natural group
length
colour
width
shape
venation
chromosome number
stems
leaves
UPGMA
ratio
multivariate analysis
number
phenotype
morphology
crossability
flowers
embryology
hairs
genotype
anatomy
secondary chemistry
parsimony
RAPDs
F-statistics
monophyletic
intron
nucleus
chloroplast
bootstrap
SNPs
RFLPs
gene
PCR
SSRs
spacer
Bayesian inference
paraphyletic
AFLPs
microsatellites
mitochondrion
maximum likelihood
Types of DNA
Plants have THREE genomes:
1) Nucleus
2) Chloroplast
3) Mitochondrion
T
A
C
G
C
G
T
A
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.
atpE
atpB
rbcL
large single
copy region
matK
rpl2
rpl2
16S
23S
16S
23S
small
scr
psbA
trnH
Mitochondrial DNA
Animal
Plant
14-26 kb
150-2500kb
Circular, usually
homogeneous among
cells
No recombination,
inheritance maternal
Set of different-sized circles, which
arise from processes that
interconvert between mother circle &
subgenomic circles
Mutation rates high at
sequence level;
substitutions
Rapid evolution in gene order
but slower at sequence level
(ca x100 slower than in
animals)
No recombination,
inheritance maternal
Main sources of DNA evidence




Control centres
 turn genes on & off 100
Genes
80
 single-copy
60
 multi-copy
40
20
 code for proteins
0
Inter-genic spacers
 non-coding sequences
between genes
Introns
 non-coding sequences
within genes
Non-coding
Coding
transposons & retroviruses
Gene structure
upstream
enhancer
promoter
TATA
box
exon 1
5’ UTR
exon 2
intron 1
exon 3
intron 2
• Exons are composed of
start, amino acid & stop
codons.
• Highly conserved regions.
• Useful at higher
taxonomic levels, e.g.
genus & above.
spacer
3’ UTR
• 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.
Multi-copy genes: rDNA
5.8S
25S
18S
IGS


ITS2
sometimes problem with concerted evolution.
Coding regions (nS) highly conserved


ITS1
Tandem repeats: 100s to 1000s of copies.
Nuclear genome: biparental inheritance.


25S
18S gene of soyabean shares 75% nucleotide
homology with yeast.
ITS & IGS regions highly variable.
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
Approaches
Phylogeny 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
Genepool & population
phenomena
st 0.447
st 0.468
st 0.555
st 0.390
st 0.287
st 0.289
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.
Fragment patterns in hybrids
nuclear DNA
probe
4
12
7
9
5
enzyme 1 fragments
AA
AB
BB
19 ---
enzyme 1
12
7
enzyme 2 fragments
AA
AB
BB

---
-----
14 ---
---
9
---
---
5
4 ---
-----
-----
-----

enzyme 2
Different patterns
are the result of
gains/losses of
restriction sites or
inversions.
Co-dominant in
nuclear DNA: good
for detecting
hybrids.
RAPD
Randomly Amplified Polymorphic DNA
gel
indiv A
A
B
--
--
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)
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
Microsatellites
(SSRs: Simple Sequence Repeats)
(GA)7
pri.
flanking
GAGAGAGAGAGAGA
pri.
flanking
GAGAGAGAGA
flanking
flanking
pri.
pri.
(GA)5






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
Summary
Type 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 (genepool 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)