Phenotyping, Genotyping, and Selections within Italian Local Landraces of Romanesco Globe ...

15 Pages·2017·1.25 MB·English

diversity
Article
Phenotyping, Genotyping, and Selections within
Italian Local Landraces of Romanesco
Globe Artichoke
PaolaCrinò1andMarioAugustoPagnotta2,*
1 ItalianNationalAgencyforNewTechnologies,EnergyandSustainableEconomicDevelopment(ENEA),
C.R.Casaccia,BiotechnologiesandAgroindustryDivision,ViaAnguillarese301,00123Roma,Italy;
[email protected]
2 DepartmentofAgriculturalandForestrySciences(DAFNE),UniversitàdegliStudidellaTuscia,
ViaS.C.deLellis,01100Viterbo,Italy
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:MichaelWink
Received:25November2016;Accepted:23February2017;Published:1March2017
Abstract: TenItalianglobeartichokeclonesbelongingtotheRomanescotypologywerecharacterized
inthewesterncoastalareaofItaly(Cerveteri,Rome),usingacombinationofmorphological(UPOV
descriptors), biochemical (HPLC analysis), and molecular (AFLP, ISSR, and SSR markers) traits.
Significant differences among clones were found for many of the quantitative and qualitative
morphological traits. Multivariate analyses (Principal Component Analysis) showed that, of the
47morphologicaldescriptorsassessed,four(i.e.,plantheight,centralflower-headweight,earliness,
and total flower-head weight) presented a clear grouping of the clones. Biochemical analyses
showedthattheclonessignificantlydifferedinthepolyphenolicprofilesoftheflower-head,with
thesuggestionthatsomeofthese,suchasS2,S3,S5,andS18,aremoresuitableforthefreshmarket.
The clones, clustered by a UPGMA dendrogram based on 393 polymorphic AFLP and ISSR loci,
showedthattheclonesweregeneticallyseparatedfromeachother. Thishighlightstheimportance
ofcharacterizing,evaluating,andconservingautochthonousgermplasmforfutureplantbreeding
activities. Overall,thesestudiesresultedintheidentificationoftwonewclones,selectedonthebasis
of flower-head morphology and earliness. These clones, named Michelangelo and Raffaello, are
registeredontheItalianNationalRegisterofVarieties(DMn. 6135,3/29/2013G.U.91,18April2013).
Keywords: Cynaracardunculus;geneticdiversity;germplasmselection;nutraceuticalcompounds;
varietalrelease
1. Introduction
The globe artichoke [Cynara cardunculus L. var. scolymus (L.) Fiori] is a diploid species
(2n=2x=34), traditionally grown for its immature flower-heads and used in dishes across the
Mediterraneanregion[1,2]. Morerecently,theglobeartichokehasalsoattractedattentionduetoits
potentialasabiomasscrop[3,4]andforitsnutraceuticalcontent[5,6]. Asof2013,globalartichoke
production was 1793 Mt of heads over an area of 130,676 ha [7]. The top producers were Italy
(548kt/46,954ha),Egypt(391kt/16,213ha),andSpain(200kt/15,400ha)[7].
InItaly,theglobeartichokeisaneconomicallyimportanthorticulturalcrop,especiallyinregions
suchasSicily(165Mt/14,832ha), Apulia(148Mt/15,610ha), andSardinia(114Mt/13,528ha)[8].
Italyalsohousestherichestcollectionsofglobeartichokeautochthonousgermplasm; theprimary
cultivatedgenepool[9]includingfarmervarieties/landraces,traditionallypropagatedvegetativelyvia
offshoots. Thiscultivatedgermplasmwastraditionallydifferentiatedonthebasisofheadmorphology
(Romaneschi, Violetti, Catanesi, Spinosi) [10] or head appearance, and harvest time (autumn and
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Diversity2017,9,14 2of15
spring,or“re-bloom”and“spring”floweringtypes)[11,12]. Manytraditionallandraces,however,do
notfallintothesebroadvarietalgrouptraits.
Italianproductionismainlybasedonlocalheterogeneousgenotypes,whichrepresentexamples
offarmerselectioncarriedouton-farm,andarenamedbasedonthegeographicalareaofcultivation,
without genetic or origin identification [13]. For example, in the Latium region, the Romanesco
landracesCastellammareandCampagnanowerewidelycultivatedinnon-specializedsmall-holdings.
In 2002, these landraces were added to the PGI (Protected Geographical Indication) ‘Romanesco
ArtichokeofLatium’(Reg. ECn. 2006/2002),thevarietalregistrationplatform. Despitetheadded
valueofthiscertification,andthehighlysought-afterqualityoftheheads,preferredfortraditional
recipes,theselandracesarebeingrapidlyreplacedbynewmicropropagatedartichokecultivarsand
seed-propagatedF1hybrids,whicharemoreproductiveandmatureearlier,asthemarketrequests[14].
Inparticular,thecloneC3,derivedfromthetraditionalpopulationCastellammare,andselectedfor
itsearlinessintheLatiumregion, iswidelycultivated[15]. Thishasledtoasignificanterosionof
Castellammarepopulationsandacorrespondinglossofdiversity[16]. ThetraditionalRomanesco
germplasmcontainshighlevelsofgeneticvariationwithineachlandrace[13,17–21]. Manyofthese
landraces are highly resistant to endemic diseases, such as the Artichoke latent Virus (ArLV) and
VerticilliumdahliaeKleb.[14,18]. Thishighlightstheimportanceofconservingthelandraces,astheir
highlevelsofheterogeneityallowthemtorespondtoabioticandbioticstressesandadapttolow-input
farmingsystems.
In Italy, more in-depth studies are needed for the genetic improvement of these traditional
landraces of Romanesco types, especially in terms of varietal release. Preliminary studies on the
characterization of traditional Romanesco genetic resources have assessed the complementarity
between molecular and morphological approaches [16,18,22]. However, plant breeding programs
aimedattherecoveryandreleaseofnewRomanescoregisteredvarietieshavenotbeencarriedout.
Indeed,inItaly,itismainlytheseed-propagatedF1hybrids(SIANcode/Hybridname:3062/Ademaro,
3284/Almiro,3061/Amos,3285/Ernesto,3058/Istar,3064/Napoleone,3300/Omero,3066/Rinaldo,
3282/Romano,3283/Verdiano,and3059/Zenone)thathavebeenplacedontheNationalRegisterof
HorticulturalVarietiesofItalianMinistryofAgriculture, FoodandForestryPolicies(Commission
Directive2006/124/EC).
Taking into account the above issues and the requirements for several well-defined quality
standards for the release of new varieties (European Directive No. 184/1/2004), autochthonous
germplasm belonging to the Romanesco typology was evaluated for two years [18], within the
framework of the CYNARES project (financial support from the European Commission, DG for
AgricultureandRuralDevelopment,underCouncilRegulationECNo870/2004).
Thesamegeneticmaterialwasassessed: (i)molecularly,utilizingISSR,AFLP,andSSRmarkers;
(ii) morphologically, through the use of UPOV (International Union for the Protection of New
VarietiesofPlants)descriptors;and(iii)biochemically,usingHPLCanalysis. Theobjectiveswereto:
(a) investigate the existing genetic diversity within and among these clones under morphological,
biochemical, and molecular profiles; and (b) select clones for varietal release and inclusion on the
ItalianRegisterofNationalVarieties.
2. MaterialsandMethods
2.1. ExperimentalFieldandPlantMaterial
Ten clones, selected on the basis of previous data [18], and representative of Romanesco
germplasm, were assessed in this study. In particular, clones from the north and south of Rome
(Figure1),withdifferenttimesofheading,wereused. TheclonesS17,S22,andS23wereearlyheading
specimens; S18, S20, and S21 were medium heading specimens; and S2, S3, S5, and S11 were late
heading specimens. All clones were planted in Cerveteri, Latium (Italy) (41◦59(cid:48) N 12◦01(cid:48) E 30 m
a.s.l.),inanexperimentalfarmstationbelongingtotheRegionalAgencyfortheDevelopmentand
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Diversity 2017, 9, 14 3 of 15
the Innovation of Agriculture (ARSIAL), where they were evaluated for two subsequent growing
seasons. Landraceswereplantedinarandomizedblockdesignwithfourreplicates,witheachtest
seasons. Landraces were planted in a randomized block design with four replicates, with each test
plot consisting of twenty plants. The planting density was 7700 plants·ha−1 (inter and intra-row
plot consisting of twenty plants. The planting density was 7700 plants·ha−1 (inter and intra-row
distancesof1.30and1.00m,respectively). Fieldexperimentswereconductedunderlowresource
distances of 1.30 and 1.00 m, respectively). Field experiments were conducted under low resource
inputs(oneirrigation/yearinAugustconsistingof60mmofwater,organicfertilizationwith50kgof
inputs (one irrigation/year in August consisting of 60 mm of water, organic fertilization with 50 kg
N·ha−1,andwithoutherbicidesandgibberellicacid),asperlocalagronomicalpractices. Duringthe
of N·ha−1, and without herbicides and gibberellic acid), as per local agronomical practices. During the
trials,meteorologicaldatawerecollectedbyameteorologicalstationsetupattheexperimentalfarm.
trials, meteorological data were collected by a meteorological station set up at the experimental farm.
Thefirstgrowingseasonexhibitedmaximumandminimumairtemperatures(expressedasaverage
The first growing season exhibited maximum and minimum air temperatures (expressed as average
values)rangingfrom30.6to4.8◦CbetweenAugustandMay,whiletemperaturesoverthesecond
values) ranging from 30.6 to 4.8 °C between August and May, while temperatures over the second
growingseasonrangedfrom29.1to5.9◦C.Therecordedvalueforrainfallwas549.6mmfromAugust
growing season ranged from 29.1 to 5.9 °C. The recorded value for rainfall was 549.6 mm from August
toMayofthefirstyearoftheassessment,and921.8mminthesecondyear. Soilcharacteristicswere
to May of the first year of the assessment, and 921.8 mm in the second year. Soil characteristics were
asfollows: 62%sand,23%clay,and15%silt;pH6.3;organicmatter,1.24%;totalnitrogen,0.08%;P,
as follows: 62% sand, 23% clay, and 15% silt; pH 6.3; organic matter, 1.24%; total nitrogen, 0.08%; P,
24ppm;K,355ppm;andCE,0.16mS.
24 ppm; K, 355 ppm; and CE, 0.16 mS.

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growing seasons.
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Table1.Forty-sevenmorphologicaldescriptorsutilizedforclonecharacterization.
No. Descriptor No. Descriptor
1. Plant:height(includingcentralflower-head) 25. Centralflower-head:shapeoftip
2. Plant:numberoflateralshootsonmainstem 26. Firstflower-headonlateralshoot:length
3. Mainstem:height(excludingcentralflower-head) 27. Firstflower-headonlateralshoot:diameter
Mainstem:distancebetweencentralflower-headand
4. 28. Firstflower-headonlateralshoot:size
youngestwelldevelopedleaf
Mainstem:diameter(atabout10cmbelowcentral
5. 29. Firstflower-headonlateralshoot:shape
flower-head)
6. Leaf:attitude 30. Outerbract:lengthofbase
7. Leaf:longspines 31. Outerbract:widthofbase
8. Leaf:length 32. Outerbract:thicknessatbase
9. Leaf:incisions 33. Outerbract:mainshape
10. Leaf:numberoflobes, 34. Outerbract:shapeofapex
11. Lobe:shapeoftip(excludingterminallobe) 35. Outerbract:depthofemargination
12. Lobe:numberofsecondarylobes 36. Outerbract:colour(externalside)
13. Lobe:shapeoftipofsecondarylobes 37. Outerbract:hueofsecondarycolour
14. Leafblade:shapeincrosssection 38. Outerbract:sizeofspine
15. Leafblade:intensityofgreencolour 39. Outerbract:mucron
Centralflower-head:anthocyanincolorationofinner
16. Leafblade:hueofgreencolour 40.
bracts
17. Leafblade:intensityofgreyhue 41. Centralflower-head:densityofinnerbracts
18. Leaf:hairinessonupperside 42. Receptacle:diameter
19. Leafblade:blistering 43. Receptacle:thickness
20. Petiole:anthocyanincolorationatbase 44. Receptacle:shapeinlongitudinal
21. Centralflower-head:length 45. Mainheadweight
22. Centralflower-head:diameter 46. Totalnumberofheads
23. Centralflower-head:size 47. MainHeaddateofmaturity
24. Centralflower-head:shapeinlongitudinalsection
2.3. DNAExtractionandMolecularMarkerAnalysis
Oneplantperlandracewasrandomlyselectedandmolecularlyassessed. ThetotalgenomicDNA
was isolated from young leaves and extracted from 100 mg of frozen tissue, using the Plant DNA
ExtractionKit(EURxLtd. 80-297GdanskPoland). TheDNAqualitywascheckedbyelectrophoresis
ina1%agarosegelandstainedwithethidiumbromide. TheDNAconcentrationwasdetermined
throughspectrophotometry.
MolecularanalysiswasrunusingAFLPs,ISSRs,andSSRsmarkers.
Seven AFLP primer combinations were used: EcoACC/MseCTA, EcoACG/MseCTT,
EcoAGC/MseCTT, MseAC/PstCA, MseAC/PstCG, MseGC/PstCA, and MseGC/PstCG.
Amplifications were carried out according to Vos et al. [24], and consisted of using MseI and
PstItodigestthetemplateDNA.Non-selectiveprimerswereusedforpre-amplification. FourISSR
primers, selected from the British Columbia University (810, 834, 841, and 857), were also used.
PCR reactions were performed in a final volume of 10 µL, containing 10 ng DNA, 0.3 µM primer,
100µMdNTP,10mMTris-HCl(pH9.0), and1UnitofTaqpolymerase. Theamplificationregime
was 94◦C/5min, followed by 39 cycles of 96 ◦C/1 min, 43–54 ◦C/1 min (see Ta in Table 2), and
72◦C/2min,endingwithanextensionstepof72◦C/10min.
Thirteen SSR primers, developed by Acquadro et al. [25] (CMAL11, CMAL117, CMAL24,
CMAL06, and CMAL108), [26] (CDAT01, CLIB02, and CLIB12), [27] (CMAFLP18), and
Sonnanteetal.[28](CsPal03,CsPal02,CsEST03,andCsCiCaCa05),wereused. Primers,sequences,
andtheiramplificationconditionswereselectedinaccordancewiththeauthorswhodevelopedthe
primers(seeabove);theannealingtemperaturesarereportedinTable2.
The forward primers for all of the 24 markers were labeled with either FAM or HEX. PCR
amplificationswereresolvedina3130xlsequencermachine(AppliedBiosystems,FosterCity,CA,
USA)andreadbyGenemap3.1. Software(AppliedBiosystems). Primerreproducibilitywaschecked
byrepeatingtheamplificationstwice.
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Table2.ISSR,AFLP,andSSRusedforgermplasmcharacterization.
MolecularMarkers Ta NPB He Ho PIC
EcoACC/MseCTA 5 0.37
EcoACG/MseCTT 3 0.45
EcoAGC/MseCTT 4 0.34
AFLP MseAC/PstCA 81 0.31
MseAC/PstCG 68 0.27
MseGC/PstCA 95 0.28
MseGC/PstCG 67 0.28
810(GA) T 43 4 0.24
8
827(AG) G 52 1 0.41
ISSR 8
841(GA) YC 45 30 0.20
8
857(AC) YG 54 35 0.22
8
CMAL11 54 2 0.32 0.40 0.268
CMAL117 60 2 0.48 0.60 0.365
CMAL24 60 4 0.66 0.90 0.592
CMAL06 60 2 0.50 0.80 0.375
CMAL108 60 2 0.10 0.10 0.091
CDAT01 54 3 0.56 1.00 0.442
SSR CLIB02 59 2 0.50 0.90 0.373
CLIB12 55 2 0.50 0.90 0.373
CMAFLP18 55 2 0.50 0.90 0.373
CsPal03 52 3 0.55 1.00 0.442
CsPal02 60 2 0.10 0.10 0.091
CsEST03 62 2 0.38 0.50 0.305
CsCiCaCa05 55 2 0.10 0.10 0.091
Ta: annealingtemperature; NPB:numberofpolymorphicbands; He: expectedheterozygosity; Ho: observed
heterozygosity;PIC:polymorphicinformationcontent.
2.4. BiochemicalAnalysis
2.4.1. SolventsandReagents
Ethanol solvent and reagent grade formic acid (96%) were purchased from Scharlau Co.
(Barcelona, Spain). Deionized water was made using a Milli-Q water purifying system,
purchased from Millipore Co. (Bedford, MA, USA). Apigenin, luteolin, luteolin-7-O-glucoside
(cynaroside), 1,3-O-dicaffeoylquinic acid (cynarin), and 1,5-O-dicaffeoylquinic acid were obtained
fromExtrasynthese(Lyon,France);while3-O-caffeoylquinicacid(chlorogenicacid)wasobtainedfrom
SigmaChemicalCo. (St. Louise,MO,USA).
2.4.2. PolyphenolExtractionandHPLCAnalysis
At least three primary flower-heads per replicate were harvested during the commercial
maturationstageforeachclone,inthespringofthefirstgrowingseason.Allsampleswereimmediately
weighed, freeze-dried,andgroundedtoa1mmdiameterfinepowder. Extractionwasperformed
usingAcceleratedSolventExtraction(ASE),accordingtothemethodsdescribedbyCiancolinietal.[6].
PolyphenolanalysiswascarriedoutusingaDionexHPLCchromatograph,equippedwithan
UVdetectorHP1100. HPLCseparationwasperformedaspreviouslydescribedbyCiancolinietal.[6].
Chromatograms were recorded at 325 nm for chlorogenic acid, 1,3-O-dicaffeoylquinic acid, and
1,5-O-dicaffeoylquinicacid,andat350nmforluteolin,apigenin,andcynaroside. Allsampleswere
analyzedinduplicate.
2.5. StatisticalAnalysis
MorphologicalandbiochemicaldatawereanalyzedthroughanANOVArunningGeneralized
LinearModel(GLM),PrincipalComponentAnalysis(PCA),andClusteranalysesusingSPSSsoftware
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version15.0. MeanseparationswereperformedbyaTukeytest. Significancewasacceptedatp≤0.05
level. Shapiro-WilkandKurtosistestswereusedtoassessthenormalityoftheobservations. Basedon
themorphologicaltraits,asimilaritydendrogramwasconstructedusinganagglomerativehierarchical
clusteranalysis.
Polymorphic bands for each ISSR and AFLP marker (between 50 and 500 bp) were recorded;
eachsignificantpeakwasassumedtorepresentasinglelocus,withtwopossiblealleles(presenceand
absence). Profileswereconvertedtoa1/0matrixforstatisticalanalyses,whilebanddimensionswere
consideredforSSRmarkers. Statisticsofthemoleculardata,suchasnumberofpolymorphicbands
(NPB),expectedheterozygosity(He),observedheterozygosity(Ho),andpolymorphicinformation
content (PIC), were computed for each clone, in accordance with Pagnotta et al. [29], utilizing
GenAlEx [30] or Power Marker [31]. The marker matrix was also used to compute Nei’s genetic
distances [32] between clones, and then used to conduct a cluster analysis and to draw an
UPGMAdiagram.
Thenumbersofcommonversusrarealleleswerealsoanalyzedtoobtainanassessmentofthe
richnesswithineachlandrace. Rarealleleswerecategorizedasthoseallelespresentinlessthan5%of
eachlandrace.
A Mantel test was applied to verify the statistical relationship between the distance matrices
obtained by the genetic and morphological traits [33], as well as between biochemical and
morphologicalmatrices. APearson’scorrelationwasusedtoevaluatetheManteltest(significance
levelofp≤0.05).
3. Results
3.1. MorphologicalCharacterization
Onthebasisofmorphologicaldata,aclusteranalysiswasappliedandasimilaritydendrogram
was generated (Figure 2), which showed significant differences among clones. The dendrogram
obtainedallocatedthe10clonesintotwomainclusters: thefirstclustergroupedtheclonesS2,S3,S5,
andS11;andtheclonesS17,S20,S18,S22,S23,andS21wereincludedinthesecondgroup. Inthe
twoidentifiedclusters,theclonesweremainlydividedonthebasisoftheirearliness/latenessand
geographicalorigin(southvs. northofRome,seeFigure1). Thisisconsistentoverthetwoyears(data
unshown),althougheightoutofthe15traitshavesignificantclone-by-yearinteractions(Table3).
For 15 of the 47 quantitative and qualitative traits measured, significant differences among
clones were found, highlighting high levels of genetic variability within the germplasm (Table 3).
Withreferencetomorphologicaltraits,S5,S18,andS20showedthehighestvaluesforplantheight.
TheclonesS3,S11,S18,S20,andS21exhibitedthelongestdistancebetweentheyoungestleafonthe
mainfloralstemandthecentralflower-head. S11,S17,S18,S22,andS23revealedthebiggestmain
floralstemdiameter. TheclonesS2,S17,andS23werecharacterizedbytheshortestheadlength,while
S3, S17, S18, and S20 had the smallest head diameter. Regarding the head shape, S20 showed the
mostelongatedheads,whileS2,S17,S22,andS23providedthemostellipticalheads. Concerning
theheadcolor,theclonesS11,S18,andS21showedthemostintensepurple-coloredstripedheads,
while S5, S17, S22, and S23 were characterizedby themost green-coloredheads. Inboth growing
seasons, S17 budded the earliest with regard to the appearance of the central flower-head, while
S2, S3, S5, andS11producedtheircentralflower-headslater. Thehighestvalueforthenumberof
totalheadsproducedwasseenforS22, andS23, S17, S21, S22, andS23yieldedthehighestvalues
whenexpressedaskgofheadsperplant(Table3). TheANOVAtestrevealedsignificantdifferences
betweenyearsfor13traitsandsignificantclone-per-yearinteractionsforeighttraitsoutofthe15traits,
showingsignificantdifferencesamongclones(Table3). Theprincipalcomponentanalysisundertaken,
basedonthemorphologicaldatasetsofclonesofthesametypology,showedthatonly37.61%ofthe
totalvarianceisexplainedbythefirstthreecomponents(Figure3). Nevertheless,thefirstPCfactor
(15.84%ofvariance)includedthefollowingprimarytraits: plantheight,mainfloralstemheight,head
Diversity 2017, 9, 14 7 of 15
Diversity 2017, 9, 14 7 of 15
first PC factor (15.84% of variance) included the following primary traits: plant height, main floral
Dfiirvsetr sPityC2 0f1a7c,t9o,r1 4(15.84% of variance) included the following primary traits: plant height, main f7loofr1a5l
stem height, head shape index, receptacle shape, time of the central flower-head appearance, and
stem height, head shape index, receptacle shape, time of the central flower-head appearance, and
time of the primary flower-head appearance. The second factor explained 13.29% of the variance and
time of the primary flower-head appearance. The second factor explained 13.29% of the variance and
sinhcalpuediendd: ethx,er decisetpatnaccel ebsehtwapeee,nt itmhee foirfstth feuclleyn tdreavleflloowpeedr- hleeaafd anapdp tehaer acenncter,aaln fdlotwimere-hoefatdh,e tphrei mmaariyn
included: the distance between the first fully developed leaf and the central flower-head, the main
flstoewme rd-hiaemadetaepr,p ceeanratrnacle f.lTohweesre-hcoenadd fdaicatmoreetxerp,l areinceedpt1a3c.l2e9 %diaomftehteerv,a arniadn cceenantrdali nflcoluwdeerd-h:ethaed dwisetaignhcte.
stem diameter, central flower-head diameter, receptacle diameter, and central flower-head weight.
bTehtew teheinrdt hfaecfitorsr t(f8u.4ll8y%d eovf evlaorpiaendclee)a efnatnadiletdhe thceen ntruamlflboerw oefr -lhaetearda,l tshheomotas,i ntosttaelm nudmiabmeert eorf, fcleonwtrearl-
The third factor (8.48% of variance) entailed the number of lateral shoots, total number of flower-
flhoeawdesr,- haneadd wdeiiagmhet toefr ,threec ehpetaadcsl.e diameter,andcentralflower-headweight. Thethirdfactor(8.48%of
heads, and weight of the heads.
variance)entailedthenumberoflateralshoots,totalnumberofflower-heads,andweightoftheheads.

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Figure 2. Similarity dendrogram of globe artichoke clones based on an agglomerative hierarchical
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cluster analysis of the morphological data listed in Table 1. The axis reports the Rescaled Distance
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Figure3.DistributionoftheanalyzedgenotypesagainstthefirstthreePrincipalComponentfunctions.
Figure 3. Distribution of the analyzed genotypes against the first three Principal Component
Figure 3. Distribution of the analyzed genotypes against the first three Principal Component
functions.
functions.
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Table3. Differencesinsomemorphologicaltraits(meansovertwoyears)ofthe10Romanescolandracesanalyzed. Plantheight(PH,cm),distancebetweenthe
youngestleafonthemainfloralstemandthecentralflower-head(DHL,cm),stemdiameter(MSD,cm),headlength(HeadL,cm),headdiameter(HeadD,cm),head
shape(ShapeIndex),headtipshape(ShapeTip),bractcolour(ColBract),diameterofthereceptacle(RecD,cm),receptaclethickness(RecThick,cm),receptacleshape
(RecShape),headweight(HeadWg),timeofheadmaturity(TimeHead),totalheadnumber(HeadN),andtotalheadweight(TotHeadW,g)
Clones PH DHL MSD HeadL HeadD ShapeIndex ShapeTip ColBract RecD RecThick RecShape HeadW TimeHead HeadN TotHeadW
S2 68.6d,e 23.4b,c 2.80a,b 7.72b,c 9.93a,b 0.78e 3.54a 3.00b,c 4.88a 0.69b,c 1.96c,d 358.4a,b 191.5a 8.08d 1291.7e
S3 71.4c,d 29.6a,b 2.66b,c 8.34a,b 9.38b,c 0.89a,c 2.54b,d 3.08b,c 4.20a,b 0.57c 2.29a,c 326.8a,b 186.5a 10.67a,d 1342.1e
S5 85.1a,b 27.5b,c 2.54c 8.52a,b 9.47a,c 0.90a,b 2.46c,d 2.67c,d 4.62a,b 0.61c 2.61a 340.3a,b 188.6a 9.5b,d 1323.8e
S11 79.8b,c 29.5a,b 2.75a,c 8.48a,b 9.51a,c 0.89a,c 1.67e 3.67a,b 4.38a,b 0.64c 2.54a,b 354.6a,b 188.7a 10.88a,d 1399.0d,e
S17 54.1f 25.2b,c 2.93a 7.28c 9.13b,c 0.80b,e 3.38a 2.25d 4.68a,b 0.90a,b 1.13e 336.9a,b 148.7e 10.58a,d 1820.8a,d
S18 86.4a,b 35.0a 2.94a 7.86a,c 9.33b,c 0.84a,d 2.96a,d 3.50a,b 4.85a 0.64c 1.58d,e 355.2a,b 183.0a,b 9.21c,d 1687.1b,e
S20 90.7a 34.6a 2.61b,c 8.21a,c 8.79c 0.93a 3.00a,c 3.21b,c 4.01b 0.60c 2.13b,c 314.2b 183.5a,b 11.38a,c 1647.6c,e
S21 72.0c,d 29.4a,b 2.64b,c 8.76a 9.91a,b 0.88a,c 2.33d 3.92a 4.31a,b 0.78a,c 1.96c,d 378.4a 175.1b,c 12.50a,b 2174.0a
S22 59.9e,f 23.1b,c 2.97a 8.09a,c 10.32a 0.78e 3.08a,c 2.58c,d 4.89a 0.94a 1.58d,e 359.1a,b 166.1c,d 13.54a 1908.1a,c
S23 54.2f 21.3c 2.85a,b 7.65b,c 9.61a,c 0.79c,e 3.17a,b 2.54c,d 4.80a 0.99a 1.58d,e 329.9a,b 161.8d 13.42a 2107.0a,b
Std.Er. 1.62 1.44 0.39 0.20 0.18 0.02 0.14 0.13 0.14 0.04 0.09 13.68 2.082 0.596 89.719
Y *** *** *** ** ** *** ns *** ns *** *** *** *** *** ***
CxY *** *** ns ns *** ns ns *** ns *** ** ns *** * ns
Differentlettersindicatestatisticallysignificantdifferencesamongclonesatp≤0.05(ANOVAanalysis,Tukeytest);*,**,and***indicatesignificantdifferencesatp≤0.05,p≤0.01,andp
≤0.001,respectively,andnsstandsfornon-significant.
Diversity2017,9,14 9of15
3.2. DNAMarkerCharacterization
Themolecularmarkersoftheprimersused,differedintheirabilitytodetectgeneticvariation,as
shownbythedifferencesinthenumberofbands,geneticdiversity(He),andPIC(Table2). Thiswas
alsofoundwithinthesamemarkertype: theISSR827markerdetectedthehighestgeneticdiversity,
whereasISSR841amplifiedthelowestone;theEcoACG/MseCTTprimerpairshowedthehighest
geneticdiversity,whereastheMseAC/PstCG,inspiteofthehighnumberofbands,hadthelowest
Hevalue. ThemostinformativeSSRmarkerwasCMAL24,withthehighestgeneticdiversityandthe
highestPICvalue,whiletheleastinformativewereCMAL108,CsPal02,andCsCiCaCa05,withaPIC
ofonly0.091.
ThenumberofprivateallelesforeachpopulationrangedfromfourinS11,to41inS20(Table4).
Table4.Privateallelespresentineachoftheclonesanalyzed.
Clones N.PrivateAlleles PrivateBands
S11 4 MgcPca/10,MgcPca/202,841/302,857/168
MacPca/107,MgcPca/131,MgcPca/159,MgcPca/228,MgcPca/267,
S17 7
MgcPcg/9,857/277
MacPcg/4,MacPca/12,MacPca/54,MacPca/150,MacPcg/24,MacPcg/63,
S18 6 MacPcg/86,MgcPca/30,MgcPca/39,MgcPca/43,MgcPca/60,MgcPca/180,
MgcPcg/3,MgcPcg/22,MgcPcg/35,MgcPcg/151
810/4,857/172,MacPca/24,MacPcg/95,MacPcg/228,MgcPca/5,
S2 9
MgcPca/73,MgcPcg/15,MgcPcg/94
MacPca/50,MacPca/32,MacPca/167,MacPca/194,MacPca/215,
MacPca/236,MacPca/238,MacPcg/16,MacPcg/48,MacPcg/137,MgcPca/4,
S22 20
MgcPca/80,MgcPca/87,MgcPca/107,MgcPca/201,MgcPca/252,
MgcPcg/8,MgcPcg/105,MgcPcg/122,MgcPcg/126
MacPca/275,MacPcg/115,MgcPca/68,MgcPca/268,MgcPcg/31,
S23 8
MgcPcg/82,857/41,857/191
MacPcg/23,MgcPcg/26,MacPcg/234,MgcPca/78,MgcPcg/17,MgcPcg/20,
S3 15 MgcPcg/48,EaccMcta/91,810/5,841/32,841/34,841/220,841/223,
857/74,857/76
MacPca/70,MacPca/133,MacPca/134,MgcPca/117,MacPca/222,
S5 14 MgcPcg/154,MgcPcg/167,841/51,841/53,841/131,857/62,857/193,
857/262,857/275
841/76,841/194,841/195,841/330,857/315,857/321,857/323,MacPcg/26,
MacPcg/37,MacPcg/179,MgcPca/27,MgcPca/32,MgcPca/41,MgcPca/75,
S21 20
MgcPca/133,MgcPca/193,MgcPca/240,MgcPcg/40,
MgcPcg/138,MgcPcg/191
857/2,MacPca/5,MacPca/15,MacPca/35,MacPca/48,MacPca/73,
MacPca/74,MacPca/158,MacPca/193,MacPca/278,MacPcg/9,MacPcg/18,
MacPcg/25,MacPcg/34,MacPcg/38,MacPcg/44,MacPcg/45,MacPcg/46,
S20 37 MacPcg/92,MacPcg/145,MgcPca/17,MgcPca/49,MgcPca/53,MgcPca/97,
MgcPca/141,MgcPca/169,MgcPca/207,MgcPca/210,MgcPcg/135,
MgcPcg/153,MgcPcg/162,MgcPcg/168,MgcPcg/178,MgcPcg/182,
MgcPcg/19,MgcPcg/36,MgcPcg/71
The UPGMA diagram based on Nei’s genetic distance between clones, obtained with both
dominant and co-dominant DNA markers, is shown in Figure 4. The dendrogram revealed three
clusters: afirsttightgroupwithS3,S5(latefromsouthRome),S17andS18(fromtheC3selection);a
secondgroupwithS2,S11,andS23;andathirdgroupwithS20,S21,andS22,highlightingdistinctand
separategenotypes. Hence,Figure4reflectsonlypartoftheagro-morphologicaldifferencesamong
Romanescoclones(alsoshowninFigure2). Inparticular,S22andS20,belongingtothethirdgroup,
showedsimilarheadyieldsandnumberoflateralshoots. Incontrast,S2andS11weregroupedas
late-maturingclones,accordingtothetimeoftheappearanceofthecentralflower-headandbythe
growing/collectingarea.
Diversity 2017, 9, 14 10 of 15
as late-maturing clones, according to the time of the appearance of the central flower-head and by
tDhieve rgsritoyw20i1n7g,9/,c1o4llecting area. 10of15

FFiigguurree 44.. UUPPGGMMAA ddeennddrrooggrraamm ooff tthhee NNeeii’’ss ggeenneettiicc ddiissttaannccee aammoonngg 1100 cclloonneess oobbttaaiinneedd wwiitthh ddoommiinnaanntt
aanndd ccoo--ddoommiinnaanntt mmoolleeccuullaarr mmaarrkkeerrss .
AA ssiiggnniiffiiccaanntt ccoorrrreellaattiioonn bbeettwweeeenn ggeenneettiicc ssiimmiillaarriittyy aanndd mmoorrpphhoollooggiiccaall ttrraaiitt mmaattrriicceess wwaass
iilllluussttrraatteedd bbyy tthhee MMaanntteell tteesstt rreessuulltt ((rr == −−00.2.299227799; ;aat tpp ≤≤ 0.005.0)5. ).
33..33.. BBiioocchheemmiiccaall CChhaarraacctteerriizzaattiioonn
PPoollyypphheennooll pprrooffiilleess, ,ddeetetermrminineded byb yHHPLPCLC anaanlyalsyiss,i so,f otfhet hperipmraimrya rfylowfloerw-heer-ahdesa dosf tohfe t1h0e
R10omRoamneasnceos cclooncleosn, easre, agrievegniv ienn TianbTlea b5l.e T5h.eT bhieocboimocpoomupnodusn cdysnacyrinna, raipni,gaepniigne,n ainnd, alnudteolulitne,o wline,rew neoret
dneottedcteetdec tined ainnya nofy tohfet hseamsapmlepsl easnaanlyazlyezde. dH.oHwoewveevr,e rs,tsattaistitsictiacla dldififfefreernencecse saammoonngg ggeennoottyyppeess wweerree
ffoouunndd uunnddeerr tthhee bbiioocchheemmiiccaall pprrooffiillee ooff tthhee hheeaaddss,, wwhheenn aasssseessssiinngg ootthheerr bbiiooccoommppoouunnddss.. IInn ppaarrtticicuulalarr,,
SS33 aanndd SS55c lcolonnesesw wereerfeo ufonudntdo ctoo nctoanintatihne thhieg hheisgthceosntt ecnotnotefn1t, 5o-Of -1d,5ic-Oaf-fdeoicyalqffueionyilcqauciindi.cT ahceisde. cTlohnesees,
cinloandeds,i tiino nadtodSit2io1n,S t2o, SS1271,, SS128, ,Sa1n7d, SS2128,, wanedre Sa2ls2o, wchearrea catlesroi zcehdarbaycttehreizheidgh beys ttchoen hteingthoesftc hcolonrtoegnet noicf
cahcliodr.oSg5eannicd aSc2i3d.c lSo5n aensdco Sn2t3a icnleodneths ecohnigtahiensetdle tvheels hoigfhcyensta rleovsiedlse ,o(fT acybnlea5ro).sSid2e,,S (3T,aSb5l,ea n5)d. SS21,8 Sc3lo, nSe5s,
asnhdow Se1d8 cvlaolnueess sohfothweetdo tvaallpuoelsy pofh tehneo ltsomtale apsoulyrepdh:e1n3o.8ls4 ,m14e.a0s1u,r1e5d.2: 71,3a.8n4d, 1144..2001,g 1k5g.2-17,D aMnd, r1e4s.p2e0c gti vkegl-y1 .
DThMe,M reasnpteeclttivesetlyre. sTuhlets Mofanthteelb teiosct hreesmuilctas loafn thdem boiorcphheomloigciacla alntrda mitomrapthriocleosgsihcaolw treadita msalitgrhictelys sphooswitievde
as isglnigifihtclayn ptocsoirtrivelea stiiognnibfiectawnte econrtrheelaltaiotenn beestswoefegne tnhoet ylapteenpersosd oufc gtieonnotaynpde tphreod1,u5c-Otio-dni acanfdfe tohyel q1,u5i-nOic-
dacicidaffceoonytelqnuti(nPieca arcsiodn c’soncoternretl a(Ptieoanrscoone’fsfi ccoiernretlra=tio0n.6 c4o6e,fpfi≤cie0n.0t 5r) =. 0.646, p ≤ 0.05).
TTaabbllee 55.. BBiioocchheemmiiccaall cchhaarraacctteerriizzaattiioonn ((iinn gg··kkgg−−11 DDMM)) ooff tthhee hheeaaddss ooff tthhee 1100 RRoommaanneessccoo cclloonneess. .
1,5-O- Total Total
Chlorogenic Total Total
Clones 1,5-O-DDiciacfaffefoeyolqyulqinuiicnic Chlorogenic Cynaroside Caffeolyquinic Measured
Clones Acid AcidAcid Cynaroside Caffeolyquinic Measured
Acid AciAdscids PolPyoplhyepnholesnols
S2S020 4.17d4.17 d 3.623d.62 d 0.460b.46 b 7.797e.79 e 8.258e.25 e
S2S121 5.99c5.99 c 6.526a.,5b2 a,b 0.340c.,3d4 c,d 12.5112b.5,c1 b,c 12.8152b.,8c5 b,c
S2S2 6.44b6,c.44 b,c 7.007a.00 a 0.400b.,4c0 b,c 13.4143a.4,b4 a,b 13.8143a.,8b4 a,b
S3S3 7.05a7,b.05 a,b 6.746a.74 a 0.310d.31 d 13.7193a.7,b9 a,b 14.0114a.,0b1 a,b
S5 7.38a 7.30a 0.59a 14.68a 15.27a
S5 7.38 a 7.30 a 0.59 a 14.68 a 15.27 a
S11 5.96c 5.93b 0.43b 11.89c 12.32c
S1S711 3.49e5.96 c 6.635a,.b93 b 0.420b.,c43 b 10.1121d.89 c 10.5142d.32 c
S1S817 6.60b,3c.49 e 7.326.a63 a,b 0.280.d42 b,c 13.9120a.,b12 d 14.2010a,.b54 d
S2S218 4.936e.60 b,c 6.697a,.b32 a 0.410b.,c28 d 11.1632.c92 a,b 12.0134.c20 a,b
S2S322 3.53e4.93 e 4.426.c69 a,b 0.550.a41 b,c 7.9151e.62 c 8.5012e.03 c
Std.Error 0.265 0.266 0.030 0.527 0.535
S23 3.53 e 4.42 c 0.55 a 7.95 e 8.50 e
DiffSetrden. tElrerttoerr sindicatestatistic0a.2ll6y5s ignificantdiffe0re.2n6c6es amongclo0n.e0s3a0t p≤0.05(AN0O.5V2A7 analysis,Tuk0ey.5t3e5st ).
Different letters indicate statistically significant differences among clones at p ≤ 0.05 (ANOVA
4. Discussion
analysis, Tukey test).
This study characterized 10 autochthonous clones of Romanesco type through assessing a
combinationofmorphological,biochemical,andmoleculartraits. Thiscomplementaritywasused