Molecular Characterization of Mutant Germplasm A Manual Prepared by the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture Vienna 2010 FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization FOREWORD 1 FOREWORD Plantbiotechnologyapplicationsmustnotonlyrespondtothechallengeofimprovingfood securityandfosteringsocio-economicdevelopment,butindoingso,promotethe conservation,diversificationandsustainableuseofplantgeneticresourcesforfoodand agriculture.Nowadaysthebiotechnologytoolboxavailabletoplantbreedersoffersseveral newpossibilitiesforincreasingproductivity,cropdiversificationandproduction,while developingamoresustainableagriculture.Thistrainingcoursefocusesononeofthemost promising set of techniques used in modern crop improvement programmes, i.e. on molecular markers.Thesearerapidlybeingadoptedbyplantbreedersandmolecularbiologistsas effective and appropriate tools for basic and applied studies addressing biological components in agricultural production systems. Their use in applied breeding programmes can range from facilitatingtheappropriatechoiceofparentsforcrosses,tomapping/taggingofgeneblocks associatedwitheconomicallyimportanttraits(oftentermedquantitative trait loci (QTLs)). Gene tagging and QTL mapping in turn permit marker-assisted selection (MAS) in backcross, pedigree,andpopulationimprovementprogrammes,therebyfacilitatingmoreefficient incrementalimprovementofspecificindividualtargettraits.Andthroughcomparative genomics,molecularmarkerscanbeused in ways that allow us to more effectively discover and efficiently exploit biodiversity and the evolutionary relationships between organisms. Substantialprogresshasbeenmadeinrecentyearsinmapping,taggingandisolatingmany agriculturallyimportantgenesusingmolecularmarkersdueinlarge part to improvements in thetechniquesthathavebeendevelopedtohelpfindmarkersofinterest.Amongthe techniquesthatareparticularlypromisingareRestrictionFragmentLengthPolymorphism (RFLPs),AmplifiedFragmentLengthPolymorphism(AFLPs),RandomAmplified Polymorphic DNA (RAPDs), Microsatellites and PCR based DNA markers such as Sequence CharacterizedAmplifiedRegions(SCARs)orSequenceTaggedSites(STS).These techniqueshelpindirectselectionofmanydesiredcharacterssimultaneouslyusingF2and back-cross populations, near isogenic lines, doubled haploids and recombinant inbred lines. Intherecentpast,theworldofclassicalMendeliangeneticshasenteredanewage,namely that of genomics, which means the study of structure of genes and their function. A great deal of DNA sequence information is now available in particular from model species such as rice andArabidopsis,butthefunctionsofthederivedgenesaremostlyunknown.Concentrated researcheffortsarethereforebeingmadetofillthisso-calledphenotypegap.Induced mutationscombinedwithmolecularmarkertechnologyareplayinganimportantroleinthis field,leadingtoareinforceddemandformutagenizedplantmaterialinwhichcertain charactershavebeenchangedduetoknockoutmutationsoftheresponsiblegenes.Using molecular and genetic tools, a mutated character can then be associated with a DNA sequence of previously unknown function. Recent reports on the homology of genes and the gene order betweenforinstancethegrassgenomes(synteny)suggestthattheknowledgeacquiredwill also be useful for identification and isolation of genes from under-utilised crops. The first print of this manual on selected molecular marker techniques was prepared using the handoutsandothermaterialsdistributedtoparticipantsoftheFAO/IAEAInterregional TrainingCourseon"Mutantgermplasmcharacterisationusingmolecularmarkers"thatwas FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization FOREWARD 2 hosted by the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture at the Plant Breeding Unit (PBU) of the Agriculture and Biotechnology Laboratory at the IAEA LaboratoriesinSeibersdorf,Austria.MessrsJ.Bennetzen(USA),K.Devos(UK),G.Kahl (Germany),U.Lavi(Israel),M.Mohan(ICGEB)andS.Nielen(FAO/IAEA)contributed protocolstothefirstprintversion.TheircontributionsandthoseofMessrsP.Gustafson (USA),B.Forster(UK),M.Gale(UK)andR.Adlam(UK)andM.MaluszynskiandS. Nielen of the Joint Programme in the compilation, layout and editing of the manual are deeply appreciated. These protocols have been particularly useful for course participants and other investigators in molecular biology for carrying out assays relating to many molecular marker techniques such asgenomicDNAisolation,restrictionanalysisofgenomicandplasmidDNA,gel electrophoresis,SoutherntransferofgenomicDNA,non-radioactiveDNAhybridization, silverstaining,RFLP,AFLP,SSR,ISSRandRAPDanalysis,andretrotransposon-based marker systems. Sincethefirstissueofthissetofprotocolsin2001,severalmodificationshavebeenmade based on validated protocols in use at the PBU. These modified protocols along with two new modules, Targeting Induced Local Lesions IN Genomes (TILLING) and Population Genetics are included in this revised version. Thus, this revised version reflects the continuing efforts in the Joint Programme to provide an all embracing suite of tools to facilitate the enhancement of capacityininducedcropmutagenesisinMember States. The contributions of Messrs B. Till (FAO/IAEA) and J Fernandez-Manjarres (Colombia), respectively to these two new modules are gratefully acknowledged. Several staff members of the PBU have also invested substantial efforts in revising this manual and these efforts are acknowledged. This second version is retaining the loose-leaf format of the first issue in the expectation that other molecular marker systems and protocols will be added. We would very much appreciate suggestionsandcomments,whichcouldfurtherimproveandenrichthecontentsofthis manual.CorrespondenceshouldbeaddresseddirectlytoMr.PJLLagoda,Head,Plant Breeding and Genetics Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture,P.O.Box100,Vienna,Austria,Telephone:+431260021626;email P.Lagoda@iaea.org. ThemanualwillalsobeavailableontheFAO/IAEAJointDivisionwebsitehttp://www-naweb.iaea.org/nafa/pbg/public/manuals-pbg.html.AhardcopywithattachedCD-ROMwill bedistributed,freeofcharge,tointerestedscientistsfromFAOandIAEAMemberStates. Requests for the manual should be sent to Ms. K. Allaf, Plant Breeding and Genetics Section, JointFAO/IAEADivisionofNuclearApplicationinAgriculture,P.O.Box100,Vienna, Austria,Telephone:+431260021621orbyemail:K.Allaf@iaea.org.Itisstronglyadvised thatrequestsbemadeasindividualsinordertofacilitatetheinclusionofalluserscontact informationinamailinglistforthedistributionofsubsequentrevisionsandotherrelated learning resources. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization CONTENTS 1 CONTENTS Foreword Foreword 1 List of acronyms List of Acronyms 1 1.INTRODUCTION Introduction11.1.Marker techniques Introduction31.2.Ideal genetic markers Introduction41.3.Marker application suitability Introduction51.4.Implementation Introduction91.5.Requirements Introduction101.6.Comparison of different marker systems Introduction11 2.DNA ISOLATION PROTOCOL DNA Isolation 12.1.Isolation of total DNA from leaf or other plant material DNA Isolation 12.1.1.Materials required DNA Isolation 12.1.2.Method DNA Isolation 12.2.Quantification of nucleic acids DNA Isolation 42.2.1.Equipment required DNA Isolation 42.3.References DNA Isolation 5 2.4Solutions/chemicals needed DNA Isolation 6 2.5Solutions to be prepared DNA Isolation 7 26The roles played by different reagents in DNA extraction 27Bench protocol for DNA isolation 3.RESTRICTION DIGESTION PROTOCOL Restriction Digestion 1 4.RFLP RFLP 14.1.Protocol RFLP 24.1.1.Agarose gel electrophoresis RFLP 24.1.2.Southern blotting and hybridization RFLP 24.1.2.1.Southern blotting RFLP 44.1.2.2.DNA:DNA hybridisation using the DIG system RFLP 64.1.2.3.Labelling the probe and dot blot/quantification RFLP 84.1.2.4.Hybridization RFLP 104.1.2.5.Washing method RFLP 114.1.2.6.Detection RFLP 124.1.2.7.Membrane rehybridization method RFLP 144.2.References RFLP 154.3.Solutions/chemicals needed RFLP 16 5.SSR SSR 15.1.Protocol SSR 25.1.1.PCR reaction mixSSR 2 5.1.2.PCR amplification SSR 35.1.3.Separation of the amplification products in agarose gelSSR 3 FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization CONTENTS 2 5.1.4.Denaturing gel electrophoresis SSR 45.1.4.1.Assembling the glass plate sandwich SSR 4 5.1.4.2.Casting gel SSR 5 5.1.4.3.Setting up the operation SSR 6 5.1.4.4.Polyacrylamide gel running conditions SSR 7 5.1.4.5.Silver-staining SSR 7 5.2.References SSR 105.3.Solutions/chemicals needed SSR 11 6.ISSR ISSR 16.1.Protocol ISSR 26.1.1.Prepare 20 l reaction mix ISSR 26.1.2.PCR amplification ISSR 26.1.3.Separation and visualization of the amplification products ISSR 36.1.4.Gel running conditions ISSR 36.1.5.Silver-staining ISSR 36.1.6.Primers available in the FAO/IAEA, Plant Breeding Unit ISSR 4 6.2.References ISSR 6 6.3.Solutions/chemicals needed ISSR 7 7.AFLP AFLP 17.1.Protocol AFLP 2 7.1.1.Restriction of genomic DNA and ligation of adapters to the DNA fragmentsAFLP 2 7.1.2.Pre-amplification AFLP 3 7.1.2.1.PCR pre-amplification AFLP 3 7.1.2.2.Check step AFLP 4 7.1.3.Selective amplification PCR AFLP 5 7.1.3.1.Selective PCR amplification AFLP 5 7.1.3.2.Gel electrophoresis AFLP 6 7.1.3.3.Silver staining AFLP 6 7.2.Production of single primer, linear PCR products AFLP 6 7.3.PCR amplification to produce single stranded DNA AFLP 7 7.4.Required enzymes and primers sequences AFLP 8 7.4.1.Restriction enzymes: AFLP 8 7.4.2.Preparation of Adapters AFLP 8 7.5.Solutions/chemicals needed AFLP 9 7.6.Sequence information of adapters and primers used for AFLP a AFLP 107.7.References AFLP 12 8.RAPD RAPD 18.1.Protocol RAPD 28.1.1.Master stock mixture RAPD 28.2.References RAPD 3 9.REMAP & IRAP REMAP & IRAP 1FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization CONTENTS 3 9.1.Protocol REMAP & IRAP 29.1.1.Prepare a 50 l reaction mix REMAP & IRAP 39.1.2.PCR amplification REMAP & IRAP 39.1.3.Separation and visualization of the amplification products REMAP & IRAP 49.2.References REMAP & IRAP 49.3.Solutions/chemicals needed REMAP & IRAP 5 10.SNPs (only description no protocol!) SNPs 10.1. References SNPs 11.TILLING TILLING 111.1.Wet bench protocols TILLING 111.1.1.PCR reaction TILLING 2 11.1.2.PCR amplification TILLING 2 11.1.3.Agarose gel analysis TILLING 3 11.1.4.Heteroduplex digestion TILLING 3 11.1.5.Sample purification and volume reduction TILLING 4 11.1.6.Preparing sephadex spin plates TILLING 5 11.1.7.Preparing Li-Cor gels TILLING 5 11.1.8.Loading samples onto membrane combs TILLING 6 111.9.Running Li-Cor gels TILLING 6 11.2.Computational tools TILLING 7 11.2.1.Selecting the best region to screen, designing primers and placing a TILLING order TILLING 7 11.3.Data analysis TILLING 10 11.4.Additional info TILLING 17 11.4.1.List of consumables and equipment TILLING 17 11.6.Frequently asked questions TILLING 19 11.6.Additional protocols TILLING 21 11.5.1.Sequencing TILLING 21 11.7.EMS mutagenesis of Arabidopsis seed TILLING 21 11.7.1.Materials TILLING 21 11.7.2.Standard size batch TILLING 21 11.7.3.A note on technique TILLING 21 11.7.4.DNA extraction TILLING 23 11.8.References TILLING 24 12.POPULATION GENETICS Population genetics 1 12.1.Reading and coding genetic data Population genetics 2 12.1.1.Presence/absence coding of dominant data Population genetics 212.1.2.Allele size coding for microsatellites Population genetics 3 12.1.3.Categorical coding Population genetics 5 12.1.4.Presence/absence coding of codominant data Population genetics 6 12.1.5.Formatting dominant data as codominant Population genetics 8 12.1.6.Notes on formatting diploid data with spread sheets Population genetics 8 12.1.7.Transforming data types using software Population genetics 9 FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization CONTENTS 4 12.1.8.The FSTAT data file Population genetics 10 12.2.Genetic diversity Population genetics 11 12.3.Genetic structure Population genetics 14 12.3.1.Neis population genetics parameters: Gst family Population genetics 15 12.3.2.Sewall Wrights Fstatistics Population genetics 15 12.4.Population and individual divergence and phylogenetic trees Population genetics 17 12.5.Web ressources and software non exhaustive Population genetics 18 12.6.List of selected references non-exhaustive Population genetics 21 12.7.Some key concepts Population genetics 23 12.8.Equations Population genetics 24 Appendices Appendices 1 A.1.General DNA extraction techniques Appendix 1 1A.1.1.Phenol/chloroform extraction Appendix 1 1A.1.2.Ethanol precipitation Appendix 1 1A.1.3.Solutions Appendix 1 2 A.2.Polymerase Chain Reaction protocol Appendix 2 1A.2.1.References Appendix 2 4 A.3.Plant genome database contact information Appendix 3 1 A.4.Acronyms of chemicals & buffers Acronyms of chemicals & buffers 1 FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization LIST OF ACRONYMS 1 LIST OF ACRONYMS AFLPAmplified Fragment Length Polymorphism CAPSCleaved Amplified Polymorphic Sequences ESTExpressed Sequence Tag IPCRInverse Polymerase Chain Reaction IRAPInter-Retrotransposon Amplified Polymorphism ISSRInter-Simple Sequence Repeat amplification PCRPolymerase Chain Reaction RAPDRandom Amplified Polymorphic DNA REMAPRetrotransposon-Microsatellite Amplified Polymorphism RFLPRestriction Fragment Length Polymorphism SCARSequence Characterized Amplified Region SNPSingle Nucleotide Polymorphism SSCPSingle Stranded Conformation Polymorphism SSRSimple Sequence Repeat STSSequence Tagged Site TILLINGTargeting Induced Local Lesions IN Genomes FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 1 1. INTRODUCTION Molecularmarkershavealreadyplayedamajorroleinthegeneticcharacterizationand improvementofmanycropspecies.Theyhavealsocontributedtoand greatly expanded ourabilitiestoassessbiodiversity,reconstructaccuratephylogeneticrelationships,and understandthestructure,evolutionandinteractionofplantandmicrobialpopulations. MolecularmarkerssystemsrevealvariationingenomicDNAsequence.Thefirst generationofmolecularmarkers,RFLP,werebasedonDNA-DNAhybridisationand wereslowandexpensive.Theinventionofthepolymerasechainreaction(PCR)to amplifyshortsegmentsofDNAgaverisetoasecondgenerationoffasterandless expensivePCR-basedmarkers,whicharethemainfocusofthisreview.Itisalready clear, however, that the technology, and future editions of this publication, will continue tomoveonasnewdetectionsystemsaredevelopedinthesearchforevermoreefficient and cost effective markers for the breeders of the 21st century. Restrictionfragmentlengthpolymorphisms(RFLPs)werethefirstgenerationof hybridization-based markers with substantial impact in agricultural biotechnology. These weresubsequentlyfollowedbyamplification-basedtechnologiesderivedfromthe polymerase chain reaction (PCR). Notably, the use of arbitrary oligonucleotide primers in theamplificationreactionfacilitatedthestudyofpreviouslyuncharacterizedgenomes. The most common of these PCR-based DNA techniques are Amplified Fragment Length Polymorphism(AFLP),SimpleSequenceRepeat(SSR),Inter-SimpleSequenceRepeat (ISSR) and Randomly Amplified Polymorphic DNA (RAPD). Other techniques have also been developed and are widely used in genetic analysis. Molecularmarkersarebeingusedextensivelytoinvestigatethegeneticbasisof agronomic traits and to facilitate the transfer and accumulation of desirable traits between breeding lines. A number of techniques have been particularly useful for genetic analysis. Forexample,collectionsofRFLPprobeshavebeen very versatile and important for the generation of genetic maps, construction of physical maps, the establishment of syntenic relationshipsbetweengenomes,andmarkerassistedbreeding.Numerousexamplesof specificgenesthathavebeenidentifiedastightlylinkedtoRFLPmarkersareavailable fortheimprovementofspecificagronomictraitsinalmostallmajorcrops.Specific examples include viral, fungal and bacterial resistance genes in maize, wheat, barley, rice, tomatoesandpotatoes.Additionalexamplesincludeinsectresistancegenesinmaize, wheat and rice as well as drought and salt tolerance in sorghum. These markers often used inconjunctionwithbulkedsegregantanalysisanddetailedgeneticmaps,provideavery efficientmethodofcharacterizingandlocatingnaturalandinducedmutatedallelesat genes controling interesting agricultural traits. Markers have also been used to identify the genesunderlyingquantitativevariationforheight,maturity,diseaseresistanceandyield invirtuallyallmajorcrops.Inparticular,thePCR-basedtechniqueshavebeenusefulin theassessmentofbiodiversity,thestudyofplantandpathogenpopulationsandtheir interactions;andidentificationofplantvarietiesandcultivars.AmplifiedDNA techniqueshaveproducedsequence-taggedsitesthatserveaslandmarksforgeneticand physicalmapping.Itisenvisionedthatemergingoligonucleotide-basedtechnologies FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 2 derived from the use of hybridization arrays, the so-called DNA chips and oligonucleotide arrays, will become important in future genomic studies. However, many ofthesearestillunderdevelopment,areproprietary,orrequiretheuseofexpensive equipment,andarethereforenotyetsuitablenorcost-effectiveforadequatetransferto developingcountries.Clearly,theinitialtransferoftechnologyhasonlyinvolveda selectedgroupoftechniquesthatarewellestablishedand/orseemtohaveabroad application(e.g.,RFLP,SSR,ISSR,AFLP,RAPD,IRAPandREMAPandSNPs). However,techniquesarecontinuouslychangingandevolving,sotechnologytransfer needstokeeppacewithcurrentdevelopmentsingenomics.Capacityforhandling molecular marker data has been identified as a bottleneck to the integration of molecular techniquesingermplasmmanagement.Amoduleonpopulationgenetics,dealing specifically with the analysis of molecular marker data, has been added in this issue of the manual. Newdevelopmentsingenomicresearchhavegivenaccesstoanenormousamountof sequence information as well as new insights on the function and interaction of genes and theevolutionoffunctionaldomains,chromosomesandgenomes.Inthiscontext, functionalandcomparativegenomicscanhelpincomparativegeneticmappingand linkage analysis of useful agricultural traits. Future DNA marker techniques, such as the useofoligonucleotidearrays,arelikelytobesequencebased.Comparativeanalysesof sequence information in the growing databases now publicly available on the World Wide Web will be an invaluable resource for genetic characterisation of exotic crop germplasm. This will have an increasingly important role in prospection and conservation endeavours. Theeaseforthedetectionofmutationeventsatthemolecularlevelisalsobeing significantlyenhancedthroughtheexploitationofthereadilyavailablesequence information of target regions of the genome that control traits. A reverse genetics strategy, TargetingInducedLocalLesionsINgenomes(TILLING),forexample,providesahigh throughputplatformforqueryingmutantpopulationsformutationsinpre-determined genome regions. A module on the protocols for TILLING has therefore been added to this manual. The following pages in this section present, in very synthetic and basic form, information onthemorewidelyusedmarkersystems,theirimplementation,applicationsuitability, criticalrequirementsandgeneralcomparisonofdifferentmarkersystems.The informationisgeneralinnatureasmanyofthesetechniquesareflexibleandcanbe adjusted to suit various requirements. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 3 1.1. Marker Techniques Marker/techniquePCR-basedPolymorphism (abundance) Dominance RFLPNoLow-MediumCo-dominant RAPDYesMedium-HighDominant SSRYesHighCo-dominant ISSRYesHighDominant AFLPYesHighDominant IRAP/REMAPYesHighCo-dominant Additional marker systems not covered in the course MorphologicalNoLowDominant/Recessive/Co-dominant Protein/isozymeNoLowCo-dominant STS/ESTYesHighCo-dominant/Dominant SNPYesExtremely HighCo-dominant SCARS/CAPSYesHighCo-dominant MicroarrayYesHigh 1.2.IdealGeneticMarkers(highlydependentonapplicationandspecies involved) -No detrimental effect on phenotype -Co-dominant in expression -Single copy -Economic to use -Highly polymorphic -Easily assayed -Multi-functional -Highly available (Un-restricted use) -Genome-specific in nature (especially when working with polyploids) -Can be multiplexed -Ability to be automated FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 4 1.3. Marker Application Suitability RFLPComparative maps Framework maps Genetic maps Breeding Varietal/line identification (multiplexing of probes necessary) Marker-assisted selection F1 identification Diversity studies Novel allele detections Gene tagging Bulk segregant analysis Map-based gene cloning RAPDGenetic maps F1 identification Varietal/line identification (multiplexing of primers necessary) Breeding Bulk segregant analysis Diversity studies Marker-assisted selection Seed testing Map-based gene cloning SSRFingerprinting Varietal/line identification (multiplexing of primers necessary) Framework/region specific mappingGenetic maps F1 identification Comparative mapping Breeding Bulk segregant analysis Diversity studies Novel allele detections Marker-assisted selection High-resolution mapping Seed testing Map-based gene cloning FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 5 ISSRFingerprinting Varietal/line identificationGenetic maps F1 identification Gene tagging Breeding Bulk segregant analysis Diversity studies Marker-assisted selection High-resolution mapping Seed testing AFLPFingerprinting Very fast mapping Region-specific marker saturation Varietal identification Genetic maps F1 identification Gene tagging Breeding Bulk segregant analysis Diversity studies Marker-assisted selection High-resolution mapping Map-based gene cloning IRAP/REMAPFingerprinting Varietal identification F1 identification Gene tagging Bulk segregant analysis Diversity studies Marker-assisted selection High-resolution mapping Seed testing FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 6 Additional marker systems not covered in the course MorphologicalGenetic maps Alien gene introduction Varietal/line identification F1 identification Novel phenotypes Breeding Protein and Genetic maps IsozymeQuality trait mapping Varietal/line identification (multiplexing of proteins or isozymes necessary) F1 identification Breeding Seed testing STS/ESTFingerprinting Varietal identification Genetic maps F1 identification Gene tagging and identification Bulk segregant analysis Diversity studies Marker-assisted selection Novel allele detection High-resolution mapping Map-based cloning SNPGenetic maps F1 identification Breeding Gene tagging Alien gene introductionBulk segregant analysis Diversity studies Novel allele detections Marker-assisted selection High resolution mapping FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 7 SCARS/CAPSFramework mapping Can be converted to allele-specific probes F1 identification Gene tagging Bulk segregant analysis Diversity studies Marker-assisted selection Map-based cloning MicroarrayFingerprinting Sequencing Transcription Varietal identification Genetic maps F1 identification Gene tagging and identification Bulk segregant analysis Diversity studies Marker-assisted selection High-resolution mapping FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 8 1.4. Implementation Marker/techniquesDevelopment costsRunning costs per data point Portability (Lab/Crops) RFLPMediumHighHigh/High RAPDLowLowLow/Low SSRHighMediumHigh/Low ISSRLowLowHigh/Low AFLPMedium-HighLowHigh/Low IRAP/REMAPHighMediumHigh/Low Additional marker systems not covered in the course MorphologicalDependsDependsLimited to breeding aims Protein and isozymeHighMediumHigh/High SCARS/CAPSHighMediumHigh/Low STS/ESTHighMediumMedium/High SNPHighMedium-LowUnknown MicroarrayMediumLowUnknown 1.5. Requirements Marker/techniqueAmount/quality of DNA DNA Sequence Required Radioactive detection Gel system RFLPHigh/HighNoYes/NoAgarose RAPDLow/LowNoNoAgarose SSRLow/MediumYesNoAcrylamide/Agarose ISSRLow/MediumYes/NoNoAcrylamide/Agarose AFLPLow/HighNoYes/NoAcrylamide IRAP/REMAPLow/MediumYesNoAcrylamide/Agarose Additional marker systems not covered in the course MorphologicalNoNoNoNone Protein/isozymeNoNoNoAgarose/Acrylamide STS/ESTLow/HighYesYes/NoAcrylamide/Agarose SNPLow/HighYesNoSequencing required MicroarrayLow/HighYesNoNoneSCARS/CAPSLow/HighYesYes/NoAgarose FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 9 1.6. Comparison of Different Marker Systems MarkerAdvantagesDisadvantages RFLP- Unlimited number of loci- Labour intensive - Co-dominant- Fairly expensive - Many detection systems- Large quantity of DNA needed - Can be converted to SCARs- Often very low levels of polymorphism - Robust in usage- Can be slow (often long exposure times) - Good use of probes from other species - Needs considerable degree of skill - Detects in related genomes - No sequence information required RAPD- Results obtained quickly- Highly sensitive to laboratory changes - Fairly cheap- Low reproducibility within and between laboratories - No sequence information required - Cannot be used across populations nor across species - Relatively small DNA quantities required - Often see multiple loci - High genomic abundance- Dominant - Good polymorphism - Can be automated SSR- Fast- High developmental and startup costs - Highly polymorphic- Species-specific - Robust- Sometimes difficult interpretation because of stuttering - Can be automated- Usually single loci even in polyploids - Only very small DNA - Co-dominant - Multi-allelic - Multiplexing possible - Does not require FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 10 MarkerAdvantagesDisadvantages radioactivity ISSR- Highly polymorphic- Usually dominant - Robust in usage- Species-specific - Can be automated AFLP- Small DNA quantities required - Evaluation of up to 100 loci - No sequence information required - Marker clustering - Can be automated- Dominant - Can be adapted for different uses, e.g. cDNA-AFLP - Technique is patented - Can be technically challenging IRAP/ REMAP - Highly polymorphic depends on the transposon - Alleles cannot be detected - Robust in usage- Can be technically challenging - Can be automated - Species-specific Additional marker systems not covered in the course Morpho-logical - Usually fast- Few in number - Usually cheap- Often not compatible with breeding aims - Need to know the genetics Protein- Fairly cheap- Often rare and Isozyme - Fairly fast analysis- Often different protocol for each locus - Protocol for any species- Labour intensive - Co-dominant- Sometimes difficult to interpret - No sequence information required STS/EST- Fast- Sequence information required FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization INTRODUCTION 11 MarkerAdvantagesDisadvantages - cDNA sequences- Substantially decreased levels of polymorphism - Non-radioactive - Small DNA quantities required - Highly reliable - Usually single-specific - Can be automated SNP- Robust in usage- Very high development costs - Polymorphism are identifiable - Requires sequence information - Different detection methods available - Can be technically challenging - Suitable for high throughput - Can be automated SCARS/- Co-dominant- Very labour intensive CAPS- Small DNA quantities required - Highly reliable - Usually single locus- Species-specific Microarray - Single base changes- Very high development and start-up costs - Highly abundant- Portability unknown - Highly polymorphic - Co-dominant - Small DNA quantities required - Highly reliable - Usually single locus - Species-specific - Suitable for high throughput - No gel system - Can be automated FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization DNA ISOLATION PROTOCOL 1 2. DNA ISOLATION PROTOCOL There are many DNA isolation methodsand the decision as to which one to use depends on the conditions in the particular laboratory as well as the plant tissues from which DNA will be isolated. In general however, the underlying principlesarethesameandinvolvesequentialstepsformacerationofthe tissuestoreleasetheDNA,proceduresforprotectingtheintegrityofthe DNA and for eliminating othercellularcontents in orderto ensure that pure DNA that is free of contaminants is extracted. The final step usually involves the precipitation of the DNA. TheprotocolpresentedbelowhasbeenroutinelyusedintheFAO/IAEA InterregionalTrainingCourseandinthePlantBreedingUnitoftheJoint FAO/IAEA Agriculture and Biotechnology Laboratory with excellent results. YieldsofgoodqualityDNAsuitableformostmolecularbiologyassaysare usuallyachieved.Thisprotocolhastheadvantagethatrelativelyverysmall amount of plant samples and less time are needed. 2.1. Isolation of total DNA from leaf or other plant materials ThemethodutilizedintheCourseissuitablefortheisolationofhigh molecularweightDNAfromleaforotherplanttissue.Thisprocedure involvesthebreakdownofcellwallstoreleasecellularcomponentsand disruptingofmembranestoreleaseDNAintotheextractionbuffer.The extractionbuffercontainingthedetergentsodiumdodecylsulphate(SDS) ensuresthattheDNAisreleasedfromthecellnuclei,and ethylenediaminetetraacetic acid (EDTA) protects the DNA from endogenous nucleases.DNAisseparatedfromproteinsandpolysaccharidesby ammoniumacetate,cetyl-trimethylammoniumbromide(CTAB)insodium chloride(NaCl),andchloroformextraction.DNAissubsequently precipitated by the addition of isopropanol. 2.1.1. Materials Required In addition to frozen tissue the following is needed: -Mortar and pestle,-Liquid nitrogen, -Water bath (65C), -Centrifuge for 2 ml eppendorf tubes, and -Table top microcentrifuge FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 2 2.1.2. Method Tissue collection NOTE:Weargloves,goggles,andlabcoatatalltimesforsafetyandto prevent contamination. Thereareasmanywaystocollecttissue,astherearelaboratories.A suggestionistocollectonlyyoungtissuefromanypartoftheplant.The older the tissue, the harder it is to obtain an adequate grinding for good DNA extraction.Ifyouarecollectingtissuefromplantsthatarenotinthesame room as the mortar/pestle (or some form of tissue grinder that can be cooled with liquid nitrogen) and the liquid nitrogen or dry ice, then you need to take care and place the tissue in liquid nitrogen as fast as possible. If the plants are in the glasshouse or the field you should take a Styrofoam container of liquid nitrogen to the plants. You also need to take tissue-collecting containers (e.g. tubeswithlids).Onceyoulabelthesecontainersandcollectthetissue,you should immediately place them into the liquid nitrogen until you remove the tissue tothe mortar forgrinding. You might also find it easier to grind the tissue in the mortarif you placea small quantity of acid washed sand in the mortar. The sand helps the grinding of the tissue. Generally a finer grinding will increase your DNA yield from the extraction. Steps for DNA isolation NOTE:Weargloves,goggles,andlabcoatatalltimesforsafetyandto prevent contamination. The following are the sequential steps for DNA isolation: 1.Preheattheextractionbuffer(0.1MTris-HCl,pH8;0.05EDTA,pH 8.0; 1.25 % SDS) to 65C in a water bath. 2.CoolthemortarandpestlewithN2 liquidandadd1-2gtissuetothe mortar.Grindtissuequickly but carefully, to a fine powder(do not let the tissue thaw). NOTES:-Ifyouareusingsmallerquantitiesoftissue,adjustthevolumesof solutions accordingly. -Makesurealltheequipmentcontainingtissueismaintained at20C using liquid nitrogen or dry ice. -If you are using a mortar/pestle, this is also the time to add acid washed sand to help in the grinding. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 3 3.Transferthefrozenpowder(100-120mg)tothe2mleppendorftubes using (for e.g.) a self-made spatula from filter paper dipped into liquid nitrogen.Add500lofpreheatedextractionbuffertoeachtubeand add10lofRNase(100mg/ml)andvortextomixwell.Leaveto incubateinwaterbathmaintainedat65Cfor30min.Vortextomix well and return to water bath twice in the course of these 30 minutes. NOTE:Donotre-usethespatula.Ametalonecanbeusedonlyifitis thoroughlycleanedinethanolaftereveryuse.Careisneededinorderto avoid cross-contamination. 4.Letthesolutioncooldowntoroomtemperature(takesabout15 minutes in the fridge), then add 300l of 6M ammonium acetate which had been stored at 4C. Mix well by vortex and then keep them in the fridge (at 4C) for 15 minutes. 5.Centrifugethetubesfor5minutesatspeedof13,000rpmatroom temperature. 6.Transfer the supernatant (the upper aqueous solution of approximately 700l)tofreshmicrofugetubesandadd50lCTAB(10%,in0.7M NaCl) to each tube and mix gently. 7.Add700lchloroform-isoamylalcohol(24:1)andswirlorinverttube gently to avoid mechanical damage to the DNA. 8.Centrifugefor5minutesatspeedof13,000rpm.Transferupper aqueous supernatant to new eppendorf tube. This upper phase contains the DNA. NOTE:Repeatchloroform:isoamylacoholextractionbyadding700l chloroform:isoamylacohol if the supernatant is not clear enough 9.Add 2/3rd volume of ice-cold isopropanol (~500l). Swirl or invert tube gently to avoidmechanical damage to the DNA and allow the DNA to precipitate for 15 min at -20C or leave standing on ice for 30 minutes 10.Centrifuge the samples for 20 minutes at maximum speed (13,000 rpm) in order to pellet the DNA. The DNA pellets should now be visible. 11.Draintheliquidcarefully,add1000lof70%ethanolandleavefor3 minutes. Centrifuge for 10 minutes at 10,000 rpm. 12. Drain the alcohol and add 1000 l of 90% ethanol, centrifuge at 10,000 rpm for 10 min and drain the alcohol and dry the pellet remaining at the bottom of the centrifuge tube (e.g. in a flow bench or on the bench for 15 minutes). 13. Re-suspendpelletin100lTEandleavetodissolveat4Cinthe refrigerator for at least 30 minutes. 14. Spin down the un-dissolved cellular debris by centrifuging the tube for 10 min at 13,000 rpm. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 4 15. Transfer the supernatant into a new tube and store at 4C for immediate use or -20C for long term storage. 2.2. QUANTIFICATION OF NUCLEIC ACIDS 2.2.1. Equipment Required Spectrophotometer with Hg-lamp for providing UV-light, quartz cuvette. Althoughfluorometricassaysareavailablethatofferimprovedsensitivity andarespecificforDNA,nucleicacidsareroutinelyquantified spectrophotometrically by measuring the absorbance at 260 nm (A260 nm) - the aromaticringsabsorbextremely strongly and rathercharacteristically with a peak between 255 and 260 nm. Tyrosine and tryptophan confer absorption at 280 nm to proteins. Thus the A260nm: A280nm ratio of a nucleic acid extractshould exceed 1.5 if it is to be considered protein-free. If the DNA appears to beimpure,removeanyremainingproteinsbyphenol/chloroformextraction (seeA.1.).Nucleicacidextractsaregenerallydiluted100to500-foldwith water before assay. Extinction coefficients for various nucleic acids yield: |DNA|=A260nm x 50 x dil. factorg/ml |RNA|=A260nm x 40 x dil. factorg/ml |Oligonucleotides|=A260nm x 30 x dil. factorg/ml Ifaspectrophotometerisnotavailable,agoodestimationoftheDNA quantitycanbeachievedbyusingagarosegelelectrophoresiswherethe extractedDNAalongwithadilutionseriesofastandardDNA(i.e.DNA fromphagelambda)isruninanagarosegel.Afterstainingwithethidium bromidethegelisexposedtoUV-light.Bycomparisonofthebands intensitywitheachother,theunknownDNAconcentrationscanbe estimated.However,oneneedstobeextremelycarefultoloadthesame amountofDNAandstandardsinthelanes.Withoutthisyoucannot accurately compare the samples. A further advantage of this procedure is that DNA integrity can be checked at the same time. High quality DNA should give a sharp, high molecular weight band, whereas sheared or DNase-digested DNA results in no bands. Sheared orDNase-digestedDNAshouldnotbeusedforfurtheranalysis,sincethe fragmentation is only arbitrary. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 5 2.3. REFERENCES Doyle, JJ and Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf, Phytochem. Bull. 19, 11-15Sumar A., Ahmet D., and Gulay Y., 200. 3 Isolation of DNA for RAPD Analysis from Dry leaf Materials of Some Hesperis L. Speciments.. Plant molecular Biology Reporter 21 pp461a-461f. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 6 2.4. REAGENTS NEEDED The following reagents are required: ReagentMolecular formulaFormula weight Sodium dedocyl Sulfate (SDS) C12H25NaO4S:288.4 Ethylenediaminetetraacetic acid (EDTA) C10H14N2O8Na2, 2H2O 372.23 Tris[Hydroxymethyl]aminomethane (TRIS Base) C4H11NO3:FW :121.1 Hexadecyltrimethylammonium Bromide (Cetyl-trimethylammonium Bromide) CTAB C19H42NBr364.5 Ammonium acetate C2H7NO277.08 Chloroform CHCl3119.39 Isoamylalcohol /Isopentyle alcohol 3-methyle 1-butanol C5H12O88.15 Isopropanol/ Isopropyle alcohol C3H8O60 RNase A (DNase free) (10 mg/ml) Ethanol C2H5OH46.07 Sodium Chloride NaCl40.00 FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 7 2.5. SOLUTIONS TO BE PREPARED Theexperimenterwillhavetopreparethefollowingsolutionsusingthe recipes provided: IngredientsVolume Final concentrationsE Ex xt tr ra ac ct ti io on n b bu uf ff fe er r 0.5M EDTA 100ml 0.05M EDTA, pH8.0 M Tris (pH:8)100ml 0 0. .1 1M M Tris-HCl, pH 8.0 SDS (10%)125ml 1.25%Water 1000ml10% CTAB buffer CTAB100g 10% NaCl40.95g 0.7MH2O1000ml6M-Ammonium acetate Ammonium acetate115.62g 6MH2O250 ml 70% EthanolEthanol (molecular grade) 350ml70% H2O150 ml 90% EthanolEthanol (molecular grade) 450 ml90% H2O50 ml TE buffer 0.5 M EDTA2ml 1mM EDTA1M Tris (pH :8)10ml 10mM Tris-HCl8.0) Water1000ml 2.6.THEROLESPLAYEDBYDIFFERENTREAGENTSINDNA ISOLATION.ThereagentsusedinDNAextractionplaydifferentcriticalroles;theseare tabulated below. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 8 ReagentFunction Extraction Buffer SDS and CTABDetergenttodiscombobulatelipidlayersand makethemembranesdissolvebindswiththe positivechargesofthechromosomalproteinsto releasetheDNAintosolutionformswhite precipitateswithpolysaccharides,denatured proteinsandcellwalldebristhatoften contaminate DNA. EDTAChelatesthecationsMg2+andCa2+,andthereby preventsthedegradationandrandomnickingof high-mol-wtDNAbyDNAses,whichare dependent on these cations for activity TrisMaintainsaconstantpH(betweenpH6-8)to preventhydrolysisatlowerpHanddenaturation of DNA at higher pH NaClNaClkeepstheDNAinitsdoublehelixform; otherwiseitwoulddenatureandbecome ssDNA |-mercaptoethanol (to be added fresh) Anantioxidantandpreventsoxidationof polyphenols(polyphenolsbindcovalentlyto DNAgivingitabrowncolorandmakingit useless for most research applications) RNAse (10mg/ml)Enzyme RNAse removes any residual RNA DNA precipitation Chloroform:isoamylalcohol (24:1) Chloroformisanorganiccompound.Allthe cellularcompoundswhicharesolublein chloroform(lipids,proteins)willbedissolved intothechloroform.DNAisnotsolublein chloroformandwillremaindissolvedinthe aqueous (water) layer. Isoamylalcoholreducesfoamingandfacilitates the separation of phases. Isopropanol Causes the solution to become more hydrophobic. Polarmolecules(likeDNA)precipitateoutof solution. Ammonium acetateThesaltsolutioncontributespositivelycharged FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 9 atoms that are attracted to the negative charge of DNA, effectively neutralizing the DNAs electric charge.ThisneutralizationallowstheDNA molecules to aggregate with one another.Washing EthanolWhen ethanol is added, the DNA clumps together andprecipitatesatthewater/ethanolinterface because the DNA is not soluble in ethanol. FAO/IAEA Interregional Training Course on Mutant Germplasm CharacterizationDNA ISOLATION PROTOCOL 10 2.7. BENCH PROTOCOL FOR DNA ISOLATION (it is recommended to print this page, which summarises the detailed protocol, and keep handy during the process) 1. Add 500l of preheated extraction buffer to each tube and add 10 l of RNase (100mg/ml) and vortex to mix well. 2. Incubate in water bath maintained at 65C for 30 min. Vortex to mix well and return to water bath twice in the course of these 30 minutes. 3.Cooldowntoroomtemperature(takesabout15minutesinthe refrigerator). 4. Add 300l of 6M ammonium acetate which had been stored at 4C. Mixwellbyvortexingandthenkeeptheminthefridge(at4C)for15 minutes. 5.Centrifugefor5minutesatmaximumspeed(13,000rpm)atroom temperature. 6. Transfer the supernatant to fresh microfuge tubes and add 50l CTAB (10%, in 0.7M NaCl) to each tube and mix gently. 7. Add 700l chloroform-isoamylalcohol (24.1) and swirl or invert tube. 8. Centrifuge for 5 minutes at maximum speed of 13,000 rpm. 9. Transfer top aqueous phase to new eppendorf tube. 10.Add2/3rdvolumeofice-coldisopropanol(~500l).Swirlorinvertand allowprecipitatingfor15minat-20Corleavetostandonicefor30 minutes. 11. Centrifuge for 20 minutes at maximum speed to pellet DNA on bottom of the tube. 12. Drain the liquid carefully.Keep the pellet on the bottom of the tube. Add 1000l 70% ethanol and leave for 3 minutes. 13. Centrifuge for 10 minutes at 10,000 rpm. 14. Drain the alcohol and add 1000 l of 90 % ethanol, keep pellet in tube. 15 Centrifuge at 10,000 rpm for 10 min. 16.Drainthealcoholanddrythepelletremainingatthebottomofthe centrifuge tube (e.g. in a flow bench for 15 minutes). 17.Re-suspendpelletin100lTEandleavetodissolveat4Cinthe refrigerator for at least 30 minutes. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization RESTRICTION DIGESTION 1 3. RESTRICTION DIGESTION PROTOCOL Restriction enzymes are produced by various bacterial strains. In these bacterial strains they areresponsibleforlimitingattackfromcertainbacteriophages.Theyactbycutting (restricting)thephageDNAatasequence-specificpoint,therebydestroyingphage activity. Sequence-specific cutting is a fundamental tool in molecular biology. DNA fragments can be ligated back together (recombined) by T4 DNA ligase. Many restriction enzymes have beenclonedandareavailableinacommerciallypureform.Theyarenamedaftertheir bacterial origin: e.g. EcoRI from E. coli. The known restriction enzymes recognize four or six bases (eight in the case of very rare cutters like NotI and SfiI). Recognition sequences are almost always palindromic where the first half of the sequence is reverse-complementary to the second: e.g. the XbaI site is 5 T C T A G A 3 3 A G A T C T 5 The position of the actual cut is enzyme dependent and symmetrical on the opposite strand: 5 TC T A G A 3 3 A G A T CT 5 leaving cohesive termini (sticky ends) at the 5 end: 5 T 3 3 A G A T C 5 Thecommerciallyavailablerestrictionenzymesaresuppliedwiththeappropriate restriction buffers (10 x concentrated). The enzymes are adjusted to a specific activity per l, usually 10 U/l. (1 Unit is the amount of enzyme needed to cut 1 g of lambda DNA in one hour at 37C). Atypicalrestrictiondigestionisperformedusingbetween20land100lreaction volumeper5g and more of plant DNA. For purified plasmid DNA 2 U per g DNA is sufficient, for plant DNA 4 U per g should be used. For example: digestion of 5 g DNA in 40 l reaction volume: Restriction buffer (10x)4 l DNA 1 g/l5 l Doubled distilled H20 29 l Enzyme (10 U/l)2 l Incubate for at least 1 hour at 37C. The restriction enzyme can be inactivated by heating to 65C for 10 minutes or by adding 1.0 l 0.5 M EDTAFAO/IAEA Interregional Training Course on Mutant Germplasm Characterization RFLP 1 4. RFLP RFLP definition: The variation(s) in the length of DNA fragments produced by a specific restrictionendonucleasefromgenomicDNAsoftwoormoreindividualsofaspecies (Kahl, 2001). Restrictionfragmentlengthpolymorphism(RFLP)technologywasfirstdevelopedinthe 1980sforuseinhumangeneticapplicationsandwaslaterappliedtoplants.Bydigesting totalDNAwithspecificrestrictionenzymes,anunlimitednumberofRFLPscanbe generated.RFLPsarerelativelysmallinsizeandareco-dominantinnature.Iftwo individualsdifferbyaslittleasasinglenucleotideintherestrictionsite,therestriction enzymewillcuttheDNAofonebutnottheother.Restrictionfragmentsofdifferent lengthsarethusgenerated.AllRFLPmarkersareanalyzedusingacommontechnique. However, the analysis requires a relatively complex technique that is time consuming and expensive. The hybridization results can be visualized by autoradiography (if the probes are radioactivelylabelled),orusingchemiluminesence(ifnon-radioactive,enzyme-linked methodsareusedforprobelabellinganddetection).Anyofthevisualizationtechniques will give the same results. The visualization techniques used will depend on the laboratory conditions. Figure4.1.TheschemedepictsenzymedigestionofDNAintofragmentsandtheir subsequent gelseparationandthedetectionofallelicvariationinvarietiesAandB(Withpermission,K. Devos). FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization RFLP 2 Figure4.2.Anautoradiographdetectingparent(P1andP2)andhomozygous(1and2), respectively and heterozygous (H) F2 segregation (With permission, M.D. Gale). 4.1. PROTOCOL 4.1.1. Agarose Gel Electrophoresis Agaroseisagalactose-basedpolymer,widelyusedinanalyticaland preparative electrophoretic separation of linear nucleic acids in the size range above100bp.DNAappliedtoanagarosegel,whichisexposedtoan electrical field, migrates towards the anode, since nucleic acids are negatively charged. The smaller the molecules the faster they run through the gel matrix (Figures4.1,4.2,and4.3).Migrationisinverselyproportionaltothelogof thefragmentlength.Inordertodeterminethelengthoftheseparated fragments in the gel a molecular weight fragment ladder control is placed in a lane alongside the experimental samples. Restricted genomic DNA is usually separatedina0.81.0%gelwhereasgelswithahigherconcentrationof agarose(23%)areneededforseparationofsmallDNAfragments( Save as > text (tab delimited) (*.txt) > OK > Yes. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 20 -SaveonsamediskandfolderasthePopulations.exefile(Torunthe programme,thetextfile(.txt)mustbeinsamefolderas Populations.exe. (4). Step 4:-Formatting in NOTEPAD: -Open NOTEPAD-From File menu, locate the saved .txt file, open file -Put cursor in front of first locus and hit backspace so that it is now in the first column, second row -Highlight all entries by select all in Edit menu -Cut (the entries) -Paste in Word -Select All-Edit>Replace>Infindwhatboxtype^tandinreplacewith box,hitthespacebaronce.Selectreplacealloption.Allthetabs have been replaced. (It helps to have the paragraph icon on in order to see that there are only single spaces). -Deletethedots(afterthefiguresinthefirstrowandafterpopand insertcommaeachsamplename.Makesurethattherearenospaces within a sample name. -Select all entries -Cut (the entries) -Paste again in NOTEPAD -Putthecursorinfrontofthefirstdataineachrowandhitbackspace (thespacebetweenthecommaafterthesamplenameandthefirst score is deleted) -Save (Use a simple file name one word). -Savethe.txtfileinthesamefolderastheProgramme, Populations.exe. (4). Step 4:Running the Programme Openprogramandchoosesequentiallybyenteringthecorresponding numbers and hitting Enter: -Computeindividualsdistance+tree(whendatahasonlyone population) No. 1 -Type the exact name of .txt file from last saving in the space provided. The.txtextensionmustbeincludedinthename.Thenameisalso case sensitive. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 21 -Phylogenetic tree of individuals with bootstraps on locus No. 3 -Neis standard genetic distance, Ds (1972) No. 2 -UPGMA No. 1 -10000 -Enter desired name for output file with .tre extension -WaitfortheProgrammetofinishrunning.Theoutputfilewiththe .tre extension is now deposited in the same folder as the programme, Populations.exe -Double click on the output file with the .tre extension in order to see the resulting dendrogram. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 22 12.6. REFERENCES Cavalli-Sforza,L.L.;Edwards,A.W.F.,1967:Phylogeneticanalysis:modelsand estimation procedures. Am. J. Hum. Genet. 18, 233-257. ChakrabortyRandDanker-HopfeH,1991.Analysisofpopulationstructure:A comparativestudyofdifferentestimatorsofWright'sfixationindices.In'Statistical MethodsinBiologicalandMedicalSciences.'EdC.R.RaoandR.Chakraborty, Elsevier Science Publishers. Cockerham CC, 1969. Variance of gene frequencies. Evolution. 23:72-84. Cockerham CC, 1973. Analysis of gene frequencies. Genetics. 74:679-700. CockerhamCCandWeirBS,1993.Estimationofgene-flow from F-statistics.Evolution. 47:855-863. El Mousadik A and Petit RJ, 1996. High level of genetic differentiation for allelic richness amongpopulationsoftheargantree[Arganiaspinosa(L.)Skeels]endemicto Morocco. Theor. Appl. Genet. 92:832-839. Excoffier L 2001. Analysis of population subdivision. In Handbook of statistical genetics, Balding, Bishop & Cannings (Eds) Wiley & Sons, Ltd. FisherR,1954.StatisticalMethodsforResearchWorkers.12thEdition,Oliver&Boyd, Edinburgh. 356pp.Goodman SJ, 1997. Rst Calc: a collection of computer programs for calculating estimates of genetic differentition from microsatellite data and a determining their significance. Molecular Ecology 6: 881-885. Glaubitz,J.C.(submitted)CONVERT:Auser-friendlyprogramtoreformatdiploid genotypic data. Molecular Ecology. For commonly used population genetic software packages. Molecular Ecology Notes.Goudet J, 1995. FSTAT (vers. 1.2): a computer program to calculate F-statistics. J. Hered. 86: 485-486.Goudet J, Raymond M, Demeeus T and Rousset F, 1996. Testing differentiation in diploid populations. Genetics. 144:1933-1940. HartlDL,ClarckAG(1997)PrinciplesofPopulationGenetics.ThirdEdition.Sinauer Associates. Nei, M. (1972) Genetic distance between populations. Am. Nat. 106:283-292. Nei M, 1973.Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA. 70:3321-3323. Nei M, 1988. Molecular Evolutionary Genetics. Columbia University Press, New York. NeiMandChesserRK,1983.Estimationoffixationindicesandgenediversities.Ann. Hum. Genet. 47:253-259. PamiloP,1984.Genotypiccorrelationandregressioninsocialgroups:multiplealleles, multiple loci and subdivided populations. Genetics. 107:307-320. PetitRJ,ElMousadik,AandPonsO,1998.Identifyingpopulationsforconservationon the basis of genetic markers. Conservation Biology. 12:844-855. QuellerDCandGoodnightKF,1989.Estimatingrelatednessusinggeneticmarkers. Evolution. 43:258-275.RaymondM.&RoussetF,1995.GENEPOP(version1.2):populationgeneticssoftware for exact tests and ecumenicism. Journal of Heredity, 86, 248-249. RaymondMandRoussetF,1995. An exact test for population differentiation.Evolution. 49:1280-1283. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 23 ReynoldsJ,WeirBSandCockerhamCC,1983.Estimationofthecoancestrycoefficient: Basis for a short-term genetic distance. Genetics. 105:767-779. Rousset F, 1996. Equilibrium Values of Measures of Population Subdivision For Stepwise Mutation Processes. Genetics 142:1357-1362. RoussetF,1997.GeneticdifferentiationandestimationofgeneflowfromF-statistics under isolation by distance. Genetics 145:1219-1228. Saitou, N and M Nei, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425. SlatkinM,1993.Isolationbydistanceinequilibriumandnon-equilibriumpopulations. Evolution 47:264-279. SlatkinM,1995.Ameasureofpopulationsubdivisionbasedonmicrosatelliteallele frequency. Genetics. 139:457-462. SlatkinMandBartonNH,1989.Acomparisonofthreemethodsforestimatingaverage levels of gene flow. Evolution 43:1349-1368. Sokal RR and Rohlf FJ, 1981. Biometry. 2nd Edition. Freeman & Co. WeirBSandCockerhamCC,1984.EstimatingF-statisticsfortheanalysisofpopulation structure. Evolution 38:1358-1370. Weir BS, 1996. Genetic data analysis II. Sinauer Publ., Sunderland, MA. WhitlockMCandMcCauleyD,1999.Indirectmeasuresofgeneflowandmigration: Fst1/(4Nm+1). Heredity. 82: 117-125. WrightS,1969.Evolutionandthegeneticsofpopulations.Vol.2.Thetheoryofgene frequencies. University of Chicago Press. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 24 12.7. SOME KEY CONCEPTS Alleles: All possible forms of a gene. Gene:Aunitofinheritance,anon-recombiningsegmentofDNA.Agiven location on a chromosome Genotype:Thecombinationofthetwohomologousallelescarriedonthe two chromosomes of a diploid individual at a given locus. Haplotype:Aparticularcombinationofallelesatdifferentlociona chromosome. Heterozygosity: The probability of an individual to have two different alleles at a given locus (the probability of being heterozygote). Homozygosity: The probability of an individual to be homozygote at a given locus. Homozygote: The fact that an individual has two identical alleles at a given locus. Locus: A given location on a chromosome, a non-recombining segment of a chromosome (usually interchanged with gene)Phenotype:Thevisible(physicalorgenetical)stateofanindividual.The relationbetweenthegenotypeandthephenotypecanbecomplexandwill usuallydependonthedegreeofdominanceandtheinteractionofdifferent alleles at a single or multiple loci. Polymorphism: the fact that there exist different alleles at a given locus in a population. Population: A group of interbreeding individuals living together in time and space. It is usually a subdivision of a species. Sample: A collection of individuals or of genes drawn from a population. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization POPULATION GENETICS 25 12.8. EQUATIONS FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDICES 1 APPENDICES FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 1 1 A.1. GENERAL DNA EXTRACTION TECHNIQUES A.1.1. Phenol/Chloroform Extraction NOTE:Weargloves,goggles,andlabcoatatalltimesforsafetyandto prevent contamination. Removes protein from DNA preparations. Advisable for example if A260nm: A 280nm(fromthespectrophotometerreadings)oftheDNAarebelow1.6. Phenol extraction requires subsequent ethanol precipitation of the DNA. Phenol:freshlydistilledandequilibratedwith20%0.5MTris-Base.Prepare a mixture of phenol/chloroform/isoamylalcohol (PCI) (25:24:1). NOTE: Use caution as phenol is toxic. S-.1. The DNA sample is mixed with an equal volume of PCI, vortexed, and centrifugedforabout5minutes.Removetheupperaqueousphase avoiding contamination with protein from interphase and transfer it to a fresh reaction tube. S.2.Remainingtracesofphenolintheaqueousphaseareextractedwith 1 volume of chloroform/isoamylalcohol (24:1). Vortex and centrifuge for 5 minutes. Transfer the upper phase carefully to a fresh reaction tube. A.1.2. Ethanol Precipitation NOTE: Wear glasses at all time for safety. S.1.Determinevolume of the sample, add 0.1volume 3 M sodium acetate and 2.5 volumes cold ethanol (96%). Mix well and leave at 20C for 2 hours. S.2.Centrifugefor15minutes(inmicrocentrifugeat>12,000rpm), preferably at 4C. S.3. Carefullyremoveethanolandwashpelletwithcold70%ethanolto remove salt from the sample centrifuge for 5 minutes.S.4 Dry DNA pellet in vacuum centrifuge or air dry in flow bench. S.5. Dissolve DNA in TE buffer or sterile double distilled H2O (ddH20). - Step FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 12 A.1.3. Solutions - 1.5 x CTAB extraction buffer (1 liter): CTAB15.0 g 1 M Tris (pH 8.0) 75 ml 0.5 M EDTA30 ml NaCl61.425 g ddH20to 1 litre - 10% CTAB (1 litre) CTAB100 g NaCl (0.7M)40.95 g ddH20to 1 litre - -mercaptoethanol,- Chloroform:isoamylalcohol (24:1), - Isopropanol,- Ethanol 96% and 70%- sodium acetate (3 M) - TE buffer10 mM Tris HCl 1 mM EDTA (pH 8.0) FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 2 1 A.2. POLYMERASE CHAIN REACTION PROTOCOL The polymerase chain reaction (PCR) is basically a technique forin vitro amplification of specificDNAsequencesbythesimultaneousprimerextensionofcomplementaryDNA strands.TheprincipleofprimerextensionisillustratedinFigureA.2.1foroneDNA strand. The primer binds to its complementary sequence of the single stranded target DNA andthepolymeraseextendstheprimerin5-3directionbyusingthecomplementary DNA as a template. For a PCR reaction, two primers are used, one binding to the lower strand(forwardprimer)andonebindingtotheupperstrand(reverseprimer).Thus, the requirementsforthereactionare:templateDNA,oligonucleotideprimers,DNA polymerase, deoxynucleotides (to provide both energy and nucleosides for DNA synthesis), and a buffer containing magnesium ions. In general the DNA sequence of both ends of the regiontobeamplifiedmustbeknowntobeabletosynthesizeproperprimer oligonucleotides.ThePCRreactionisacyclicprocess,whichisrepeated25to35times. One cycle consists of three basic steps with characteristic reaction temperatures: S-.1.DenaturationofthedoublestrandedDNAtomakethetemplateaccessibleforthe primers and the DNA polymerase (94C, 30 seconds). S.2.Annealingofprimerstocomplementarysequenceontemplate(between45and 60C, depending on the primer sequences, 30 seconds). S.3.Extension of primers by DNA-polymerase (72C - the optimum temperature of Taq DNA-polymerase -, 1 minute per kilobase of template to be amplified). FigureA.2.1.Primerextension.DNApolymeraseextendsaprimerbyusinga complementary strand as a template (McPherson et al., 1991). - Step FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 2 2 Bymultiplerepetitionofthiscyclethenumberoftemplatemoleculesincreases.This resultsinexponentialamplificationoftheDNAsequencethatisborderedbythetwo primers used (Figure A.2.2). FigureA.2.2.SchematicdiagramofPCR.Byusingprimerpairsaandb(shortblacklines) annealed to complementary strands of DNA (long black lines), two new strands (shaded lines) are synthesizedbyprimerextension.Iftheprocessisrepeated,boththesampleDNAandthenewly synthesized strands can serve as templates, leading to an exponential increase of product which has its ends defined by the position of the primers (McPherson et al., 1991). SuccessfulperformanceofaPCRexperimentisdependentonanumberofdifferent factors; some of them have to be determined empirically. - The selection of the primers is a very important step. They should be long enough to be specific,notannealagainstthemselvesbyfolding(avoidpalindromicsequences),nor should the forward primer anneal with the reverse primer. Furthermore the G/C content of theprimersshouldbesimilarandtheyshouldhavesimilarmeltingtemperatures(Tm). SeveralcomputerprogramsareavailableontheInternettohelptofindthebestprimer pairs for a given sequence. Try the addresses below- submit the DNA sequence and some required parameters and you will get a list of possible primers: http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 2 3 http://www.nwfsc.noaa.gov/protocols/oligoTMcalc.html -Theannealingtemperaturemustbedeterminedempiricallyandisdependentfromthe Tmsoftheprimers.Aruleofthumb(Wallacerule)providesafirstorderapproximation for Tm of oligonucleotides that have 20 bases or less: Tm = 2C (A + T) + 4C (C + G) The annealing temperature is a few degrees lower than Tm. -PCRisextremelysensitive!Thuscontaminationofsamplesandsolutionswithminimal amountsofforeignDNA,orthewrongPCRprogrammecanresultinunspecificPCR products. Always include controls without template DNA in order to check if there is any contamination in your nucleotides, primers, etc. A typical PCR experiment is given in the table below. In the FAO/IAEA course, PCR was demonstratedbyamplifyinga1050bpsequenceofthericeretrotransposonTos17 accession number D88394: Forward Primer 1 (100 pmol/l):Reverse Primer 2 (100 pmol/l): Reaction volume: 50 l Stock solutionslFinal conc./amount 10 x PCR buffer (15 mM MgCl2)5.0 l1 x PCR buffer (1.5 mM MgCl2) Primer 1 (100 pmol/l)0.5 l1 pmol Primer 2 (100 pmol/l)0.5 l1 pmol dNTP mix (10 mM)1 l0.2 mM DNA template (100 ng/l)1 l100 ng Taq DNA Polymerase (5 U/l)0.5 l2.5 U H2041.5 l- NOTE: It is very important to prepare a master mix corresponding to the number of desired samples that contains all the reagents except for the template DNA. Mix well and add the appropriate amount of the master solution to single reaction vials containing the individual templateDNAsamplesyouwishanalyzed.Thisproceduresignificantlyreducesthe numberofpipettingsteps,avoidserrorsderivedfrompipettingsmallamountsofliquid, and finally ensures that every tube contains the same concentrations of reagents. FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 2 4 For amplification of the Tos17 sequence the PCR machine was programmed as follows: Step 1Initial denaturation94C(4:00 minutes) Step 2Denaturation94C(0:30 minute) Step 3 Primer annealing56C(0:30 minute) Step 4 Primer extension72C(1:10 minutes) Step 5 CyclingRepeat steps 2-429 times Step 6 Final extension72C(6:00 minutes) Step 7 Hold4C(hold) NOTE:ThePCRprogrammecanvaryfromprimertoprimersetandspeciestospecies with the annealing temperature being the most variable step. A.2.1. REFERENCES McPherson,M.,P.Quirke,andGTaylor,1991.PCR:APracticalApproach.Oxford University Press, New York. A.3. PLANT GENOME DATABASE CONTACT INFORMATION (Taken from an IAEA-TECDOC on Radioactively LabelledDNA Probes For Crop Improvement VIENNA SEPTEMBER 6-8, 1999). DATABASECROPSCURATORE-MAIL ADDRESSDATABASE ADDRESS AAtDBArabidopsisDavid Flandersflanders@genome.stanford.eduhttp://genome-www.stanford,edu/Arabidopsis/ AlfagenesAlfalfa (Medicago sativa) Daniel Z. SkinnerDzolek@ksu.ksu.eduhttp://naaic.org/ Bean GenesPhaseolus and Vigna Phil McCleanmcclean@beangenes.cws.ndsu.nodak.eduhttp://probe.nalusda.gov:8300/cgi-bin/browse/beangenes ChlamyDBChlamydomonas reinhardtii Elizabeth H. Harrischlamy@acpub.duke.eduhttp://probe.nalusda.gov:8300/cgi-bin/browse/chlamydb CoolGenesCool season food legumes Fred Muehlbauermuehlbau@wsu.eduhttp://probe.nalusda.gov:8300/cgi-bin/browse/coolgenes CottonDBGossypium speciesSridhar Madhavanmsridhar@tamu.eduhttp://probe.nalusda.gov:8300/cgi-bin/browse/cottondb GrainGenesWheat, barley, rye and relatives Olin Andersonoandersn@pw.usda.govhttp://probe.nalusda.gov:8300/cgi-bin/browse/graingenes MaizeDBMaizeMary Polaccomaryp@teosinte.agron.missouri.eduhttp://www.agron.missouri.edu/ MilletGenesPearl milletMatthew CouchmanMatthew.Couchman@bbsrc.ac.ukhttp://jiio5.jic.bbsrc.ac.uk:8000/cgi-bin/ace/search/millet. PathoGenesFungal pathogens of small-grain cereals Henriette Gieseh.giese@risoe.dkhttp://probe.nalusda.gov:8300/cgi-bin/browse/pathogenes RiceGenesRiceSusan McCouchsrm4@cornell.eduhttp://genome.cornell.edu/rice/ RiceGenome Project Ricehttp://www.staff.or.jp SolGenesSolanaceaeMolly Kylemmk9@cornell.eduhttp://genome.cornell.edu/solgenes/welcome.html SorghumDBSorghum bicolorRussel Kohel/Bob Klein nus6389@tam2000.tamu.eduhttp://probe.nalusda.gov:8300/cgi-bin/browse/sorghumdb SoybaseSoybeansDavid Grantdgrant@iastate.eduhttp://129.186.26.94/ TreeGenesForest treesKim Marshallkam@s27w007.pswfs.govhttp://dendrome.ucdavis.edu/index.html National Center for Genome Resources Varioushttp://www.ncgr.org/ FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization APPENDIX 3 1 A.3. PLANT GENOME DATABASE CONTACT INFORMATION A.4. ACRONYMS OF CHEMICALS & BUFFERS AMPPD4-Methoxy-4-(3-phosphatephenyl)spirol(1,2-dioxetan-3,2-adamantan) BCIP5-Bromo-4-Chloro-3-Indolyl Phosphate CSPDChemiluminescence substrate (a registered trademark of Tropix Inc., USA) CTABHexadecyltrimethylammonium Bromide ddH2ODouble distilled Water DIGDigoxygenin N2 liquidLiquid Nitrogen NBTNitro Blue Tetrazolium PCIPhenol/Chloroform/Isoamylalcohol (25:24:1) SDSSodium Dodecyl Sulfate SSCSaline-Sodium Citrate Buffer TBETris-Borate-EDTA Buffer TETris-EDTA Buffer TEMEDN,N,N,N-Tetramethylenediamine TRIS[Tris(hydroxymethyl)aminomethane] FAO/IAEA Interregional Training Course on Mutant Germplasm Characterization ACRONYMS OF CHEMICALS & BUFFERS 1