b. less precise, but more efficient at detecting variation

Transcription

1 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 B. Allozymes / Isozymes a. separation by size, shape, & charge b. less precise, but more efficient at detecting variation 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

2 1. Enzyme structure a. monomers (28%) O b. dimers (43%) OO c. tetramers (24%) 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 2. null alleles b. non-intermediate position c. unequal staining intensity 3. overlapping isozymes (isoloci) 4. lack of codominance 5. post-translational modification (ghost bands) F. To help us out: a. epigenetic causes b. protease degradation 1. conserved isozyme number known 2. isozyme localization a. organellar

3 b. cytosolic 3. genetic analysis a. controlled crosses II. Measuring Genetic Variation A. Sampling strategy b. progeny analysis of seed c. analysis of haploid tissue 1. plant material a. living b. fresh frozen -80 C 2. sample size 1- > is typical 3. outcrossing species a. higher expected gene flow b. fewers pops. but more individuals 4. inbreeding species B. Enzyme sampling a. lower expected gene flow b. more pops. (fewer individuals) cultivated Poaceae 50 loci (upper limit for the method) for range wide surveys: enzymes = 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 /

4 locus polymorphic locus Ap A 3. frequency of each allele p, q, r = # of each allele total number of alleles (2n) n = # of individuals 4. observed heterozygosity (H o ) = # of heterozygotes n 5. expected heterozygosity (H e ) = 2pq + 2pr + 2qr 6. fixation index (F) = 1 - H o He 1 = no heterozygotes 0 = H-W expectations -1 = all heterozygotes 1/ 0.5 III. Distribution of Genetic Diversity A. Hierarchical F-statistics (Wright, 1978) H I = heterozygosity of an individual in a subpopulation "average heterozygosity of all the genes in an individual" H S = expected heterozygosity in a randomly mating subpopulation H T = expected heterozygosity in a randomly mating total population F IS = H S - H I F IT = H T - H I H S H T F ST = H T - H S = 1 - (Hs / Ht ) H T ( 1-F IT ) = ( 1-F ST ) ( 1-F IS ) F IT = total deviation from expected frequencies under H-W equilibrium

5 range -1 to 1 F IS = deviations from H-W expectations within populations range -1 to 1 F ST = deviations from H-W expectations due to population subdivision "percentage of genetic variability due to differences among subpopulations" G ST = F ST for multiple alleles at locus (Nei, 1973) F ST = 0 "all subpops. in H-W equil. w/ the same allele frequencies & no subpopulation structuring" F ST = 0.20 "one migrant per generation" Nm = (1- F ST ) / 4 F ST "absolute # of individuals exchanged between subpops. / generation" F ST = 1.0 "all subpopulations are monomorphic and different from each other"

6 B. Examples 1. Kincaid s lupine a. G ST = b. consistent with recent population fragmentation 2. Astragalus spp. (annual) a. G ST = b. historical vs. proximal factors 3. pines a. G ST = 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" J xy I = (JxxJyy) 1/2 Jxx = p i 2 = probability that 2 randomly chosen are identical ( = the homozygosity in population X ) alleles Jyy = q i 2 = probability that 2 randomly chosen alleles are identical ( = the homozygosity in population Y)

7 Jxy = p i q i = 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. ( ) 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: Frequency parsimony Swofford & Berlocher 1987 Syst. Zool. 36: