CZECH FLAX AND HEMP COLLECTION CZECH FLAX AND HEMP COLLECTION MARTIN PAVELEK MARTIN PAVELEK AGRITEC, RESEARCH, BREEDING AGRITEC, RESEARCH, BREEDING AND SERVICES LTD AND SERVICES LTD AND SERVICES LTD. AND SERVICES LTD. SUMPERK, THE CZECH REPUBLIC SUMPERK, THE CZECH REPUBLIC
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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 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
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,
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
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
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
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
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
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
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
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 ☺☺