length

width

crossability

chromosome number

phenotype genotype

colour shape

number

ratio

multivariate analysis

UPGMA

leaves

stems

hairs

venation

flowers

morphology

anatomy

secondary chemistryembryology

natural group

parsimony

maximum likelihood

Bayesian inference

RAPDs

AFLPs

RFLPsbootstrap

chloroplast

mitochondrion

nucleus

gene

intronspacer

PCR

SNPs

microsatellites

SSRs

monophyletic

paraphyletic

F-statistics

Types of DNAPlants have THREE genomes:1) Nucleus2) Chloroplast3) Mitochondrion

A

G

T

C

A T

GC

Nuclear DNA

Large size, ca 10x106

kb in flowering plants Linear arrangement,

as chromosomes Inheritance biparental Recombination

Chloroplast DNA Small, 120-220 kb Circular, usually with

inverted repeat No recombination Inheritance usually

maternal in angiosperms, paternal in gymnosperms

Constant gene order in all green plants.

rpl2

16S23S

rpl2

16S23S

rbcL

atpB

atpE

large single copy region

small scr

matK

psbA

trnH

Mitochondrial DNAAnimal 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

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)

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

List

eria

Sacc

haro

myc

es

Arabi

dops

is

Droso

phila

Homo

sapi

ens

020406080

100

Non-coding Coding

transposons & retroviruses

Gene structure

• 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.

upstream enhancer promot

er

TATA box

5’ UTR

3’ UTR

exon 1

exon 2

exon 3

intron 1

intron 2

spacer

Multi-copy genes: rDNA

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.

25S 25S18S

5.8S

IGS ITS1 ITS2

18S

IGS

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

ApproachesPhylogeny reconstruction, systematics Sequencing

Genepool & population level phenomena RFLPs ‘Fingerprinting’

RAPDs AFLPs

Microsatellites Allozymes (protein products of genes)

Sequence: electropherogram

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

st 0.447

st 0.555

st 0.468

st 0.390

st 0.287

st 0.289

Genepool & population phenomena

RFLPsRestriction 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.

Fragment patterns in hybrids

Different patterns are the result of gains/losses of restriction sites or inversions.

Co-dominant in nuclear DNA: good for detecting hybrids.

enzyme 1

enzyme 2

probe

7 12

4 5 9

enzyme 1 fragments AA AB BB

19 --- ---

12 --- ---

7 --- ---

enzyme 2 fragments AA AB BB

14 --- ---

9 --- ---

5 --- ---4 --- --- ---

nuclear DNA

RAPDRandomly Amplified Polymorphic DNA

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)

indiv A

indiv B

gelA B

-- --

--

AFLPsAmplified 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

Microsatellites(SSRs: Simple Sequence Repeats)

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

GAGAGAGAGAGAGA

(GA)7

GAGAGAGAGA

(GA)5

pri. flanking

pri. flanking

flanking pri.

flanking pri.

SummaryType of study Type of DNA Preferred marker

Gene diversity & breeding system

nrDNA co-dominant markers: microsats, allozymes

Genotype diversity, clonality, individuality

nrDNA 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 all

Inter-specific hybridisation

nr + cp/mt DNA microsats, allozymes, RFLPs, AFLPs

Systematics (above sp. level)

nr + cp/mt DNA sequences, (AFLPs)