5 Molecular markersSequenze di proteine o DNA facilmente distinguibili, la cui eredità può essere verificata e associata con tratti ereditati indipendentemente dall’ambiente :a) Polimorfismi di proteineb) Polimorfismi di DNA
6 Molecular markers Sequenze (SNPs) Microsatelliti (SSRs) Multi-locus fingerprintsAFLP (Amplified Fragment Length Polymorphism)Potere di risoluzioneRAPD (random amplified polymorphic DNA)DNA cloroplastico PCR-RFLPallozimi (elettroforesi di proteine)
7 Polimorfismi di proteine Proteine di riserva del semeIsozimi
9 IsozimiStarch gel of the isozyme malate dehydrogenase (MDH). The numbers indicate first the MDH locus, and next the allele present (ie is locus 3 allele 18). Some bands are heterodimers (intralocus or interlocus).
12 Image from UV light table Image from computer screen
13 Co-dominant marker Dominant marker Polymorphism -Parent 1 : one band -Parent 2 : a smaller band-Offspring 1 : heterozygote = both bands-Offspring 2 : homozygote parent 1P 2P 1O 2O 1Gel configurationDominant markerPolymorphismParent 1 : one band-Parent 2 : no band-Offspring 1 : homozygote parent 1-Offspring 2 : ????P 2Gel configurationP 1O 1O 2
14 Dominant versus Co-dominant No distinction between homo- and heterozygotes possibleNo allele frequencies availableAFLP, RAPDCo-dominant:homozygotes can be distinguished from heterozygotes; allele frequencies can be calculatedmicrosatellites, SNP, RFLPs
15 Desirable properties for a good molecular marker* Polymorphic* Co-dominant inheritance* Occurs throughout the genome* Reproducible* Easy, fast and cheap to detect* Selectivity neutral* High resolution with large number of samples
16 Basis for DNA marker technology Restriction EndonucleasesPolymerase chain reaction (PCR)DNA-DNA hybridizationDNA sequencing
17 RFLP based markers*Examine differences in size of specific DNA restriction fragments*Require pure, high molecular weight DNA*Usually performed on total cellular genome
18 Endonucleases and restriction sequences AAATCGGGACCTAATGGGCC ATTTAGGGCAATTCCAAGGAYFGInd 1Ind 2
21 Advantages and disadvantages of RFLP Time consumingExpensiveUse of radioactive probesAdvantagesReproducibleCo-dominantSimple
22 DNA/DNA Hybridization DenaturationElevated temperatureKnown DNA sequence
23 Polymerase Chain Reaction Powerful technique for amplifying DNAAmplified DNA are then separated by gel electrophoresis
24 PCR based methods 1. Reactions conditions *Target DNA ( or template) *Four nucleotides (dATP, dCTP, dGTP, dTTP)1. Reactions conditions*Target DNA ( or template)*Reaction buffer containing the co-factor MgCl2*One or more primers*Thermostable DNA polymerase
25 = an enzyme that can synthesize DNA at 2. Use of DNA polymerase= an enzyme that can synthesize DNA atelevated temperatureex : Taq = enzyme purified from hot spring bacterium : Thermus aquaticus3. Thermal cycle*Denaturing step - one to several min at º C*Annealing step - one to several min at º C*Elongation step - one to several min at 72 º C4. Repetitiontypically 20 to 50 times average 35 times
26 AFLP Markers Most complex of marker technologies Involves cleavage of DNA with two different enzymesInvolves ligation of specific linker pairs to the digested DNASubsets of the DNA are then amplified by PCR
27 AFLP Markers The PCR products are then separated on acrylamide gel 128 linker combinations are readily availableTherefore 128 subsets can be amplifiedPatented technology
31 AFLP Markers Technically demanding Reliable and stable Moderate cost Need to use different kits adapted to the size of the genome being analyzed.Like RAPD markers need to be converted to quick and easy PCR based marker
32 RAPD MarkersThere are other problems with RAPD markers associated with reliabilityBecause small changes in any variable can change the result, they are unstable as markersRAPD markers need to be converted to stable PCR markers.How?
33 RAPD Markers The polymorphic RAPD marker band is isolated from the gel It is used a template and re-PCRedThe new PCR product is cloned and sequencedOnce the sequence is determined, new longer and specific primers can be designed
34 RAPD Fast and easy method for detecting polymorphisms Domimant markers Amplifies anonymous stretches of DNA using arbitrary primersFast and easy method for detecting polymorphismsDomimant markersReproducibility problems
35 RAPD Polymorphisms among landraces of sorghum Sequences of 10-merRAPD primersName SequenceOP A08 5’ –GTGACGTAGG- 3’OP A15 5’ –TTCCGAACCC- 3’OP A 17 5’ –GACCGCTTGT- 3’OP A19 5’ –CAAACGTCGG- 3’OP D02 5’ –GGACCCAACC- 3’MRAPD gel configuration
36 SSR repeats and primers GGT(5)SequenceGCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTCATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTGGCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGAATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGTAAGAAACCTCTTCCTGTTAGATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCTGTGGAACCA…..GAGGGCTGATGAGGTGGATAATCTTATGGCGGTTCTCGTG
37 SSR polymorphisms AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG P1 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCGP2P1P2Gel configuration
39 SSR scoring for F 5:6 pop from the cross Anand x NN97AnandM
40 4. SNPs (Single Nucleotide Polymorphisms) Hybridization using fluorescent dyesSNPs on a DNA strandAny two unrelated individuals differ by one base pair every1,000 or so, referred to as SNPs.Many SNPs have no effect on cell function and thereforecan be used as molecular markers.
41 DNA sequencingSequencing gelSequencerSequencing graph
42 Types of traits =types of markers Single gene trait: seed shapeMultigenic trait; ex: plant growth =Quantitative Trait LociMFGMFG
43 USES OF MOLECULAR MARKER Measure genetic diversityMappingTagging
44 Genetic DiversityDefine appropriate geographical scales for monitoring and management (epidemology)Establish gene flow mechanismidentify the origin of individual (mutation detection)Monitor the effect of management practicesmanage small number of individual in ex situ collectionEstablish of identity in cultivar and clones (fingerprint)paternity analysis and forensic
46 Gotcha! early selection of the good allele fingerprints seeds, plantlets
47 A linear order of genes or DNA fragments MappingThe determination of the position and relative distances of gene on chromosome by means of their linkageGenetic mapA linear arrangement of genes or genetic markers obtained based on recombinationPhysical mapA linear order of genes or DNA fragments
48 It contains ordered overlapping cloned DNA fragment Physical MappingIt contains ordered overlapping cloned DNA fragmentThe cloned DNA fragments are usually obtained using restriction enzyme digestion
49 QTL MappingA set of procedures for detecting genes controlling quantitative traits (QTL) and estimating their genetics effects and location To assist selection
50 Marker Assisted Selection Breeding for specific traits in plants and animals is expensive and time consumingThe progeny often need to reach maturity before a determination of the success of the cross can be madeThe greater the complexity of the trait, the more time and effort needed to achieve a desirable result.
51 MASThe goal to MAS is to reduce the time needed to determine if the progeny have traitThe second goal is to reduce costs associated with screening for traitsIf you can detect the distinguishing trait at the DNA level you can identify positive selection very early.
52 Developing a MarkerBest marker is DNA sequence responsible for phenotype i.e. geneIf you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNADevelop specific primers to gene that will distinguish the two forms
53 Developing a Marker If gene is unknown, screen contrasting populations Use populations rather than individualsNeed to “blend” genetic differences between individual other than trait of interest
54 Developing MarkersCross individual differing in trait you wish to develop a markerCollect progeny and self or polycross the progenyCollect and select the F2 generation for the trait you are interested inSelect individuals in the F2 showing each trait
55 Developing Markers Extract DNA from selected F2s Pool equal amounts of DNA from each individual into two samples - one for each traitScreen pooled or “bulked” DNA with what method of marker method you wish to useMethod is called “Bulked Segregant Analysis”
56 Marker DevelopmentOther methods to develop population for markers exist but are more expensive and slower to developNear Isogenic Lines, Recombinant Inbreeds, Single Seed DecentWhat is the advantage to markers in breeding?
57 Reducing Costs via MAS Example disease resistance 10000 plantsGreenhouse space or field plots$ $10000Time 4 months (salary)$10 - $15000total cost = $15 - $25,000
58 Reducing Costs via MAS PCR-based testing @ $5 sample $50,000 - costs more?Analysis of trait not easily phenotypedE.g: Cadmium in Durum wheat10000 plants need to reach maturityCadmium accumulates in seed
59 Reducing costs via MAS $15 - 25 growing costs + analysis Atomic $15 per sample$150,000 + growing costsPCR analysis still $50000Savings in time and money increase as more traits are analyzedMany biochemical tests cost >$50 sample
60 Marker Assisted Breeding MAS allows for gene pyramiding - incorporation of multiple genes for a traitPrevents development of biological resistance to a geneReduces space requirements - dispose of unwanted plants and animal early
61 QTL study Types of population used for molecular markers studies: Trait188.8.131.52.52.3M. 1132M. 213M. 3132P.1P.2I.1I.2I.3I.4Statistical programs used in molecular marker studies* SAS* ANOVA * Mapmaker* CartographerTypes of population used for molecular markers studies:F2, RILs, Backcrosses (MILs), DH.