociatedwiththeithreplication,and
thekthgenotype,whichisassumedtobenormallyandindependentlydistributed.For
acrossenvironments,Yijkisthetraitresponse,Envjisthejthenvironment,Repi(Envj)is
theeffectofithreplicationinthejthenvironment,andEnvj×Genkistheenvironmentand
genotypeinteraction.Theresultinganalysisproducedtheadjustedtraitphenotypic
valuesintheformofBLUPwithinandacrossenvironments.TheBLUPmodelconsiders
genotypesasrandomeffects.Broadsenseheritability(H2)andrepeatability(H)were
calculatedaccordingtoAlemuetal.[45]usingMETARsoftware.
H= σ 2g
σ 2g + σ 2e /reps (within the environment)
H= σ
2
g
σ 2g + σ 2 ge /env + σ 2 e/(reps × env) (across environment)
Plants2022,11,228813of15
whereσ
2gandσ
2earethegenotypicanderrorvariance,σ
2geisthegenotypeby
environmentinteractionvariance,repisthenumberofreplicates,andenvisthenumber
ofenvironments.
QTLanalysiswasperformedusingtheinclusivecompositeintervalmapping(ICIM)
functionimplementedinQTLIciMappingsoftware[46].ALODthresholdvalue≥2.5
indicatesthepresenceofQTL(selectedby1000permutationteststoobtaina0.05genome
wideprobabilitylevelofTypeIerror,withasearchstepof1cm).QTLswerenamedby
placinga“q”atthebeginningofthetrait“HD”,followedbythechromosomenumber.
FormorethanoneQTLonthesamechromosome,asecondidentifierwasplacedafterthe
chromosomenumberreportedpreviously[47].
5.Conclusions
Atotaloffourteensignificant(LOD≥2.5)HDQTLs(qHD2a,qHD4a,qHD4b,qHD5a,
qHD6a,qHD6b,qHD7b,qHD7c,qHD8a,qHD10a,qHD10b,qHD11a,qHD12a,andqHD12b)
weredetectedinthe76CSSLpopulationsconcerningthepositionandintrogression
segmentsunderfourdifferentenvironments.WefocusedonqHD7bforfurtherresearch
duetoitsstability.AsecondaryF2(BC5F2)populationwasdevelopedbybackcrossing
CSSL52withrecurrentparentZH9308,andqHD7bwasnarroweddowntothe912.7kb
region,flankedbymarkersRM5436andRM5499using2995individualsfromthe
secondaryF2:3(BC5F2:3)population.TheCSSL52alleleattheqHD7blocusnegatively
regulatesHDunderSDandLDconditions.Sequencingandexpressionanalysis
demonstratedthatOs07g0261200encodesGhd7,asuitablecandidategeneforqHD7b.The
ghd7mutantgeneratedthroughCRISPR/Cas9promotedtheheadingdateandvalidated
Ghd7asaputativecandidategeneforHD.FurtherstudyonqHD7bwillcontributetoMAS
andthedevelopinglatematuringvarietiesthatcanbeusedindiversegeographical
regions.
SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:
www.mdpi.com/article/10.3390/plants11172288/s1,FigureS1:BreedingschemeforQTL
identificationandfinemapping,FigureS2:GGEBiplotofdaystoheadingfrom76CSSLstestedin
fourenvironments,FigureS3:Linkagemapof76CSSLsderivedfromZH9308×XQZBusing120
polymorphicmarkers,FigureS4:AschematicrepresentationofZH9308,XQZB,andCSSL52plants
toshowdifferencesonchromosome7nearqHD7b,FigureS5:Agronomictraitphenotypesof
ZH9308andCSSL52,FigureS6:MeasurementofagronomictraitsofZH9308andCSSL52,Figure
S7:PhenotypesandgenotypesofZH9308,CSSL52,andsecondaryF2(BC5F2)population,FigureS8:
FrequencydistributionofheadingdateinthesecondaryF2(BC5F2)populationunderHainanand
Hangzhouconditions,FigureS9:TheexpressionlevelsofOs07g0261200inZH9308andCSSL52,
TableS1:ChromosomewiseSNPmarkersandgeneticmaplengthofriceCSSLpopulation,Table
S2:Pearsoncorrelationcoefficientsamongheadingdateandyieldrelatedtraits,TableS3:
EvaluationofheadingdateQTLqHD7bunderHainanandHangzhouconditions,TableS4:
Candidategeneswithin912.7kbphysicalregionsofqHD7bonchromosome7,TableS5:
PolymorphicDNAmarkersusedinheadingdateQTLanalysisin76CSSLsandthesecondaryF2
(BC5F2)population,TableS6:DNAmarkersusedinQTLanalysisandfinemappingofqHD7b,Table
S7:Markersusedforsequencing,qRTPCR,andCRISPR.
AuthorContributions:Conceptualization,W.W.,Y.Z.,S.C.andL.C.;methodology,W.W.;software,
A.S.;investigation,A.S.,L.S.,L.L.,A.I.,Z.Y.,Q.Y.,G.B.A.,P.X.,R.M.K.,J.L.andX.S.;resources,Y.Z.;
writingoriginaldraftpreparation,A.S.;writingreviewandediting,A.S.andW.W.;supervision,
W.W.;projectadministration,W.W.;fundingacquisition,W.W.Allauthorshavereadandagreed
tothepublishedversionofthemanuscript.
Funding:ThisresearchwassupportedbygrantsfromtheNationalKeyR&DProgramofChina
(2020YFE0202300), theNationalNaturalScienceFoundationofChina(31871604,32071996,and
31961143016),theFundamentalResearchFundsofCentralPublicWelfareResearchInstitutions
(CPSIBRFCNRRI202102),HainanYazhouBaySeedLab(B21HJ0219),andtheAgriculturalScience
andTechnologyInnovationProgramoftheChineseAcademyofAgriculturalSciences(CAAS
ASTIP2013CNRRI).
Plants2022,11,228814of15
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Notapplicable.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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