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multivariate analysis chromosome number hairs number phenotype

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1 multivariate analysis chromosome number hairs number phenotype
natural group length morphology colour shape ratio width venation multivariate analysis chromosome number hairs number phenotype genotype stems crossability geniculate awns sympetalous corollas anatomy leaves flowers secondary chemistry UPGMA embryology

2 parsimony RAPDs PCR chloroplast F-statistics bootstrap SNPs RFLPs monophyletic intron SSRs nucleus spacer Bayesian inference gene mitochondrion AFLPs paraphyletic maximum likelihood microsatellites

3 Types of DNA Plants have THREE genomes: Nucleus Chloroplast
Mitochondrion T A C G C G T A

4 Nuclear DNA Large size, ca 10x106 kb in flowering plants
Linear arrangement, as chromosomes Inheritance biparental Recombination

5

6 Chloroplast DNA Small, 120-220 kb
atpE Small, kb Circular, usually with inverted repeat No recombination Inheritance usually maternal in angiosperms, paternal in gymnosperms Constant gene order in all green plants. atpB rbcL large single copy region matK psbA rpl2 16S 23S rpl2 16S 23S trnH small scr

7 Mitochondrial DNA Animal Plant 14-26 kb 150-2500kb
Circular, usually homogeneous among cells Set of different-sized circles, which arise from processes that interconvert between mother circle & subgenomic circles No recombination, inheritance maternal Mutation rates high at sequence level; substitutions Rapid evolution in gene order but slower at sequence level (ca x100 slower than in animals)

8 Main sources of DNA evidence
Control centres turn genes on & off Genes single-copy multi-copy code for proteins Inter-genic spacers non-coding sequences between genes Introns non-coding sequences within genes transposons & retroviruses

9 Gene structure upstream enhancer TATA box exon 1 exon 2 exon 3 spacer
promoter 5’ UTR intron 1 intron 2 3’ UTR Exons 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
IGS ITS1 ITS2 IGS Tandem repeats: 100s to 1000s of copies. Nuclear genome: biparental inheritance. sometimes problem with concerted evolution. Coding regions (nS) highly conserved 18S 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 trees often only two genes (or regions) studied [out of ca 25,000 genes present] Data from the different genomes may or may not be congruent each genome tells its own story, which may not be that of the whole organism

12 Approaches Phylogeny reconstruction, systematics Sequencing
Genepool & population level phenomena RFLPs ‘Fingerprinting’ RAPDs AFLPs Microsatellites Allozymes (protein products of genes)

13 Sequencing Dideoxynucleotide method OH  H = dideoxynucleotide
deoxythymidine triphosphate P OH T O OH  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-labelled one of the 4 dideoxynucleotides, i.e. ddTTP, ddATP, ddCTP or ddGTP DNA polymerase Start reaction. ddTTP A T * T A G C A G and A T C G T * ddATP A * T A G C A G ddCTP A T C * T A G C A G and A T C G T C * ddGTP A T C G * T A G C A G In 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 ddGTP C T G C T A

16 Sequence: electropherogram

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!!! bootstrap jacknife decay index N.B. Likelihood ≠ probability. Probabilities sum to 1, likelihoods do not.

18 Phylogenetic definitions
A B C A B C A B C A B C Monophyletic 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. AB monophyletic defined by a synapomorphy BC paraphyletic defined by a symplesiomorphy BC paraphyletic defined by a false synapomorphy BC polyphyletic defined by a false synapomorphy

19 Residue problem: paraphyly
Chamerion Epilobium Zauschneria DNA–ITS phylogeny

20 Sequencing: pros & cons
large amounts of easily scored, robust data inter-taxon comparisons easy universal primers mean prior sequence knowledge unnecessary screen one ‘locus’ at a time (time-consuming but now automated) technical problems: alignments, 2y structure, etc. heterozygosity, requires cloning to resolve

21 Genepool & population phenomena
st 0.447 st 0.555 st 0.468 st 0.390 st 0.287 st 0.289

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 DNA probe enzyme 1 enzyme 2 7 12 4 5 9 enzyme 1 fragments AA AB BB enzyme 2 fragments AA AB BB Different patterns are the result of gains/losses of restriction sites or inversions. Co-dominant in nuclear DNA: good for detecting hybrids.

24 Mapping When 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 digests a b c 17 __ __ 10 __ 7 __ Double digests a+b b+c a+c 15 __ __ __ 6 __ 4 __ 3 __ 2 __ 1 __ Use single and double digests b a c 1 2 4 10 Data 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 DNA when divergence is great, RFLP fragments are too complex to analyse: need to use mapping approach applications: 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 used Logarithmic migration of fragments means small changes in large fragments are hard to detect Some restriction enzymes are sensitive to methylation

28 RAPD Randomly Amplified Polymorphic DNA
gel A B -- -- -- indiv A indiv B arbitrary 10bp primers target sequences flanked by inverted repeat primer sites permits multiple annealing throughout all three genomes coding & non-coding regions; single- & multi-copy DNA inherited 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 genotypes applications: gene diversity, clonality, population structure

30 RAPD: pros & cons simple problems of reproducibility PCR-based
no prior sequence info needed non-destructive can screen large number of loci cheap problems of reproducibility product competition product homology genome sampling non-independence of loci estimates of population differentiation may be inflated

31 AFLPs Amplified Fragment Length Polymorphsims
cut DNA with pair of enzymes: one rare cutter & one common cutter attach known DNA sequences to the products amplify products using the known sequences as priming sites rather like RAPDs but much more reproducible dominant inheritance selective 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
DNA restriction x2 digestion double-stranded adaptor ligation PCR1: preselective amplification PCR2: selective common rare A+pr selective 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+pr pr+G(N)3*

33 AFLP properties number of fragments determined by no. bases in selective primer (1 base  more fragments than 2 or 3 bases) scored as diallelic loci usually dominant inheritance applications: gene diversity, clonality, population structure, hybridisation

34 AFLPs: pros & cons reproducible (long primers used) PCR-based
no prior sequence info needed non-destructive can screen large number of loci: per run technically demanding product homology? genome sampling non-independence of loci estimates of population differentiation may be inflated

35 Microsatellites (SSRs: Simple Sequence Repeats)
GAGAGAGAGAGAGA (GA)7 GAGAGAGAGA (GA)5 pri. flanking Short (1-6bp), tandem repeats (10-50 copies) Mono- to tetra-nucleotides, e.g. (AT)n Random distribution assumed Primers designed for conserved flanking regions Variation in repeat number  polymorphism Co-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 analysis abundant uniformly distributed thro’ genome mutation rates high  large no. alleles/locus time-consuming to develop primers primer-pairs often species-specific stutter bands make interpretation hard homoplasy between alleles may be high few loci sampled

38 Summary Type of study Type of DNA Preferred marker
Gene diversity & breeding system nrDNA co-dominant markers: microsats, allozymes Genotype diversity, clonality, individuality high resolution markers: microsats, RAPDs, AFLPs Population structure & gene flow nr or cp/mt DNA all Phylogeography (gene-pool structure) cp/mt DNA sequences, RFLPs Speciation nr + cp/mt DNA Inter-specific hybridisation microsats, allozymes, RFLPs, AFLPs Systematics (above sp. level) sequences, (AFLPs)


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