6 Chloroplast DNA Small, 120-220 kb atpESmall, kbCircular, usually with inverted repeatNo recombinationInheritance usually maternal in angiosperms, paternal in gymnospermsConstant gene order in all green plants.atpBrbcLlarge single copy regionmatKpsbArpl216S23Srpl216S23StrnHsmall scr
7 Mitochondrial DNA Animal Plant 14-26 kb 150-2500kb Circular, usually homogeneous among cellsSet of different-sized circles, which arise from processes that interconvert between mother circle & subgenomic circlesNo recombination, inheritance maternalMutation rates high at sequence level; substitutionsRapid evolution in gene order but slower at sequence level (ca x100 slower than in animals)
8 Main sources of DNA evidence Control centresturn genes on & offGenessingle-copymulti-copycode for proteinsInter-genic spacersnon-coding sequences between genesIntronsnon-coding sequences within genestransposons & retroviruses
9 Gene structure upstream enhancer TATA box exon 1 exon 2 exon 3 spacer promoter5’ UTRintron 1intron 23’ UTRExons are composed of start, amino acid & stop codons.Highly conserved regions.Useful at higher taxonomic levels, e.g. genus & above.Introns are non-coding regions within a gene.Spacers are non-coding regions between genes.Both potentially highly variable regions.Useful at genus level and below, sometimes down to population level.Introns: transcribed to precursor mRNA, but later removed by a process called splicing.After splicing, the mRNA consists only of exon-derived sequences, i.e. those that are translated into a protein.
10 Multi-copy genes: rDNA IGSITS1 ITS2IGSTandem repeats: 100s to 1000s of copies.Nuclear genome: biparental inheritance.sometimes problem with concerted evolution.Coding regions (nS) highly conserved18S gene of soyabean shares 75% nucleotide homology with yeast.ITS & IGS regions highly variable.
11 Making inferences from the data Gene trees vs species or organism treesoften only two genes (or regions) studied [out of ca 25,000 genes present]Data from the different genomes may or may not be congruenteach genome tells its own story, which may not be that of the whole organism
12 Approaches Phylogeny reconstruction, systematics Sequencing Genepool & population level phenomenaRFLPs‘Fingerprinting’RAPDsAFLPsMicrosatellitesAllozymes (protein products of genes)
13 Sequencing Dideoxynucleotide method OH H = dideoxynucleotide deoxythymidine triphosphatePOHTOOH H = dideoxynucleotide(stops sequencing reaction)
14 Sequencing reaction T A G C A G single-stranded target DNA template Divide the template into four samples. To each sample add:all 4 deoxynucleotides (G, C, A, T, one of which is dye- or radio-labelledone of the 4 dideoxynucleotides, i.e. ddTTP, ddATP, ddCTP or ddGTPDNA polymeraseStart reaction.ddTTPA T *T A G C A GandA T C G T *ddATPA *T A G C A GddCTPA T C *T A G C A GandA T C G T C *ddGTPA T C G *T A G C A GIn each strand of new DNA the last base is a ddNTP because it terminates chain.
15 Pattern of gel fragments Newly synth DNA is isolated & run out on a gel. Radio-labelling -> visualised.bases ddTTP ddATP ddCTP ddGTPCTGCTA
17 Phylogenetic systematics parsimony. Identifies tree with minimium number of mutations (character-state changes).maximum likelihood. Identifies tree that has the highest probability of producing the observed data, given a particular model of evolution.Bayesian inference. Like maximum likelihood but much more sophisticated. Hurts the brain!ALL TREES CAN BE TESTED STATISTICALLY!!!bootstrapjacknifedecay indexN.B. Likelihood ≠ probability. Probabilities sum to 1, likelihoods do not.
18 Phylogenetic definitions ABCABCABCABCMonophyletic groups contain all the descendants of a common ancestor; defined by shared, derived character states.Paraphyletic groups share a common ancestor but do not contain all of its descendants. They are usually defined by shared, ancestral character states.Polyphyletic groups do not have a common ancestor.ABmonophyleticdefined by a synapomorphyBCparaphyleticdefined by a symplesiomorphyBCparaphyleticdefined by a false synapomorphyBCpolyphyleticdefined by a false synapomorphy
20 Sequencing: pros & cons large amounts of easily scored, robust datainter-taxon comparisons easyuniversal primers mean prior sequence knowledge unnecessaryscreen one ‘locus’ at a time (time-consuming but now automated)technical problems: alignments, 2y structure, etc.heterozygosity, requires cloning to resolve
22 RFLPs Restriction Fragment Length Polymorphisms Use restriction enzymes to cut DNA at recognition sites (usually 6b long).Separate fragments on an agarose gel.Stain fragments with ethidium bromide & view with UV.
23 Fragment patterns in hybrids nuclear DNAprobeenzyme 1enzyme 2712459enzyme 1 fragmentsAA AB BBenzyme 2 fragmentsAA AB BBDifferent patterns are the result of gains/losses of restriction sites or inversions.Co-dominant in nuclear DNA: good for detecting hybrids.
24 MappingWhen divergence is great, RFLP fragments are too complex to analyse.Need to map.Cloned fragments from one genome are used to probe Southern blots of different enzyme digests of that (or another) genome.
25 Mapping example Use single and double digests Single digestsa b c17 __ __10 __7 __Double digestsa+b b+c a+c15 ____ __6 __4 __3 __2 __1 __Use single and double digestsbac12410Data scored as site mutations; ordered.Can be used for phylogenetic purposes.
26 RFLP properties uniparental inheritance in plastid DNA biparental, co-dominant inheritance in nuclear DNAwhen divergence is great, RFLP fragments are too complex to analyse: need to use mapping approachapplications: detecting hybridisation, analysis of genepool structure (phylogeography)
27 RFLP pros & cons Robust, repeatable data PCR-based Capable of detecting much variation if enough enzyme combinations usedLogarithmic migration of fragments means small changes in large fragments are hard to detectSome restriction enzymes are sensitive to methylation
28 RAPD Randomly Amplified Polymorphic DNA gelA B-- ----indiv Aindiv Barbitrary 10bp primers target sequences flanked by inverted repeat primer sitespermits multiple annealing throughout all three genomescoding & non-coding regions; single- & multi-copy DNAinherited as a dominant (cannot distinguish htz from hmz)
29 RAPD properties each prime site treated as +/- (diallelic) inheritance dominant (primer site present in homozygote + heterozygote but not homoz 2)presence/absence of primer sites due to many possible causes (substitutions, indels, secondary structure between prime sites)identifies multi-locus genotypesapplications: gene diversity, clonality, population structure
30 RAPD: pros & cons simple problems of reproducibility PCR-based no prior sequence info needednon-destructivecan screen large number of locicheapproblems of reproducibilityproduct competitionproduct homologygenome samplingnon-independence of lociestimates of population differentiation may be inflated
31 AFLPs Amplified Fragment Length Polymorphsims cut DNA with pair of enzymes: one rare cutter & one common cutterattach known DNA sequences to the productsamplify products using the known sequences as priming sitesrather like RAPDs but much more reproducibledominant inheritanceselective amplification of an arbitrary subset of restriction fragments generated by double-digestion with a common/rare-cutting exzyme pair
32 AFLPs Amplified Fragment Length Polymorphsims DNArestrictionx2 digestiondouble-stranded adaptor ligationPCR1: preselectiveamplificationPCR2: selectivecommonrareA+prselective amplification of an arbitrary subset of restriction fragments generated by double-digestion with a common/rare-cutting exzyme pair[primers complementary to adaptor, plus extra base pair][primers as in PCR1, plus up to 3 extra base pairs, labelled]pr+G*(N)3A+prpr+G(N)3*
33 AFLP propertiesnumber of fragments determined by no. bases in selective primer (1 base more fragments than 2 or 3 bases)scored as diallelic lociusually dominant inheritanceapplications: gene diversity, clonality, population structure, hybridisation
34 AFLPs: pros & cons reproducible (long primers used) PCR-based no prior sequence info needednon-destructivecan screen large number of loci: per runtechnically demandingproduct homology?genome samplingnon-independence of lociestimates of population differentiation may be inflated
35 Microsatellites (SSRs: Simple Sequence Repeats) GAGAGAGAGAGAGA(GA)7GAGAGAGAGA(GA)5pri.flankingShort (1-6bp), tandem repeats (10-50 copies)Mono- to tetra-nucleotides, e.g. (AT)nRandom distribution assumedPrimers designed for conserved flanking regionsVariation in repeat number polymorphismCo-dominant inheritance
36 Microsatellite properties Homologous chromosomes may have different repeat lengths, hence inheritance is co-dominant.SSRs abundant across genome (but commoner in animals than in plants)applications: population level studies, esp. gene flow
37 Microsatellite pros & cons co-dominant inheritance allows full genetic analysisabundantuniformly distributed thro’ genomemutation rates high large no. alleles/locustime-consuming to develop primersprimer-pairs often species-specificstutter bands make interpretation hardhomoplasy between alleles may be highfew loci sampled
38 Summary Type of study Type of DNA Preferred marker Gene diversity & breeding systemnrDNAco-dominant markers: microsats, allozymesGenotype diversity, clonality, individualityhigh resolution markers: microsats, RAPDs, AFLPsPopulation structure & gene flownr or cp/mt DNAallPhylogeography (gene-pool structure)cp/mt DNAsequences, RFLPsSpeciationnr + cp/mt DNAInter-specific hybridisationmicrosats, allozymes, RFLPs, AFLPsSystematics (above sp. level)sequences, (AFLPs)