CZECH FLAX AND HEMP COLLECTIONCZECH FLAX AND HEMP COLLECTION

MARTIN PAVELEKMARTIN PAVELEKAGRITEC, RESEARCH, BREEDING AGRITEC, RESEARCH, BREEDING

AND SERVICES LTDAND SERVICES LTDAND SERVICES LTD.AND SERVICES LTD.SUMPERK, THE CZECH REPUBLICSUMPERK, THE CZECH REPUBLIC

OUTLINE OF THE LECTUREOUTLINE OF THE LECTUREOUTLINE OF THE LECTUREOUTLINE OF THE LECTURE

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONCZECH FLAX COLLECTIONCZECH FLAX COLLECTIONBASIC INFORMATIONBASIC INFORMATIONSTRUCTURE OF THE CZECH FLAX COLLECTIONSTRUCTURE OF THE CZECH FLAX COLLECTIONSTRUCTURE OF THE CZECH FLAX COLLECTIONSTRUCTURE OF THE CZECH FLAX COLLECTIONDOCUMENTATION SYSTEMDOCUMENTATION SYSTEMMETHODS OF EVALUATIONMETHODS OF EVALUATIONMETHODS OF EVALUATIONMETHODS OF EVALUATIONSEVERAL INFORMATIONS ABOUT HEMP SEVERAL INFORMATIONS ABOUT HEMP COOLECTIONCOOLECTIONCOOLECTIONCOOLECTIONCONCLUSIONSCONCLUSIONS

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONBASIC INFORMATIONBASIC INFORMATIONBASIC INFORMATIONBASIC INFORMATION

–– MANAGED IN AGRITEC LTD COMPANYMANAGED IN AGRITEC LTD COMPANYFINANCIALLY SUPPORTED BY THE CZECHFINANCIALLY SUPPORTED BY THE CZECH–– FINANCIALLY SUPPORTED BY THE CZECH FINANCIALLY SUPPORTED BY THE CZECH MINISTRY OF AGRICULTUREMINISTRY OF AGRICULTURECOORDINATED BY THE CENTRAL GENEBANKCOORDINATED BY THE CENTRAL GENEBANK–– COORDINATED BY THE CENTRAL GENEBANK COORDINATED BY THE CENTRAL GENEBANK IN PRAGUEIN PRAGUE--RUZYNE IN THE FRAMEWORK RUZYNE IN THE FRAMEWORK OF „NATIONAL PROGRAMME ON PLANTOF „NATIONAL PROGRAMME ON PLANTOF „NATIONAL PROGRAMME ON PLANT OF „NATIONAL PROGRAMME ON PLANT GENETIC RESOURCES CONSERVATION AND GENETIC RESOURCES CONSERVATION AND UTILIZATION“UTILIZATION“

–– DESCRIBED BY 25 PASSPORT AND 55 DESCRIBED BY 25 PASSPORT AND 55 SPECIAL DESCRIPTORS IN THE SPECIAL DESCRIPTORS IN THE FRAMEWORK OF THE NATIONALFRAMEWORK OF THE NATIONALFRAMEWORK OF THE NATIONAL FRAMEWORK OF THE NATIONAL DOCUMENTATION SYSTEM EVIGEZDOCUMENTATION SYSTEM EVIGEZ

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONBASIC INFORMATIONBASIC INFORMATIONBASIC INFORMATIONBASIC INFORMATION

2081 genotypes are maintained till the end 20092081 genotypes are maintained till the end 2009

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONECOGEOGRAPHICAL REPRESENTATIONECOGEOGRAPHICAL REPRESENTATION

1536

ASIA

AUSTRALIA

NORTHAMERICASOUTHAMERICAAFRICA

88514813982 EUROPE

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONO OO OSTRUCTURE BASED ON ORIGINSTRUCTURE BASED ON ORIGIN

26%26%Landraces

24%

50% BreedingmaterialVarieties24% Varieties

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONSTRUCTURE BASED ON TYPE OF USESTRUCTURE BASED ON TYPE OF USE

Fibre

Mixedtypes

39%Linseed

53%

8%

CZECH DOCUMENTATION SYSTEMCZECH DOCUMENTATION SYSTEM

CZECH DOCUMENTATION SYSTEMCZECH DOCUMENTATION SYSTEM

http://genbank.vurv.cz/genetic/resources/asp2/default_a.htm

CZECH DOCUMENTATION SYSTEMCZECH DOCUMENTATION SYSTEMcontributing institutescontributing institutescontributing institutescontributing institutes

CZECH FLAX COLLECTIONCZECH FLAX COLLECTIONCONTEMPORARY SITUATION IN DESCRIPTIONCONTEMPORARY SITUATION IN DESCRIPTION

2081 ACCESSIONS ARE INCLUDED INTO THE 2081 ACCESSIONS ARE INCLUDED INTO THE COLLECTION NOWCOLLECTION NOWCOLLECTION NOWCOLLECTION NOW2040 ACCESSIONS (98.03 %) ARE DESCRIBED 2040 ACCESSIONS (98.03 %) ARE DESCRIBED BY PASSPORT DESCRIPTORSBY PASSPORT DESCRIPTORSBY PASSPORT DESCRIPTORSBY PASSPORT DESCRIPTORS700 ACCESSIONS (33,64 %) ARE DESCRIBED 700 ACCESSIONS (33,64 %) ARE DESCRIBED BY SPECIAL DESCRIPTORSBY SPECIAL DESCRIPTORSBY SPECIAL DESCRIPTORS BY SPECIAL DESCRIPTORS

DNA ANALYSISDNA ANALYSISGENERAL SCHEME BEING USED BY FLAXGENERAL SCHEME BEING USED BY FLAX

ISOLATION OF DNA FROM PLANT ISOLATION OF DNA FROM PLANT MATERIAL (LEAVES SEEDS)MATERIAL (LEAVES SEEDS)MATERIAL (LEAVES, SEEDS)MATERIAL (LEAVES, SEEDS)PURIFICATION OF ISOLATED DNAPURIFICATION OF ISOLATED DNAPCR AMPHLIFICATION WITH RAPD PCR AMPHLIFICATION WITH RAPD PRIMERSPRIMERSSEPARATION OF AMPHLIFICATION SEPARATION OF AMPHLIFICATION PRODUCT BY ELECTROPHORESIS ON 2 % PRODUCT BY ELECTROPHORESIS ON 2 % AGAROSIS GELAGAROSIS GELAGAROSIS GELAGAROSIS GELPHOTODOCUMENTATION AND PHOTODOCUMENTATION AND ELECTROPHOREOGRAMS EVALUATION BYELECTROPHOREOGRAMS EVALUATION BYELECTROPHOREOGRAMS EVALUATION BY ELECTROPHOREOGRAMS EVALUATION BY COMPUTERCOMPUTER

DNA ANALYSISDNA ANALYSISsampling of plant materialsampling of plant material

15 plants were 15 plants were sampled from everysampled from everysampled from every sampled from every accessionaccessionRAPD and ISSR RAPD and ISSR ddmethods were usedmethods were usedCTAB CTAB –– DNA isolation, DNA isolation, DNA bankDNA bank

MMoleolecular markers usedcular markers usedMMoleolecular markers usedcular markers used

NowadaysNowadays: : RAPDRAPDRAPD RAPD ISSRISSR

FutureFuture::mimiccrosatelitrosatelittete (SSR) repeti(SSR) repetitiontionmimiccrosatelitrosatelitte te (SSR) repeti(SSR) repetitiontionretrotransposonretrotransposonesesIRAP IRAP (Inter(Inter--Retrotransposone Amplified Polymorphism)Retrotransposone Amplified Polymorphism)(( p p y p )p p y p )RBIP RBIP (Retrotransposone Based Inzertion Polymorphism)(Retrotransposone Based Inzertion Polymorphism)

Molecular methodsMolecular methodsRAPD markers for flax genotypizationRAPD markers for flax genotypizationRAPD markers for flax genotypizationRAPD markers for flax genotypization

40 RAPD primers (Operon) were tested40 RAPD primers (Operon) were testedMaterial: 12 chosen flax and linseed genotypesMaterial: 12 chosen flax and linseed genotypes508 markers were found/104 polymorphic (average508 markers were found/104 polymorphic (average508 markers were found/104 polymorphic (average 508 markers were found/104 polymorphic (average 10/2)10/2)

OPD20OPD20 OPW08OPW08

ISSR markers for flax genotypizationISSR markers for flax genotypizationISSR markers for flax genotypizationISSR markers for flax genotypization

• 12 ISSR primers were tested12 ISSR primers were tested

•• low polymorphism was foundlow polymorphism was found

CT 9CT 9 AG 9AG 9 808 GA808 GA

Example of Example of ISSR aISSR andnd RAPD analRAPD analysisysispp yy

AdvantagesAdvantages::* * ssimplicity of realizationimplicity of realizationp yp y* * not necessary to know not necessary to know

sequencesequence* robustn* robustnessess robustn robustnessess

DisadvantagesDisadvantages::gg* * low level low level polymorpolymorphismphism* * low information valuelow information value** lowlow reproducibilitreproducibilityy* * lowlow reproducibilitreproducibilityy* * pproblroblems ems multiplex PCR multiplex PCR * * difficult evaluationdifficult evaluationISSR RAPDISSR RAPDISSR RAPDISSR RAPD

1212 primers testedprimers tested 40 s40 setsets

AFLP analAFLP analysis of flax ysis of flax genotypgenotypesesyy g ypg yp

EcoRI-MseI

IMAGE ANALYSISIMAGE ANALYSIScolour image formatg

*tiff; resolution of 8bit per channel512 x 512 spatial resolution256 grays for each colour

Software NIS Elements AR 2 30 (Laboratory Imaging LtD PragueSoftware NIS Elements AR 2.30 (Laboratory Imaging LtD. Prague, Czech Republic)

conversion of colour image to binary form (object and background)modification by software editor (mathematical morphology) erosion,modification by software editor (mathematical morphology) erosion,

dilatace etc. measurements of parameters

Image analysis Image analysis -- software LUCIA 4.61software LUCIA 4.61

AreaMaxFeret

Flower morphology description and evaluation

Gentiana variety Gentiana variety –– scan original picture of flower petalsscan original picture of flower petalsyy g p pg p p

Gentiana variety Gentiana variety –– binary picturebinary picture

Image analysis Image analysis –– obtained resultsobtained results

digital photopgraphs of two sets X13 accessions per digital photopgraphs of two sets X13 accessions per g p pg p pg p pg p p350 accessions for each one were made350 accessions for each one were madeflower morphology at 430 accessions were flower morphology at 430 accessions were documented: corolla flower petals flower sepalsdocumented: corolla flower petals flower sepalsdocumented: corolla, flower petals, flower sepals, documented: corolla, flower petals, flower sepals, stamens, anthersstamens, anthersdigital photographs of stem and bolls were made atdigital photographs of stem and bolls were made atdigital photographs of stem and bolls were made at digital photographs of stem and bolls were made at 700 accessions 700 accessions –– above perspective, perspective above perspective, perspective from the side, section of boll from the side, section of boll

f ff fparametres of image analysis for chosen parametres of image analysis for chosen characteristics will be calculated and used for characteristics will be calculated and used for accessions identificationaccessions identification –– finding of similarity orfinding of similarity oraccessions identification accessions identification finding of similarity or finding of similarity or dissimilarity, duplicates detectiondissimilarity, duplicates detection

Photodocumentation of flowers for image Photodocumentation of flowers for image analysisanalysisanalysisanalysis

700 genotypes 700 genotypes from flax collection from flax collection were evaluated for were evaluated for image analysisimage analysis P 6906P 6906

11--196196--12 Selection RA12 Selection RA

Ottawa C.I. 355

Photodocumentation of stem and generative Photodocumentation of stem and generative organs for image analysisorgans for image analysisorgans for image analysisorgans for image analysis

Digital photographs of stemDigital photographs of stemand bolls and bolls -- above above

ti titi tiperspective, perspective perspective, perspective from the side, section of bollfrom the side, section of boll

350 X13 accessions350 X13 accessionsanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor and analysis according to the ACCENAME descriptor and

image analysis for duplicate identificationimage analysis for duplicate identification

ACCENUMBACCENUMB ACCENAMEACCENAME ORGCTYORGCTY USETYPEUSETYPE01700170 BUCK 114BUCK 114 ARGARG 3300940094 BUCK 114BUCK 114 ARGARG 3300940094 BUCK 114BUCK 114 ARGARG 33

01700170 00940094

350 X13 accessions350 X13 accessionsanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor and analysis according to the ACCENAME descriptor and

image analysis for duplicate identificationimage analysis for duplicate identification

ACCENUMBACCENUMB ACCENAMEACCENAME ORGCTYORGCTY USETYPEUSETYPE00270027 FLEISCHMANNUVFLEISCHMANNUV HUNHUN 3300590059 FLEISCHMANNUVFLEISCHMANNUV HUNHUN 1 ?1 ?

00270027 00590059

350 X13 accessions350 X13 accessionsanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor and analysis according to the ACCENAME descriptor and

image analysis for duplicate identificationimage analysis for duplicate identification

ACCENUMBACCENUMB ACCENAMEACCENAME ORGCTYORGCTY USETYPEUSETYPE00610061 MAROCMAROC MARMAR 3302690269 MAROCMAROC MARMAR 33

00610061 02690269

350 X13 accessions350 X13 accessionsanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor andanalysis according to the ACCENAME descriptor and analysis according to the ACCENAME descriptor and

image analysis for duplicate identificationimage analysis for duplicate identification

ACCENUMBACCENUMB ACCENAMEACCENAME ORGCTYORGCTY USETYPEUSETYPE00240024 LA PREVISIONLA PREVISION ARGARG 3301710171 LA PREVISIONLA PREVISION ARGARG 33

00240024 01710171

SEED TYPE identification by using IMAGE ANALYSIS Correlation of NUTRITIVE COMPOUNDS with SIZE, SHAPE and COLOUR CHARACTERISTICS

Smýkalová I. 1, Pavelek M.2, Bjelková M. 2, Hýbl M. 3, Horáček J 1 Hampl D 4 Krulikovská T 5Horáček J. , Hampl D. , Krulikovská T.

1Department of Biotechnology, 2Department of Technical Crops, 3Department of Grain p gy, p p , pLegumes, Agritec Plant Research, Ltd. Zemědělská 16, Šumperk, Czech Republic; 4

National Plant Variety Office, ÚKZÚZ, Hroznová 2, 656 06 Brno, Czech Republic, 5Department of Fermentative Chemistry and Bioengineering, Institut of Chemical

T h l P T h i ká 5 166 28 P C h R bliTechnology Prague, Technická 5, 166 28 Prague, Czech Republic

The aims of the study:

utilization of DIA in germplasm collections of pea and flax

seeds as studied objects ….. testing sets

evaluation of size a shape (quantitative characteristics) by DIA parameters

utilization of scanner for acquisition of colour images

evaluation of colour (qualitative characteristics ) by DIAevaluation of colour (qualitative characteristics ) by DIA parameters

statistical analysis of the DIA datastatistical analysis of the DIA data

clustering of varieties based on the DIA results

correlation analysis of DIA data and compounds

Material and Methods

Plant materialDry mature seeds of model plants

Fl /li d ll l d i d d i l fFlax/linseed - small oval seeds pointed to one end in colour range from yellow to dark brown Pea – large round shape seeds with colour between yellow and gray green

Analysis of 16 varieties (seeds collection of Agritec Ltd., Šumperk)

Flax/linseeFlax/linseedd

*characterizatio*characterizationn

PeaPea characterizationcharacterization

AA LL (2 7%) llLL (2 7%) ll AlAl S (53 81%) llS (53 81%) ll

*Low linolenic genotypes(LL) linseed typehigh linolenic genotypes

AmonAmon LL (2.7%) yellowLL (2.7%) yellow AlanAlan S (53.81%) yellowS (53.81%) yellow

BonetBonet HL (50.3%) brownHL (50.3%) brown BohatýrBohatýr S (56.16%) yellowS (56.16%) yellow

JantarJantar LL (2.1%) yellowLL (2.1%) yellow HeroldHerold S (56.76%) yellowS (56.76%) yellow

JitkaJitka HL (55 2%) brownHL (55 2%) brown JanusJanus S (50 15%) yellowS (50 15%) yellow

(HL) flax or linseed

JitkaJitka HL (55.2%) brownHL (55.2%) brown JanusJanus S (50.15%) yellowS (50.15%) yellow

LolaLola HL (4.1%) brownHL (4.1%) brown RomeoRomeo S (53.27%) yellowS (53.27%) yellow

MarylinMarylin HL (57.8%) brownHL (57.8%) brown SmaragdSmaragd S (51.75%) greenS (51.75%) green

RinaRina HL (61 4%) brownHL (61 4%) brown TyrkysTyrkys S (54 02%) greenS (54 02%) greenRinaRina HL (61.4%) brownHL (61.4%) brown TyrkysTyrkys S (54.02%) greenS (54.02%) green

OmegaOmega HL (54.2%) yellowHL (54.2%) yellow ZekonZekon S (50.62%) greenS (50.62%) green

Scheme of work on/with binary image

colour image segmentation by treshold setting +colour image segmentation by treshold setting + supplement

F11 Programme editor: clean, erosion etc. F5 measure

Evaluation of shape and size of seeds by flatbed scanner Canon 4400F (Canon Inc., USA)

Test of stability: comparison of 2 types of scanner: 4400F and 8800F ofCannon

calibration of colour perception; 100dpi; 0-255 (white); 0.08mm/px; MiniColorChecker (X-Rite Inc.USA) white colour (R=243, G=243, B=242)( ) ( , , )

3x288 seeds per flax/linseed genotype ; 3x237 seeds per pea genotype

FLAX PEA

Quantitative and qualitative parameters images from scanner Canon type 4400Ffrom scanner Canon type 4400F

AreaArea - main criterion of the object size.PerimeterPerimeter dimension of total bo de calc lated f om 4 p ojection inPerimeterPerimeter - dimension of total border; calculated from 4 projection indirections 0, 45, 90 a 135o based on Crofton´s equal:π*(Pr0+Pr45+Pr90+Pr135)/4EqDiameterEqDiameter - criterion of size derived from area; mean of circle abotarea coincident with the are of object: sqrt(4*Plocha/ π)MaxFeret,MaxFeret, MinFeretMinFeret - maximum/minimum from Feret´s diameter,projection length of the object at the angle = {0o, 10o, 20o, …, 180o}ElongationElongation ratio MaxFeret/MinFeretCircularityCircularity - the most useful criterion of size evaluation derived from theCircularityCircularity the most useful criterion of size evaluation derived from thespace and the perimeter; Circularity = ;space/perimeter2*ּת*4 1 = circleand other shapes < 1MeanRedMeanRed MeanGreenMeanGreen MeanBlueMeanBlue - means intensity of colourMeanRed,MeanRed, MeanGreen,MeanGreen, MeanBlueMeanBlue means intensity of colourchannelsMeanSaturationMeanSaturation - mean of colour saturationHueTypicaHueTypical most widely used (typical) hue of colour derived fromHueTypicaHueTypical – most widely used (typical) hue of colour derived frommaximum in hue histogram

Qualitative evaluation of colour characteristics of seeds by macro-opticsseeds by macro optics

Pentax Cosmicar equipped with digital camera DS-U1 (5 Mpixel)Nikon (Nikon Instruments, CS-Optoteam, Prague)M nsellColor table Mini ColorChecker (X Rite Inc USA)MunsellColor table Mini ColorChecker (X-Rite Inc.USA)Measurements: coordinates L,a,b in CIE-LAB colour space

2 5 l f fl d 50 100 d f th bl k2.5g sample of flax seeds or cca 50-100 seeds of pea - on the blackvelour Colour of flax:

(1) yellowColour of pea:(1) light yellow ( ) y

(2) olive green (3) light brown(4) brown

( ) g y(2) yellow-pink(3) waxing(4) yellow-green(4) brown

(5) black-brown(6) dark brown

(4) yellow green (5) dark-green (6)gray-green (7)light brown(7)light brown(8) brown(9) black

method for evaluation of objects colouring

Colour deviation: ΔEa,b ΔEa,b=(ΔL2+Δa2+Δb2)1/2

Chemical analytical methodsAnalysis of fatty acids in flax seedsAnalysis of fatty acids in flax seedsby gas chromatography according to technical norm ČSN ISO5508Determination of total starch in pea seedsDetermination of total starch in pea seedsby NIRS (Near Infra-Red Spectroscopy); calibration of deviceusing commercial kit Megazyme „Total starch“, assay methodAOAC 996 11AOAC 996.11

Statistical analysis of dataSoftware NIS Elements AR 2.30 (Laboratory Imaging LtD.Prague, Czech Republic)- automatic export of data to Excel (Microsoft Office 2007,p ( ,

USA)ANOVA Statistica ver.8.0 (StatSoft Inc., USA)– variability of each parameter among genotypesvariability of each parameter among genotypesEvaluating computing system MATLAB R2009a– correlation analysis (one DIAparametr x content ofcompounds in seed)compounds in seed)- clustering from all DIA parameters

Differences in a group of evaluated varieties

15 measured parameters

FLAX/LINSEED:AMON 13 t t P i t d M F tAMON - 13 parameters except Perimetr and MaxFeretJANTAR - Cirularity, HueTypical, aBONET – ElongationFinal dendrogram indicate 2 clusters yellow-seeded (Amon, Jantar)and brown-seeded (Bonet, Jitka, Marylin, Rina, Lola).

PEA:more variable parameter Circularity in comparison to flax;dendogram of 2 different clusters green-seeded (Tyrkys, Zekon,dendogram of 2 different clusters green seeded (Tyrkys, Zekon,Smaragd) and yellow-seeded (Herold, Romeo, Bohatýr, Janus, Alan)

Histogram of MeanRed, MeanGreen and MeanBlue generated from the DIA data

ParameterParameter MeanMean SDSD MinMin MaxMax

MeanRedMeanRed 185.21185.21 7.27.2 165.71165.71 199.06199.06

MeanGreenMeanGreen 142.96142.96 8.628.62 120.41120.41 162.36162.36

MeanBlueMeanBlue 99.1599.15 9.599.59 74.4374.43 121.23121.23

Sample was separately analyzed for each seed → for each variety means, minimum, maximum, standard deviation and histogram from

measured values

Qualitative evaluation of coloring of seedsColour calibration sets

to selection of standard/references varietyto selection of standard/references varietyto acquire typical „calibration seed“

Advance: L a b and means of 100 seeds of the selected varietyAdvance: L,a,b and means of 100 seeds of the selected varietythe seeds, which corresponded to the most close means of the100 seeds (the lowest of ΔEa,b) were selected as colour markert lib ti l tto calibration colour set

For flax : 4 different clusters – yellow, ligth, medium and darkb ll d b DIA5brown anallyzed by DIA5.

For pea: 2 categories – yellow (brown-yellow, orange and yellow)and green category: ligth green, olive green, green8.

BUT: colour of the seeds is more variable character

8 Public notice no. 329/1997Sb (law no. 110 Ministry of Agriculture of CR).

samplesampleAmonAmon

Amon1Amon1 Amon2Amon2AmonAmon33

LL aa bb LL aa bb LL aa bb7878..784784 5.4865.486 28.1628.16 78.7678.76 5.4585.458 28.3728.37 79.5079.50 5.1265.126 28.0528.05

C lib tiC lib tiNo.No.

Calibration Calibration setset

11 70.79370.793 4.1254.125 26.4626.46 71.4171.41 3.9993.999 25.5625.56 71.5871.58 4.0214.021 25.7525.7522 64.05064.050 5.3315.331 26.1826.18 64.3964.39 5.2495.249 25.9525.95 64.9564.95 5.2265.226 25.9625.9633 59.50759.507 13.3913.39 30.0930.09 60.1560.15 13.17413.174 30.3930.39 60.1660.16 13.10413.104 30.4230.4244 59.74959.749 7.2237.223 18.8418.84 60.3460.34 7.1127.112 18.6118.61 60.2860.28 7.0617.061 18.5318.5355 54.80454.804 12.2712.27 23.3823.38 55.2055.20 12.1512.15 23.7523.75 54.9154.91 12.2912.29 22.9822.9866 46 85246 852 7 5037 503 14 3314 33 47 2947 29 7 3057 305 13 9713 97 47 2647 26 7 0787 078 14 5714 5766 46.85246.852 7.5037.503 14.3314.33 47.2947.29 7.3057.305 13.9713.97 47.2647.26 7.0787.078 14.5714.57Results Results ΔΔEa,bEa,b

yellowyellow olive greenolive green yellowyellow--brownbrown graygray--brownbrown brownbrown blackblack--brownbrown

Amon1Amon1 8.2818.281 14.86614.866 20.92520.925 21.26421.264 25.37725.377 34.85434.854

Amon2Amon2 8.0048.004 14.57414.574 20.25620.256 20.92220.922 24.93124.931 34.65934.659

Amon3Amon3 8.3138.313 14.69914.699 21.05621.056 21.52921.529 26.10926.109 34.99434.994

AVG AmonAVG Amon 8.1998.199 14.71314.713 20.74620.746 21.23821.238 25.47225.472 34.83634.836

RESULTS of testing set: genotype AMON …. yellow type of flax

Colour qualitaties of seeds is important distinctive characteristics for:

determination of colour levelcreate clusters

In a case of coincident colour level of seeds (2 ≥ varieties):In a case of coincident colour level of seeds (2 ≥ varieties):quantitative evaluation of size and shape of seeds

Si ifi t ff t th l f tSignificat effect on the value of parameters:??year (age of seeds)

quality of seeds (diseases, maturity etc.)conditions of storing

In the study:ybrown genotype Marylin (flax)calculated ΔEa,b for independent 3 years: 2005; 2007; 2008ΔEa,b = 3,467; 3,656; 3,837ΔEa,b 3,467; 3,656; 3,837RESULTS: Effect of year on the brown colour of seeds in flax isirrelevantFor future:For future:!! nevertheless to have conversion coeficient to filtrate theeffect !!

Correlation of DIA data with compounds

Flax/linseed: Mean values of colour characteristics MeanRed, MeanGreen, MeanBlue (left) and content of fat and fatty acidsMeanGreen, MeanBlue (left) and content of fat and fatty acids

oleic, linolic and linolenic (right)

Evaluating computing system MATLAB R2009aEvaluating computing system MATLAB R2009a – correlation analysis (parametr x compounds in seed)

Significant correlation between content of fat in seeds and some of DIA parameters

FLAX /LINSEEDParametrParametr correlationcorrelation

fatfat xxparameterparameter

pp--valuevalue minimumminimum maximummaximum

/

AreaArea 0.7510.751 0.0520.052 --0.0040.004 0.96210.9621EqDiameterEqDiameter 0.7650.765 0.0450.045 0.0290.029 0.9630.963PerimeterPerimeter 0.3810.381 0.4000.400 --0.5220.522 0.8810.881MaxFeretMaxFeret 0.5140.514 0.2380.238 --0.3900.390 0.9140.914MinFeretMinFeret 0.5200.520 0.2310.231 --0.3830.383 0.9150.915CircularityCircularity --0.0430.043 0.9270.927 --0.7710.771 0.7340.734ElongationElongation 0.31920.3192 0.4850.485 --0.5710.571 0.8640.864MeanIntensityMeanIntensity 0.8620.862 0.0130.013 0.3100.310 0.9790.979MeanRedMeanRed 0.8620.862 0.0130.013 0.3100.310 0.9790.979MeanGreenMeanGreen 0.8550.855 0.0140.014 0.2870.287 0.9790.979MeanBlueMeanBlue 0.8390.839 0.0180.018 0.2320.232 0.9760.976HueTypicalHueTypical 0.8290.829 0.0210.021 0.2030.203 0.9740.974MeanSat ationMeanSat ation 0 5560 556 0 1950 195 0 3400 340 0 9230 923MeanSaturationMeanSaturation 0.5560.556 0.1950.195 --0.3400.340 0.9230.923MeanBrightnesMeanBrightnesss 0.8620.862 0.0130.013 0.3100.310 0.9790.979LL 0 8630 863 0 0120 012 0 3130 313 0 9800 9800.8630.863 0.0120.012 0.3130.313 0.9800.980aa --0.8530.853 0.0150.015 --0.9780.978 --0.2800.280bb 0.8420.842 0.0180.018 0.2430.243 0.9760.976FatFat 11 11 11 11

Conclusions

• to objective and just and comprehensive description of genotypegenotype

• to explore results of both, quantitative (Area, EqDiameter etc ) and qualitative (L a b) DIA of seedsetc.) and qualitative (L,a,b) DIA of seeds

• statistical analysis of DIA data provided by computing (MATLAB R2009 t )programme (MATLAB R2009a etc.)

• function and automatized evaluating technology

• utillization in descriptions and originality in germplasm collections

• quick searching accessions in databases of collection

Hemp varieties trials in 2010Hemp varieties trials in 201016 foreign varieties included16 foreign varieties included16 foreign varieties included16 foreign varieties included

1.1. Bialobrzeskie  (POL)Bialobrzeskie  (POL)2.2. JUSO JUSO –– 31 (UKR)31 (UKR)3.3. Ferimon (FRA)Ferimon (FRA)4.4. Monoica (HUN)Monoica (HUN)

( )( )5.5. Fibrol (HUN)Fibrol (HUN)6.6. Futura 75 (FRA)Futura 75 (FRA)7.7. Fedora 19 (FRA)Fedora 19 (FRA)88 F li (FRA)F li (FRA)8.8. Felina (FRA)Felina (FRA)

99 Santhica 27 (FRA)Santhica 27 (FRA)9.   9.    Santhica 27 (FRA)Santhica 27 (FRA)10.  Beniko (POL)10.  Beniko (POL)11.11. Epsilon 68 (FRA)Epsilon 68 (FRA)12.12. KFKF‐‐TC (HUN)TC (HUN)12.12. KFKF TC (HUN)TC (HUN)13.13. Chamaeleon (FRA)Chamaeleon (FRA)14.14. Tygra (POL)Tygra (POL)15.15. Finola (FIN)Finola (FIN)( )( )16.16. Uniko B (HUN)Uniko B (HUN)

Hemp varieties maintained in genebank in Hemp varieties maintained in genebank in AGRITEC Ltd. ŠumperkAGRITEC Ltd. ŠumperkAGRITEC Ltd. ŠumperkAGRITEC Ltd. Šumperk

–– 19 varieties are included19 varieties are included

–– Slovakia Slovakia –– 2:2:RASTISLAVICKÉ, KRAJOVÉ Z ČERVENIKARASTISLAVICKÉ, KRAJOVÉ Z ČERVENIKA

–– HungaryHungary –– 2:2:–– Hungary Hungary –– 2:2:FIBROL, MONOICA FIBROL, MONOICA

–– Poland Poland –– 3:3:BENIKO, SILESIA, BIALOBRZEZSKIEBENIKO, SILESIA, BIALOBRZEZSKIE

–– Ukraine Ukraine –– 3:3:JUSO 11 JUSO 32 JUSO 34JUSO 11 JUSO 32 JUSO 34JUSO 11, JUSO 32, JUSO 34JUSO 11, JUSO 32, JUSO 34

–– Romaina Romaina –– 1:1:LOVRIN 110LOVRIN 110

–– France France –– 7:7:FEDORA 17, FUTURA 75, FELINA 32, FERIMON 12, FEDORA 17, FUTURA 75, FELINA 32, FERIMON 12, EPSILON SANTHICA 27 CHAMAELEONEPSILON SANTHICA 27 CHAMAELEONEPSILON, SANTHICA 27, CHAMAELEONEPSILON, SANTHICA 27, CHAMAELEON

–– Finland Finland –– 1: 1: FINOLAFINOLA

CONCLUSIONSCONCLUSIONS

2081 ACCESSIONS ARE MAINTAINED IN THE CZECH 2081 ACCESSIONS ARE MAINTAINED IN THE CZECH FLAX COLLECTION AT THE END 2009 COVERING ASFLAX COLLECTION AT THE END 2009 COVERING ASFLAX COLLECTION AT THE END 2009 COVERING AS FLAX COLLECTION AT THE END 2009 COVERING AS MUCH GENETIC DIVERSITY OF MUCH GENETIC DIVERSITY OF LINUM USITATISSIMUMLINUM USITATISSIMUML. AS POSSIBLEL. AS POSSIBLENEW PROGRESSIVE METHODS OF FLAX GENETIC NEW PROGRESSIVE METHODS OF FLAX GENETIC RESOURCES ARE BEING DEVELOPED BASED ON DNA RESOURCES ARE BEING DEVELOPED BASED ON DNA METHODS RAPD AND ISSRMETHODS RAPD AND ISSRMETHODS RAPD AND ISSRMETHODS RAPD AND ISSRWE ARE GOING TO VERIFY THE UTILIZATION OF THE WE ARE GOING TO VERIFY THE UTILIZATION OF THE LAST MODERN METHODS LIKE MICROSATELITTE (SSR) LAST MODERN METHODS LIKE MICROSATELITTE (SSR) REPETITION, RETROTRANSPOSONE, REPETITION, RETROTRANSPOSONE, IRAPIRAP AND RBIPAND RBIP(Inter(Inter--Retrotransposone Amplified PolymorphismRetrotransposone Amplified Polymorphism,,Retrotransposone Based Inzertion Polymorphism)Retrotransposone Based Inzertion Polymorphism)Retrotransposone Based Inzertion Polymorphism)Retrotransposone Based Inzertion Polymorphism)

CONCLUSIONSCONCLUSIONS –– TO BE CONTINUEDTO BE CONTINUEDCONCLUSIONS CONCLUSIONS TO BE CONTINUEDTO BE CONTINUED

IMAGE ANALYSIS LIKE THE NEW LASTIMAGE ANALYSIS LIKE THE NEW LASTIMAGE ANALYSIS LIKE THE NEW LAST IMAGE ANALYSIS LIKE THE NEW LAST METHOD OF FLAX GENETIC RESOURCES METHOD OF FLAX GENETIC RESOURCES EVALUATION HAS BEEN USED FOREVALUATION HAS BEEN USED FOREVALUATION HAS BEEN USED FOR EVALUATION HAS BEEN USED FOR DISTINCTION OF ACCESSIONS AND DISTINCTION OF ACCESSIONS AND DUPLICATE SEARCH SINCE 2005DUPLICATE SEARCH SINCE 2005

Thank you for your attention Thank you for your attention ☺☺