I. Proteins
A. Electrophoretic detection
1. SDS PAGE
polyacrylamide gel electrophoresis
a. denaturing conditions
b. separation by size (MW)
2. Isoelectric focusing
a. pH gradient
b. separation by charge
3. Starch gel electrophoresis
a. separation by size, shape, & charge
b. less precise, but more efficient at detecting variation
B. Allozymes / Isozymes
Allele = Different forms of a gene occurring at a particular locus on
homologous chromosomes
Allozyme = Different molecular forms of an enzyme that are encoded by
different alleles at a particular locus
Isozyme = Different molecular forms of an enzyme that are encoded at
different loci
C. Rationale for genetic analysis of allozymes
1. Mendelian inheritance
2. Codominant expression
3. Complete penetrance
4. Variation in mobility results from amino
acid change
D. Genetic interpretation
1. Enzyme structure
a. monomers (28%)
b. dimers (43%)
c. tetramers (24%)
O
OO
88
2. Homozygotes (one allele)
-> Homomeric Protein
3. Heterozygote (two alleles)
-> Heteromeric Protein
4. Interlocus Heteropolymers
(two isozymes interract)
E. Interpretational Difficulties
1. non-random heteropolymer formation
a. absence of interlocus heteropolymers
organellar vs. cytoplasmic expression
b. non-intermediate position
c. unequal staining intensity
2. null alleles
3. overlapping isozymes (isoloci)
4. lack of codominance
5. post-translational modification (ghost bands)
a. epigenetic causes
b. protease degradation
F. To help us out:
1. conserved isozyme number known
2. isozyme localization
a. organellar
b. cytosolic
3. genetic analysis
a. controlled crosses
b. progeny analysis of seed
c. analysis of haploid tissue
II. Measuring Genetic Variation
A. Sampling strategy
1. plant material
a. living
b. fresh frozen -80 C
2. sample size 1- >100 20-60 is typical
3. outcrossing species
a. higher expected gene flow
b. fewers pops. but more individuals
4. inbreeding species
a. lower expected gene flow
b. more pops. (fewer individuals)
B. Enzyme sampling
cultivated Poaceae 50 loci (upper limit for the method)
for range wide surveys:
10-12 enzymes = 14-24 loci
gene flow studies in a pop.: 1-3 loci, but polymorphic
C. Levels of variation
1. Percent polymorphic loci
P
0.99 or 0.95 level
2. Average number of alleles /
locus
polymorphic locus
A
Ap
3. frequency of each allele
p, q, r
=
# of each allele
total number of alleles (2n)
n = # of individuals
4. observed heterozygosity (Ho)
=
# of heterozygotes
n
5. expected heterozygosity (He)
6. fixation index
=
2pq + 2pr + 2qr
(F)
=
1-
1 = no heterozygotes
0 = H-W expectations
-1 = all heterozygotes 1/ 0.5
Ho
He
III. Distribution of Genetic Diversity
A. Hierarchical F-statistics (Wright, 1978)
HI = heterozygosity of an individual in a subpopulation
"average heterozygosity of all the genes in an individual"
HS = expected heterozygosity in a randomly mating subpopulation
HT = expected heterozygosity in a randomly mating total population
FIS = HS - HI
--------HS
FST =
FIT = HT - HI
--------HT
HT - HS
---------- = 1 - (Hs / Ht )
HT
( 1-FIT ) = ( 1-FST ) ( 1-FIS )
FIT = total deviation from expected frequencies under H-W equilibrium
range -1 to 1
FIS = deviations from H-W expectations within populations
range -1 to 1
FST = deviations from H-W expectations due to population subdivision
"percentage of genetic variability due to differences among subpopulations"
GST = FST for multiple alleles at locus (Nei, 1973)
FST = 0
FST = 0.20
"all subpops. in H-W equil. w/ the same allele frequencies & no
subpopulation structuring"
"one migrant per generation"
Nm = (1- FST ) / 4 FST
"absolute # of individuals exchanged between subpops. / generation"
FST = 1.0
"all subpopulations are monomorphic and different from each other"
B. Examples
1. Kincaid’s lupine
a. GST = 0.119
b. consistent with recent population fragmentation
2. Astragalus spp. (annual)
a. GST = 0.0 - 0.725
b. historical vs. proximal factors
3. pines
a. GST = 0.0 - 0.10
b. gene conservation and reforestation
C. Non-neutrality of allozyme markers
1. Known from particular loci (e.g. Adh, Aat, Udp)
2. solution: use multiple independent loci
IV. Genetic Identity (Similarity)
A.
Nei (1972) "the probability that a randomly chosen allele from each of 2
different populations will be identical, relative to the probability that 2 randomly
chosen alleles from the same population will be identical"
I=
J xy
------------(JxxJyy) 1/2
Jxx = ∑ pi2 = probability that 2 randomly chosen
are identical
(=
the homozygosity in population X )
alleles
Jyy = ∑ qi2 = probability that 2 randomly chosen
are identical
( = the homozygosity in population Y)
alleles
Jxy =
∑ piqi = probability that 2 alleles are
identical when one is chosen from
pop. X and one from pop. Y
Varies from 0 (no alleles in common) to 1 (identity)
B. Plant Averages (Crawford, 1990)
I = 0.95 for conspecific pops. (.90 -1.0)
I = 0.67 for congeneric spp.
C. Standard Genetic Distance:
D = -ln I
"mean number of codon substitutions per locus"
varies between 0 [no distance] to infinity
D. Phenetic analysis of genetic distance
1. UPGMA or Neighbor Joining
2. PCA principle coordinates analysis
E. Cladistic analysis of allozymes
limited by tokogeny
1. Alleles as characters [binary chax.]
2. Loci as characters [multistate chax.]
problems of polymorphism parsimony
Kornet & Turner 1999 Syst. Biol. 48: 365-379.
3. Frequency parsimony
Swofford & Berlocher 1987 Syst. Zool. 36:293-325.