Characterization of the Lsi1 Homologs in Cucurbita moschata and C. ficifolia for Breeding of Stock Cultivars Used for Bloomless Cucumber Production

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Characterization of the Lsi1 Homologs in Cucurbita moschata and C. ficifolia for Breeding of Stock

Cultivars Used for Bloomless Cucumber Production

Jaemin Jung^1†, Joonyup Kim^3†, Bingkui Jin¹, Youngmi Choi¹, Chang Oh Hong², Hyun Ho Lee², Youngwhan Choi¹, Jumsoon Kang¹, and Younghoon Park^1,3*

1Department of Horticultural Bioscience, Pusan National University, Miryang 50463, Korea

2Department of Life Science and Environmental Biochemistry, Pusan National University, Miryang 50463, Korea

3Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Korea

*Corresponding author: [email protected]

Bloomless cucumber fruits are commercially produced by grafting onto the pumpkin stocks (Cucurbita moschata) to restricted silicon (SiO²) absorption. Inhibition of silicon absorption in bloomless stocks is conferred by a mutant allele of the CmLsi1 homologous to Lsi1 in rice.

In this study, we characterized the Lsi1 homologs in pumpkin (C. moschata) and its cold- tolerant wild relative C. ficifolia (‘Heukjong’) in order to develop a DNA marker for selecting a bloomless trait and to establish the molecular basis for breeding bloomless stock cultivars of C. ficifolia. A Cleaved amplified polymorphic sequence (CAPS) marker (CM1-CAPS) was designed based on a non-sysnonymous single nucleotide polymorphism (SNP, C>T) of the CmLsi1 mutant-type allele, and its applicability for Marker-assisted selection (MAS) was confirmed by evaluating three bloom and five bloomless pumpkin stock cultivars. Quantitative RT-PCR of the CmLsi1 for these stock cultivers implied that expression level of the CmLsi1 gene does not appear to be associated with the bloom/bloomless trait and may differ depending on plant species and tissues. A full length cDNA of the Lsi1 homolog [named CfLsi1(B⁺)]

of ‘Heukjong’ (C. ficifolia), was cloned and sequence comparison between CmLsi1(B⁺) and CfLsi1(B⁺) revealed that there exists total 24 SNPs, of which three were non-synonymous.

Phylogenetic analysis of CfLsi1(B⁺) and Lsi1 homologs further revealed that CfLsi1(B⁺) is closesly related to Nodulin 26-like intrinsic proteins (NIPs) and most similar to CpNIP1 of C.

pepo than C. moschata.

This work was supported by a grant (710001- 07-5) from the Vegetable Breeding Research Center through Agriculture, Food and Rural Affairs Research Center Support Program, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Korea. Further, this research was supported by the National Agricultural Genome Program (Project No. PJ010438022015), Rural Development Administration.

HORTICULTURAL SCIENCE and TECHNOLOGY 35(3):333-343, 2017

URL: http://www.kjhst.org pISSN : 1226-8763 eISSN : 2465-8588

This is an Open-Access article distributed under the terms of the Creative Commons Attribution NonCommercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

OPEN ACCESS Received:

Accepted:

March 2, 2017 April 24, 2017

Abstract

Additional key words: cucurbitaceae, Lsi2, Low silicon rice 1, marker-assisted breeding, transporters, RACE PCR

Introduction

Cucumbers (Cucumis sativas L.), ^theCucurbitaceae, âre^fruitsând^vegetables^withâ^totalânnual productionof75milliontons (FAO, 2014). TotalproductionofdomesticcucumberinKoreais271,040

†The authors contributed equally to this manuscript.

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tonsperyear (^KOSIS, ²⁰¹⁵), ôf^which²⁸.⁴^tonsîsêxported^to^Japan (ÛNI-^PASS, ²⁰¹⁵). ^FormarketabilityinJapan, ^cucumber fruitwithoutbloom (bloomlesscucumber) isinevitablebecauseitdisplaysacleanandshinyappearanceonthesurfaceofthe fruit (^Choiêtâl., ²⁰¹³; ^Mitaniêtâl., ²⁰¹¹). În^majorÂsian^countriesîncluding^Japanând^Korea, ôriental^pumpkin (C. moschata Duch.) isusedasastockingraftingofcucumberforpreventionofFursariumwiltdisease, maintenanceofvegetativegrowth, andimprovementofcoldtolerance. ^For^producing^cucumbersêxported^to^Japan, ^therefore, â^specific^typeôf^pumpkin^stock (bloomlessstock) thatpreventsthecucumberfruitfrombloomingmustbeused (Seoetal., 2004; Choietal., 2013). Theseedsfor bloomlessstockareimportedmainlyfromJapan, ^while^no^domestic^stock^cultivarsârecommerciallyavailabletodate.

Forproductionofwintergreenhousecucumber, awildtypepumpkincultivar ‘Heukjong’, (C. ficifolia Bouche.) iswidely usedasastockduetoitsnatureofcold-^toleranceôverôther^non-^tolerant^pumpkin^stocks (^Tachibana, ¹⁹⁸⁸; ^Seoêtâl., ²⁰⁰⁴).

However, ‘Heukjong’ producesbloomingraftingofthecucumberandisinadequateforcultivatingbloomlesscucumbersinthe winterseason. Furthermore, ^despite^the^necessity^for^cold-^tolerant^bloomless^stock^cultivars, introgressionofthebloomlesstrait into C. ficifolia from C. moschata isnotstraightforwardduetotheinfeasibilityofcross-fertilizationbetweenthesetwospecies (^Robinson^and^Decker-^Walters, ¹⁹⁹⁷).

Thebloomofcucumberfruitisgeneratedwhenabsorbedsilicon (SiO²) isexudedwithwaterontotheskinandthendried (^Yamamotoêtâl., ¹⁹⁸⁹). Â^gene^thatfacilitatestheuptakeofsilicon, Low silicon rice 1 (Lsi1), wasfirstidentifiedinrice (^Maêt al., 2006), anditshomologousgeneswerealsoidentifiedinmanyotherplantsincludingmaizeandbarley (Chibaetal., 2009; Mitanietal., ²⁰⁰⁹). ^Thereâre^two^typesôftransportersthatmediatesiliconabsorption, â^channel-^typetransporter (ôrânînflux transporter) andaneffluxtransporter (MaandYamaji, 2015). Inrice, the Lsi1 geneencodesachannel-typetransporterthat facilitatespassivetransportofsiliconacrosstheplasmamembranebetweenapoplastandtheplantcell (^Yamajiêtâl., ²⁰⁰⁸), whileaneffluxtransportismediatedbythe Lsi2 genethatbelongstoanuncharacterizedaniontransporterfamily (Maand Yamaji, ²⁰¹⁵). În^response^to^silicon, êxpressionôf Lsi1 ^was^down-^regulatedⁱⁿ^riceând^cucumber (^Maêtâl., ²⁰⁰⁶; ^Sunêtâl 2017), butwasunalteredforitshomologousgenesinmaizeandbarley (Chibaetal., 2009; Mitanietal., 2009). Recently, itwas reportedthatpumpkinhomologs, CmLsi1 ând CmLsi2, âreâssociated^with^the^transportôf^silicon (^Mitaniêtâl., ²⁰¹¹; ^Mitani- Uneoetal., 2011). Furthermore, abloomlesspumpkincultivar ‘Super-unryu’ possessesamissensemutation (P>L) atposition 242aminoacidthatislinkedtothetransportactivityofsilicon (^Mitaniêtâl., ²⁰¹¹).

DNAmarkershavebeenemployedtoidentifyDNApolymorphismsandanalyzegenotypesandgeneticlinkagebetween genesandtraits (^Leeând^Chung, ²⁰¹¹; ^Kwonând^Choi, ²⁰¹³). În^modern^breeding^strategies, ^Marker-âssisted^selection (^MAS) isthemostpopularapplicationoftheDNAmarker. MASshortensthetimerequiredforbreeding, isnotinfluencedby environmentalfactors, ând^can^greatlyîncreasereliabilityandprecisionincomparisontophenotype-^based^selections (^Tanksley, 1983; CollardandMackill, 2008; Jonahetal. 2011). Althoughithasbeenreportedformutationaleffectof CmLsi1 relatedtothe bloomlesstrait (^Mitaniêtâl., ²⁰¹¹), â^DNA^marker^that^can^beûsed^toânalyzeâllelicôr^mutational^variationsôf^the CmLsi1 geneisnotpubliclyavailable.

Here, ^we^developedâ^molecular^marker^for^selectionôfâ^bloomless^trait^basedôn^nucleotidepolymorphismsof CmLsi1. ^We clonedthehomologousgeneof Lsi1 in C. ficifolia toestablishthemolecularbasisforbreedingofcold-tolerantbloomlessstock cultivarsof C. ficifolia..

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Materials and Methods

Plant Materials

Threecultivarsofbloompumpkinstocks [‘Heukjong’ (KyungNongSeed, Busan, Korea), ‘Odaetojwa’ (KyungNongSeed, Busan, ^Korea), ând ‘Ârirang’ (^Koregon, Ânseong, ^Korea)] ând^five^cultivarsôf^bloomless^pumpkin^stocks [(‘Ôhmai^Stock’ (KyungNongSeed, Busan, Korea), ‘OhmaiSummerStock’ (KyungNongSeed, Busan, Korea), ‘Union’ (Koregon, Anseong, Korea), ‘^Nunbusyeo’ (^Koregon, Ânseong, ^Korea), ‘^Newtype’ (^Koregon, Ânseong, ^Korea)] ^wereûsed^for^thedeterminationof siliconuptakeandgeneanalysis.

Gene-Based DNA Marker

Extraction of DNA: GenomicDNAwasextractedfromyoungleavesofthetwo-trueleafstageoftheplant. Collectedleaf tissueswereplacedintoa1.⁵^mL^micro-^centrifuge^tube^with^beadsând⁶⁰⁰^μLôf^DNAêxtraction^bufferând^subjected^to Tissuelyser (TissueLyserII, QIAGEN, Venlo, Netherlands) forhomogenization. Sampleswerelysedina65°Cwaterbathfor45 minandfurtherincubatedbyadding200μLof7.⁵^Mâmmoniumâcetateônîce^for¹⁵ - ²⁰^min. Încubated^samples^were separatedbycentrifugationfor10minat14,240xg. Thesupernatantofcentrifugedsampleswasplacedina1.5mLtube containing5μL (⁵^mg ∙ ^mL^-1) ôf^glycogen^solutionând⁶⁰⁰^μLôfisopropanolina1.⁵^mL^tubeândcentrifugedfor10minat 14,240xg. Afterdecantingthesupernatant, remainingpelletwaswashedusing300μLof 70% EtOH. Thewashedpelletwas resuspendedin200μLof ⁰.¹^M^Tris. ConcentrationofDNAwasmeasuredusingaNanodrop1000spectrophotometer (^Thermo Scientific, Waltham, MA, USA) and20ng ∙ μL^-1wasusedforPCRreactions.

Development of CAPS Marker: CodingDNASequences (CDS) ofwild-typeallele CmLsi1(B⁺) (AB551949) andmutant- typeallele CmLsi1 (^B^-) (ÂB551950) ôf^the C. moschata Lsi1 wereobtainedfromNCBIandalignedforcomparisonusing CLUSTALW (www.genome.jp/tools/clustalw) andconfirmedfortheSNPofthemutantallele (Fig. 1). IdentifiedSNPswere searchedforrestrictionsites (^NEBcutter^V².⁰, ^NEB^®, Îpswich, ^MA, ÛSA) ^to^generateâ^CleavedÂmplifiedPolymorphic Sequence (CAPS) markerusingPrimerpremier5 (PREMIERBiosoft, PaloAlto, CA, USA) (Fig. 1). PCRconditionsfor genotypingconsistedof1μLoftemplateDNA (²⁰^ng ∙ ^μL^-1), ⁰.⁵^μLôf^forwardând^reverse^primer (¹⁰^pmol), ⁰.¹^μLôf^Taq polymerase (5U ∙ μL^-1, eTaqSolg ™, SolGent, Daejeon, Korea), 0.2μLofdNTPs (Solg ™, SolGent, Daejeon, Korea), 1μLof 10Xbuffer (^Solg ™, ^SolGent, ^Daejeon, ^Korea) ând⁶.⁷^μLôf^ddH²Ôⁱⁿâ^total^volumeôf¹⁰^μL. ^Reactions^wereîncubatedât 95°Cfor2min, andcycled35timesasfollows: denaturationat94°Cfor15sec, annealingat58°Cfor30sec, extensionat72°C for1minusingaPCRcycler (^T100 ™, ^BIO-^RAD, ^Hercules, ^CA, ÛSA). Âfter^the^last^cycle, ^reactions^wereîncubatedât⁷²°^C for3min. RestrictiondigestionofPCRproductswasperformedbyadding0.3μLof Hae III (10,000U ∙ mL^-1, Time-Saver ™, NEB^®, Îpswich, ÛSA), ¹.⁵^μLôf^10X^buffer (^CutSmart ™, ^NEB^®, Îpswich, Massachusetts, ÛSA), ³.²^μLôf^ddH²Ô^to^the^PCR productandincubatedat37°Cfor1h. Digestedproductswererunona2% agarosegelat70Vfor40minandstainedwithEtBr, andcheckedfortheresultsusingGelImageAnalysisSystem (^CoreBioⁱ-^MAXTM, ^Davinch-^K, ^Seoul, ^Korea).

Analysis of CmLsi1 Gene Expression

RNA Extraction and cDNA Synthesis: ^Young^leavesând^roots^fromât^least^three^plants^per^cultivar^were^collectedând

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immediatelyfrozeninliquidnitrogen. ^Total^RNA^wasêxtractedûsingâ^Seed/^Fruit^Kit (^Ribospin™, ^GeneAll^®, ^Seoul, ^Korea) accordingtothemanufacturer’sinstructions. QuantitatedRNAusingNanodrop1000 (ThermoScientific, Waltham, MA, USA) wasdilutedto10ng ∙ ^μL^-1ândûsed^for^cDNA^synthesis^withân^RT^Premixture^kit (HyperScript ™, ^GeneAll^®, ^Seoul, ^Korea) followingthemanufacturer’sinstructions.

Quantitative PCR (qPCR): Gene-specificprimers (Q- ^Fând^R) for CmLsi1 (B⁺) (AB551949) weredesignedusingPrimer premier5 (^Table¹). Quantitativereal-^time^RT-^PCR (^qPCR) ^was^performedⁱⁿâ^Real-^time^PCR^cycler (LightCycler^®480, Roche, Basel, Switzerland) for ‘Heukjong’, ‘Odaetojwa’, ‘OhmaiStock’, and ‘OhmaiSummerStock’. Reactionswereperformed byadding1μLofcDNA (¹⁰^ng ∙ ^μL^-1), ⁰.⁵^μLôfêach^forwardând^reverse^primer (¹⁰^pmol), ⁵^μLôf^2X^SYBR^GreenÎ^Master (Roche, Basel, Switzerland), and3μLofddH2Oinatotalvolumeof10μL. CmACTIN wasusedasareferencegeneasdescribed byObreroetal. (²⁰¹¹) (^Table¹). Âll^reactions^wereîncubatedât⁹⁵°^C^for⁵^min, ând^cycled^for⁴⁵^timesâs^follows: ⁹⁵°^C ^for¹⁰ sec, 55°Cfor20sec, 72°Cfor1min. Relativequantitationofthe Lsi1 transcriptto CmACTIN genewasperformedusingthe 2^-ΔΔCTmethod (^Livakând^Schmittgen, ²⁰¹¹).

Cloning of Lsi1 Homolog in C. ficifolia

5’ and 3’ RACE: Thefull-lengthcDNAsequenceof CmLsi1 homologousgenefrom C. ficifolia wasobtainedusingRACE 5'/³' ^Kit (^SMARTer^®, ^Takara, ^Kusatsu, ^Japan). ^Total^RNA^wasêxtractedâsôutlinedâboveând^RACE^reactions^were^performed accordingtothemanufacturer’sinstructions. Gene-specificprimersweredesignedfrom CmLsi1 (B⁺) CDS (AB551949) with primerlengthsof23 - ²⁸^bp, ^GC^contentsôf⁵⁰ - ⁷⁰%, ^T^m>⁷⁰°^Când¹⁵^bp⁵'- ênd^primerôverlap^sequence (GATTACGCCAAGCTT).

Cloning and Sequencing of CfLsi1: RACEreactionswerecheckedonanagarosegelandpurifiedtheproductusingagel extractionkit (ExpinTM, GeneAll^®, Seoul, Korea). PurifiedRACEproductwasclonedintoT-EasyVectorSystemI (pGEM, Progma, ^Madison, ^WI, ^USA) ^and^verified^the^sequence^by^the^dye^terminator^method (^Genotech, ^Daejeon, ^Korea).

Sequence Alignment and Phylogenetic Analysis

Thenucleotidesequenceof CfLsi1 ^wasûsedâsâ^query^sequence^to^conduct^BLAST^searches^toîdentify^homologsôf^the CfLsi1 fromNCBI (www.ncbi.nlm.nih.gov). Todeterminetherelationshipbetweensearchedsequences, aphylogenetictree (^dendrogram) ôf^homologs^was^generated^with^MEGA⁵ (^Tamuraêtâl., ²⁰¹¹) ûsing^the^Neighbor-^Joining (^NJ) âlgorithmând Tamura-Neiparametermodelwith1,000bootstrapreplicates.

Table 1. PCR primers used in this study.

Primer name Primer Sequence (5’- 3’) Tm (°C⁾ Enzyme

CAPS CM1_CAPS_F GACATTAGGACCTGCAATGG 58.2 HaeIII

CM1_CAPS_R CTTTCACCTTCACCAACGTC 58.1

RACE PCR Cm1_3'RACE GATTACGCCAAGCTTGGCGGCATTGAACGGGAGCGATGTG 76.4

Cm1_5'RACE GATTACGCCAAGCTTACCGATACCAAAGCCGTGGGAGAGCTG 75.7

qPCR Q_F GTTGGTTCTGCCGTGTGTAT 55

Q_R TTTGAGTGAAAATGAGTGAGGAG 55

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Measurement of Silicon Uptake

Seedsofeightcommercialcultivars (^seeâbove) ^were^sownⁱⁿ^Baroker^potting^soil (^Seoul^Bio, Êumseong, ^Korea) ⁱⁿâ⁵⁰ - holedtray. After25daysofsowing, seedlingsweretransplantedinaplasticpot (15cm × 13cm × 19cm) andgrownfor30days inagreenhouseatPusanNationalUniversity (^Miryang, ^Korea). ^Whiletransplanting, ^the^soil^collected^fromâ^paddy^fieldôf^the AgricultureResearchStationatPusanNationalUniversitywasair- driedandsifted, and2.4kgof thesoilwasfilledintheplastic pot. ^Three^plantsôfêight^cultivars^were^grownⁱⁿâ^completely^randomized^design^with^three^biological^replicates. ^Bulk^density ofsoilwas1.37g ∙ cm^-1, and576mLofdistilledwaterwasaddedsothatthewatercontentinsoilwas60% oftheporevolume withaparticledensityof2.⁶⁵^g ∙ ^cm^-1. Âftertransplanting, ^the^silicate^fertilizer (^NonepongÊco, ^Nousbo, ^Suwon, ^Korea) ^was dissolvedin100mLoftapwaterandaddedattherateof0 (untreatedcontrol), 100and200mgSi ∙ kg^-1ineachpot. Throughout thewholegrowth, ^nitrogen (¹².⁸^mg ∙ ^kg^-1), ^phosphoricâcid (⁸.⁷⁵^mg ∙ ^kg^-1) ând^potassium (¹².⁷^mg ∙ ^kg^-1) ând^compost (¹^g ∙ ^kg^-1) weresupplementedwiththesameamount, andwaterwasaddedtocompensatethedecreasedamountbyweighingthepotonce everyfourdays. Âfter³⁰^daysôftransplanting, ^whole^plants^were^collected^from^soilând^driedⁱⁿâ^dryôvenât⁷⁰°^C^for⁷²^hând thenpulverized. Onegramofthepulverizedsamplewaslysedinthelysisbuffer (H²SO⁴:HClO:H²O = 5: 9:1, Samchun, Pohang, Korea) ând^then^the^contentôf^silicon^wasânalyzed^withÎCP-ÂES (^GBC^model^X-¹⁰⁰, ^GBC, ^Melbourne, Âustralia) .

Results and Discussion

Development of CmLsi1 Marker

CodingDNASequences (CDS) ofwild-typeallele (B⁺) andmutant- typeallele (B^-) ofthe CmLsi1 wereusedforalignmentto confirmthenon-^synonymous^SNP (^C>^T) ^missense^mutation (^P>^L). ^To^designâ^CAPS^marker^for^theîdentified^SNPs, restriction enzymesiteswithinthesequenceweresearchedandrecognitionsitesof Hae IIIwasselectedforthestudy. APCRprimerset CM1-^CAPS(^Table¹) ^was^designed^spanning^the Hae IIIrestrictionsitetocuttheamplifiedproduct (²⁰¹^bp) înto¹⁴⁰^bpând⁶¹ bpinthemutant-typeallele(Fig. 1). GenotypinganalysisoftheeightstockcultivarsusingtheCM1-^CAPS^marker^revealed^that bloomandbloomlesstraitswerecorrelatedwitheachgenotype (^Fig. ²). ^Forâ^bloom^F¹^cultivar ‘Ârirang’, ^markerânalysis indicatedthatitisheterozygousgenotypefor CmLsi1 (YamajiandMa, 2007), whichsuggeststhatoneoftheparentallinesof

‘Ârirang’ ^wasâ^bloomless^cultivar^homozygous^for^the^mutantâlleleôf CmLsi1. ^From^this, ^we^confirmedûseôf^the^CM1- CAPSmarkerforMAS-assistedbreedingofbloomlesstrait.

Fig. 1.Coding DNA sequence (867 bp) of the CmLsi1(B⁺) (AB551949). PCR primers for CM1_CAPS marker (yellow) and quantitative real time RT-PCR (red) are color-coded. Restriction site used in this study for HaeIII is boxed and the SNP (C/T) to CmLsi1(B^-) (AB551950) is red-colored.

769 846 ATAAAGGACTTTGGGTGTACTTTGTTGGGCC/TGGTTACAGGAACCCTATTAGGGGCATGGTCATATAAGTTCATAC

CAACCGATACCAAAGCCGTGGGAGAGCTGGCAGGTTTGGCAGTTGGTTCTGCCGTGTGTATCACATC 617 694 CATCTTGGCTGGACCCGTATCAGGTGGGTCGATGAACCCTGTGAGGACATTAGGACCTGCAATGGCAAGTGATAATT

GTGCCAGTGATAAACCTGTGCACTTAATTTCTCCTCACTCATTTTCACTCAAACTTCGAAGAATGTCAAGATCTGAC GTTGGTGAAGGTGAAAGATGA 857

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Transcripts of CmLsi1 in Bloom and Bloomless Stock Cultivars

Expressionlevelsofendogenous CmLsi1 genewereexaminedinleafand roottissuesfrom bloom (‘Heukjong’ and

‘Ôdaetojwa’) ând^bloomless (‘Ôhmai^Stock’ ând ‘Ôhmai^Summer^Stock’) ^cultivarsûsingQuantitativerealtimeRT- ^PCR (^qRT- PCR) (Fig. 3). Fromthetissuestestedforrepresentativecultivars, relativeexpressionof CmLsi1 washigherinrootthanleafof thesecultivars (^Fig. ³). ^Theêxpression^patternôf Lsi1 ^was^consistent^with^the^result^from^rice (Oryza sativa) andcucumber (Cucumis sativus), whichshowedhigherexpressionof Lsi1 inrootthanleaf (Maetal., 2006; Sunetal., 2017). Amongcultivars ofpumpkins (C. moschata), ithasbeenshownthatexpressionof Lsi1 ^geneⁱⁿâ^bloomless^cultivar ‘^Super-ûnryu’ ^was^higher^than abloomcultivar ‘Sintosa’ (Mitanietal., 2011). Curiously, itwasalsoreportedthatexpressionof CmLsi1 inshootapexwas Fig. 2. Agarose gel (2%) image showing the result of CM1_CAPS genotyping for the CmLsi1 gene in eight stock cultivars.

Lane 1, ‘Heukjong’; 2, ‘Odaetojwa’; 3, ‘Arirang’; 4, ‘Nunbusyeo’; 5, ‘Ohmai Stock’; 6, ‘Ohmai Summer Stock’; 7, ‘Union’; and 8,

‘Newtype’.

Bloom Bloomless

2

1 3 4 5 6 7 8 M

200 bp

100 bp

Leat

Heukjong Odaetojwa Ohmai Ohmai Heukjong Odaetojwa Ohmai Ohmai

Stock Stock

Summer Summer

Root

Relative to Heukjong Leaf

0 5 10 15 20 25 30